Antibody-drug conjugates containing toxins bearing polar groups and uses thereof

Abstract

The present disclosure relates to drugs and toxins functionalized with at least one saccharide, sulfate, or sulfonate; drug conjugates comprising said drug or toxin and a cleavable linker; and targeting conjugates comprising said drug or toxin, a cleavable linker, and a targeting moiety. The present disclosure also relates to methods of treating cancer, autoimmune diseases, and inflammatory diseases using the compounds and conjugates of the present disclosure.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 292,101, filed December 21, 2021, the entirety of which is incorporated herein by reference. [Background technology]

[0002] Cleavable drug conjugates have the ability to combine the binding specificity of antibodies or other targeting groups with the potency of chemotherapeutic agents. This technology enhances the efficacy of therapeutic agents and reduces the risk of side effects, as targeting allows drugs to be precisely delivered to targeted cancer cells and released under specific conditions while minimizing collateral damage to healthy cells. However, traditional therapies have shown non-selective drug uptake into normal and cancer cells, resulting in limited therapeutic efficacy. Non-selective uptake is mainly due to the hydrophobicity of the linker-drug, and research has been conducted to reduce the hydrophobicity of the linker-drug, but so far the results have been limited. Summary of the Invention

[0003] Disclosure Summary In certain embodiments, the compound represented by formula (VII) or (VIII): [ka] Alternatively, provided herein is a drug conjugate comprising a pharma- ceutically acceptable salt thereof, and a linker group; During the ceremony: A is a heterocycle; Each R a ' and R b ' is independently halogen, amino, hydroxyl, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; 2 Germinal R b’are optionally joined together to form oxo or =CH 2 or two R b ' together with the intervening atoms optionally completes a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R c ' is sulfonate, sulfate, hydroxyl, amino, or thiol; R d ' is -L"-Gly, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; However, at least one R c ' is a sulfonate or sulfate, or at least one R d '-L ’’’ -Gly; R e ' is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; m is an integer selected from 0 to 3; n is an integer selected from 0 to 8 depending on the valence; Ring Cy is selected from aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; [ka] is a single or double bond; X' is a halogen; X″ is —NR—, —S—, or —O—; R is hydrogen or alkyl; Each R a " and R b " is independently halogen, amino, hydroxyl, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, alkenyl, alkynyl, =O, carboxyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L''') r -X”-Gly; d is an integer selected from 0 to 4; r is an integer from 0 to 1; each L" is a bond or a linker; R e " is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; p is an integer selected from 0 to 4; DBD is the DNA binding domain; L″ is a bond or a linker; and Gly is a monosaccharide, disaccharide, or oligosaccharide.

[0004] In certain aspects, provided herein are targeted drug conjugates, including drug conjugates comprising a compound of the present disclosure, a linker group, and a targeting moiety.

[0005] In certain embodiments, the targeted drug conjugate has formula (XII), (XIII) or (XIV): [ka] or a pharma- ceutically acceptable salt thereof; where TM is a targeting moiety.

[0006] In certain aspects, provided herein are drug conjugates comprising an active agent and a linking group; wherein the active agent is substituted with a polar group. In some embodiments, the polar group is selected from a saccharide, a sulfate, or a sulfonate.

[0007] In some embodiments, the drug conjugate comprising an active agent and a linking group has the formula (I): [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: Z' is a coupling group; Ar is aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; Y'1 is an N, O, or S atom that is arranged to bond to TG when y is 1 -(CR b 2 ) y N(R a )-, -(CR b 2 ) y O- or -(CR b 2 ) y S-, When activated, TG reacts with SO 2 In response to (Q) q -(L') w Replace X-SO 2 and a trigger group that generates an N, O, or S atom capable of forming a 5- or 6-membered ring containing an intervening atom of Ar; X is -O-, -C(R b ) 2 - or -N(R c )-and; L', if present, is a heteroatom selected from O, S, and N. 2 The spacer portion binds to L' and SO 2 is selected such that cleavage of the bond between facilitates release of the active agent; each Q is independently a saccharide, sulfate, or sulfonate substituted active agent; q is an integer selected from 1 to 3; w and y are each independently 0 or 1; and R a , R b and R c are each independently hydrogen or C 1-6 alkyl; or two R b complete a 3- to 5-membered ring together with the atoms to which they are attached; However, when w is 0, q is 1.

[0008] In certain embodiments, the drug conjugate comprises a compound of formula (IIIa) or (IIIb): [ka] Or a pharma- ceutically acceptable salt thereof.

[0009] In a further embodiment, the drug conjugate is a compound represented by formula (V): [ka] Or a pharma- ceutically acceptable salt thereof.

[0010] In a further embodiment, the drug conjugates of the present disclosure include a targeting moiety conjugated to any one of the drug conjugates of formula (VI): [ka] Provided herein is a targeted drug conjugate comprising: where TM is a targeting moiety.

[0011] In a further embodiment, the drug conjugate of the present disclosure comprises a targeting moiety conjugated to the formula (VIb): [ka] Provided herein is a targeted drug conjugate comprising: During the ceremony: TM is a targeting moiety; R is hydrogen or a hydroxy protecting group; X is -C(O)-, -NH-, -O-, or -S-; Q is a sugar, sulfate, or sulfonate substituted active agent; T is [ka] and; n is an integer selected from 0 and 1; Y is hydrogen, haloC 1 -C 8alkyl, halogen, cyano, or nitro; z is an integer selected from 1 to 3, and when z is an integer of 2 or more, Y may be the same or different; z1 is an integer selected from 0 or 1; W 1 teeth [ka] and; W 2 teeth [ka] and; W a1 and W a2 are each independently -NH-, -C(=O)-, or -CH 2 - and; W a3 and W a4 are each independently -NH-, -C(=O)-, or -CH 2 -, -C(=O)NH-, -NHC(=O)-, or triazolylene; W b1 is an amide bond or a triazolylene; L is W a2 an amino acid, peptide, or amide bond as a linker connecting a and Z; Z is a single bond, -W a5 -(CH 2 ) a2 -W b2 -(CH 2 ) a3 -W a6 -, or -W a7 -(CH 2 ) a4 -CR'R”-X''-; R' is C 1 -C 8 Alkyl or TM-W a8 -Q 3 -W c1 -(CH 2 ) a5 - and; R” is TM-W a8-Q 3 -W c1 -(CH 2 ) a5 - and; Q 1 and Q 3 are each independently -(CH 2 ) a6 -(X 1 CH 2 CH 2 ) b1 -(CH 2 ) a7 - and; X 1 and X 3 are each independently -O-, -S-, -NH-, or H-CH 2 - and; X'' is -NHC(=O)-(CH 2 ) a8 -W a9 - or -C(=O)NH-(CH 2 ) a8 -W a9 - and; 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-; Q 2 is a linear or branched, saturated or unsaturated alkylene having 1 to 50 carbon atoms that satisfies any one of the following (i) to (iii): (i) at least one -CH in the alkylene 2 - is substituted with one or more heteroatoms selected from -NH-, -C(=O), -O-, and -S-; (ii) the alkylene group contains at least one arylene or heteroarylene group; (iii) Alkylene is C 1 -C 20Alkyl, C 6 -C 20 Aryl C 1 -C 8 Alkyl, -(CH 2 ) s1 COOR 3 , -(CH 2 ) s1 COR 3 , -(CH 2 ) s2 CONR 4 R 5 , and -(CH 2 ) s2 NR 4 R 5 is further substituted with one or more selected from the group consisting of: The arylene or heteroarylene of (ii) above may be further substituted with nitro; R 3 , R 4 , and R 5 are each independently hydrogen or C 1 -C 15 is alkyl; X 2 is -O-, -S-, -NH-, or -CH 2 - and; U 1 is attached to B' at the asterisk (*) by a linking group selected from the following structures: [ka] R is C 1 -C 10 Alkyl, C 6 -C 20 Aryl or C 2 -C 20 is heteroaryl; TM and B' are each independently a ligand or protein that has the property of selectively targeting a drug to a particular organ, tissue or cell, i.e., binding to a receptor; a1, a2, a3, a4, a5, a6, a8, b1, p1, p2, p3, and p4 each independently represent an integer selected from 1 to 10; a7, y, s1, s2, and s4 are each independently an integer selected from 0 to 10; and R 1 and R 2 are each independently hydrogen, C 1 -C 8 Alkyl or C 3 -C 8 It is cycloalkyl.

[0012] In a further aspect, provided herein is a targeted drug conjugate comprising a targeting moiety conjugated to a drug conjugate of the present disclosure, the targeting moiety being of formula (VIc): [ka] During the ceremony, TM is the targeting moiety; G is a glucuronic acid moiety or a derivative thereof; Q is a sugar, sulfate, or sulfonate substituted active agent; W is an electron withdrawing group; Z is hydrogen, C 1 -C 8 is alkyl, halogen, cyano, or nitro; n is an integer selected from 1 to 3, and when n is an integer of 2 or more, each Z(s) is the same or different from each other; L is a linker connecting TM and W; and R 1 and R 2 are each independently hydrogen, C 1 -C 8 Alkyl or C 3 -C 8 It is cycloalkyl.

[0013] In certain aspects, provided herein is a targeted drug conjugate of formula (VId) comprising a targeting moiety conjugated to a drug conjugate of the present disclosure: TM-L 1 -(A a -W w -Y y -Q 1-4 )p (VId); Wherein TM is a targeting moiety; L 1 is the ligand moiety; Q is a sugar, sulfate, or sulfonate substituted active agent; -A a -W w -Y y - is a linker moiety; A is an optional stretcher section; a is an integer selected from 0 to 3; Each W is independently a glucuronide unit having one of the following formulas: [ka] Su is a sugar moiety; Each R is independently hydrogen, halogen, -CN, or -NO 2 and; w is an integer selected from 1 to 2; Y is an optional self-immolative spacer moiety; y is an integer selected from 0 to 2; and p is an integer selected from 1 to 20. In yet a further aspect, provided herein is a compound of formula (VII) or (VIII): [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony, A is a heterocycle; Each R a ' and R b ' is independently halogen, amino, hydroxyl, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; 2 Germinal R b ' may optionally be taken together to represent oxo or =CH2 or two R b ' together with the intervening atoms optionally completes a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R c ' is sulfonate, sulfate, hydroxyl, amino, or thiol; R d ' is -L"-Gly, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; However, at least one R c ' is a sulfonate or sulfate, or at least one R d '-L ” -Gly; R e ' is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; m is an integer selected from 0 to 3; n is an integer selected from 0 to 8 depending on the valence; Ring Cy is selected from aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; [ka] is a single or double bond; X' is a halogen; X″ is -NR-, -S-, or -O-; Each R a " and R b " is independently halogen, amino, hydroxyl, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, alkenyl, alkynyl, =O, carboxyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L''') r -X”-Gly; d is an integer selected from 0 to 4; r is an integer selected from 0 to 1; each L" is a bond or a linker; R e " is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; p is an integer selected from 0 to 4; DBD is the DNA binding domain; L″ is a bond or a linker; and Gly is a monosaccharide, disaccharide, or oligosaccharide.

[0014] In certain embodiments, the compound has formula (VII): [ka] Or a pharma- ceutically acceptable salt thereof.

[0015] In a further embodiment, the compound is a compound of formula (VIII): [ka] Or a pharma- ceutically acceptable salt thereof.

[0016] In certain aspects, provided herein is a drug conjugate comprising any one of the disclosed compounds and a linker group. In certain embodiments, the drug conjugate comprises a compound of formula (IX), (X), or (XI): [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: Z' is a coupling group; Ar is aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; Y' is -(CR b 2 ) y N(R a )-, -(CR b 2 )y O- or -(CR b 2 ) y S- and y is 1, an N, O, or S atom is positioned to be bonded to TG; When activated, TG reacts with SO 2 In response to (Q) q -(L') w Replace X-SO 2 and a trigger group that generates an N, O, or S atom capable of forming a 5- to 6-membered ring containing an intervening atom of Ar; X is -O-, -C(R b ) 2 - or -N(R c )-and; L', if present, is a heteroatom selected from O, S, and N. 2 The spacer portion binds to L' and SO 2 is selected such that cleavage of the bond between facilitates release of the active agent; w is an integer selected from 0 to 1; r is an integer from 0 to 1; Z 2 is a linking group; Z 3 is a linking group; R a , R b and R c are each independently hydrogen or lower alkyl; y is an integer selected from 0 to 1; t is an integer from 1 to 5; and e is an integer from 1 to 5.

[0017] In a further aspect, provided herein is a targeted drug conjugate comprising any one of the drug conjugates provided herein and a targeting moiety. In certain embodiments, the drug conjugate is a compound of formula (XII), (XIII) or (XIV): [ka] or a pharma- ceutically acceptable salt thereof; where TM is a targeting moiety.

[0018] In yet another aspect, the present disclosure provides a method for treating cancer, comprising administering to a subject in need thereof any one of the compounds, drug conjugates, targeted drug conjugates, or pharmaceutical compositions provided herein.In certain embodiments, the cancer is selected from leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung cancer, bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, bladder cancer, kidney cancer, renal pelvis cancer, oral cancer, pharyngeal cancer, uterine cancer, or melanoma.

[0019] In yet a further aspect, the present disclosure provides a method for treating an autoimmune or inflammatory disease, comprising administering to a subject in need thereof any one of the compounds, drug conjugates, targeted drug conjugates, or pharmaceutical compositions provided herein.In certain embodiments, the autoimmune or inflammatory disease is selected from B cell-mediated autoimmune or inflammatory diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), idiopathic thrombocytopenic purpura (ITP), Waldenstrom's hypergammaglobulinemia, Sjogren's syndrome, multiple sclerosis (MS), or lupus nephritis. [Brief description of the drawings]

[0020] [Figure 1A] 1 shows the in vivo efficacy of T-2-AB, T-3-AB, and T-4-AB in the JIMT-1 xenograft model. [Figure 1B] 1 shows the in vivo efficacy of T-103-AB, T-104-AB, T-116-AB, and T-117-AB in the JIMT-1 xenograft model. [Figure 2A] FIG. 1 shows plasma stability of seco-MCBI-HAI duocarmycin payloads, where A shows the unsubstituted toxin-linker conjugate and B shows the sugar-substituted toxin-linker conjugate. [Figure 2B]FIG. 1 shows plasma stability of seco-DUBA duocarmycin payloads, where C shows the unsubstituted toxin-linker conjugate and D shows the sugar-substituted toxin-linker conjugate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Provided herein are compounds, drug conjugates, and targeted drug conjugates that are useful for the treatment of cancer and / or autoimmune or inflammatory diseases.The compounds, drug conjugates, and targeted drug conjugates can generally be derived from toxin payloads modified with sugars, sulfates, or sulfonates.The compounds, drug conjugates, and targeted drug conjugates can significantly reduce the non-specific uptake of drugs.

[0022] Certain components of the technology disclosed herein, including cleavable linker technology and targeting moieties, are further described in WO2019 / 008441, WO2019 / 229536, WO2020 / 141459, WO2020 / 141460, PCT / IB2021 / 000445, U.S. Pat. No. 16,472,983, U.S. Pat. No. 14,898,932, and U.S. Pat. No. 11,996,009, each of which is incorporated by reference in its entirety.

[0023] definition Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the nomenclatures used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

[0024] The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification, unless otherwise indicated. See, for example, "Principles of Neural Science", McGraw-Hill Medical, New York, NY (2000); Motulsky, "Intuitive Biostatistics", Oxford University Press, Inc. (1995); Lodish et al., "Molecular Cell Biology, 4th ed.", WH Freeman & Co., New York (2000); Griffiths et al., "Introduction to Genetic Analysis, 7th ed.", WH Freeman & Co., NY (1999); and Gilbert et al., "Developmental Biology, 6th ed.", Sinauer Associates, Inc., Sunderland, MA (2000).

[0025] Chemical terms used herein, unless otherwise defined herein, are used according to conventional usage in the art, as exemplified in "The McGraw-Hill Dictionary of Chemical Terms", Parker S., Ed., McGraw-Hill, San Francisco, Calif. (1985).

[0026] All of the above, as well as any other publications, patents, and published patent applications mentioned in this application are specifically incorporated herein by reference. In case of conflict, the present specification, including its specific definitions, will control.

[0027] The term "agent" is used herein to refer to a compound (such as an organic or inorganic compound, a mixture of compounds), a biopolymer (such as a nucleic acid, an antibody, including portions thereof, as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portions thereof, such as a peptide, lipid, carbohydrate), or an extract made from biological materials such as a cell or tissue of a bacteria, a plant, a fungus, or an animal (especially a mammal). Agents include, for example, agents of known structure and agents of unknown structure. The ability of such agents to inhibit AR or promote AR degradation may make them suitable as "therapeutic agents" in the methods and compositions of the present disclosure.

[0028] "Patient," "subject," or "individual" are used interchangeably and refer to either a human or non-human animal. These terms include mammals, such as humans, primates, livestock animals (including cows, pigs, etc.), companion animals (e.g., dogs, cats, etc.), and rodents (e.g., mice and rats).

[0029] "Treating" a condition or patient refers to taking measures to obtain beneficial or desired results (including clinical results). As used herein, and as is well understood in the art, "treatment" is an approach to obtain beneficial or desired results (including clinical results). Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether detectable or undetectable, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), prevention of disease spread, delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or total). "Treatment" can also mean prolonging survival compared to the expected survival if not receiving treatment.

[0030] The term "prevention," when used in reference to a condition (e.g., a disease such as local recurrence (e.g., pain), cancer, a complex syndrome such as heart failure, or any other medical condition), is art-recognized and well understood in the art and includes administration of a composition that reduces the frequency of or delays the onset of symptoms of a medical condition in a subject compared to subjects to which the composition is not administered. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment compared to an untreated control population, and / or delaying the onset of detectable cancerous growths in a treated population compared to an untreated control population, for example, by a statistically and / or clinically significant amount.

[0031] "Administering" or "administration" of a substance, compound, or agent to a subject can be accomplished by one of a variety of methods known to those of skill in the art. For example, the compound or agent may be administered intravenously, intraarterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (e.g., by absorption through the skin's channels). The compound or agent may also be suitably introduced by rechargeable or biodegradable polymeric or other devices (e.g., patches and pumps) or formulations that provide sustained, slow, or controlled release of the compound or agent. Administration may also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0032] The appropriate method of administering a substance, compound, or agent to a subject also depends, for example, on the age and / or health of the subject and the chemical and biological properties of the compound or agent (e.g., solubility, digestibility, bioavailability, stability, and toxicity). In some embodiments, the compound or agent is administered to the subject orally, for example, by ingestion. In some embodiments, the orally administered compound or agent is in a sustained or slow release formulation or is administered using such a slow or sustained release device.

[0033] As used herein, the term "co-administration" refers to any administration form of two or more different therapeutic agents, where the second agent is administered while the previously administered therapeutic agent is active in the body (e.g., the two agents are active simultaneously in the patient, which may include a synergistic effect of the two agents). For example, the different therapeutic compounds may be administered in the same formulation or in separate formulations, and may be administered simultaneously or sequentially. Thus, an individual undergoing such treatment may benefit from the combined effect of the different therapeutic agents.

[0034] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or agent is an amount that, when administered to a subject, has the intended therapeutic effect of the drug or agent. The full therapeutic effect does not necessarily occur in one dose, but may occur only after a series of doses. Thus, a therapeutically effective amount may be administered in one or more doses. The exact effective amount required for a subject depends, for example, on the subject's size, health and age, and the nature and extent of the condition being treated, e.g., cancer or MDS. Those skilled in the art can easily determine the effective amount for a given situation by routine experimentation.

[0035] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes examples when the event or circumstance occurs as well as examples when it does not occur. For example, "optionally substituted alkyl" refers to the fact that the alkyl can be substituted as well as the fact that the alkyl is not substituted.

[0036] It is understood that the substituents and substitution patterns on the compounds of the present disclosure can be selected by those skilled in the art to provide chemically stable compounds that can be easily synthesized from readily available starting materials by techniques known in the art, as well as those methods described below. When a substituent is itself substituted with two or more groups, it is understood that these multiple groups can be on the same carbon or on different carbons, so long as a stable structure results.

[0037] As used herein, the term "optionally substituted" means any of the following: hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH 2 -O-alkyl, -OP(O)(O-alkyl) 2 or -CH 2 -OP(O)(O-alkyl) 2 "Optionally substituted" refers to the replacement of 1-6 hydrogen atoms in a given structure with a specified substituent, including, but not limited to, 1-4 hydrogen atoms in a given structure with the above substituent. More preferably, 1-3 hydrogen substituents are replaced with the above substituent. It is understood that the substituents may be further substituted.

[0038] As used herein, the term "alkyl" includes, but is not limited to, C 1 -C 10 Straight chain alkyl group or C 1 -C 10 It refers to saturated aliphatic groups, including branched chain alkyl groups. Preferably, the "alkyl" group is 1 -C 6 Straight chain alkyl group or C 1 -C 6 It refers to a branched chain alkyl group. Most preferably, the "alkyl" group is 1 -C 4 Straight chain alkyl group or C 1 -C 4It refers to a branched chain alkyl group. Examples of "alkyl" include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neo-pentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl, or 4-octyl. An "alkyl" group may be optionally substituted.

[0039] The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0040] The term "acylamino" is art-recognized and refers to an amino group substituted with an acyl group and may be represented, for example, by the formula hydrocarbylC(O)NH-.

[0041] The term "acyloxy" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)O-, preferably alkylC(O)O-.

[0042] The term "alkoxy" refers to an alkyl group having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0043] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.

[0044] The term "alkyl" refers to saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In preferred embodiments, a straight-chain or branched-chain alkyl group has 30 or fewer carbon atoms in its backbone (e.g., C for straight chain). 1-30 , C for branched chain 3-30), more preferably no more than 20. The term "lower alkyl" refers to alkyl groups having 1 to 6 carbon atoms.

[0045] Furthermore, the term "alkyl," as used throughout the specification, examples, and claims, is intended to include both unsubstituted and substituted alkyl groups, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl.

[0046] "C x-y " or "C x -C y The term "alkyl" when used in combination with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups containing x to y carbons in the chain. 0 Alkyl denotes hydrogen when the group is in a terminal position and a bond when the group is internal. 1-6 An alkyl group, for example, contains 1 to 6 carbon atoms in the chain.

[0047] The term "alkylamino," as used herein, refers to an amino group substituted with at least one alkyl group.

[0048] The term "alkylthio," as used herein, refers to a thiol group substituted with an alkyl group and may be represented by the general formula alkylS-.

[0049] As used herein, the term "amide" refers to the group [ka] In the formula, R 9 and R 10 each independently represents hydrogen or a hydrocarbyl group, or R 9 and R 10 together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure.

[0050] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines and their salts, for example, a moiety that may be represented by: [ka] In the formula, R 9 , R 10 , and R 10 Each ' independently represents hydrogen or a hydrocarbyl group, or R 9 and R 10 together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure.

[0051] As used herein, the term "aminoalkyl" refers to an alkyl group substituted with an amino group.

[0052] The term "aralkyl," as used herein, refers to an alkyl group substituted with an aryl group.

[0053] The term "aryl" as used herein includes substituted or unsubstituted monocyclic aromatic groups 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, where two or more carbons are common to two adjacent rings, and at least one of the rings is aromatic, for example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0054] The term "carbamate" is art-recognized and refers to the group: [ka] In the formula, R 9 and R10 independently represent hydrogen or a hydrocarbyl group.

[0055] The term "carbocyclylalkyl," as used herein, refers to an alkyl group substituted with a carbocycle group.

[0056] The term "carbocycle" includes 5-7 membered monocyclic rings and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated, and aromatic rings. Carbocycles include bicyclic molecules in which one, two, or more than two atoms are shared between the two rings. The term "fused carbocycle" refers to a bicyclic carbocycle in which each ring shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated, and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated, and aromatic bicyclic rings is included in the definition of carbocycle, as long as valences permit. Exemplary "carbocycles" include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene, and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene, and bicyclo[4.1.0]hept-3-ene. A "carbocycle" may be substituted at any one or more positions that may bear a hydrogen atom.

[0057] The term "carbocyclylalkyl," as used herein, refers to an alkyl group substituted with a carbocycle group.

[0058] The term "carbonate" is art-recognized and includes the moiety -OCO 2 - refers to the group.

[0059] The term "carboxy" as used herein refers to a group of the formula -CO2 H.

[0060] The term "ester" as used herein means an ester group consisting of -C(O)OR 9 R 9 represents a hydrocarbyl group.

[0061] The term "ether" as used herein refers to a hydrocarbyl group linked to another hydrocarbyl group via oxygen. Thus, the ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. Ethers can be symmetrical or asymmetrical. Examples of ethers include, but are not limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethers include "alkoxyalkyl" groups, which can be represented by the general formula alkyl-O-alkyl.

[0062] The terms "halo" and "halogen" as used herein mean halogen and include chloro (Cl), fluoro (F), bromo (Br), and iodo (I).

[0063] The terms "hetaralkyl" and "heteroaralkyl," as used herein, refer to an alkyl group substituted with a hetaryl group.

[0064] The terms "heteroaryl" and "hetaryl" include substituted or unsubstituted aromatic monocyclic ring structures, preferably 5-7 membered rings, more preferably 5-6 membered rings, 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 in which the other cyclic rings can be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0065] The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0066] The term "heterocyclylalkyl," as used herein, refers to an alkyl group substituted with a heterocycle group.

[0067] The terms "heterocyclyl", "heterocycle", and "heterocyclic" refer to a substituted or unsubstituted non-aromatic ring structure, preferably a 3- to 10-membered ring, more preferably a 3- to 7-membered ring, which ring structure contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heterocyclyl" and "heterocyclic" also include polycyclic ring systems having two or more cyclic rings, where two or more carbons are common to two adjacent rings and at least one of the rings is heterocyclic, for example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

[0068] The term "hydrocarbyl" as used herein refers to a group that is bonded through a carbon atom that does not have =O or =s substituents, and that usually has at least one carbon-hydrogen bond and a predominantly carbon backbone, but may optionally contain heteroatoms. Thus, groups such as methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered hydrocarbyl for the purposes of this application, while substituents such as acetyl (having =O substituent on the bonded carbon) and ethoxy (bonded through an oxygen rather than a carbon) are not considered hydrocarbyl. Hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

[0069] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group.

[0070] The term "lower" when used in conjunction with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups having 10 or fewer atoms in the substituent, preferably 6 or fewer atoms. For example, "lower alkyl" refers to an alkyl group having 10 or fewer carbon atoms, preferably 6 or fewer carbon atoms. In certain embodiments, an acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituent as defined herein is a lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, respectively, whether appearing alone or in combination with other substituents, such as in the descriptions of hydroxyalkyl and aralkyl (where, for example, atoms in an aryl group are not counted when counting the carbon atoms of an alkyl substituent).

[0071] The terms "polycyclyl," "polycycle," and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl) in which two or more atoms are common to two adjacent rings, e.g., the rings are "fused rings." Each ring of the polycycle can be substituted or unsubstituted. In certain embodiments, each ring of the polycycle contains from 3 to 10 atoms, preferably from 5 to 7 atoms, within the ring.

[0072] The term "sulfate" is art-recognized and includes the moiety -OSO 3 H group, or a pharma- ceutically acceptable salt thereof.

[0073] The term "sulfonamide" is art-recognized and can be represented by the general formula [ka] is represented by In the formula, R 9 and R 10 independently represent hydrogen or hydrocarbyl.

[0074] The term "sulfoxide" is art-recognized and refers to the group --S(O)--.

[0075] The term "sulfonate" is art-recognized and refers to a compound having the group SO 3 H, or a pharma- ceutically acceptable salt thereof.

[0076] The term "bisulfite" is art-recognized and refers to the group -OS(O)OH, or a pharma- ceutically acceptable salt thereof.

[0077] The term "sulfate" is art-recognized and includes the moiety -OSO 3 H group, or a pharma- ceutically acceptable salt thereof.

[0078] The term "sulfone" is art-recognized and refers to the group -S(O) 2 - refers to.

[0079] The term "substituted" refers to a moiety having a substituent replacing a hydrogen on one or more carbons of the backbone. It will be understood that "substituted" or "substituted with" includes the implicit proviso that such substitution results in a stable compound that does not spontaneously undergo transformation, e.g., by rearrangement, cyclization, elimination, and the like, subject to the permissible valence of the substituted atom and substituent. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In one broad aspect, 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 disclosure, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valence of the heteroatom. Substituents include any of the substituents described herein, 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. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if desired.

[0080] The term "thioalkyl," as used herein, refers to an alkyl group substituted with a thiol group.

[0081] The term "thioester" as used herein refers to the group -C(O)SR9 , or -SC(O)R 9 where R 9 represents a hydrocarbyl.

[0082] The term "thioether" as used herein is the equivalent of an ether where the oxygen is replaced with a sulfur.

[0083] The term "urea" is art-recognized and has the general formula: [ka] may be expressed as In the formula, R 9 and R 10 independently represent hydrogen or hydrocarbyl.

[0084] As used herein, the term "modulate" includes inhibiting or suppressing a function or activity (such as cell proliferation), as well as enhancing a function or activity.

[0085] "Pharmaceutically acceptable salt" or "salt" is used herein to refer to an acid addition salt or a base addition salt which is suitable or compatible with the treatment of a patient.

[0086] The term "pharmaceutical acceptable acid addition salts" as used herein refers to any non-toxic organic or inorganic salt of any base compound represented by formula I. Examples of inorganic acids that form suitable salts include hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Examples of organic acids that form suitable salts include mono-, di-, and tricarboxylic acids, such as glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, benzoic acid, phenylacetic acid, cinnamic acid, and salicylic acid, as well as sulfonic acids such as p-toluenesulfonic acid and methanesulfonic acid. Either mono- or di-acid salts may be formed, and such salts may exist in either hydrated, solvated, or substantially anhydrous form. In general, acid addition salts of compounds of formula I are more soluble in water and various hydrophilic organic solvents than their free base forms, and generally exhibit higher melting points. The selection of an appropriate salt will be known to one of skill in the art. Other pharmaceutically unacceptable salts, such as oxalates, may be used for laboratory use or for subsequent conversion to a pharmaceutically acceptable acid addition salt, such as for isolating the compounds of the present disclosure.

[0087] As used herein, the term "pharmaceutically acceptable base addition salt" refers to any non-toxic organic base addition salt or non-toxic inorganic base addition salt of any acid compound represented by formula I or any of its intermediates. Examples of inorganic bases that form suitable salts include lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or barium hydroxide. Exemplary organic bases that form suitable salts include aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine, and picoline or ammonia. The selection of suitable salts is known to those skilled in the art.

[0088] Many of the compounds useful in the methods and compositions of the present disclosure have at least one stereocenter in their structure. This stereocenter may exist in either R or S configuration, and the R and S designations are used according to the rules described in Pure Appl.Chem.(1976),45,11-30. The present disclosure contemplates all stereoisomeric forms, such as enantiomeric and diastereomeric forms, of the compounds, salts, prodrugs, or mixtures thereof, including all possible mixtures of stereoisomers. For example, see WO01 / 062726.

[0089] In certain embodiments, the compound of the present disclosure may be racemic.In certain embodiments, the compound of the present disclosure may be enriched with one enantiomer.For example, the compound of the present disclosure may have enantiomeric excess of more than about 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or even more.

[0090] As is commonly understood in the art, a single bond drawn without stereochemistry does not indicate the stereochemistry of the compound. The compound of formula I provides an example of a compound where the stereochemistry is not indicated.

[0091] In certain embodiments, the compositions or compounds of the present disclosure may be enriched to provide mainly one enantiomer of a compound. An enantiomerically enriched composition or compound may, for example, contain at least 60 mole percent of one enantiomer, or more preferably at least 75, 90, 95, or even 99 mole percent. In certain embodiments, a compound enriched in one enantiomer is substantially free of other enantiomers, where substantially free means that the substance in question accounts for, for example, less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% compared to the amount of other enantiomers in the composition or compound mixture. For example, if a composition or compound contains 98 grams of a first enantiomer and 2 grams of a second enantiomer, it is considered to contain 98 mole percent of the first enantiomer and only 2% of the second enantiomer.

[0092] Furthermore, certain compounds containing alkenyl groups can exist as Z (zusammen (together)) or E (entgegen (opposite)) isomers. In each case, the present disclosure includes both mixtures and the separate individual isomers.

[0093] Some compounds may also exist in tautomeric forms. Such forms, although not explicitly shown in the formulae set forth herein, are intended to be included within the scope of the present disclosure.

[0094] "Prodrug" or "Pharmaceutically acceptable prodrug" refers to a compound that is metabolized, e.g., hydrolyzed or oxidized, in the host after administration to form a compound of the present disclosure (e.g., a compound of formula I). ​​Typical examples of prodrugs include compounds that have a biologically labile or cleavable (protecting) group on a functional group of an active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to yield an active compound. Examples of prodrugs that use esters or phosphoramidates as biologically labile or cleavable (protecting) groups are disclosed in U.S. Patent Nos. 6,875,751, 7,585,851, and 7,964,580, the disclosures of which are incorporated herein by reference. Prodrugs of the present disclosure are metabolized to yield a compound of formula I. The present disclosure includes within its scope prodrugs of the compounds described herein. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in “Design of Prodrugs” Ed. H. Bundgaard, Elsevier, 1985.

[0095] As used herein, the terms "logarithm of solubility", "LogS" or "logS" are used in the art to quantify the aqueous solubility of a compound. The aqueous solubility of a compound has a significant effect on its absorption and distribution properties. Poor solubility often leads to poor absorption. The LogS value is the unit-removed logarithm (base 10) of the solubility measured in moles / liter.

[0096] The term "glycosyl" as used herein refers to a monovalent substituent formed from any naturally occurring sugar, its metabolite / xometabolite, its prodrug, or combinations thereof. The term includes both linear and branched oligosaccharides and polysaccharides, as well as alpha and beta structures, or any combinations thereof. The preferred chain length of polysaccharides is 1 or 2 (i.e., monosaccharides or disaccharides). In certain preferred embodiments, glycosyl refers to a substituent formed from glucose, fucose, galactose, mannose, xylose, galatosamine, glucuronic acid, galacturonic acid, mannuric acid, sialic acid, iduronic acid, neuraminic acid, derivatives thereof, or combinations thereof.

[0097] Toxin Payload Many toxin payloads are suitable for use in the presently disclosed conjugates. In certain embodiments, the toxin payload is selected from a chemotherapeutic agent substituted with a saccharide, sulfate, or sulfonate, or a toxin substituted with a saccharide, sulfate, or sulfonate. In further embodiments, the active agent is a chemotherapeutic agent substituted with a saccharide, sulfate, or sulfonate. In yet further embodiments, the toxin payload is independently selected from an immunomodulatory compound substituted with a saccharide, sulfate, or sulfonate, an anticancer agent substituted with a saccharide, sulfate, or sulfonate, an antiviral agent substituted with a saccharide, sulfate, or sulfonate, an antibacterial agent substituted with a saccharide, sulfate, or sulfonate, an antifungal agent substituted with a saccharide, sulfate, or sulfonate, or an antiparasitic agent substituted with a saccharide, sulfate, or sulfonate.

[0098] In still further embodiments, the toxin payload is independently selected from a benzodiazepine substituted with a saccharide, sulfate, or sulfonate, a duocarmycin substituted with a saccharide, sulfate, or sulfonate, an auristatin substituted with a saccharide, sulfate, or sulfonate, a tubulysin substituted with a saccharide, sulfate, or sulfonate, SN-38 substituted with a saccharide, sulfate, or sulfonate, PNU substituted with a saccharide, sulfate, or sulfonate, or exatecan substituted with a saccharide, sulfate, or sulfonate, or amanitin substituted with a saccharide, sulfate, or sulfonate.

[0099] In still further embodiments, the toxin payload may be functionalized with one or more functional groups selected from -C(O)-, -O-, -NH-, -S-, and -C(O)O-. In further embodiments, the functional group is functionalized with a saccharide, sulfate, or sulfonate. In some embodiments, the toxin payload may include a modifying moiety attached to a saccharide via a functional group selected from an ester, amide, thio, carbamate, oxime, hydrazone, and the like. In some implementations, the toxin payload may include a modifying moiety attached to a polar group such as a sulfonate (see, e.g., WO2006 / 111759A1), sulfate, sulfite, and the like.

[0100] In yet a further aspect, provided herein is a compound of formula (VII) or (VIII): [ka] Alternatively, a pharma- ceutically acceptable salt thereof is provided; During the ceremony: A is a heterocycle; Each R a ' and R b ' is independently halogen, amino, hydroxyl, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; 2 Germinal R b' may optionally be taken together to represent oxo or =CH 2 or form two R b ' together with the intervening atoms optionally completes a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R c ' is sulfonate, sulfate, hydroxyl, amino, or thiol; R d ' is -L"-Gly, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; However, at least one R c ' is a sulfonate or sulfate, or at least one R d '-L ” -Gly; R e ' is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; m is an integer selected from 0 to 3; n is an integer selected from 0 to 8 depending on the valence; Ring Cy is selected from aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; [ka] is a single or double bond; X' is a halogen; X″ is —NR—, —S—, or —O—; Each R a " and R b " is independently halogen, amino, hydroxyl, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, alkenyl, alkynyl, =O, carboxyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L''') r -X”-Gly; d is an integer selected from 0 to 4; r is an integer selected from 0 to 1; each L''' is a bond or a linker; R e " is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; p is an integer selected from 0 to 4; DBD is the DNA binding domain; L″ is a bond or a linker; and Gly may be a monosaccharide, disaccharide, or oligosaccharide.

[0101] In certain embodiments, each L''' is C 10 -C 100 and optionally containing one or more double bonds and / or triple bonds. In a further embodiment, each p and each d is independently an integer from 0 to 1.

[0102] In yet a further embodiment, the compound has formula (VII): [ka] or a pharma- ceutically acceptable salt thereof.

[0103] In yet further embodiments, A is a 5- to 6-membered heterocycle. c In a further embodiment, R′ is hydroxyl. d ' is hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocycloalkyl, C 6-10 In yet further embodiments, R d In yet a further embodiment, the compound is: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony, [ka] is a single bond or a double bond.

[0104] In certain embodiments, R a ' is halogen, amino, hydroxyl, alkoxy, cyano, nitro, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 4-10 membered heterocycloalkyl, C 6-10 In a further embodiment, two geminal R b ' together =CH 2 In still further embodiments, two R b Together with the intervening atoms, R′ completes a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. In still further embodiments, two R b Together with the intervening atoms, R ′ completes an aryl or heteroaryl. In certain embodiments, two R b ' together with the intervening atom completes the aryl.

[0105] In a further embodiment, R e ' is hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocycloalkyl, C 6-10 In yet further embodiments, R e ' is hydrogen, C 1-6 Alkyl, or C 3-10 In yet a further embodiment, R e ' is hydrogen.

[0106] In certain embodiments, the compound is: [ka] Alternatively, it is selected from a pharma- ceutically acceptable salt thereof. In a further embodiment, the compound has formula (VIII): [ka] Alternatively, it may be represented by a pharma- ceutically acceptable salt thereof. In yet further embodiments, Cy is phenyl. In yet further embodiments, Cy is pyrrolidine or pyrrole. In certain embodiments, the compound is (VIIIa) or (VIIIb): [ka] Alternatively, it may be represented by a pharma- ceutically acceptable salt thereof. In a further embodiment, DBD-(L''') r The -X”-Gly unit is as follows: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: Y" is C or N; X″ is selected from —NR—, —S—, or —O—; R is hydrogen or alkyl; r is an integer selected from 0 to 1; Each R b " is independently halogen, amino, hydroxyl, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L''') r -X”-Gly; R k is alkyl, preferably C 1 -C 3 is alkyl; q is an integer selected from 0 to 3; and [ka] is a single bond or a double bond. In still further embodiments, the compound has formula (VIIIc), (VIIId), (VIIIe), or (VIIIf): [ka] Alternatively, it may be represented by a pharma- ceutically acceptable salt thereof.

[0107] In still further embodiments, each R a " is independently halogen, amino, hydroxyl, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, =O, carboxyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and each R b " are independently halogen, amino, hydroxyl, alkoxy, cyano, nitro, C 1-6 Alkyl, Heteroalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 4-10 membered heterocycloalkyl, C 6-10 aryl, or 5- to 10-membered heteroaryl.

[0108] In certain embodiments, X' is Cl. In further embodiments, X' is Br. In still further embodiments, Y" is C. In certain embodiments, Y" is N. In further embodiments, R e In a further embodiment, R′ is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. e " is hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocycloalkyl, C 6-10 In yet further embodiments, R e ' is hydrogen, C 1-6 Alkyl, or C 3-10 In certain preferred embodiments, R e" is hydrogen. In certain embodiments, the compound is: [ka] [ka] Alternatively, it is selected from a pharma- ceutically acceptable salt thereof.

[0109] In a further embodiment, L''' is a bond. In yet a further embodiment, L''' is: [ka] is a linker selected from During the ceremony: R a "'' is hydrogen, halogen, amino, hydroxyl, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, =O, carboxyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and Each R b "'' is independently hydrogen, halogen, amino, hydroxyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and h is an integer selected from 0 to 4 depending on the atomic valence. In still further embodiments, L''' is [ka] It is.

[0110] In certain embodiments, Gly is a monosaccharide. In further embodiments, Gly is a monosaccharide selected from glucose, glucuronic acid, fucose, and galactose. In yet further embodiments, Gly is [ka] and Optionally, one or more -OH groups are masked with a protecting group.

[0111] In still further embodiments, Gly is [ka] It is.

[0112] In certain embodiments, Gly is a disaccharide. In further embodiments, Gly is a disaccharide comprising glucose, glucuronic acid, fucose, galactose, or a combination thereof. In yet further embodiments, Gly is [ka] and Optionally, one or more -OH groups are masked with a protecting group.

[0113] In certain embodiments, Gly is [ka] It is.

[0114] In certain embodiments, X″ is attached to Gly at the anomeric position.

[0115] Drug Conjugates In certain embodiments, the compound represented by formula (VII) or (VIII): [ka] Alternatively, provided herein is a drug conjugate comprising a pharma- ceutically acceptable salt thereof, and a linker group, During the ceremony: A is a heterocycle; Each R a ' and R b' is independently halogen, amino, hydroxyl, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; 2 Germinal R b ' may optionally be taken together to represent oxo or =CH 2 or form two R b ' together with the intervening atoms optionally completes a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R c ' is sulfonate, sulfate, hydroxyl, amino, or thiol; R d ' is -L"-Gly, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; However, at least one R C’ is a sulfonate or sulfate, or at least one R d’ Ga-L ’’’ -Gly; R e ' is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; m is an integer selected from 0 to 3; n is an integer selected from 0 to 8 depending on the valence; Ring Cy is selected from aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; [ka] is a single or double bond; X' is a halogen; X″ is —NR—, —S—, or —O—; R is hydrogen or alkyl; Each R a " and R b" is independently halogen, amino, hydroxyl, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, alkenyl, alkynyl, =O, carboxyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L''') r -X”-Gly; d is an integer selected from 0 to 4; r is an integer from 0 to 1; each L''' is a bond or a linker; R e is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; p is an integer selected from 0 to 4; DBD is the DNA binding domain; L″ is a bond or a linker; and Gly is a monosaccharide, disaccharide, or oligosaccharide.

[0116] In certain embodiments, each L''' is C 10 -C 100 and optionally containing one or more double bonds and / or triple bonds. In a further embodiment, each p and each d is independently an integer from 0 to 1.

[0117] In still further embodiments, the drug conjugate comprises a compound of formula (VII): [ka] Or a pharma- ceutically acceptable salt thereof. In yet further embodiments, A is a 5- to 6-membered heterocycle. c In a further embodiment, R′ is hydroxyl. d ' is hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocycloalkyl, C 6-10In yet further embodiments, R d In yet a further embodiment, the compound is: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony, [ka] is a single bond or a double bond.

[0118] In certain embodiments, R a ' is halogen, amino, hydroxyl, alkoxy, cyano, nitro, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 4-10 membered heterocycloalkyl, C 6-10 In a further embodiment, two geminal R b ' together =CH 2 In still further embodiments, two R b Together with the intervening atoms, R′ completes a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. In still further embodiments, two R b Together with the intervening atoms, R ′ completes an aryl or heteroaryl. In certain embodiments, two R b ' together with the intervening atom completes the aryl.

[0119] In a further embodiment, R e ' is hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocycloalkyl, C 6-10 In yet further embodiments, R e ' is hydrogen, C 1-6Alkyl, or C 3-10 In yet a further embodiment, R e ' is hydrogen. In certain embodiments, the compound is: [ka] Alternatively, it is selected from a pharma- ceutically acceptable salt thereof.

[0120] In a further embodiment, the compound has formula (VIII): [ka] Alternatively, it may be represented by a pharma- ceutically acceptable salt thereof.

[0121] In yet further embodiments, Cy is phenyl. In yet further embodiments, Cy is pyrrolidine or pyrrole. In certain embodiments, the compound has formula (VIIIa) or (VIIIb): [ka] Alternatively, it may be represented by a pharma- ceutically acceptable salt thereof.

[0122] In a further embodiment, DBD-(L''') r The -X”-Gly unit is as follows: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: Y" is C or N; X″ is selected from —NR—, —S—, or —O—; R is hydrogen or alkyl; r is an integer selected from 0 to 1; Each R b" is independently halogen, amino, hydroxyl, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L''') r -X”-Gly; R k is alkyl, preferably C 1 -C 3 is alkyl; q is an integer selected from 0 to 3; and [ka] is a single bond or a double bond.

[0123] In still further embodiments, the compound has formula (VIIIc), (VIIId), (VIIIe), or (VIIIf): [ka] Alternatively, it may be represented by a pharma- ceutically acceptable salt thereof.

[0124] In still further embodiments, each R a " is independently halogen, amino, hydroxyl, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, =O, carboxyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and each R b " are independently halogen, amino, hydroxyl, alkoxy, cyano, nitro, C 1-6 Alkyl, Heteroalkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 4-10 membered heterocycloalkyl, C 6-10 aryl, or 5-10 membered heteroaryl. In certain preferred embodiments, at least one R aIn certain embodiments, X′ is alkoxy, such as methoxy, ethoxy, or propoxy. In certain embodiments, X′ is Cl. In further embodiments, X′ is Br. In still further embodiments, Y″ is C. In certain embodiments, Y″ is N. In further embodiments, R e In a further embodiment, R′ is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. e " is hydrogen, C 1-6 Alkyl, C 3-10 Cycloalkyl, 3-10 membered heterocycloalkyl, C 6-10 In yet further embodiments, R e " is hydrogen, C 1-6 Alkyl, or C 3-10 In certain preferred embodiments, R e " is hydrogen.

[0125] In certain embodiments, R a In a further embodiment, p is 1, d is 0, and R a " is at the 8-position, i.e., the compound has the formula (VIIIg) or (VIIIh): [ka] or a pharma- ceutically acceptable salt thereof. In certain embodiments of formula (VIIIg) or (VIIIh), R a " is alkoxy, e.g., methoxy, ethoxy, or propoxy, preferably methoxy. In certain such embodiments, the compound is [ka] [ka] Alternatively, it is selected from a pharma- ceutically acceptable salt thereof.

[0126] In a further embodiment, L''' is a bond. In yet a further embodiment, L''' is: [ka] is a linker selected from During the ceremony, R a "'' is hydrogen, halogen, amino, hydroxyl, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, =O, carboxyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and Each R b "'' is independently hydrogen, halogen, amino, hydroxyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and h is an integer selected from 0 to 4 depending on the atomic valence.

[0127] In still further embodiments, L''' is [ka] It is.

[0128] In certain embodiments, Gly is a monosaccharide. In further embodiments, Gly is a monosaccharide selected from glucose, glucuronic acid, fucose, and galactose. In yet further embodiments, Gly is [ka] and Optionally, one or more -OH groups are masked with a protecting group.

[0129] In still further embodiments, Gly is [ka] It is.

[0130] In certain embodiments, Gly is a disaccharide. In further embodiments, Gly is a disaccharide comprising glucose, glucuronic acid, fucose, galactose, or a combination thereof. In yet further embodiments, Gly is [ka] and Optionally, one or more -OH groups are masked with a protecting group.

[0131] In certain embodiments, Gly is [ka] It is.

[0132] In certain embodiments, X″ is attached to Gly at the anomeric position.

[0133] In certain aspects, provided herein is a drug conjugate comprising any one of the disclosed compounds and a linker group. In certain embodiments, the drug conjugate is represented by formula (IX), (X), or (XI): [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: Z' is a coupling group; Ar is aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; Y' is -(CR b 2 ) y N(R a )-, -(CR b 2 ) y O- or -(CR b 2 ) y S- and y is 1, an N, O, or S atom is positioned to be bonded to TG; When activated, TG reacts with SO 2In response to (Q) q -(L') w Replace X-SO 2 and a trigger group that generates an N, O, or S atom capable of forming a 5- to 6-membered ring containing an intervening atom of Ar; X is -O-, -C(R b ) 2 - or -N(R c )-and; L', if present, is a heteroatom selected from O, S, and N. 2 The spacer portion binds to L' and SO 2 is selected such that cleavage of the bond between facilitates release of the active agent; w is an integer selected from 0 to 1; r is an integer from 0 to 1; Z 2 is a linking group; Z 3 is a linking group; R a , R b and R c are each independently hydrogen or lower alkyl; y is an integer selected from 0 to 1; t is an integer from 1 to 5; and e is an integer from 1 to 5.

[0134] In certain embodiments, Z 3 is the following: [ka] Selected from; During the ceremony: X 5 is -O- or -NR x - and; Y 1 CR y , or N; R x and R y are each independently hydrogen or C 1-6 is alkyl; each b is independently an integer from 1 to 3; and and c is an integer from 1 to 5. In a further embodiment, Z 3 is the following: [ka] is selected from. In yet a further embodiment, Z 2 is methylene. In still further embodiments, Z 2 teeth, [ka] and During the ceremony: Y 5 CR Y1 or N, but Y 5 is N; R Y1 is H, hydroxyl, amino, amide, or (CH 2 ) y (R Y1a ) and; R Y1a is amino (e.g., secondary or tertiary amino), aryl (e.g., phenyl), or heteroaryl; and y is an integer having a value from 1 to about 10.

[0135] In certain embodiments, Z 2 teeth, [ka] It is.

[0136] In a further embodiment, Z 2 is the following: [ka] and During the ceremony: Y 6 CR Y2 or N; R Y2 is H or alkyl, preferably lower alkyl; R Z2 is (CH 2 ) z R Z2a and; R Z2a is amino (preferably tertiary amino), aryl (e.g., phenyl), or heteroaryl; and z is an integer having a value from 0 to about 10.

[0137] In yet a further embodiment, Z 2 is the following: [ka] It is.

[0138] In a further embodiment, Ar is aryl. In yet a further embodiment, Ar is C 6-10 In yet further embodiments, Ar is aryl. In still further embodiments, Ar is phenyl. In certain embodiments, Ar is heteroaryl. In further embodiments, Ar is 5-10 membered heteroaryl. In still further embodiments, Y' is -(CR b 2 ) y N(R a )-or-(CR b 2 ) y In yet a further embodiment, Y' is -(CR b 2 ) y In certain embodiments, y is 0. In further embodiments, y is 1. In still further embodiments, X is -O-, C(R b )(R c )-or-N(R c )-. In yet further embodiments, X is -O-. In certain embodiments, L' is a spacer moiety and forms an -O-, -OC(O)-, -OC(O)O-, -NHC(O)O-, or -OC(O)NH- bond (including a heteroatom of the active agent).

[0139] In certain embodiments, L' is [ka] Selected from; During the ceremony, X 4 is absent or forms an -O-, -OC(O)-, -OC(O)O-, or -OC(O)NH- bond (including the heteroatom of Q); X 1 is -O- or -NR a - and; X 2 is -O-, -OC(O)-, -OC(O)O-, or -OC(O)NH-; X 3 is -OC(=O)-; w' is an integer having a value of 1, 2, 3, 4, or 5; 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 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; and u is an integer having a value of 1 to about 10.

[0140] In still further embodiments, L' is [ka] It is.

[0141] In certain aspects, provided herein is a drug conjugate comprising any one of the toxin payload compounds of the present disclosure (an active agent) and a linking group; wherein the active agent is substituted with a polar group. In some embodiments, the polar group is selected from a saccharide, a sulfate, or a sulfonate.

[0142] In some embodiments, the drug conjugate comprising an active agent and a linking group has the formula (I): [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: Z' is a coupling group; Ar is aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; Y' is -(CR b 2 ) y N(R a )-, -(CR b 2 ) y O- or -(CR b 2 ) y S- and y is 1, an N, O, or S atom is positioned to be bonded to TG; Upon activation, TG reacts with SO 2 In response to (Q) q -(L') w Replace X-SO 2 and a trigger group that generates an N, O, or S atom capable of forming a 5- to 6-membered ring containing an intervening atom of Ar; X is -O-, -C(R b ) 2 - or -N(R c )-and; L', if present, is a heteroatom selected from O, S, and N. 2 The spacer portion binds to L' and SO 2 is selected such that cleavage of the bond between facilitates release of the active agent; Each Q is independently a saccharide, sulfate, or sulfonate substituted active agent. q is an integer selected from 1 to 3; w and y are each independently 0 or 1; and R a , R b and R c are each independently hydrogen or C 1-6 alkyl; or two R b complete a 3- to 5-membered ring together with the atoms to which they are attached; However, when w is 0, q is 1.

[0143] In certain embodiments, each Q is independently selected from chemotherapeutic agents substituted with sugars, sulfates, or sulfonates, or toxins substituted with sugars, sulfates, or sulfonates.In further embodiments, the active agent is a chemotherapeutic agent substituted with sugars, sulfates, or sulfonates.In still further embodiments, each Q is independently selected from immunomodulatory compounds substituted with sugars, sulfates, or sulfonates, anticancer agents substituted with sugars, sulfates, or sulfonates, antiviral agents substituted with sugars, sulfates, or sulfonates, antibacterial agents substituted with sugars, sulfates, or sulfonates, antifungal agents substituted with sugars, sulfates, or sulfonates, or antiparasitic agents substituted with sugars, sulfates, or sulfonates.

[0144] In still further embodiments, each Q is independently selected from a benzodiazepine substituted with a saccharide, sulfate, or sulfonate; a duocarmycin substituted with a saccharide, sulfate, or sulfonate; an auristatin substituted with a saccharide, sulfate, or sulfonate; a tubulysin substituted with a saccharide, sulfate, or sulfonate; SN-38 substituted with a saccharide, sulfate, or sulfonate; PNU substituted with a saccharide, sulfate, or sulfonate; or exatecan substituted with a saccharide, sulfate, or sulfonate; or amanitin substituted with a saccharide, sulfate, or sulfonate.

[0145] In certain embodiments, Q may be a modifying moiety attached to the sugar via a linking group. In still further embodiments, Q may be a modifying moiety attached to the sugar via a functional group selected from -C(O)-, -OH, -NH-, -SH, -COH, -COOH, etc. In still further embodiments, Q may be a modifying moiety attached to the sugar via a functional group selected from ester, amide, thio, carbamate, oxime, hydrazone, etc.

[0146] In certain embodiments, Q may comprise a modifying moiety attached to the sugar via a functional group selected from ester, amide, thio, carbamate, oxime, hydrazone, etc. In yet further embodiments, Q may comprise a modifying moiety attached to a polar group such as a sulfonate, sulfate, sulfite, etc.

[0147] In certain embodiments, each Q is independently represented as: [ka] During the ceremony: Z 2 is a linking group; Z 3 is a linking group; t is an integer from 1 to 5; e is an integer from 1 to 5; and each Q' is independently a modified benzodiazepine. In further embodiments, each Q′ is independently selected from the formula (IIa) or (IIb): [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: each A is a heterocycle; Each R a' is independently halogen, amino, hydroxyl, alkoxy, cyano, nitro, alkyl (preferably lower alkyl), alkenyl, alkynyl, cycloalkyl, aryl, or heterocycle, preferably a 5- or 6-membered ring; optionally fused to or substituted by one or more aryl or heteroaryl rings; Each R b ' is independently halogen, amino, hydroxyl, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heterocycle, preferably a 5- or 6-membered ring; optionally fused to or substituted by one or more aryl or heteroaryl rings; 2 Germinal R b ' may optionally be taken together to represent oxo or =CH 2 or form two R b ', together with the intervening atoms, optionally completes a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, and optionally one or more R f ' is substituted, Each R f ' is independently halogen, hydroxyl, -O-Gly, cyano, nitro, alkyl, haloalkyl, cycloalkyl, carboxyl, amino, aminoalkyl (-CH 2 NH 2 , -CH 2 NH(Me), or -CH 2 N(Me) 2 ), aryl, or heteroaryl; R c ' is sulfonate, sulfate, hydroxyl, amino, or thiol; R d '-L ” -Gly, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; However, at least one R c ' is a sulfonate or sulfate, or at least one R d '-L ”-Gly; m is an integer selected from 0 to 3; n is an integer selected from 0 to 8 depending on the valence; and Gly is glycosyl, preferably Gly is a monosaccharide, disaccharide, or oligosaccharide.

[0148] As will be understood, in formula (IIb), R b ' example (or two geminal R's joined together as above) b ') serves as a point of attachment to the remainder of the conjugate (e.g., where Q is [ka] Under the definitions provided below, the remainder of the conjugate is R b ' may be understood as a substituent of that (or those) instance(s) of R b The instance(s) are selected from the above-listed substituents that can be divalent, such as amino, alkoxy, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heterocyclic rings, preferably 5- or 6-membered rings, optionally fused or substituted with one or more aryl or heteroaryl rings. Two geminal R b When the ' are taken together to form a point of attachment, they are also potentially divalent substituents, e.g., =CH 2 or, together with the intervening atoms, optionally one or more R f 'Complete a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl substituted with '.

[0149] In certain embodiments, the drug conjugate has formula (IIIa) or (IIIb): [ka] Alternatively, it may be represented by a pharma- ceutically acceptable salt thereof.

[0150] In some embodiments herein, the drug conjugate is represented by formula (IIIa) or (IIIb), wherein: each A is a heterocycle; Each R a ' is independently halogen, amino, hydroxyl, alkoxy, cyano, nitro, alkyl (preferably lower alkyl), alkenyl, alkynyl, cycloalkyl, aryl, or heterocycle, preferably a 5- or 6-membered ring; optionally fused to or substituted by one or more aryl or heteroaryl rings; Each R b ' is independently halogen, amino, hydroxyl, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heterocycle, preferably a 5- or 6-membered ring; optionally fused to or substituted by one or more aryl or heteroaryl rings; 2 Germinal R b ' optionally taken together to represent oxo or =CH 2 or form two R b ', together with the intervening atoms, optionally completes a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, and optionally one or more R f ' is substituted, Each R f ' is independently halogen, hydroxyl, -O-Gly, cyano, nitro, alkyl, haloalkyl, cycloalkyl, carboxyl, amino, aminoalkyl (-CH 2 NH 2 , -CH 2 NH(Me), or -CH 2 N(Me) 2 ), aryl, or heteroaryl; R c ' is sulfonate, sulfate, hydroxyl, amino, or thiol; R d '-L ”-Gly, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; However, at least one R c ' is a sulfonate or sulfate, or at least one R d '-L ” -Gly; m is an integer selected from 0 to 3; n is an integer selected from 0 to 8 depending on the valence; and Gly is glycosyl, preferably Gly is a monosaccharide, disaccharide, or oligosaccharide.

[0151] In some embodiments herein, where the context is clear, formulas (IIIa) and (IIIb) are referred to as formulas (IX) and (X), respectively.

[0152] In certain embodiments, A is a 5-6 membered heterocycle. c In yet a further embodiment, R c In yet a further embodiment, R′ is a sulfonate or sulfate. d '-L ” In certain preferred embodiments, R d ' is hydrogen.

[0153] In certain embodiments, each Q′ is independently: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony, [ka] is a single bond or a double bond.

[0154] In a further embodiment, R a ' is halogen, amino, hydroxyl, alkoxy, cyano, nitro, C1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-10 Cycloalkyl, 4-10 membered heterocycloalkyl, C 6-10 In yet a further embodiment, one R b ' is an alkyl group or two geminal R b In still further embodiments, two R ′ together form an alkenyl group. b ' taken together with the intervening atoms completes a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; preferably the aryl or heteroaryl is optionally joined to one or more R f ' is a 6-membered aryl or heteroaryl substituted with .

[0155] In certain embodiments, two R b ' taken together with the intervening atoms completes an aryl or heteroaryl; preferably the aryl or heteroaryl is optionally joined to one or more R f In a further embodiment, two R b In yet a further embodiment, two R' are joined together with the intervening atoms to complete an aryl. b ' together with the intervening atoms completes a heteroaryl.

[0156] In certain embodiments, each Q′ is independently: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony, [ka] is a single bond or a double bond; and Here, g is an integer from 0 to 4.

[0157] In certain embodiments, Z 3 is the following: [ka] Selected from; During the ceremony: X 5 is -O- or -NR x - and; Y 1 CR y , or N; R y is hydrogen or C 1-6 is alkyl; each b is independently an integer from 1 to 3; and and c is an integer from 1 to 5. In a further embodiment, Z 3 is selected from: [ka]

[0158] In yet a further embodiment, Z 2 is methylene. In yet a further embodiment, Z 2 teeth, [ka] During the ceremony: Y 5 CR Y1 or N, but Y 5 is N, R Y1 is H, hydroxyl, amino, amide, or (CH 2 ) y (R Y1a ) and; R Y1a is amino (e.g., secondary or tertiary amino), aryl (e.g., phenyl), or heteroaryl; and y is an integer having a value from 1 to about 10. In certain embodiments, Z 2 teeth, [ka] It is.

[0159] In a further embodiment, Z 2 is the following: [ka] and During the ceremony: Y 6 CR Y2 or N; R Y2 is H or alkyl, preferably lower alkyl; R Z2 is (CH 2 ) z R Z2a and; R Z2a is amino (preferably tertiary amino), aryl (e.g., phenyl), or heteroaryl; and z is an integer having a value from 0 to about 10.

[0160] In yet a further embodiment, Z 2 is the following: [ka] It is.

[0161] In yet another embodiment, each Q independently represents a group of formula (IV): [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: Ring Cy is selected from aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; [ka] is a single or double bond; X' is a halogen; X″ is selected from —NR—, —S—, or —O—; Each R a " and R b” is independently halogen, amino, hydroxyl, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, alkenyl, alkynyl, =O, carboxyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L''')rX''-Gly; p is an integer selected from 0 to 4; d is an integer selected from 0 to 4; r is an integer from 0 to 1; DBD is the DNA binding domain; each L''' is a bond or a linker, and Gly is a monosaccharide, disaccharide, or oligosaccharide.

[0162] In still further embodiments, each L''' is C 10 -C 100 and optionally containing one or more double and / or triple bonds. In still further embodiments, each p and each d is independently an integer from 0 to 1. In certain embodiments, Cy is phenyl. In further embodiments, Cy is pyrrolidine or pyrrole.

[0163] In certain embodiments, each Q independently represents a group of formula (IVa) or (IVb): [ka] Or a pharma- ceutically acceptable salt thereof.

[0164] In a further embodiment, DBD-(L''') r The -X”-Gly unit is as follows: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: Y" is C or N; X″ is selected from —NR—, —S—, or —O—; Each R b " is independently halogen, amino, hydroxyl, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L''') r -X”-Gly; R k is alkyl or hydroxyalkyl, preferably C 1 -C 3 is alkyl; q is an integer selected from 0 and 3; and [ka] is a single bond or a double bond.

[0165] In still further embodiments, each Q is independently selected from the group of formula (IVc), (IVd), (IVe), (IVf), (IVg), (IVh), (IVi), or (IVj). [ka] [ka] Or a pharma- ceutically acceptable salt thereof.

[0166] In preferred embodiments, each Q is independently selected from the formula (IVc), (IVd), (IVe), or (IVf): [ka] Alternatively, it is selected from the group of pharma- ceutically acceptable salts thereof.

[0167] In certain embodiments, the drug conjugate has formula (V): [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: Z' is a coupling group; Ar is aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; Y' is -(CR b 2 ) y N(R a )-, -(CR b 2 ) y O- or -(CR b 2 ) y S- and y is 1, an N, O, or S atom is positioned to be bonded to TG; Upon activation, TG reacts with SO 2 In response to (Q) q -(L') w Replace X-SO 2 and a trigger group that generates an N, O, or S atom capable of forming a 5- to 6-membered ring containing an intervening atom of Ar; X is -O-, -C(R b ) 2 - or -N(R c )-and; L', if present, is a heteroatom selected from O, S, and N. 2 The spacer portion binds to L' and SO 2 is selected such that cleavage of the bond between facilitates release of the active agent; and r is an integer from 0 to 1.

[0168] In some embodiments herein, where the context is clear, formula (V) is referred to as formula (XI).

[0169] In a further embodiment, Cy is phenyl. In yet a further embodiment, Cy is selected from pyrrolidine or pyrrole. In yet a further embodiment, the drug conjugate has the formula (Va) or (Vb): [ka] Alternatively, it may be represented by a pharma- ceutically acceptable salt thereof.

[0170] In certain embodiments, DBD-(L''') r The -X”-Gly unit is as follows: [ka] or a pharma- ceutically acceptable salt thereof; During the ceremony: Y" is C or N; Each R b " is independently halogen, amino, hydroxyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R k is alkyl or hydroxyalkyl, preferably C 1 -C 3 is alkyl; q is an integer selected from 0 and 3; and [ka] is a single bond or a double bond.

[0171] In certain embodiments, the drug conjugate has formula (Vc), (Vd), (Ve), or (Vf): [ka] [ka] Alternatively, it is selected from the group of pharma- ceutically acceptable salts thereof.

[0172] In a preferred embodiment, the drug conjugate has formula (Vc) or (Vd): [ka] Alternatively, it may be represented by a pharma- ceutically acceptable salt thereof.

[0173] In certain embodiments, each R a” are independently hydrogen, halogen, amino, hydroxyl, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, alkenyl, alkynyl, =O, carboxyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L''') r -X″-Gly; b " is independently hydrogen, halogen, amino, hydroxyl, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L''') r -X”-Gly.

[0174] In further embodiments, X' is Cl. In yet further embodiments, X' is Br. In still further embodiments, Y" is C. In certain embodiments, Y" is N.

[0175] In certain embodiments, Q is: [ka] [ka] Alternatively, it is selected from a pharma- ceutically acceptable salt thereof.

[0176] In a further embodiment, L''' is a bond. In yet a further embodiment, L''' is: [ka] is a linker selected from During the ceremony: Each R a "'' is independently hydrogen, halogen, amino, hydroxyl, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, =O, carboxyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and Each R b ''' is independently hydrogen, halogen, amino, hydroxyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; h is an integer selected from 0 to 4 depending on the valence; and [ka] is the connection point with the adjacent functional group.

[0177] In still further embodiments, L''' is [ka]

[0178] It is. In certain embodiments, Gly is a monosaccharide. In further embodiments, Gly is a monosaccharide selected from glucose, glucuronic acid, fucose, and galactose. In yet further embodiments, Gly is [ka] and Optionally, one or more -OH groups are masked with a protecting group.

[0179] In still further embodiments, Gly is [ka] It is.

[0180] In certain embodiments, Gly is a disaccharide. In further embodiments, Gly is a disaccharide comprising glucose, glucuronic acid, fucose, galactose, or a combination thereof. In yet further embodiments, Gly is [ka] and Optionally, one or more -OH groups are masked with a protecting group.

[0181] In still further embodiments, Gly is [ka] It is.

[0182] In certain embodiments, X" or L" is attached to Gly at the anomeric position. In further embodiments, Ar is aryl. In yet further embodiments, Ar is C 6-10 In yet further embodiments, Ar is aryl. In still further embodiments, Ar is phenyl. In certain embodiments, Ar is heteroaryl. In further embodiments, Ar is 5-10 membered heteroaryl. In still further embodiments, Y' is -(CR b 2 ) y N(R a )-or-(CR b 2 ) y In yet a further embodiment, Y' is -(CR b 2 ) y In certain embodiments, y is 0 or 1. In further embodiments, y is 1. In still further embodiments, X is -O-, C(R b ) 2 -or-N(R c )-. In yet further embodiments, X is -O-. In certain embodiments, L' is a spacer moiety and forms an -O-, -OC(O)-, -OC(O)O-, or -OC(O)NH- bond (including a heteroatom of the active agent).

[0183] In certain embodiments, Q-(L') w -teeth, [ka] Selected from; During the ceremony: X 4is absent or forms an -O-, -OC(O)-, -OC(O)O-, or -OC(O)NH- bond (including the heteroatom of Q); X 1 is -O- or -NR a - and; X 2 is -O-, -OC(O)-, -OC(O)O-, or -OC(O)NH-; X 3 is -OC(=O)-; w' is an integer having a value of 1, 2, 3, 4, or 5; 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 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; and u is an integer having a value of 1 to about 10.

[0184] In a further embodiment, Q-(L') w -teeth, [ka] is selected from.

[0185] In certain embodiments, the drug conjugate comprises: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or a pharma- ceutically acceptable salt thereof.

[0186] In certain embodiments, the drug conjugate comprises: [ka] [ka] [ka] [ka] [ka] is not selected from.

[0187] In certain embodiments, the drug conjugate is not a compound disclosed in U.S. Patent Application Publication No. 2022 / 0047717.

[0188] Targeted Drug Conjugates In certain aspects, provided herein are targeted drug conjugates, including drug conjugates comprising a compound of the present disclosure, a linker group, and a targeting moiety.

[0189] In certain embodiments, the targeted drug conjugate has formula (XII), (XIII) or (XIV): [ka] or a pharma- ceutically acceptable salt thereof; where TM is a targeting moiety.

[0190] In certain embodiments, the drug conjugates of the present disclosure further comprise a targeting moiety. In further embodiments, provided herein is a targeted drug conjugate of formula (VI) comprising a targeting moiety conjugated to any one of the drug conjugates of the present disclosure: [ka] where TM is a targeting moiety.

[0191] In certain embodiments, the targeted drug conjugate comprises a compound of formula (XII), (XIII) or (XIV): [ka] or a pharma- ceutically acceptable salt thereof; where TM is a targeting moiety.

[0192] In a further aspect, provided herein is a targeted drug conjugate of formula (VIb) comprising a targeting moiety conjugated to a drug conjugate of the present disclosure: [ka] During the ceremony: TM is the targeting moiety; R is hydrogen or a hydroxy protecting group; X is -C(O)-, -NH-, -O-, or -S-; Q is a sugar, sulfate, or sulfonate substituted active agent; T is [ka] and; n is an integer selected from 0 and 1; Y is hydrogen, haloC 1 -C 8 alkyl, halogen, cyano, or nitro; z is an integer selected from 1 to 3, and when z is an integer of 2 or more, Y's may be the same or different; z1 is an integer selected from 0 or 1; W 1 teeth [ka] and; W 2 teeth [ka] and; W a1 and W a2 are each independently -NH-, -C(=O)-, or -CH 2 - and; W a3 and W a4 are each independently -NH-, -C(=O)-, or -CH 2 -, -C(=O)NH-, -NHC(=O)-, or triazolylene; W b1 is an amide bond or a triazolylene; L is W a2 an amino acid, peptide, or amide bond as a linker connecting a and Z; Z is a single bond, -W a5 -(CH 2 ) a2 -W b2 -(CH 2 ) a3 -Wa6 -, or -W a7 -(CH 2 ) a4 -CR'R”-X''-; R' is C 1 -C 8 Alkyl or TM-W a8 -Q 3 -W c1 -(CH 2 ) a5 - and; R” is TM-W a8 -Q 3 -W c1 -(CH 2 ) a5 - and; Q 1 and Q 3 are each independently -(CH 2 ) a6 -(X 1 CH 2 CH 2 ) b1 -(CH 2 ) a7 - and; X 1 and X 3 are each independently -O-, -S-, -NH-, or H-CH 2 - and; X'' is -NHC(=O)-(CH 2 ) a8 -W a9 - or -C(=O)NH-(CH 2 ) a8 -W a9 - and; 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-; Q 2is a linear or branched, saturated or unsaturated alkylene having 1 to 50 carbon atoms that satisfies any one of the following (i) to (iii): (i) at least one -CH in the alkylene 2 - is substituted with one or more heteroatoms selected from -NH-, -C(=O), -O-, and -S-; (ii) the alkylene group contains at least one arylene or heteroarylene group; (iii) Alkylene is C 1 -C 20 Alkyl, C 6 -C 20 Aryl C 1 -C 8 Alkyl, -(CH 2 ) s1 COOR 3 , -(CH 2 ) s1 COR 3 , -(CH 2 ) s2 CONR 4 R 5 , and -(CH 2 ) s2 NR 4 R 5 and is further substituted with one or more selected from the group consisting of: The arylene or heteroarylene in (ii) above may be further substituted with nitro; R 3 , R 4 , and R 5 are each independently hydrogen or C 1 -C 15 is alkyl; X 2 is -O-, -S-, -NH-, or -CH 2 - and; U 1 is attached to B' at the asterisk (*) by a linking group selected from the following structures: [ka] R is C 1 -C 10 Alkyl, C6 -C 20 Aryl or C 2 -C 20 is heteroaryl; TM and B' are each independently a ligand or a protein having the property of selectively targeting a drug to a specific organ, tissue, or cell, i.e., the property of binding to a receptor; a1, a2, a3, a4, a5, a6, a8, b1, p1, p2, p3, and p4 each independently represent an integer selected from 1 to 10; a7, y, s1, s2, and s4 are each independently an integer selected from 0 to 10; and R 1 and R 2 are each independently hydrogen, C 1 -C 8 Alkyl or C 3 -C 8 It is cycloalkyl. In yet a further aspect, provided herein is a targeted drug conjugate of formula (VIc) comprising a targeting moiety conjugated to a drug conjugate of the present disclosure: [ka] During the ceremony: TM is the targeting moiety; G is a glucuronic acid moiety or a derivative thereof; Q is a sugar, sulfate, or sulfonate substituted active agent; W is an electron withdrawing group; Z is hydrogen, C 1 -C 8 is alkyl, halogen, cyano, or nitro; n is an integer selected from 1 to 3, and when n is an integer of 2 or more, each Z(s) is the same or different from each other; L is a linker connecting TM and W; and R 1 and R 2 are each independently hydrogen, C 1 -C 8 Alkyl or C 3-C 8 It is cycloalkyl.

[0193] In certain aspects, provided herein is a targeted drug conjugate of formula (VId) comprising a targeting moiety conjugated to a drug conjugate of the present disclosure: TM-L 1 -(A a -W w -Y y -Q 1-4 ) p (VId); Wherein TM is a targeting moiety; L 1 is the ligand moiety; Q is a sugar, sulfate, or sulfonate substituted active agent; -A a -W w -Y y - is a linker moiety; A is an optional stretcher section; a is an integer selected from 0 to 3; Each W is independently a glucuronide unit having one of the following formulas: [ka] Su is a sugar moiety; Each R is independently hydrogen, halogen, -CN, or -NO 2 and; w is an integer selected from 1 to 2; Y is an optional self-immolative spacer moiety; y is an integer selected from 0 to 2; and p is an integer selected from 1 to 20.

[0194] Active Agent Release As mentioned above, in certain embodiments, the compounds and conjugates disclosed herein may release one or more active agents through a chemical reaction that activates a trigger group followed by an intramolecular cyclization reaction. In certain embodiments, the chemical reaction is a physicochemical reaction and / or a biochemical reaction.

[0195] In some embodiments, the compounds and conjugates disclosed herein include a nucleophilic functional group (Y or Y') introduced at an atom on Ar adjacent to X (e.g., O). Generally, the nucleophilic functional group is masked by a trigger group (TG), as described in more detail below. Upon activation, the trigger group releases the nucleophilic functional group and catalyzes intramolecular cyclization to a nearby SO 2 moiety to ultimately release one or more compounds of formula (II), (IIa), or (IIb). In some such embodiments, the one or more active agents are released through an intramolecular cyclization reaction following a chemical, physicochemical, and / or biochemical reaction (see, e.g., Reaction Scheme 1), or the active agents are released via 1,6-elimination or 1,4-elimination following an intramolecular cyclization reaction (see, e.g., Reaction Scheme 2).

[0196] For example, Y is -Y'-TG and Q is directly SO 2 In the case of an active agent that is conjugated to a group, the active agent may be released by the mechanism shown in Reaction Scheme 1: [ka] Q is [ka] In this case, Q 1 can be released by the mechanism shown in Reaction Scheme 2: [ka]

[0197] In some embodiments, Q 1is an active agent that, when released, comprises at least one functional group selected from -C(O)-, -OH, -NH-, -SH, -COH, and -COOH. According to these embodiments, Q, as further described herein, 1 is conjugated to a compound as described herein through a functional group selected from -C(O)-, -OH, -NH-, -SH, -COH, and -COOH, e.g., ester, amide, thioester, carbamate, urea, oxime, hydrazone, and the like. In some such embodiments, Q 2 But, Q 1 is used instead of Q 2 is a drug that contains an amine group. 2 is an activator capable of binding to an ammonium unit. 2 Q 2 Upon release, it may dissociate in its original form bearing the amine groups, where the active agent may be a drug, a toxin, an affinity ligand, a detection probe, or a combination thereof.

[0198] In some embodiments, the compounds and conjugates disclosed herein are chemically and physicochemically stable. 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.

[0199] Trigger group (TG) In some embodiments, the conjugates of the present disclosure include a trigger group (TG). A TG is a group that is cleavable, preferably selectively cleavable, by a chemical reaction, such as a biological reaction. In general, the trigger group serves to mask the nucleophilicity of the Y' group, thereby providing stability to the compounds and conjugates disclosed herein (e.g., by preventing self-immolation or intramolecular cyclization before the conjugate reaches the target location or experiences a predetermined trigger condition). Upon activation, the trigger group releases the nucleophilic Y group, which undergoes self-immolation or intramolecular cyclization as described above.

[0200] In some embodiments, the TG comprises a sequence (e.g., a peptide sequence) or moiety recognized by TEV, trypsin, thrombin, cathepsin B, cathepsin D, cathepsin K, caspase 1, matrix metalloproteinase (MMP), etc., which may be hydrolyzed by an enzyme (e.g., oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, etc.), and / or may comprise a moiety selected from sulfate, phosphodiester, phospholipid, ester, β-galactose, β-glucose, fucose, oligosaccharide, etc.

[0201] 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).

[0202] 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).

[0203] In some embodiments, the TG may contain a reactive chemical moiety or functional group that can be cleaved by photoirradiation (e.g., 2-nitrobenzyl derivatives, phenacyl esters, 8-quinolinylbenzenesulfonates, coumarins, phosphotriesters, bis-arylhydrazones, or bimanbi-thiopropionic acid derivatives).

[0204] 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, levulinic acid, nitro, or 4-nitrobenzyl derivatives).

[0205] 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, trityls, vinyl ethers, polyketals, and 2-(diphenylphosphino)benzoic acid alkyl derivatives, alkyl esters, 8-hydroxyquinoline esters, and picolinic acid esters).

[0206] 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).

[0207] In certain preferred embodiments, the TG comprises a sugar that can be cleaved under acidic or enzymatic conditions. In certain preferred embodiments, the trigger group comprises -NO, which can be cleaved under reducing conditions. 2 In certain preferred embodiments, the trigger group is a -boronate, which can be cleaved under reducing conditions. In certain preferred embodiments, the trigger group is an ester, which can be cleaved under acidic, basic or enzymatic conditions. In certain preferred embodiments, the trigger group is a hydrazone, which can be cleaved under nucleophilic or acidic conditions. In certain preferred embodiments, the trigger group is a hydroxylamine, which can be cleaved under reducing conditions.

[0208] Sugar trigger group In some embodiments, the compounds and conjugates disclosed herein include a sugar trigger group, for example a trigger group selected from the following: [ka] Including, In the formula, each R 21 are independently hydrogen or OR 21is 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 group is, but is 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-1 0-oxo)anthryl ether, trimethylsilyl ether, isopropyldimethylsilyl ether, t-butyldimethylsilyl ether, t-butyldiphenylsilyl ether, tribenzylsilyl ether, triisopropylsilyl ether, formate ester, acetate ester, trichloroacetate ester, phenoxyacetate ester, isobutyrate ester, pivalonate ester, adamantate ester, benzoate ester, 2,4,6-trimethylbenzoate ester, methyl carbonate, 2,2,2-trichloroethyl carbonate, allyl carbonate, p-nitrophenyl carbonate, benzyl carbonate, p-nitrobenzyl carbonate, S-benzylthiocarbonate, N-phenylcarbamate ester, nitrate ester, 2,4-dinitrophenylsulfonate ester, and the like may be used in organic synthesis, including, but not limited to, the following:

[0209] In certain embodiments, the TG is a monosaccharide. In further embodiments, the TG is a monosaccharide selected from glucose, glucuronic acid, fucose, and galactose. In yet further embodiments, the TG is [ka] and Optionally, one or more -OH groups are masked with a protecting group.

[0210] In yet a further embodiment, TG is [ka] It is.

[0211] In certain embodiments, the TG is a disaccharide. In further embodiments, the TG is a disaccharide comprising glucose, glucuronic acid, fucose, galactose, or a combination thereof. In yet further embodiments, the TG is [ka] and Optionally, one or more -OH groups are masked with a protecting group.

[0212] In yet a further embodiment, TG is [ka] It is.

[0213] In certain embodiments, Y' or L' is attached to TG at the anomeric position.

[0214] Protecting Groups as Trigger Groups In some embodiments, TG is a group that can be cleaved by chemical reaction, physicochemical reaction, and / or biological reaction. In certain embodiments, TG is a protecting group. In some such embodiments, the protecting group is an amine protecting group, an alcohol protecting group, or a thiol protecting group.

[0215] Amine Protecting Groups In certain embodiments, the amine protecting group is a common 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 carbamate)methyl, alkyl carbamate, 9-fluorenylmethyl carbamate, 2,2,2-trichloroethyl carbamate, 2-trimethylsilylethyl carbamate (Teoc), t-butyl carbamate (Boc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc). , 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, benzyl carbamate, p-methoxybenzyl carbamate, p-nitrobenzyl carbamate, diphenylmethyl carbamate, acetamide, chloroacetamide, trichloroacetamide, phenylacetamide, benzamide, N-phthalimide, N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, benzenesulfenamide, o-nitrobenzenesulfenamide, triphenylmethylsulfenamide, p-toluenesulfonamide, methanesulfonamide, and the like.

[0216] Alcohol Protecting Groups In certain embodiments, the alcohol protecting group is a common 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-methoxytetrahydro ... 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, triphenyl methyl ether, α-naphthyldiphenyl methyl ether, p-methoxyphenyldiphenyl methyl ether, 9-(9-phenyl-10-oxo)anthryl ether, trimethylsilyl ether (TMS ether), isopropyldimethylsilyl ether, t-butyldimethylsilyl ether (TBDMS ether), t-butyldiphenylsilyl ether, tribenzylsilyl ether, triisopropylsilyl ether, formate ester, acetate ester, trichloroacetate ester, phenoxyacetate ester, isobutyrate ester, pivalate ester, adamantate ester, benzoate ester, 2,4,6-trimethylbenzoic acid (mesitoate) ester, methyl carbonate, 2,2,Examples of the aryl carbonate include, but are not limited to, 2-trichloroethyl carbonate, allyl carbonate, p-nitrophenyl carbonate, benzyl carbonate, p-nitrobenzyl carbonate, S-benzylthiocarbonate, N-phenyl carbamate, nitrate ester, 2,4-dinitrophenylsulfenate ester, dimethylphosphinyl ester (DMP ester), dimethylthiophosphinyl ester (MPT ester), arylmethanesulfonate, and aryltoluenesulfonate.

[0217] Thiol Protecting Groups In certain embodiments, thiol protecting groups may 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-ethyl carbamate), S-(N-methoxymethyl carbamate), and the like.

[0218] The attachment group (linking group) from the drug conjugate to the targeting moiety In some embodiments, the compounds and conjugates disclosed herein include a linking group connecting each TM and Ar via a covalent bond. Exemplary linking groups include, for example, C 10 -C 100 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 replaced by one or more heteroatoms selected from -NH-, -C(=O), -O-, -S-, and -P-; (ii) the alkylene moiety contains at least one heteroarylene; (iii) at least one amino acid moiety, glycolinkage, peptide linkage, or amide linkage is contained in the alkylene moiety; and (iv) The 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 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.

[0219] 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) The 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 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.

[0220] In other embodiments, the linking group connecting each TM and Ar comprises a functional group generated by click chemistry.

[0221] In an alternative embodiment, the linking unit comprises a reactive functional group capable of participating in a click chemistry reaction.

[0222] Click chemistry is a reaction that can be carried out under mild conditions and is highly selective for functional groups that are not usually found in biomolecules (such as azide groups, acetylene groups, etc.). Therefore, the reaction can be carried out in the presence of complex trigger 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 being affected by other functional groups present in the molecule. For example, azide-acetylene click chemistry can sometimes provide triazole moieties in high yield.

[0223] Thus, in some embodiments, the linking group connecting each TM 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 - and 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 may be an integer having a value of 1 to about 10, p may be an integer having a value of 0 to about 10, q may be an integer having a value of 1 to about 10, and L" may be a single bond.

[0224] A variety of linking groups are suitable for use with the presently disclosed drug conjugates. When a targeting moiety is not present (i.e., when the drug conjugate is not a targeted drug conjugate), the linking group comprises a terminal reactive functional group that can react with a targeting moiety. In some embodiments, Z' is [ka] [ka] [ka] is selected from During the ceremony: R za is H or methyl; R zb is -OH, =O, or =NHOH; n and m are each independently an integer selected from 1 to 10; x is an integer selected from 1 to 2; [ka] represents the bond between Z′ and the drug conjugate; [ka] is a single or double bond; Z” is [ka] is selected from.

[0225] In further embodiments, when a targeting moiety is present (i.e., the drug conjugate is a targeted drug conjugate), the linking group links the conjugate to the targeting moiety. [ka] [ka] [ka] is selected from During the ceremony, R za is H or methyl; R zb is -OH, =O, or =NHOH; [ka] is a single or double bond; n and m are each independently an integer selected from 1 to 10; x is an integer selected from 1 to 2; a″ represents the bond between Z′ and the drug conjugate; b" represents the bond between Z' and TM; and Z” is as follows: [ka] is selected from.

[0226] targeting part The compounds and conjugates of the present disclosure may further comprise one or more ligands or targeting moieties, TM. In some embodiments, the ligand or targeting moiety is any molecular recognition element capable of undergoing specific interaction with at least one other molecule through non-covalent bonds, such as, for example, hydrogen bonds, metal coordination, hydrophobic forces, van der Waals forces, π-π interactions, halogen bonds, electrostatic and / or electromagnetic effects. In certain embodiments, the TM is selected from nanoparticles, immunoglobulins, nucleic acids, proteins, oligopeptides, polypeptides, antibodies, fragments of antigenic polypeptides, repebodies, and the like.

[0227] The compounds and conjugates of the present disclosure may include one or more targeting moieties. In certain embodiments, 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 repebody. In further embodiments, the targeting moiety is an antibody selected from an intact polyclonal antibody, an intact monoclonal antibody, an antibody fragment, a single chain Fv (scFv) variant, a multispecific antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, a fusion protein containing an antigenic determinant of an antibody, and other modified immunoglobulin molecules containing an antigen recognition site.

[0228] In still further embodiments, the antibody is muromonab-CD3, abciximab, rituximab, daclizumab, palivizumab, infliximab, trastuzumab (herceptin), etanercept, basiliximab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, alefacept, omalizumab, efalizumab, tositumomab-I 131 , Cetuximab, Bevacizumab, Natalizumab, Ranibizumab, Panitumumab, Eculizumab, Rilonacept, Certolizumab pegol, Romiplostim, AMG-531, CNTO-148, CNTO-1275, ABT-874, LEA-29Y, Belimumab, TACI-Ig, Second generation anti-CD20, ACZ-885, Tocilizumab, Atolizumab, Mepolizumab, Pertuzumab, HuMax CD20, Tremelimumab (CP-675 206), Ticilimumab, MDX-010, IDEC-114, Inotuzumab ozogamicin, HuMax Selected from EGFR, aflibercept, HuMax-CD4, Ala-Ala, ChAglyCD3, TRX4, catumaxomab, IGN101, MT-201, pregovomab, CH-14.18, WX-G250, AMG-162, AAB-001, motavizumab, MEDI-524, efumugumab, aurograb, raxibacumab, third generation anti-CD20, LY2469298, and veltuzumab.

[0229] In some embodiments, a TM 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., previously engineered proteins).

[0230] In some embodiments, the TM is selected from an antibody that binds to a receptor, a hormone, a drug, an antibody analog (e.g., non-IgG), a protein, an oligopeptide, a polypeptide, etc. In certain embodiments, the TM selectively targets a drug within a particular organ, tissue, or cell. In other embodiments, the TM 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 TM 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.

[0231] Exemplary ligands or targeting moieties include, but are not limited to, 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), transferein, 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, 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 (non-metastatic glycoprotein) b), Ley, CA6, CanAng, SLC44A4 (solute carrier family 44 member 4), CEACAM5 (carcinoembryonic antigen-related cell 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 benzodiazepine receptor), MERTK (Mer receptor tyrosine kinase) receptor, CD71,LLT1 (lectin-like) transcript 1 or CLED2D), interleukin-22 receptor, sigma 1 receptor, peroxisome proliferator-activated receptor, DLL3, C4.4a, cKIT, ephrin A, CTLA4 (cytotoxic T-lymphocyte-associated protein 4), FGFR2b (fibroblast growth factor receptor 2b), N-acetylcholine receptor, gonadotropin-releasing hormone receptor, gastrin-releasing peptide receptor, bone morphogenetic protein receptor type 1B (BMPR1B), E16 (LAT1, SLC7A5), STRAP1 (prostate six-transmembrane epithelial antigen), 077 2P (CA125, MUC16), MPF (MSLN, mesothelin), Napi3b (SLC34A2), Sema5b (semaphorin 5b), ETBR (endothelin type B receptor), MSG783 (RNF124), STRAP2 (six-transmembrane epithelial antigen of prostate 2), TrpM4 (transient receptor potential cation 5 channel, subfamily M, member 4), CRIPTO (teratocarcinoma-derived growth factor), CD21, CD79b, FcRH2 (IFGP4), HER2 (ErbB2), NCA (CEACM6), MDP (DPEP1), IL20R-alpha (I N20Ra), 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, SSTR2, SSTR5, SSTR1, SSTR3, SSTR4, ITGAV (integrin, alpha 5), ​​ITGB6 (integrin, beta 6), MET, MUC1, EGFRvIII, CD33, CD19, IL2RA (integrin, beta 6) Interleukin 2 receptor), alpha), AXL, BCMA, CTA (cancer testis antigen), CD174, CLEC14A, GPR78, CD25, CD32, LGR5 (GPR49), CD133 (prominin), ASG5, ENPP3 (ectonucleotide pyrophosphatase / phosphodiesterase 3), PRR4 (proline-rich protein 4), GCC (guanylate cyclase 2C), Liv-1 (SLC39A6), CD56, CanAg, TIM-1, RG-1, B7-H4, PTK7, CD138, Claudins, Her3 (ErbB3),RON (MST1R), CD20, TNC (tenascin C), FAP, DKK-1, CD52, CS1 (SLAMF7), annexin A1, V-CAM, gp100, MART-1, MAGE-1 (melanoma antigen-encoding gene-1), MAGE-3 (melanoma-associated antigen 3), BAGE, GAGE-1, MUM-1 (multiple myeloma oncogene 1), CDK4, TRP-1 (gp75), TAG-72 (tumor-associated glycoprotein-72), ganglioside GD2, GD3, GM2, GM3, VEP8, VEP9, My1, VIM-D5, D156-22, OX40, RNAK, PD-L1, TNFR1, TNFR2, etc.

[0232] target In some embodiments, the target(s) of the molecular recognition element are specifically associated with one or more particular cell 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 that 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 greater than its average expression in a reference population. Detection or measurement of a cell type specific marker may allow the cell type(s) of interest to be distinguished 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.

[0233] 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.

[0234] 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.).

[0235] In some embodiments, the conjugates and compounds described herein comprise a targeting moiety that binds to a target specific for 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 for one or more particular tissue 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 for 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 for one or more particular developmental stages (e.g., stem cells vs. differentiated cells).

[0236] In some embodiments, the target may be a marker that is exclusively or primarily associated with one or a few cell types, one or a few diseases, and / or one or a few developmental stages. A cell type specific marker is typically expressed in that cell type at a level at least two times higher than a reference population of cells, 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, the cell type specific marker is present at a level that is at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least fifty times, at least one hundred times, or at least one thousand times greater than its average expression in the reference population. Detection or measurement of a cell type specific marker may allow one to distinguish a cell type of interest from many, most, or all other types of cells.

[0237] In some embodiments, targets include proteins, carbohydrates, lipids, and / or nucleic acids. In some embodiments, targets include proteins and / or characteristic portions thereof, such as tumor markers, integrins, cell surface receptors, transmembrane proteins, intracellular proteins, ion channels, membrane transport proteins, enzymes, antibodies, chimeric proteins, glycoproteins, and the like. In some embodiments, targets include carbohydrates and / or characteristic portions thereof, such as glycoproteins, sugars (e.g., monosaccharides, disaccharides, polysaccharides), glycocalyx (i.e., the carbohydrate-rich peripheral region on the outer surface of most eukaryotic cells). In some embodiments, targets include lipids and / or characteristic portions thereof, such as oils, fatty acids, glycerides, hormones, steroids (e.g., cholesterol, bile acids), vitamins (e.g., vitamin E), phospholipids, sphingolipids, lipoproteins, and the like. In some embodiments, targets include nucleic acids and / or characteristic portions thereof, such as DNA nucleic acids; RNA nucleic acids; modified DNA nucleic acids; modified RNA nucleic acids; nucleic acids including any combination of DNA, RNA, modified DNA, and modified RNA.

[0238] 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 receptors; LDL receptors; growth factor receptors, such as epidermal growth factor receptor family members (e.g., EGFR, Her2, Her3, Her4) or vascular endothelial growth factor receptors, cytokine receptors, cell adhesion molecules, integrins, selectins, and CD molecules. Markers can be molecules that are present exclusively or in greater amounts on malignant cells, such as tumor antigens.

[0239] Antibody-drug conjugates (ADCs) In some embodiments, TM is an antibody and Q is a drug. Thus, the compounds and conjugates disclosed herein can be used to conjugate an antibody to a drug moiety to form a targeted drug conjugate, which is an antibody-drug conjugate (ADC). Antibody drug conjugates (ADCs), like other targeted drug conjugates, may enhance therapeutic efficacy in the treatment of diseases such as 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 disclosure provides ADCs for therapeutic applications, e.g., for the treatment of cancer.

[0240] The ADC of the present disclosure comprises an antibody linked to one or more drug moieties. The specificity of the ADC is defined by the specificity of the antibody. In one embodiment, the antibody is conjugated to one or more cytotoxic drug(s) that are delivered to the interior of the cancer cells.

[0241] Examples of drugs that can be used in the ADCs of the disclosure 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 refer to the covalent attachment of an antibody to a moiety.

[0242] In certain aspects, the disclosure relates to ADCs, compositions comprising ADCs, methods of treatment, and methods of formulating ADC compositions. The ADC comprises an antibody or antibody fragment linked to a cytotoxic compound. In some embodiments, the cytotoxic compound is conjugated to the antibody via a linker. In other embodiments, the cytotoxic compound is directly attached to the antibody. Types of antibodies, linkers, and cytotoxic compounds encompassed by the disclosure are described below.

[0243] antibody The antibody of the ADC can be any antibody that typically, but not necessarily specifically, binds to an antigen expressed on the surface of a target cell of interest. In some embodiments, the antigen-bound ADC can be internalized intracellularly, although an antigen is not required. 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 is a protein that is specifically expressed on the target cell and not expressed on normal or healthy cells, or a protein that is overexpressed on the target cell, as compared to normal or healthy cells, such that the ADC selectively targets a particular cell of interest, such as, for example, a tumor cell. As will be appreciated by those skilled in the art, the particular antigen, and therefore the antibody, selected will depend on the identity of the desired target cell of interest. In certain embodiments, the antibody of the ADC is an antibody suitable for administration to humans.

[0244] Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins with the same structural characteristics. Antibodies exhibit binding specificity to a specific target, while immunoglobulins include both antibodies and other antibody-like molecules that lack target specificity. Natural 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.

[0245] 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.

[0246] The term "antibody" is used herein in the broadest sense to refer to an immunoglobulin molecule that specifically binds to or immunologically reacts with a particular antigen, and includes polyclonal, monoclonal, genetically engineered, or otherwise modified forms of antibodies, including, but not limited to, murine, chimeric, humanized, heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, 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 of a conventional antibody are combined to form one chain.

[0247] Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that can recognize and bind to a specific antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has multiple binding sites, also called epitopes, that are recognized by the CDRs of multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have two or more corresponding antibodies. Antibodies include full-length immunoglobulin molecules, or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to a target antigen or a portion thereof of interest, such targets including, but not limited to, cancer cell(s) 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.

[0248] The term "antibody fragment" refers to a portion of a full-length antibody, generally its 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 and one light chain variable domain in tight non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target-binding site on the surface of the VH-VL dimer. Often, the six CDRs confer target-binding specificity to the antibody. However, in some cases, even a single variable domain (or half of an Fv containing only three target-specific CDRs) may 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, which 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 certain embodiments, a single domain antibody is a camelized antibody (see, e.g., Riechmann, 1999, Journal of Immunological Methods 231:25-38).

[0249] Fab fragments also 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 carboxy 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 disulfide bonds at the hinge cysteines of the F(ab')2 pepsin digestion product. Further chemical coupling of antibody fragments is known to those skilled in the art.

[0250] 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 delineate the hypervariable regions of an antibody may vary depending on the context and the various definitions known in the art. Some positions within a variable domain may be considered hybrid hypervariable positions, because they may be considered to be within a hypervariable region under one set of criteria, but outside of a 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 of the other chain, contribute to the formation of the target binding site of antibodies (Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987)). In this specification, numbering of immunoglobulin amino acid residues is done according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise specified.

[0251] In certain embodiments, the antibody of the ADC of the present disclosure is a monoclonal antibody. 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 antibody of the present disclosure is present in a homogenous or substantially homogenous population. Monoclonal antibodies include intact molecules as well as antibody fragments that can specifically bind to proteins, such as, for example, Fab fragments and F(ab')2 fragments. Fab and F(ab')2 fragments lack the Fc fragment of intact antibodies, are cleared more rapidly from the circulation 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 may 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.

[0252] In most cases, antibodies are composed only of genetically encoded amino acids, although in some embodiments, non-encoded amino acids may be incorporated into specific moieties. Examples of non-encoded amino acids that can be incorporated into antibodies for use in controlling stoichiometry and binding sites, as well as methods for making such modified antibodies, are described 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.

[0253] 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 generating 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 entireties.

[0254] In certain embodiments, the antibody of the ADC described herein is a humanized antibody. A "humanized" form of a non-human (e.g., murine) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from a non-human immunoglobulin. In general, a humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin sequence. A humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), usually at least a portion of a human immunoglobulin 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; 5,693,762; and 6,180,370 (to Queen et al.); European Patent No. 239400; PCT Publication WO 91 / 09967; U.S. Pat. No. 5,225,539; European Patent No. 592106; European Patent No. 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; and US Pat. No. 5,565,332, all of which are incorporated by reference herein in their entireties.

[0255] In certain embodiments, the antibodies of the ADCs described herein are human antibodies. Fully "human" antibodies may be desirable for therapeutic treatment of human patients. As used herein, "human antibodies" include antibodies having human immunoglobulin amino acid sequences, including antibodies isolated from human immunoglobulin libraries or from animals that are transgenic for one or more human immunoglobulins and do not express endogenous immunoglobulins. Human antibodies may be generated 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 herein by reference in their entirety. Human antibodies may also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but that can express human immunoglobulin genes. See, e.g., U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; 5,939,598; 7,723,270; 8,809,051 and U.S. Patent Application Publication No. 2013 / 117871, which are incorporated herein by reference in their entireties. Additionally, companies such as Medarex (Princeton, NJ), Astellas Pharma (Deerfield, Ill.), and Regeneron (Tarrytown, NY) can be engaged to provide human antibodies against a selected antigen using technology similar to that described above. Fully human antibodies that recognize a selected epitope can be generated using a technique called "guided selection."In this approach, a selected non-human monoclonal antibody, such as a murine antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope (Jespers et al., 1988, Biotechnology 12:899-903).

[0256] In certain embodiments, the antibody of the ADC described herein is a primatized antibody. The term "primatized antibody" refers to an antibody comprising a monkey variable region and a human constant region. Methods for producing primatized antibodies are known in the art. See, 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.

[0257] 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.

[0258] 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, binding to cellular ligands or other proteins, 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. In addition, a derivative can include one or more unnatural amino acids, for example, using ambrx technology (see, e.g., Wolfson, 2006, Chem. Biol. 13(10):1011-2).

[0259] In certain embodiments, the antibodies of the ADCs described herein have sequences that are modified to alter at least one constant region-mediated biological effector function compared to the corresponding wild-type sequence. For example, in some embodiments, the antibodies can be modified to reduce at least one constant region-mediated biological effector function compared to an unmodified antibody, e.g., to reduce binding to Fc receptors (FcRs). FcR binding can be reduced by mutating the immunoglobulin constant region segment of the antibody at specific regions required for FcR interaction (see, e.g., Canfield and Morrison, 1991, J. Exp. Med 173:1483-1491; and Lund et al., 1991, J. Immunol. 147:2657-2662).

[0260] 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 compared to an unmodified antibody, e.g., to enhance FcγR interactions (see, e.g., U.S. Patent Application Publication No. 2006 / 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.

[0261] 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 specifically expressed on the surface of one or more particular types of cancer cells, as compared to one or more normal non-cancerous cell(s). Often, such tumor-associated polypeptides are more abundantly expressed on the surface of cancer cells, as 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.

[0262] Exemplary Cell Surface Receptors and TAAs Examples of cell surface receptors and TAAs that may be targeted by the antibodies of the ADCs described herein include, but are not limited to, the various receptors and TAAs listed in Table 1 below. For convenience, information relating to these antigens (all known in the art) is listed below, including the convention of identifying the name, synonyms, Genbank accession numbers, and primary reference(s) followed by the National Center for Biotechnology Information (NCBI) nucleic acid and protein sequences. The nucleic acid and protein sequences corresponding to the listed cell surface receptors and TAAs are available in public databases, such as Genbank. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0263] Exemplary Antibodies Exemplary antibodies for use with the ADCs of the disclosure include, but are not limited to, 3F8 (GD2), abagovomab (CA-125 (mimetic)), adecatumumab (EpCAM), afutuzumab (CD20), alacizumab pegol (VEGFR2), ALD518 (IL-6), alemtuzumab (CD52), altumomab pentetate (CEA), Amatuximab (mesothelin), anatumobumafenatox (TAG-72), apolizumab (HLA-DR), arcitumomab (CEA), bavituximab (phosphatidylserine), bectumomab (CD22), belimumab (BAFF), besilesomab (CEA-associated antigen), bevacizumab (VEGF-A), bivatuzumab mertansine (CD44 v6), blinatumab (CD19), brentuximab vedotin (CD30 (TNFRSF8)), cantuzumab mertansine (mucin CanAg), cantuzumab mertansine (MUC1), capromab pendetide (prostate cancer cells), carumab (MCP-1), catumaxomab (EpCAM, CD3), CC49 (Tag-72), cBR96-DOX ADC (Lewis Y antigen), cetuximab (EGFR), sitatuzumab 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 lymphoma cells), drozitumab (DR5), dusigitumab (ILGF2), ecromeximab (D3 ganglioside), eculizumab (C5), edrecolomab (EpCAM), elotuzumab (SLAMF7), ercilimab (IL-6), enavatuzumab (TWEAK receptor), enoticumab (DLL4), encituximab (5AC), epitumomab situxetan (episialin), epratuzumab (CD22), ertumaxomab ((HER2 / neu, CD3)), etancizumab (integrin αvβ3), farletuzumab (folate receptor 1), FBTA05 (CD20), ficlatuzumab (HGF), figitumumab (IGF-1 receptor),Flavotumab (TYRP1 (glycoprotein 75)), fresolimumab (TGF-1), galiximab (CD80), ganitumab (IGF-I), gemtuzumab ozogamicin (CD33), girentuximab (carbonic anhydrase 9 (CA-IX)), glemtuzumab vedotin (GPNMB), ibritumomab tiuxetan (CD20), icrucumab (VEGFR-1), igovomab (CA-125), IMAB362 (CLDN18.2), imgatuzumab (EGFR), indatuximab ravtansine (SDC1), intetumumab (CD51) , inotuzumab ozogamicin (CD22), ipilimumab (CD152), iratumumab ((CD30 (TNFRSF8)), labetuzumab (CEA), lambrolizumab (PDCD1), lexatumumab (TRAIL-R2), lintuzumab (CD33), lorvotuzumab mertansine (CD56), lucatumumab (CD40), rumiliximab ((CD23 (IgE receptor))), mapatumumab (TRAIL-R1), margetuximab (ch4DS), matuzumab (EGFR), milatuzumab (CD74), mitumomab (GD3 ganglioside) , mogamulizumab (CCR4), moxetumomab passudotox (CD22), nacolomab butafenatox (C2-42 antigen), naputumab estafenatox (5T4), narutumab (RON), natalizumab (integrin α4), necitumumab (EGFR), nesvacumab (angiopoietin 2), nimotuzumab (EGFR), nivolumab (IgG4), ocaratulumab (CD20), ofatumumab (CD20), olaratulumab (PDGF-Rα), onartuzumab (human scatter factor receptor kinase), ontucizumab (TEM1), onartuzumab (human scatter factor receptor kinase), onartuzumab (TEM2), onartuzumab (TEM1 ... Portuzumab monat (EpCAM), oregovomab (CA-125), otlertuzumab (CD37), panitumumab (EGFR), pancomab (tumor-specific glycosylation of MUC1), palsatuzumab (EGFL7), patritumab (HER3), pemtumomab (MUC1), pertuzumab (HER2 / neu), pidilizumab (PD-1), pinatuzumab vedotin (CD22), pritumumab (vimentin), racotumab (N-glycolylneuraminic acid), radletumab (fibronectin extra domain B), ramucirumab (VEGFR2),Rilotumab (HGF), Rituximab (CD20), Lobatumumab (IGF-1 receptor), Samalizumab (CD200), Satumomab pendetide (TAG-72), Seribantumab (ERBB3), Sibrotuzumab (FAP), SGN-CD19A (CD19), SGN-CD33A (CD33), Siltuximab (IL-6), Solitomab (EpCAM), Soneptizumab (Sphingosine-1-phosphate), Tabalum (BAFF), Tacatuzumab tetraxetan (Alpha-fetoprotein), Taplitumomab paptox (CD19), Tenatumomab (Tenascin-C), Teprotuzumab (CD221), TGN1412 (CD28), Ticili tuzumab (CTLA-4), tigatuzumab (TRAIL-R2), TNX-650 (IL-13), tobetumab (CD40a), trastuzumab (HER2 / neu), TRBS07 (GD2), tremelimumab (CTLA-4), tucotuzumab celmoleukin (EpCAM), ublituximab (MS4A), urelumab (4-1BB), vandetanib (VEGF), vantizumab (Frizzled receptors), boroximab (integrin α5β1), borsetuzumab mafodotin (CD70), votumumab (tumor antigen CTAA16.88), zalutumumab (EGFR), zanolimumab (CD4), and zatuximab (HER1).

[0264] 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 recombinantly express an antibody, one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody are transfected into a host cell, so that the light and heavy chains are 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 a host cell, such as the host cells 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.

[0265] 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 may be synthesized and used as a template for mutagenesis to generate antibodies as described herein using conventional mutagenesis techniques; alternatively, a DNA fragment encoding the antibody may be directly synthesized.

[0266] Once the DNA fragments encoding the wild-type Fc domain are obtained, these DNA fragments may 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 operably linked to another DNA fragment encoding another protein, such as an antibody variable region or a flexible linker. The term "operably linked" as used in this context shall mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0267] To express the Fc variant antibody, the DNA encoding the partial or full-length light and heavy chains obtained as described above is inserted into an expression vector such that the 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 linked to 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 the 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 may be inserted into separate vectors, or more typically, both genes are inserted into the same expression vector.

[0268] The antibody gene is inserted into an expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). Prior to insertion of the variant Fc domain sequence, the expression vector may already contain an antibody variable region sequence. Additionally, or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene 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 may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0269] 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, Methods in Enzymology: Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif., 1990). It will be appreciated by those skilled 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 level of expression of protein desired, etc. Suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers 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. Pat. No. 5,168,062 (by Stinski), U.S. Pat. No. 4,510,245 (by Bell et al.), and U.S. Pat. No. 4,968,615 (by Schaffner et al.).

[0270] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors may 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 in DHFR- host cells with methotrexate selection / propagation) 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 foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, etc.

[0271] Antibodies can be expressed in either prokaryotic or eukaryotic host cells. In certain embodiments, expression of the antibody is carried out in eukaryotic cells, such as mammalian host cells, for optimal secretion of properly folded and immunologically active antibodies. Exemplary mammalian host cells for expressing recombinant antibodies include Chinese Hamster Ovary (CHO cells) (described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, including DHFR-CHO cells, used with a DHFR selection 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. When a recombinant expression vector encoding an antibody gene is 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 within 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.

[0272] In some embodiments, the antibody of the ADC may be a bifunctional antibody. Such an antibody, in which one heavy chain and one light chain are specific for one antigen and the other heavy and light chains are specific for a second antigen, may be produced by crosslinking the antibody to a second antibody by standard chemical crosslinking methods. Bifunctional antibodies may also be produced by expressing nucleic acids engineered to encode the bifunctional antibody.

[0273] 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 surrounding 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.

[0274] Antibodies may also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd Edition, 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 (Rochemolecular Biologicals)).

[0275] 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.

[0276] 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, affinity, particularly by affinity for the antigen after Protein A or Protein G selection, and by size exclusion chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

[0277] 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).

[0278] General antibody preparation methods Various procedures known in the art can be used to generate polyclonal or monoclonal antibodies against a given target, such as, for example, B7-H3, a tumor-associated antigen, or other target, or against derivatives, fragments, analogs, homologs, or orthologs thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0279] Antibodies can be purified by well-known techniques such as affinity chromatography using Protein A or Protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen that is the target of the desired immunoglobulin, or an epitope thereof, can be immobilized on a column, and the immune specific antibody can be purified by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0280] In some embodiments, the antibody that can be used in the embodiments disclosed herein is a monoclonal antibody. The monoclonal antibody is generated, for example, by using the procedures described in the examples provided herein. The antibody is also generated, for example, by immunizing BALB / c mice with a combination of cell transfectants that express high levels of a given target on the surface. The hybridoma resulting from the myeloma / B cell fusion is then screened for reactivity against the selected target.

[0281] Monoclonal antibodies are prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0282] The immunizing agent typically includes a protein antigen, a fragment thereof, or a fusion protein thereof. In general, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian origin is desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp.59-103). The immortalized cell line is usually a transformed mammalian cell, particularly a myeloma cell of rodent, bovine, and human origin. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells may be cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent growth of HGPRT-deficient cells.

[0283] Preferred immortalized cell lines are those that fuse efficiently, support stable high-level antibody expression by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are mouse myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of monoclonal antibodies (see Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.51-63).

[0284] The culture medium in which the hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known to those skilled in the art. The binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis (Munson and Pollard, Anal. Biochem., 107:220 (1980)). Furthermore, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies with high specificity and high binding affinity for the target antigen.

[0285] After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

[0286] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0287] Monoclonal antibodies may be produced by recombinant DNA methods such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of a murine antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector and then transfected into host cells such as Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, resulting in the synthesis of the monoclonal antibody in the recombinant host cells. The DNA may also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (see U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)), or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide may be substituted for the constant domains of an antibody or for the variable domains of one antigen-binding site of an antibody to create a chimeric bivalent antibody.

[0288] Monoclonal antibodies that may be used in the embodiments disclosed herein include humanized or human antibodies. These antibodies are suitable for administration to humans without eliciting an immune response by the human to the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')) that are composed primarily of human immunoglobulin sequences and contain minimal sequence derived from non-human immunoglobulins. 2, or other antigen-binding subsequence of an antibody). Humanization is performed by substituting rodent CDR or CDR sequences for the corresponding sequences of a human antibody following the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)). (See also U.S. Pat. No. 5,225,539). In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found, for example, neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

[0289] A fully human antibody is an antibody molecule in which the entire sequence of both the light and heavy chains, including the CDRs, is derived from human genes. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies." Monoclonal antibodies can be prepared using trioma technology; human B-cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today 4:72); and EBV hybridoma technology to produce monoclonal antibodies (see Cole, et al., 1985: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Monoclonal antibodies may be utilized and may be produced by the use of human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by in vitro transformation of human B cells with Epstein-Barr virus (see Cole, et al., 1985 In:MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0290] Moreover, human antibodies may also be produced using additional technologies, such as phage display libraries (see Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, as well as Marks et al., Bio / Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 1998. 65-93(1995).

[0291] Additionally, human antibodies may be further produced using transgenic non-human animals that have been modified to produce fully human antibodies in response to antigen challenge rather than the animal's endogenous antibodies. (See PCT Publication WO94 / 02602). The endogenous genes encoding heavy and light chain immunoglobulins of the non-human host are disabled and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes that contain the necessary human DNA segments. Animals that provide all the desired modifications are then obtained as progeny by mating intermediate transgenic animals that have less than the full complement of modifications. An example of such a non-human animal is a mouse called Xenomouse™, disclosed in PCT Publications WO96 / 33735 and WO96 / 34096. The animal produces B cells that secrete fully human immunoglobulins. Antibodies may be obtained directly from an animal after immunization with an immunogen of interest, e.g., as a polyclonal antibody preparation, or from immortalized B cells derived from the animal, such as hybridomas that produce monoclonal antibodies. Additionally, genes encoding immunoglobulins with human variable regions may be harvested and expressed to obtain antibodies directly, or may be further modified to obtain analogs of antibodies, such as, for example, single chain Fv (scFv) molecules.

[0292] An example of a method for producing a non-human host, exemplified as a mouse, lacking expression of endogenous immunoglobulin heavy chains is disclosed in U.S. Patent No. 5,939,598. This may be obtained by a method comprising deleting J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent the formation of transcripts of the rearranged immunoglobulin heavy chain locus, the deletion being performed by a targeting vector that contains a gene encoding a selection marker; and producing a transgenic mouse from the embryonic stem cell, the somatic and germ cells of which contain the gene encoding the selection marker.

[0293] One method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. The method comprises introducing an expression vector containing a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybridoma cell. The hybrid cell expresses the antibody comprising the heavy and light chains.

[0294] As a further refinement of this procedure, methods for identifying clinically relevant epitopes on immunogens and correlation methods for selecting antibodies that specifically bind with high affinity to the relevant epitopes are disclosed in U.S. Patent Application Publication No. 2003 / 009212.

[0295] The antibodies may be expressed by vectors containing a DNA segment encoding the single chain antibody described above.

[0296] These include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, etc. Vectors include chemical conjugates such as those described in WO93 / 64701 having a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid binding moiety (e.g., polylysine), viral vectors (e.g., DNA or RNA viral vectors), fusion proteins such as those described in U.S. Pat. No. 7,186,697, which are fusion proteins containing a targeting moiety (e.g., an antibody specific for a target cell) and a nucleic acid binding moiety (e.g., protamine), plasmids, phages, etc. Vectors may be chromosomal, non-chromosomal, or synthetic vectors.

[0297] Preferred vectors include viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Moloney Murine Leukemia Virus. DNA viral vectors are preferred. These vectors include pox vectors, e.g., orthopox or avipox vectors, herpes virus vectors, e.g., herpes simplex virus type I (HSV) vectors (Geller, AI et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al., Proc Natl. Acad. Sci.: USA 90:7603 (1993); Geller, AI, et al., Proc Natl. Acad. Sci USA 87:1149 (1990), Adenovirus Vectors (LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet. 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) and Adeno-associated Virus Vectors (see Kaplitt, M ​​Get al., Nat. Genet. 8:148 (1994)).

[0298] Poxvirus vectors introduce genes into the cytoplasm of cells. Avipoxvirus vectors result in only short-term expression of the nucleic acid. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors are preferred for introducing nucleic acids into neural cells. Adenovirus vectors result in a shorter period of expression (approximately 2 months) than adeno-associated virus (approximately 4 months), which in turn results in shorter expression than HSV vectors. The particular vector selected will depend on the target cell and the condition being treated. Introduction may be by standard techniques, such as infection, transfection, transduction, or transformation. Examples of modes of gene introduction include, for example, naked DNA, CaPO 4 These include precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0299] Vectors can be used to target essentially any desired target cell. For example, stereotactic injection may be used to guide vectors (e.g., adenovirus, HSV) to the desired location. Additionally, particles can be delivered by intracerebroventricular (icv) injection using a minipump infusion system such as the SynchroMed Infusion System. A bulk flow-based method called convection has also proven effective in delivering large molecules to widespread areas of the brain and may be useful for delivering vectors to target cells. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)). Other methods that can be used may include catheter, intravenous, parenteral, intraperitoneal and subcutaneous injection, and oral or other suitable routes of administration.

[0300] A bispecific antibody is an antibody that has binding specificities for at least two different epitopes. In this case, one of the binding specificities is for a target such as B7-H3 or any fragment thereof. The second binding target is another antigen, preferably a cell surface protein or receptor or receptor subunit.

[0301] Many methods for making bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually achieved by affinity chromatography steps. Similar techniques are disclosed in WO 93 / 08829 published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0302] Bispecific and / or monovalent antibodies that may be used in the embodiments disclosed herein may be produced using any of a variety of techniques recognized in the art, including those disclosed in application WO2012 / 023053, filed August 16, 2011, the contents of which are incorporated herein by reference in their entirety. The method described in WO2012 / 023053 produces bispecific antibodies that are identical in structure to human immunoglobulins. This type of molecule is composed of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant kappa domain, and a second light chain variable region fused to a constant lambda domain. Each binding site exhibits a different antigen specificity, with contributions from both the heavy and light chains. The light chain variable region may be of the lambda or kappa family, and is preferably fused to a lambda constant domain and a kappa constant domain, respectively. This is preferred to avoid the generation of non-natural polypeptide bonds. However, it is also possible to obtain bispecific antibodies that can be used in the embodiments disclosed herein by fusing a kappa light chain variable domain to a constant lambda domain for the first specificity and a lambda light chain variable domain to a constant kappa domain for the second specificity. The bispecific antibodies described in WO2012 / 023053 are called IgG κλ antibodies or "κλ bodies" and are a new fully human bispecific IgG format. This κλ body format is advantageous compared to previous formats, since it allows affinity purification of bispecific antibodies that are indistinguishable from standard IgG molecules, with properties that are indistinguishable from standard monoclonal antibodies.

[0303] The key step of this method is to identify two antibody Fv regions (each composed of a variable light chain and a variable heavy chain domain) that share the same heavy chain variable domain and have different antigen specificities. Many methods for generating monoclonal antibodies and fragments thereof have been described. (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference). A fully human antibody is an antibody molecule in which both the light and heavy chain sequences, including CDR1 and 2, are derived from human genes. The CDR3 region may be of human origin or designed by synthetic means. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies". Human monoclonal antibodies can be prepared using trioma technology; human B cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today 4:72); and EBV hybridoma technology producing human monoclonal antibodies (see Cole, et al., 1985 In:MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies may be utilized and may be produced by using human hybridomas (Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (Cole, et al., 1985 In:MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96).

[0304] Monoclonal antibodies are produced, for example, by immunizing animals with target antigens or their immunogenic fragments, derivatives or variants. Alternatively, animals are immunized with cells transfected with vectors containing nucleic acid molecules encoding target antigens, so that the target antigens are expressed and associated with the surface of the transfected cells. A variety of suitable techniques for producing xenogeneic non-human animals are well known in the art. See, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which are incorporated herein by reference in their entirety.

[0305] Alternatively, antibodies are obtained by screening libraries containing antibody or antigen-binding domain sequences that bind to the target antigen, for example, prepared in bacteriophage as protein or peptide fusions to bacteriophage coat proteins expressed on the surface of assembled phage particles and encoding DNA sequences contained within the phage particles (i.e., "phage display libraries").

[0306] The hybridoma resulting from the myeloma / B cell fusion is then screened for reactivity to the target antigen. Monoclonal antibodies are prepared using hybridoma methods such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, mice, hamsters, or other suitable host animals are typically immunized with an immunizing agent to induce lymphocytes that produce or can produce antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0307] Although not strictly impossible, it is highly unlikely that different antibodies with the same heavy chain variable domain but directed against different antigens would be identified by chance. In fact, in most cases, the heavy chain contributes the most to the antigen-binding surface and is also the one most variable in sequence. CDR3 on the heavy chain in particular is the most diverse CDR in sequence, length and structure. Thus, two antibodies specific for different antigens will almost always possess different heavy chain variable domains.

[0308] The method disclosed in US Patent Application No. 9,926,382 overcomes this limitation and greatly facilitates the isolation of antibodies with the same heavy chain variable domain by using an antibody library in which the heavy chain variable domain is the same for all library members, thereby limiting diversity to the light chain variable domain. Such libraries are described, for example, in US Patent No. 8,921,281 and WO 2011 / 084255, each of which is incorporated herein by reference in its entirety. However, since the light chain variable domain is expressed in conjunction with the heavy chain variable domain, both domains may contribute to antigen binding. To further facilitate this process, antibody libraries containing the same heavy chain variable domain and either diverse lambda or kappa variable light chains may be used in parallel for in vitro selection of antibodies against different antigens. This approach allows the discrimination of two antibodies with a common heavy chain, one with a lambda light chain variable domain and the other with a kappa light chain variable domain (which can be used as building blocks to generate bispecific antibodies in the complete immunoglobulin format). Bispecific antibodies that may be used in the embodiments disclosed herein may be of different isotypes and their Fc portions may be modified to alter the binding characteristics to different Fc receptors, thus modifying the effector functions of the antibody and its pharmacokinetic properties. Numerous methods for modifying Fc portions have been described and are applicable to antibodies that may be used in the embodiments disclosed herein. (See, e.g., Strohl, WR Curr Opin Biotechnol 2009(6):685-91; U.S. Patent No. 6,528,624; PCT / US2009 / 0191199 filed January 9, 2009).

[0309] A common heavy chain and two different light chains are co-expressed in a single cell, allowing the assembly of bispecific antibodies that can be used in the embodiments disclosed herein. If all polypeptides are expressed at the same level and assemble equally well to form immunoglobulin molecules, the ratio of monospecific (same light chain) to bispecific (two different light chains) should be 50%. However, it is likely that the different light chains are expressed at different levels and / or do not assemble with the same efficiency. Therefore, a means of regulating the relative expression of the different polypeptides is used to compensate for their inherent expression characteristics or different propensities to assemble with a common heavy chain. This regulation can be achieved by the strength of the promoter, the use of internal ribosome entry sites (IRES) characterized by different efficiencies, or the use of other types of regulatory elements that can act at the transcriptional or translational level, and also affect the stability of the mRNA. Different promoters of different strengths include CMV (immediate early cytomegalovirus virus promoter); EF1-1α (human elongation factor 1 alpha subunit promoter); Ubc (human ubiquitin C promoter); SV40 (simian virus 40 promoter). Various IRES of mammalian and viral origin have also been described. (See, for example, Hellen CU and Sarnow P. Genes Dev 2001 15:1593-612). These IRES can vary widely in their length and ribosome recruitment efficiency. Furthermore, activity can be further tuned by introducing multiple copies of the IRES (Stephen et al. 2000 Proc Natl Acad Sci USA 97:1536-1541). Modulation of expression can be achieved by multiple sequential transfections of cells, which increases the copy number of individual genes expressing one or the other light chain, thereby modifying their relative expression. The examples provided herein demonstrate that controlling the relative expression of the different chains is important to maximize the assembly and overall yield of bispecific antibodies.

[0310] Co-expression of the heavy chain and two light chains generates a mixture of three different antibodies in the cell culture supernatant: two monospecific bivalent antibodies and one bispecific bivalent antibody. The latter needs to be purified from the mixture to obtain the molecule of interest. The method described herein greatly facilitates this purification procedure by using affinity chromatography media that specifically interact with kappa or lambda light chain constant domains, such as CaptureSelect Fab Kappa and CaptureSelect Fab Lambda affinity matrices (BAC BV, Holland). This multi-step affinity chromatography purification approach is efficient and generally applicable to antibodies that may be used in the embodiments disclosed herein. This is in sharp contrast to specific purification methods that must be developed and optimized for each bispecific antibody derived from a quadroma or other cell line expressing an antibody mixture. Indeed, if the biochemical properties of the different antibodies in the mixture are similar, their separation using standard chromatographic techniques, such as ion exchange chromatography, may be difficult or even impossible.

[0311] Other suitable purification methods include those disclosed in U.S. Patent Application Publication No. 2013 / 0317200, the contents of which are incorporated herein by reference in their entirety.

[0312] In other embodiments for generating bispecific antibodies, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. Preferably, the fusions are with an immunoglobulin heavy chain constant domain, including at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy chain constant region (CH1) containing the site necessary for light chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. For further details on the generation of bispecific antibodies, see, e.g., Suresh et al., Methods in Enzymology, 121:210 (1986).

[0313] According to another approach described in WO96 / 27011, the interface between a pair of antibody molecules may be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. A preferred interface comprises at least a portion of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensatory "cavity" of identical or similar size to the large side chain(s) is created on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end-products such as homodimers.

[0314] Techniques for generating bispecific antibodies from antibody fragments are described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

[0315] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form the antibody heterodimers. This method can also be used for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) provides an alternative mechanism for making bispecific antibody fragments. The fragments consist of a light chain variable domain (V) connected to the light chain variable domain (V) by a linker that is too short to allow pairing between the two domains on the same chain. L ) linked to a heavy chain variable domain (V H ) is included. Therefore, the V H and V L Domain complementary to another fragment V L and V H The Fv domains are paired together to form two antigen-binding sites. Another strategy for making bispecific antibody fragments using single-chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol. 152:5368 (1994).

[0316] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0317] Exemplary bispecific antibodies may bind to two different epitopes, at least one of which is derived from a protein antigen that may be used in the embodiments disclosed herein. Alternatively, the anti-antigenic arm of an immunoglobulin molecule may be combined with an arm that binds to a triggering molecule on a white blood cell, such as a T cell receptor molecule (e.g., CD2, CD3, CD28, or B7), or an Fc receptor (FcγR) for IgG, such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), to focus cellular defense mechanisms against cells expressing a particular antigen. Bispecific antibodies may also be used to direct cytotoxic agents to cells expressing a particular antigen. These antibodies have an antigen-binding arm and an arm that binds to a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds to a protein antigen as described herein and further binds tissue factor (TF).

[0318] Heteroconjugate antibodies are also within the scope of this disclosure. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (see U.S. Pat. No. 4,676,980) and to treat HIV infection (WO91 / 00360; WO92 / 200373; EP03089). It is envisioned that antibodies can be prepared in vitro using synthetic protein chemistry, including those that require crosslinking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate, as well as those disclosed, for example, in U.S. Pat. No. 4,676,980.

[0319] It may be desirable to modify the antibodies that may be used in the embodiments disclosed herein with respect to effector function, for example to enhance the effectiveness of the antibodies in treating cancer and / or other diseases and disorders associated with aberrant B7-H3 expression and / or activity. For example, a cysteine ​​residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability, and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Alternatively, an antibody may be engineered with dual Fc regions, thereby having enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)).

[0320] Conjugated antibodies The present disclosure also relates to conjugated antibodies (also referred to herein as immunoconjugates), which include antibodies, or antigen-binding fragments thereof, conjugated to a cytotoxic agent, such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

[0321] In some embodiments, the toxin is a microtubule inhibitor or derivative thereof. In some embodiments, the toxin is a dolastatin or derivative thereof. In some embodiments, the toxin is auristatin E, auristatin F, AFP, MMAF, MMAE, MMAD, DMAF, or DMAE. In some embodiments, the toxin is a maytansinoid or a maytansinoid derivative. In some embodiments, the toxin is DM1 or DM4. In some embodiments, the toxin is a nucleic acid damaging toxin. In some embodiments, the toxin is a duocarmycin or derivative thereof. In some embodiments, the toxin is a calicheamicin or derivative thereof. In some embodiments, the agent is a pyrrolobenzodiazepine or derivative thereof. In some embodiments, the agent is exatecan or derivative thereof. In some embodiments, the agent is amanitin or derivative thereof.

[0322] Enzymatically active toxins and their fragments that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include: 212 Bi, 131 I, 131 In, 90 Y, and 186 Re is an example.

[0323] Conjugates of antibodies and cytotoxic drugs can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as 2,6-diisocyanate toluene), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies (see WO 94 / 11026).

[0324] Those of skill in the art will appreciate that a wide variety of possible moieties can be attached to the resulting antibodies that can be used in the embodiments disclosed herein (see, e.g., "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0325] Coupling can be accomplished by any chemical reaction that bonds two molecules together, so long as the antibody and other moieties retain their respective activities. This binding can include many chemical mechanisms, such as covalent bonds, affinity bonds, intercalation, coordinate bonds, and complex formation. However, the preferred binding is a covalent bond. Covalent binding can be achieved by direct condensation of existing side chains or by incorporation of an external bridging molecule. Many bivalent or polyvalent linking agents are useful for binding protein molecules, such as the antibodies of the present disclosure, to other molecules. For example, representative coupling agents include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various classes of coupling agents known in the art, but is an example of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987).

[0326] Suitable linkers are described in the literature (see, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-n-hydroxysuccinimide ester). See also U.S. Patent No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives attached to antibodies via oligopeptide linkers. Particularly preferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)-toluene (Pierce Chem. Co., Catalog No. 21558G), (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Catalog No. 21651G), (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Catalog No. 2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Catalog No. 2165-G) conjugated to EDC. Chem. Co., Cat. No. 24510) (conjugated to EDC).

[0327] The above linkers contain components with different attributes, resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylic acids. Linkers containing NHS-esters are less soluble than sulfo-NHS esters. In addition, the linker SMPT contains a sterically hindered disulfide bond, which may lead to the formation of conjugates with increased stability. Disulfide bonds are generally less stable than other bonds, because they are cleaved in vitro, resulting in fewer conjugates available. In particular, sulfo-NHS may increase the stability of carbodiimide coupling. The use of carbodiimide coupling (such as EDC) in combination with sulfo-NHS results in the formation of esters that are more resistant to hydrolysis than the carbodiimide coupling reaction alone.

[0328] The antibody disclosed herein may also be formulated as an immunoliposome. Liposomes containing antibodies may be prepared by any suitable method, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77:4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with improved circulation time are disclosed in U.S. Patent No. 5,013,556.

[0329] Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibodies of the present disclosure can be conjugated to liposomes via a disulfide exchange reaction as described in Martin et al., J.Biol.Chem.,257:286-288 (1982).

[0330] Use of anti-B7-H3 antibodies It will be understood that administration of therapeutic substances according to the present disclosure will be with suitable carriers, excipients, and other agents that are incorporated into the formulation to improve transfer, delivery, tolerance, and the like. Many suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 therein by Blaug, Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipids (cationic or anionic) including vesicles (e.g., Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be suitable for treatment and therapy according to the present disclosure, provided that the active ingredients in the formulation are not inactivated by the formulation and the formulation is physiologically compatible and acceptable for the route of administration.See also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance" Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. "Lyophilization and development of solid protein pharmaceuticals" Int. J. Pharm. 203(1-2):1-60 (2000), Charman WN "Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts" J Pharm Sci. 89(8):967-78 (2000), Powell et al. "Compendium of excipients for parenteral formulations" PDA J Pharm Sci Technol. 52:238-311 (1998), and citations therein, for additional information regarding formulations, excipients and carriers well known to pharmaceutical chemists.

[0331] The therapeutic formulations of the present disclosure, including the conjugates of the present disclosure, are used to treat or alleviate symptoms associated with cancer, such as, for example, but not limited to, leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung and bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, bladder cancer, kidney and renal pelvis cancer, oral and pharyngeal cancer, uterine cancer, and / or melanoma. The present disclosure also provides methods of treating or alleviating symptoms associated with cancer. Treatment regimens can include identifying a subject, e.g., a human patient, suffering from (or at risk of developing) cancer, e.g., using standard methods.

[0332] Therapeutic formulations of the present disclosure include conjugates of the present disclosure that recognize B7-H3 and optionally a second target, and may be used to treat or alleviate symptoms associated with autoimmune and / or inflammatory diseases, such as B cell mediated autoimmune and / or inflammatory diseases, including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), idiopathic thrombocytopenic purpura (ITP), Waldenstrom's hypergammaglobulinemia, Sjogren's syndrome, multiple sclerosis (MS), and / or lupus nephritis.

[0333] The efficacy of treatment can be determined in association with any suitable method for diagnosing or treating a particular immune-related disorder. Reduction in one or more symptoms of the immune-related disorder indicates that the conjugate provides clinical benefit.

[0334] Conjugates to targets such as B7-H3, tumor-associated antigens, or other antigens may be used in methods for localizing and / or quantitating these targets, e.g., for measuring the levels of these targets in an appropriate physiological sample, for use in diagnostic methods, for use in protein imaging, etc. Conjugates specific for any of these targets, including, for example, antigen-binding domains derived from antibodies, or derivatives, fragments, analogs, or homologs thereof, may be utilized as pharmacologically active compounds (hereinafter referred to as "therapeutic agents").

[0335] The conjugates of the present disclosure can be used to isolate specific targets using standard techniques such as immunoaffinity, chromatography, or immunoprecipitation. The conjugates of the present disclosure can be used diagnostically to monitor protein levels in tissues as part of a clinical trial procedure, for example to determine the effectiveness of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include 125 I, 131 I, 35 S, or 3 Examples include H.

[0336] The conjugates of the present disclosure can be used as therapeutic agents. Such agents are usually used to treat or prevent diseases or conditions associated with abnormal expression or activation of a given target in a subject. A conjugate preparation, preferably one with high specificity and high affinity for its target antigen, is administered to a subject and will generally have an effect by binding to the target. The administration of the conjugate may disable, inhibit, or interfere with the signaling function of the target. The administration of the conjugate may disable, inhibit, or interfere with the binding of the target to the endogenous ligand to which it naturally binds.

[0337] The therapeutically effective amount of the conjugates of the present disclosure generally relates to the amount required to achieve a therapeutic goal. As mentioned above, this may be a binding interaction between an antibody and its target antigen, which in some cases interferes with the function of the target and / or the effect of an active agent conjugated to the antibody. The amount required for administration further depends on the binding affinity of the antibody to its specific antigen and / or the potency of the active agent, and also on the rate at which the administered antibody is depleted from the free volume of the other subject to which it is administered. The general range of therapeutically effective doses of the conjugates of the present disclosure may be, by way of non-limiting example, about 0.1 mg / kg body weight to about 50 mg / kg body weight. The general frequency of administration may range, for example, from twice a day to once a week.

[0338] The conjugates of the present disclosure may be administered in the form of pharmaceutical compositions for the treatment of various diseases and disorders. Principles and considerations involved in the preparation of such compositions, as well as guidance regarding the selection of ingredients, are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0339] The formulation may also contain two or more active compounds, preferably compounds with complementary activities that do not adversely affect each other, as necessary for the particular indication being treated. Alternatively, or in addition, the composition may contain an agent that enhances its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent. Such molecules are suitably present in combination in amounts effective for the intended purpose.

[0340] The active ingredient may also be encapsulated in microcapsules prepared, for example, by droplet formation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions.

[0341] The formulations to be used for in vivo administration are preferably sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0342] Sustained release preparations can also be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid ester, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, e.g., LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules for over 100 days, certain hydrogels release proteins for shorter periods of time.

[0343] The conjugates according to the present disclosure may be used as agents for detecting the presence of a given target (or a protein fragment thereof) in a sample. In some embodiments, the conjugate comprises a detectable label. The antibody may be polyclonal, or more preferably monoclonal. An intact antibody or a fragment thereof (e.g., F ab ,scFv,or F (ab)2) may be used. The term "biological sample" is intended to include tissues, cells and fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Thus, the use of the term "biological sample" includes blood and parts or components of blood, including serum, plasma, or lymph. That is, the detection methods of the present disclosure may be used to detect analyte mRNA, protein, or genomic DNA in biological samples in vitro and in vivo. For example, in vitro techniques for detecting analyte mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detecting analyte protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting analyte genomic DNA include Southern hybridization. Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in molecular Biology", Vol. 42, J.R.Crowther (Ed.) Human Press, Totowa, NJ, 1995; "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Additionally, in vivo techniques for detecting an analyte protein include introducing a labeled anti-analyte conjugate into the subject. For example, an antibody can be labeled with a radioactive marker whose presence and location in the subject can be detected by standard imaging techniques.

[0344] Pharmaceutical Compositions In certain aspects, provided herein are pharmaceutical compositions comprising a compound, drug conjugate, or targeted drug conjugate of the present disclosure. The antibody-drug conjugates can be used to deliver active agents to target cells of a subject and treat the subject using any suitable method of preparing the composition. In some aspects, the present disclosure relates to compositions (e.g., pharmaceutical compositions) comprising the antibody-drug conjugates described herein. The compositions and methods of the present disclosure may be utilized to treat an individual 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 compound of the present disclosure and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions, such as water or physiologically buffered saline, or other solvents or vehicles, such as glycols, glycerol, oils, such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly invasive routes of administration (i.e., routes such as injection or implantation that avoid transport or diffusion through epithelial barriers), the aqueous solutions are pyrogen-free or substantially pyrogen-free. Excipients may be selected, for example, to provide delayed release of the agent or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition may be in dosage unit form, such as a lyophilizate, powder, solution, injection, etc. for reconstitution.

[0345] A pharma- ceutically acceptable carrier may include, for example, a physiologically acceptable agent that acts to stabilize, increase the solubility, or increase the absorption of a compound, such as a compound of the present disclosure. 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 that includes a physiologically acceptable agent depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) may be a liposome or other polymer matrix, which may, for example, incorporate a compound of the present disclosure therein. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively easy to make and administer.

[0346] As used herein, the phrase "pharmacologically acceptable" is used to refer to those compounds, materials, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0347] Pharmaceutical compositions (preparations) can be administered to subjects by any of a number of routes of administration. For example, the compound can be simply dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable therefor can be found, for example, in U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896, and the patents cited therein.

[0348] The formulations may be conveniently provided in unit dosage form and may be prepared by any suitable method in the field of pharmacy. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form varies depending on the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form will generally be the amount of compound that produces a therapeutic effect. Generally, out of 100 percent, this amount will range from about 1 percent to about 99 percent of the active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent.

[0349] Methods of preparing these formulations or compositions include the step of bringing into association an active composition, such as a compound of the present disclosure, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0350] The phrases "parenteral administration" and "administered parenterally" as used herein refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intraocular (e.g., intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration include one or more active compounds in combination with one or more pharma- ceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into a sterile injectable solution or dispersion immediately prior to use, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0351] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate, etc. 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.

[0352] These compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial activity may be ensured by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents in the composition, such as sugars, sodium chloride, and the like. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0353] In some cases, it is desirable to delay the absorption of drugs from subcutaneous or intramuscular injections in order to prolong the effect of the drug. This can be accomplished by using a liquid suspension of crystalline or amorphous substances that are poorly water-soluble. In this case, the rate of absorption of the drug depends on the rate of dissolution, which in turn may depend on the crystal size and crystalline form. Alternatively, delayed absorption of parenterally administered drug forms can be accomplished by dissolving or suspending the drug in an oil vehicle.

[0354] Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the ratio of drug to polymer and the nature of the particular polymer used. 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.

[0355] For use in the methods of the disclosure, the active compounds may be provided per se or as pharmaceutical compositions containing, for example, 0.1-99.5% (more preferably 0.5-90%) of the active ingredient in combination with a pharma- ceutically acceptable carrier.

[0356] The introduction method may be provided by a rechargeable device or a biodegradable device.For the controlled delivery of drugs, including proteinaceous biopharmaceuticals, various sustained release polymeric devices have been developed and tested in vivo in recent years.Various biocompatible polymers, including both biodegradable and non-degradable polymers (including hydrogels), may be used to form implants for sustained release of compounds at specific target sites.

[0357] Actual dosage levels of the active ingredients in 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.

[0358] The selected dosage level will depend on a variety of factors, including the activity of the particular compound or combination of compounds, or esters, salts, or amides thereof, used, the route of administration, the time of administration, the excretion rate of the particular compound(s) used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular compound(s) used, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0359] A physician or veterinarian of ordinary skill in the art can easily determine and prescribe a therapeutically effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can begin administration of a pharmaceutical composition or compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. By "therapeutically effective amount" is meant a concentration of a compound sufficient to elicit the desired therapeutic effect. In general, it is generally understood that the effective amount of a compound varies according to 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 administered together with the compound of the present disclosure. Multiple administrations of the drug may deliver a larger total dose. Many 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).

[0360] In general, a suitable daily dose of an active compound used in the compositions and methods of the present disclosure will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0361] The patient receiving this treatment may be any animal in need of treatment, including primates, particularly humans, and other mammals such as horses, cows, pigs, sheep, cats, dogs, poultry, and pets in general.

[0362] In certain embodiments, the compounds of the present disclosure may be administered alone or in combination with another type of therapeutic agent.

[0363] Wetting agents, emulsifying agents, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition.

[0364] 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) lipid-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and (3) metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0365] The composition can be prepared in injectable form, either as a solution or suspension.Solid forms suitable for injection can also be prepared, for example, as emulsions or with the antibody-drug conjugate encapsulated in liposomes.The antibody-drug conjugate can be combined with a pharma-ceutically acceptable carrier, including any carrier that does not induce the production of antibodies harmful to the subject to which the carrier is administered.Suitable carriers typically include macromolecules that are slowly metabolized, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and the like.

[0366] The composition may also contain diluents, such as water, saline, glycerol, and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may also be present therein. The composition may be administered parenterally by injection, which may be either subcutaneous or intramuscular. In some embodiments, the composition may be administered intratumorally. The composition may be inserted (e.g., injected) into the tumor. Additional formulations are suitable for other forms of administration, such as suppositories or oral administration. Oral compositions may be administered as solutions, suspensions, tablets, pills, capsules, or sustained release formulations.

[0367] The composition may be administered in a manner compatible with the dosage and formulation. The composition preferably contains a therapeutically effective amount of the antibody drug conjugate. The dosage may vary depending on the subject being treated, the subject's health and physical condition, the degree of protection desired, and other relevant factors. The exact amount of active ingredient (e.g., antibody drug conjugate) may depend on the physician's judgment. For example, a therapeutically effective amount of the antibody drug conjugate or a composition containing it may be administered to a patient suffering from a cancer or tumor to treat the cancer or tumor.

[0368] The antibody drug conjugate according to the present disclosure or a composition comprising the same may be administered in the form of its pharma- ceutically acceptable salt. In some embodiments, the antibody drug conjugate according to the present disclosure or a composition comprising the same may be administered together with a pharma- ceutically acceptable carrier, a pharma- ceutically acceptable excipient, and / or a pharma- ceutically acceptable additive. The effective amount and type of pharma- ceutically acceptable salt, excipient, and additive may be determined using standard methods (see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th Edition, 1990).

[0369] In some embodiments, the present disclosure relates to a method for treating cancer in a subject, comprising administering to the subject a pharmaceutical composition comprising an antibody drug conjugate as described herein. In a preferred embodiment, the subject is a mammal. For example, the subject may be selected from rodents, lagomorphs, felines, canines, porcines, ovines, bovines, equines, and primates. In certain preferred embodiments, the subject is a human.

[0370] The conjugates of the present disclosure (also referred to herein as "active compounds"), as well as derivatives, fragments, analogs and homologs thereof, may be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include the conjugates and a pharma- ceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in this field, and are incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils may also be used. The use of such media and agents for pharma- ceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0371] The pharmaceutical composition of the present disclosure is formulated to suit its intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous administration, intradermal administration, and subcutaneous administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: sterile diluents, such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents, such as benzyl alcohol and methylparaben; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, and phosphates, and agents for adjusting isotonicity, such as sodium chloride or dextrose. pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0372] Pharmaceutical forms suitable for injection use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, sodium chloride, in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0373] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent with one or a combination of the above-listed components as required, followed by filtration sterilization.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other components required from the above-listed components.In the case of sterile powder for preparing sterile injectable solution, the preparation method is vacuum drying and freeze-drying (lyophilization), which produces a powder containing active ingredient plus any additional desired components from its previously sterile-filtered solution.

[0374] In certain embodiments, the active compounds are prepared with carriers that protect the compounds against rapid release from the body, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, may be used. Methods for preparing such formulations will be apparent to those skilled in the art. These materials may be commercially obtained from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions, including liposomes targeted to infected cells with monoclonal antibodies against viral antigens, may also be used as pharma-ceutically acceptable carriers. These may be prepared according to suitable methods, such as those described in U.S. Pat. No. 4,522,811.

[0375] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions in unit dosage form.Unit dosage form as used herein refers to a physically separate unit suitable as a unit dose for treating a subject; each unit contains a predetermined amount of active compound calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification of unit dosage form of the present disclosure is determined and directly depends on the inherent characteristics of active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations of the art of compounding such active compound for treating an individual.

[0376] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0377] Treatment The compounds and conjugates disclosed herein can be used in methods of inducing apoptosis in a cell.

[0378] 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 arteriosclerosis. The compounds, conjugates, and compositions described herein can be used to treat or ameliorate any of these diseases. Such treatment generally involves administering to a subject suffering from a disease a sufficient amount of the compounds, conjugates, or compositions described herein to produce a therapeutic effect. The identity of the antibody of the administered compound, conjugate, or composition depends on the disease being treated. Thus, the antibody should bind to a cell surface antigen expressed in the cell type whose inhibition is beneficial. The therapeutic effect achieved also depends on the particular disease being treated. In certain instances, the compounds and compositions disclosed herein may treat or ameliorate the disease itself or symptoms of the disease when administered as a monotherapy. In other instances, the compounds and compositions disclosed herein may be part of an overall treatment plan that includes other agents that treat or ameliorate the disease or symptoms of the disease being treated along with the inhibitors or compounds and compositions disclosed herein. Agents useful for treating or ameliorating a particular disease that can be administered adjunctively or together with the compounds and compositions disclosed herein will be apparent to those of skill in the art.

[0379] Although an absolute cure is always desirable in any treatment, it is not necessary to achieve a cure in order to obtain a therapeutic benefit. A therapeutic benefit may include halting or slowing the progression of a disease, regressing a disease without curing it, and / or improving symptoms or slowing the progression of a disease. Prolonged survival and / or improved quality of life compared to the statistical mean may also be considered a therapeutic benefit.

[0380] One particular class of disease that involves the dysregulation of apoptosis and is a major health burden worldwide is cancer. In certain embodiments, the compounds and compositions disclosed herein can be used to treat cancer. The cancer may be, for example, a solid tumor or a hematological tumor. The 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, colon 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 can be particularly useful in the treatment of cancer. This is because the antibody can be used to specifically target tumor cells, thereby potentially avoiding or ameliorating undesirable side effects and / or toxicity that may be associated with systemic administration of unbound inhibitors. One embodiment relates to a method for treating a disease involving dysregulation of intrinsic apoptosis, comprising administering to a subject having a disease involving dysregulation of apoptosis an effective amount of the compounds and compositions disclosed herein to produce a therapeutic effect, wherein the ligand of the compounds and compositions disclosed herein binds to a cell surface receptor of cells in which intrinsic apoptosis is dysregulated. One embodiment relates to a method for treating cancer, comprising administering to a subject having cancer an effective amount of the compounds and compositions disclosed herein to produce a therapeutic effect, wherein the ligand is capable of binding to a cell surface receptor or tumor-associated antigen expressed on the surface of cancer cells.

[0381] In the context of tumorigenic cancer, therapeutic benefit specifically includes, in addition to the effects discussed above, halting or slowing the progression of tumor growth, regression of tumor growth, eradication of one or more tumors, and / or improving 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.

[0382] The compounds and conjugates disclosed herein may be administered as monotherapy to provide a therapeutic effect, or may be administered adjunctively or in combination with other chemotherapeutic agents and / or radiation therapy. Chemotherapeutic agents for which the compounds and compositions disclosed herein may be utilized as adjunctive 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.

[0383] The compounds and conjugates disclosed herein, which may not be effective as a monotherapy for treating cancer, may be administered adjunctively or in combination with other chemotherapeutic agents or radiation therapy to provide therapeutic benefits. One embodiment relates to a method in which the compounds or compositions disclosed herein are administered in an amount effective to sensitize tumor cells to standard chemotherapy and / or radiation therapy. Thus, in the context of cancer treatment, "therapeutic benefits" include administering the compounds and compositions disclosed herein adjunctively or together with chemotherapy and / or radiation therapy as a means of sensitizing tumors to chemotherapy and / or radiation therapy, either in patients who have not yet started chemotherapy and / or radiation therapy, or in patients who have already started chemotherapy and / or radiation therapy but have not yet shown signs of resistance, or in patients who are beginning to show signs of resistance.

[0384] In some aspects, the present disclosure provides a pharmaceutical composition comprising an antibody drug conjugate described herein, and optionally further comprising a therapeutically effective amount of a chemotherapeutic agent.

[0385] In certain aspects, provided herein are methods of treating cancer comprising administering to a subject in need thereof one or more of the disclosed compounds, drug conjugates, targeted drug conjugates, or pharmaceutical compositions.

[0386] In certain embodiments, the cancer is selected from leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung cancer, bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, bladder cancer, kidney cancer, renal pelvis cancer, oral cancer, pharyngeal cancer, uterine cancer, or melanoma.

[0387] In a further aspect, provided herein is a method of treating an autoimmune or inflammatory disease comprising administering to a subject in need thereof one or more of the disclosed compounds, drug conjugates, targeted drug conjugates, or pharmaceutical compositions.

[0388] In certain embodiments, the autoimmune or inflammatory disease is selected from a B cell-mediated autoimmune or inflammatory disease, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), idiopathic thrombocytopenic purpura (ITP), Waldenstrom's hypergammaglobulinemia, Sjogren's syndrome, multiple sclerosis (MS), or lupus nephritis.

[0389] The present invention will be described in more detail below with reference to examples, but the following examples are merely for the purpose of aiding in the understanding of the present disclosure. The scope of the present disclosure is not limited thereto. Furthermore, unless otherwise specified, the reagents, solvents, and starting materials described herein can be easily obtained from commercial sources. EXAMPLES

[0390] Example 1: Preparation of Compound L-1 [ka]

[0391] Preparation of compound L-1-1 To a solution of dimethyl 5-hydroxyisophthalate (5 g, 23.79 mmol) in dry THF (300 mL) was added LAH (3.6 g, 95.15 mmol) at -78 °C under N 2 The reaction mixture was stirred at room temperature for 17 hours. After the reaction was complete, 15% NaOH solution (4 mL), H 2 O (8 mL) and EA (100 mL) were added, and then the reaction mixture was stirred for 1 h. The mixture was filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-1-1 (3.02 g, 82%). 1 H NMR(400MHz,DMSO-d6)δ9.21(s,1H),6.66(s,1H),6.58(s,2H),5.07(t,J=6.0Hz,2H),4.38(d,J=4.6Hz,4H)

[0392] Preparation of compound L-1-2 Compound L-1-1 (2 g, 12.97 mmol) was dissolved in HBr (5.0 mL, 33% AcOH solution) with N 2 After stirring at 60 °C for 18 h, the reaction was diluted with NaHCO 3 The reaction mixture was quenched by adding a pH-8 aqueous solution. Then, distilled water (50 mL) and EA (100 mL×2) were added to the reaction mixture. The organic layer was diluted with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure, and the residue was purified by column chromatography to give compound L-1-2 (2.9 g, 80%). 1 H NMR (400 MHz, CDCl 3 )δ6.99(s,1H),6.81(s,2H),4.85(s,1H),4.41(s,2H).

[0393] Preparation of compound L-1-3 To a solution of compound L-1-2 (1.0 g, 3.57 mmol) in DCM (35 mL), TEA (0.45 mL, 3.21 mmol) was added at room temperature and cooled with N 2 The mixture was added under atmospheric pressure. 2 F 2 Gas was introduced via balloon and the mixture was stirred at room temperature for 1 h. The mixture was then washed with DCM (50 mL) and water (30 mL) was added. The organic layer was washed with aqueous NaHCO3, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-5 (941.7 mg, 73%).

[0394] Preparation of compound L-1 A solution of compound L-1-2 (100 mg, 0.36 mmol) in dry DCM (3 mL) was added with imidazole (27 mg, 0.39 mmol) and TBDMS-Cl (59 mg, 0.39 mmol) at room temperature and N 2 After stirring for 16 h, distilled water (50 mL) and EA (100 mL) were added to the reaction mixture. The organic layer was diluted with anhydrous Na 2 SO 4The mixture was dried at 40° C., filtered and concentrated under reduced pressure, and the residue was purified by column chromatography to give compound L-1 (110 mg, 79%). 1 H NMR (400 MHz, CDCl 3 )δ7.00(s,1H),6.80(s,2H),4.41(s,4H),0.99(s,9H),0.21

[0395] Example 2 Preparation of Compound L-2 [ka]

[0396] Preparation of compound L-2-1 11-Azido-3,6,9-trioxaundecan-1-amine (Aldrich, CAS 134179-38-7, 5.0 g, 22.9 mmol) in 1,4-dioxane (100 mL) and H 2 The homogeneous solution in O (25 mL) was stirred at room temperature with N 2 Under atmosphere, NaHCO 3 (3.8 g, 45.8 mmol, 2.0 equiv.) and BOC 2 2H2O (6.0 g, 27.5 mmol, 1.2 equiv.) and then stirred for 6 h. The reaction was quenched with water (50 mL) and extracted with DCM (100 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, and the residue was purified by column chromatography (1%-3% MeOH in DCM) to give compound L-2-1 as a colorless oil (7.2 g, 99%). 1 H NMR (400 MHz, CDCl 3 )δ5.03(brs,1H),3.72-3.60(m,10H),3.98-3.52(m,1H),3.43-3.36(m,1H),3.35-3.24(m,1H),1.26(s,9H). EI-MS m / z: 319(M + +1).

[0397] Preparation of compound L-2-2 A solution of compound L-8 (2 g, 6.282 mmol) in DMF (25 mL) was added to 2 Sodium hydride (301 mg, 12.56 mmol, 60%) was added at 0° C. under atmospheric pressure. After 10 min, iodomethane (3.9 mL, 62.82 mmol) was added 2 The reaction was stirred at room temperature for 3 h under N 2 After the reaction was completed, the reaction mixture was quenched with 2N HCl (10 mL) and extracted with EA (500 mL×3). The organic layer was diluted with Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure to afford compound L-2-2 (3.3 g, quantitative) as a yellow oil which was used without further purification. 1 H NMR (400 MHz, CDCl 3 )δ3.70-3.62(m,12H),3.4(t,J=5.2Hz,4H),2.91(s,3H),1.46(s,9H).

[0398] Preparation of compound L-2 Compound L-2-2 (3.3 g, 6.282 mmol) 2 Cl 2 To the solution in (70 mL), add 4N HCl in dioxane (25 mL) at 0 °C and 2 The reaction was heated under N 2 The mixture was stirred at 0° C. under atmospheric pressure for 1 hour. After the reaction was completed, the reaction mixture was concentrated under reduced pressure to yield compound L-2 (1.8 g, 100%) as a yellow oil, which was used without further purification. 1 H NMR (400 MHz, CDCl 3 )δ3.92(t,J=4.8Hz,2H),3.73-3.69(m,10H),3.45(t,J=5.2Hz,2H),3.22-3.16(m,2H),2.77(t,J=5.6Hz,3H),2.35(brs,1H).

[0399] Example 3 Preparation of Compound L-3 [ka]

[0400] Preparation of compound L-3-1 A solution of hexaethylene glycol (5.0 g, 18.0 mmol) in anhydrous THF (20 mL) was heated at 0 °C with N 2 The mixture was treated with 1M t-BuOK (9.4 mL, 4.9 mmol), propargyl bromide (1.0 mL, 9.4 mmol) under atmospheric pressure. The reaction mixture was allowed to warm to room temperature and stirred overnight. The mixture was diluted with EA (50 mL x 2), H 2 The mixture was extracted with 20 mL of HO (20 mL). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-3-1 (2.4 g, 80%). 1 H NMR (400Hz, CDCl 3 )δ4.21(s,2H),3.7-3.6(m,24H),3.05(brs,1H),2.43(s,1H).

[0401] Preparation of compound L-3-2 A clear solution of compound L-3-1 (4.23 g, 13.2 mmol) in anhydrous DCM (45 mL) was stirred at room temperature with N 2 The reaction was treated with TEA (4.78 mL, 34.32 mmol, 2.6 equiv), pTs-Cl (5.03 g, 26.40 mmol, 2.0 equiv) under atmospheric pressure and stirred overnight. The reaction was diluted with water (50 mL) and extracted with DCM (100 mL x 2). The resulting organic layer was washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure, and the residue was purified by column chromatography to give compound L-3-2 as a colorless oil (6.09 g, 97%). 1 H NMR (400Hz, CDCl 3)δ7.80(d,J=8.4Hz,2H),7.35(d,J=8.8Hz,2H),4.22-4.20(m,J=,2H),4.16(t,J=4.8Hz,2H),3.93-3.58(m,22H),2.45(s,3H);EI-MS m / z:475(M + +1).

[0402] Preparation of compound L-3-3 A clear solution of compound L-3-2 (6.09 g, 12.83 mmol) in DMF (45 mL) was heated at room temperature with N 2 Under the atmosphere, NaN 3 (1.25 mg, 19.25 mmol, 1.5 equiv) and stirred overnight. The reaction was diluted with water (50 mL) and extracted with DCM (100 mL x 3). The resulting organic layer was washed with anhydrous Na 2 SO 4 It was dried at 40° C., filtered and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA=1:4) to give compound L-3-3 as a yellowish oil (3.52 g, 79%). 1 H NMR (400Hz, CDCl 3 )δ4.23-4.22(m2H),3.80-3.62(m,22H),3.44-3.38(m,2H),2.46-2.42(m,1H)

[0403] Preparation of compound L-3 A clear solution of compound L-3-3 (3.52 g, 10.19 mmol) in EA (24 mL) and ether (24 mL) was heated at 0 °C and N 2 The mixture was treated with 5% HCl solution (48 mL) under atmospheric pressure, triphenylphosphine (3.47 g, 13.25 mmol) was added, and the mixture was stirred overnight. 2 The mixture was diluted with 200 mL of HO (30 mL). The aqueous layer was extracted with DCM (100 mL x 3). The aqueous phase was concentrated under high vacuum to give compound L-3 (2.73 g, 75%); EI-MS m / z: 320 (M + +1).

[0404] Example 4 Preparation of Compound L-4 [ka]

[0405] Preparation of compound L-4-1 A solution of hexaethylene glycol (5.0 g, 17.71 mmol) in anhydrous DCM (178 mL) was stirred at room temperature with N 2 Under atmospheric conditions, KI (294 mg, 1.77 mmol) and Ag 2 0 (4.92 g, 19.48 mmol), p-TsCl (3.7 g, 19.48 mmol) and stirred overnight. The reaction mixture was filtered through CELITE® and the CELITE® plug was washed with DCM (100 mL). The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-4-1 (5.98 g, 73%). 1 H NMR (400Hz, CDCl 3 )δ7.80(d,J=8.4Hz,2H),7.35(d,J=8.4Hz,2H),4.16(t,J=4.8Hz,2H),3.71-3.58(m,22H),2.88(br,1H),2.45(s,3H).

[0406] Preparation of compound L-4-2 A solution of compound L-4-1 (5.98 g, 13.7 mmol) in DMF (30 mL) was heated at room temperature with N 2 Under atmospheric conditions, NaN 3 (1.34 g, 20.55 mmol) and stirred at 110° C. for 1 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-4-2 (4.1 g, 97%). 1 H NMR (400Hz, CDCl 3 )δ3.72-3.60(m,22H),3.39(t,J=4.8Hz,2H),2.78(br,1H).

[0407] Preparation of compound L-4-3 A solution of compound L-4-2 (1.9 g, 6.18 mmol) in DCM (20 mL) was stirred at room temperature for 2 h at 4°C for 1 h at 25°C. 2 The mixture was treated with triethylamine (2.0 mL, 14.22 mmol), p-TsCl (2.4 g, 12.36 mmol) under atmospheric pressure and stirred overnight. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-4-3 (2.58 g, 91%). 1 H NMR (400Hz, CDCl 3 )δ7.80(d,J=8.4Hz,2H),7.35(d,J=8.4Hz,2H),4.16(t,J=4.8Hz,2H),3.70-3.61(m,16H),3.56(s,1H),3.39(t,J=4.8Hz,2H),2.45(s,3H). EI-MS m / z: 462(M + +1).

[0408] Preparation of compound L-4-4 A solution of compound L-4-2 (1.0 g, 3.25 mmol) in EtOH (5 mL) was dissolved in H 2 Treat with 5% Pd / C (1.04 g, 0.49 mmol) under atmospheric pressure and stir for 4 h. The mixture was filtered through CELITE® to remove Pd / C and concentrated under reduced pressure. The residue was dissolved in DCM (25 mL). BOC 2 O (852.1 mg, 3.9 mmol) was added and the resulting mixture was stirred at room temperature for 3 h. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-4-4 (330 mg, 28%). 1 H NMR (400Hz, CDCl 3 )δ5.19(brs,1H),3.73(t,J=4.8Hz,2H),3.67(s,12H),3.63-3.60(m,6H),3.54(t,J=5.2Hz,2H),3.34-3.27(m,1H),1.44(s,9H). EI-MS m / z: 382(M + +1).

[0409] Preparation of compound L-4-5 A solution of compound L-4-4 (450 mg, 1.18 mmol) in anhydrous THF (10 mL) was stirred at 0 °C for 2 h at 4 °C for 1 h. 2 Treat with NaH (60% mineral oil dispersion, 47.2 mg, 1.18 mmol) under atmospheric pressure and stir for 20 min. To this was added a solution of compound L-4-4 (544.5 mg, 1.18 mmol). The reaction was allowed to warm to room temperature and stirred overnight. The reaction was allowed to cool, quenched with MeOH (5 mL) and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-4-5 (582.9 mg, 74%).

[0410] Preparation of compound L-4 Compound L-4 (quantitative, colorless oil) was synthesized in a manner similar to that of compound L-3 in Example 3. EI-MS m / z: 645(M + +1).

[0411] Example 5: Preparation of Compounds Int-1 and Int-2 [ka]

[0412] Preparation of compound Int-1-1 A solution of vanillic acid (50.0 g, 0.30 mol) in MeOH (700 mL) was diluted with SOCl 2 (207 mL, 2.85 mol) was added dropwise and N 2 After stirring at room temperature for 15 h, the reaction was diluted with saturated NaHCO 3 The pH was adjusted to 7-8 with aqueous solution, then diluted with distilled water (100 mL) and EA (400 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, and the residue was purified by column chromatography to give compound Int-1-1 (54.2 g, quantitative). 1 H NMR (400 MHz, CDCl 3)δ7.64(dd,J=6.4,1.6Hz,1H),7.55(s,1H),6.94(d,J=8.4Hz,1H),6.05(s,1H),3.95(s,3H),3.89(s,3H).

[0413] Preparation of compound Int-1-2 A solution of compound Int-1-1 (54.2 g, 0.30 mol) in DMF (200 mL) was stirred at room temperature for 2 h at 37° C. 2 CO 3 (61.6 g, 0.45 mol), benzyl bromide (39.0 mL, 0.33 mol) and stirred at 100° C. for 6 h. The reaction mixture was cooled to room temperature and diluted with distilled water (100 mL) and EA (400 mL). The organic layer was diluted with Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure, and the residue was purified by column chromatography to give compound Int-1-2 (79.8 g, 98%). 1 H NMR (400 MHz, CDCl 3 )δ7.60(dd,J=6.4,2.0Hz,1H),7.56(d,J=2.0Hz,1H),7.44-7.31(m,5H),6.89(d,J=8.4Hz,1H),5.22(s,2H),3.94(s,3H),3.88(s,3H).

[0414] Preparation of compound Int-1-3 A solution of compound Int-1-2 (79.8 g, 0.29 mol) in acetic anhydride (550 mL) was 2 Copper(II) nitrate hemi(pentahydrate) (75.0 g, 0.32 mol) was added dropwise under atmosphere at 0° C. and stirred at 0° C. for 6 hours. The reaction mixture was quenched with ice water (800 mL). The solid was filtered and washed with distilled water (100 mL) and hexane (400 mL) to obtain compound Int-1-3 (85.5 g, 92%). 1 H NMR (400 MHz, CDCl 3 )δ7.52(s,1H),7.45-7.35(m,5H),7.08(s,1H),5.22(s,2H),3.98(s,3H),3.91(s,3H).

[0415] Preparation of compound Int-1-4 To a solution of compound Int-1-3 (85.5 g, 0.27 mol) in THF (800 mL) and MeOH (300 mL) was added 2N NaOH (404 mL, 0.81 mol). After stirring at 65° C. for 5 h, the reaction was cooled to room temperature and adjusted to pH 2 by adding 2N HCl solution, then extracted with distilled water (100 mL) and EA (300 mL×2). 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 residual solid was collected and washed with hexane to give compound Int-1-4 (79.2 g, 97%). 1 H NMR (400MHz, DMSO-d6) δ7.69(s,1H), 7.47-7.35(m,5H), 7.03(s,1H), 5.24(s,2H), 3.91(s,3H).

[0416] Preparation of compound Int-1 To a solution of compound Int-1-4 (100 mg, 0.33 mmol) in anhydrous THF (500 μL) and anhydrous DCM (1.5 mL), oxalyl chloride (42.4 μL) was slowly added dropwise and 1 drop of DMF was added at 0° C. with N 2 After stirring for 30 minutes, the reaction mixture was concentrated under reduced pressure. Compound Int-1 was used directly in the next step without further purification.

[0417] Preparation of compound Int-1-5 A solution of compound Int-1-3 (5.0 g, 15.8 mmol) in DCM (300 mL) was 2 A solution of methanesulfonic acid (50 mL) in DCM (100 mL) was slowly added dropwise under atmospheric conditions at 0° C. and stirred for 2 hours. The reaction mixture was diluted with NaHCO 3 Quench with solution of H 2 The organic layer was extracted with anhydrous NaO (100 mL). 2 SO 4The mixture was dried at 40° C., filtered and concentrated under reduced pressure, and the residue was purified by column chromatography to give compound Int-1-5 (2.54 g, 71%). 1 H NMR (400 MHz, CDCl 3 )δ7.48(s,1H),7.14(s,1H),6.05(s,1H),4.02(s,3H),3.89(s,3H).

[0418] Preparation of compound Int-1-6 A solution of compound Int-1-5 (2.0 g, 8.8 mmol) in 1,4-dioxane (28 mL) under N2 atmosphere was treated with 6N NaOH solution (4.4 mL, 26.4 mmol) and stirred at 40 °C for 4 h. The reaction mixture was cooled to 0 °C and acidified with 2N HCl. The mixture was extracted with EA / H2O. The organic layer was dried over anhydrous Na2SO4, filtered, concentrated under reduced pressure, and dried in vacuum to give a white solid Int-1-6 (2.0 g, quantitative). 1H NMR (400MHz, DMSO-d6) δ10.60 (s, 1H), 7.305 (s, 1H), 7.24 (s, 1H), 3.89 (s, 3H).

[0419] Preparation of compound Int-2 N 2 A solution of compound Int-1-6 (1.87 g, 8.77 mmol) in acetic anhydride (1.0 mL, 10.5 mmol) was treated with TEA (1.8 mL, 13.1 mmol), DMAP (0.2 g, 1.75 mmol) under atmospheric conditions and stirred at room temperature for 3.5 h. The reaction mixture was diluted with EA / H 2 The organic layer was extracted with anhydrous Na 2 SO 4 The mixture was dried at rt, filtered, concentrated under reduced pressure, and dried in vacuum to give a white solid Int-2 (2.2 g of a brown solid, 49%). 1H NMR (400MHz, DMSO-d6): δ7.981(s,1H), 7.451(s,1H), 3.933(s,3H), 2.294(s,3H).

[0420] Example 6 Preparation of compound Int-TG1 [ka]

[0421] Preparation of compound Int-TG1 β-D-Galactose pentaacetate (Alfa, CAS 4163-60-4, 5.0 g, 12.81 mmol) was dissolved in 33% HBr in AcOH (20 mL) at 0 °C under N 2 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 aqueous 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, and the residue was purified by column chromatography to obtain compound Int-TG1 (5.2 g, 99%). 1 H NMR (400Hz, CDCl 3 )δ6.70(d,J=4.0Hz,1H),5.52(d,J=2.4Hz,1H),5.41(dd,J=7.6,2.8Hz,1H),5.05(d d,J=6.4,4.0Hz,1H),4.49(t,J=6.4Hz,1H),4.22-4.09(m,2H),2.16-2.01(m,12H).

[0422] Example 7 Compound Int-TG2 [ka]

[0423] Preparation of compound Int-TG2 Compound Int-TG2 was synthesized in a similar manner to that described in Example 6. Yield: 80% 1 H NMR (400 MHz, CDCl 3)δ6.654(d,J=4.0Hz,1H),5.627(t,J=10.0Hz,1H),5.252(dd,J=10.4Hz,9.6Hz,1H),4.865(dd,J=10 .0Hz,4.0Hz,1H),4.593(d,J=10.4Hz,1H),3.777(s,3H),2.113(s,3H),2.071(s,3H),2.065(s,3H).

[0424] Example 8 Preparation of compound Int-TG3 [ka]

[0425] Preparation of compound Int-TG3-1 A solution of compound Int-TG1 (18.5 g, 45.0 mmol), 4-hydroxybenzaldehyde (5.0 g, 40.9 mmol), and molecular sieves (10.0 g) in ACN (150 mL) was heated at room temperature under N 2 Under atmospheric conditions, Ag 2 0 (38.0 g, 0.164 mol) and stirred for 3 h. The reaction mixture was filtered through a pad of Celite and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG3-1 (16.0 g, 86%). 1 H NMR (400 MHz, CDCl 3 )δ9.93(s,1H),7.86(d,J=6.8Hz,2H). 7.11(d,J=6.8Hz,2H),5.52-5.47(m,2H),5.18-5.14(m,2H),4.24-4.11(m,3H),2.19(s,3H),2.07(s,6H),2.02(s,3H).

[0426] Preparation of compound Int-TG3-2 A solution of compound Int-TG3-1 (540 mg, 1.19 mmol) in anhydrous THF (15 mL) was stirred at 0° C. under N 2 Under atmosphere, NaBH 4(113 mg, 2.98 mmol) and stirred at 0° C. for 10 min. After stirring at room temperature for 4 h, the reaction was 2 The organic layer was diluted with O and EA. 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure. The residue was purified by column chromatography (EA:HEX=1:1) to give compound Int-TG3-2 (430 mg, 79%). 1 H NMR (400 MHz, CDCl 3 )δ7.30(d,J=8.8Hz,2H),6.99(d,J=8.8Hz,2H). 5.51-5.54(m,2H),5.11(dd,J=10.8Hz,1H),5.03(d,J=8.0Hz,1H),4.65(d,J=5.6 2H)4.25-4.04(m,3H),2.19(s,3H),2.07(s,3H),2.06(s,3H),2.01(s,3H).

[0427] Preparation of compound Int-TG3 A dry solution of compound Int-TG3-2 (1.0 g, 2.2 mmol) was dissolved in DMF (6.0 ml) and 2 The mixture was treated with bis(pentafluorophenyl carbonate) (1.3 g, 3.3 mmol) at room temperature under atmospheric pressure and stirred for 3 h. The reaction mixture was diluted with EA (20 mL × 2), H 2 The organic layer was extracted with anhydrous NaO (30 mL). 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure. The reaction mixture was purified by column chromatography to give compound Int-TG3 (1.4 g, 98%). 1 H NMR (400 MHz, CDCl 3 ):δ7.384(d,J=8.8Hz,2H),7.039(d,J=8.4Hz,2H),5.529-5.465(m,2H),5.280(s,2H),5.141 -5.068(m,2H),4.262-4.070(m,4H),2.195(s,3H),2.078(s,3H),2.073(s,3H),2.025(s,3H).

[0428] Example 9 Preparation of Compounds Int-TG4 and Int-TG4a [ka]

[0429] Compound Int-TG4-1 (yield 72%) was synthesized by the same method as described in Example 8. 1 H NMR (400 MHz, CDCl 3 )δ9.93(s,1H),7.86(d,J=6.8Hz,2H). 7.11(d,J=6.8Hz,2H),5.52-5.47(m,2H),5.18-5.14(m,2H),4.24-4.11(m,3H),2.19(s,3H),2.07(s,6H),2.02(s,3H).

[0430] Preparation of compound Int-TG4-2 A solution of compound Int-TG4-1 (2.06 g, 4.70 mmol) in DCM (50 mL) was treated with NaBH 4 A solution of 191 mg (5.04 mmol) of ammonium chloride (10 mL) was added dropwise and stirred for 30 min. The reaction mixture was diluted with saturated aqueous ammonium chloride (263 mL) and the aqueous layer was extracted with dichloromethane (88 mL×3) and ethyl acetate (88 mL×3). The organic layer was extracted with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure to give a white foam (2.02 g, 98%), which was used in the next step without further purification to give compound Int-TG4-2. 1 H NMR (400 MHz, CDCl 3 )δ7.31(d,J=8.8Hz,2H),6.99(d,J=8.8Hz,2H). 5.35-5.28(m,3H),5.13(d,J=7.2Hz,1H),4.64(d,J=5.2Hz,2H),4.18-4.16(m,1H),3.73(s,3H),2.12-2.04(m,9H).

[0431] Preparation of compound Int-TG4 A solution of compound Int-TG4-2 (500 mg, 1.14 mmol) in DMF (7 mL) was treated with Bis(PNP) (517 mg, 1.70 mmol), DIPEA (0.395 mL, 2.27 mmol) at room temperature under nitrogen atmosphere and stirred for 2 h. 2 The organic layer was extracted with 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 give compound Int-TG4 (645 mg, 95%) as a white foam. 1 H NMR (400 MHz, CDCl 3 )δ8.27(d,J=9.2Hz,2H),7.38-7.36(m,4H). 7.03(d,J=8.0Hz,2H),5.36-5.28(m,3H),5.24(s,2H),5.17(d,J=7.6Hz,1H),4.20-4.18(m,1H),3.73(s,3H),2.06-2.04(m,9H).

[0432] Preparation of compound Int-TG4a Yield: 98% 1 H NMR (400 MHz, CDCl 3 )δ7.54(dd,J=8.8,4.8Hz,2H),7.03(dd,J=4.4,8.8Hz,2H),5.37-5.26(m,5H ),5.18(d,J=6.8Hz,2H),4.21-4.18(m,2H),3.73(s,3H),2.09-2.06(m,9H).

[0433] Example 10 Preparation of compound Int-TG5 [ka] Compound Int-TG5 was synthesized by a method similar to that described in Example 9.

[0434] Compound Int-TG5-1 Yield 11H NMR (400 MHz, CDCl 3 ) δ 9.98 (s, 1H), 8.31 (d, J = 2.0 Hz, 1H), 8.07 (dd, J = 2.0 Hz, 8.8 Hz, 1H), 7.48 (d, J = 8.8 Hz, 1H), 5.59 (dd, J = 7.6 Hz, 10.4 Hz, 1H), 5.49 (d, J = 3.2 Hz, 1H), 5.20 (d, J = 7.6 Hz, 1H), 5.13 (dd, J = 3.6 Hz, 10.4 Hz, 1H), 4.28 - 4.09 (m, 3H), 2.20 (s, 3H), 2.13 (s, 3H), 2.10 (s, 3H), 2.04 (s, 3H).

[0435] Compound Int-TG5-2 Yield 96% 1 1H NMR (400 MHz, CDCl 3 ) δ 7.81 (d, J = 2.0 Hz, 1H), 7.52 (dd, J = 2.0 Hz, 8.8 Hz, 1H), 7.35 (d, J = 8.8 Hz, 1H), 5.54 (dd, J = 8.0 Hz, 10.4 Hz, 1H), 5.47 (d, J = 3.2 Hz, 1H), 5.10 (dd, J = 3.6 Hz, 10.4 Hz, 1H), 5.05 (d, J = 8.0 Hz, 1H), 4.73 (d, J = 6.0 Hz, 2H), 4.28 - 4.04 (m, 3H), 2.19 (s, 3H), 2.13 (s, 3H), 2.10 (s, 3H), 2.02 (s, 3H).

[0436] Compound Int-TG5 Yield 84% 11 1H NMR (400 MHz, CDCl 3 ) δ 7.89 (d, J = 2.4 Hz, 1H), 7.60 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 7.41 (d, J = 8.4 Hz, 1H), 5.56 (dd, J = 8.0 Hz, 10.4 Hz, 1H), 5.48 (d, J = 2.4 Hz, 1H), 5.32 (s, 2H), 5.13 - 5.09 (m, 2H), 4.28 - 4.07 (m, 3H), 2.19 (s, 3H), 2.13 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H).

[0437] Example 11 Preparation of Compounds Int-TG6 and Int-TG7 [ka]

[0438] Preparation of compound Int-TG6-1 A solution of 3-formyl-4-hydroxybenzoic acid (5 g, 43.06 mmol) in DMF (100 mL) was heated at room temperature with N 2 Benzyl bromide (5.1 mL, 43.06 mmol) and NaHCO 3 (2.53 g, 43.06 mmol) and stirred overnight. The reaction mixture was extracted with EA (200 mL x 2) and distilled water (100 mL). The resulting organic layer was diluted with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure, and the residue was purified by column chromatography to give compound Int-TG6-1 (2.56 g, 39%). 1 H NMR (400Hz, CDCl 3 )δ11.41(s,1H),9.95(s,1H),8.34(d,J=2.0Hz,1H),8.23(dd,J=6.4Hz,2.4Hz,1H),7.46-7.35(m,5H),7.04(d,J=9.2Hz,1H),5.37(s,2H).

[0439] Preparation of compound Int-TG6-2 A solution of compound Int-TG6-1 (1.0 g, 3.90 mmol) and compound Int-TG1 (1.6 g, 3.90 mmol) in anhydrous ACN (30 mL) was filtered with molecular sieves (8 g) and Ag 2 O (3.62 g, 15.61 mmol) at room temperature. 2 Treat under atmospheric pressure and stir for 1 h. The reaction mixture was filtered through CELITE® and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG6-2 (2.1 g, 92%). 1 H NMR (400Hz, CDCl 3)δ10.34(s,1H),8.55(d,J=2.0Hz,1H),8.26(dd,J=6.8,2.0Hz,1H),7.45-7.35(m,5H),7.17(d,J=8.8Hz,1H),5.63-5.60(m,1H),5.5 0(d,J=3.6Hz,1H),5.37(s,2H),5.23(d,J=8.0Hz,1H),5.16(dd,J=7.2,3.6Hz,1H)4.24-4.10(m,4H),2.20(s,3H),2.10-2.03(m,9H).

[0440] Preparation of compound Int-TG6-3 A solution of compound Int-TG6-2 (2.1 g, 3.58 mmol) in DCM (30 mL) was heated at 0 °C and 2 The mixture was treated with m-CPBA (2.65 g, 10.74 mmol) under atmospheric pressure and stirred for 7 h. The reaction mixture was quenched by adding saturated sodium bicarbonate solution (40 mL×2). The mixture was separated and the organic layer was washed with brine and added with Na 2 SO 4 The residue was stirred at 0 °C for 2 h at 4 °C for 3 h, dried at 4 °C, filtered, and concentrated under reduced pressure. 2 The mixture was dissolved in chloroform (5 mL) under atmospheric pressure and treated with hydrazine hydrate (261 μl, 5.37 mmol). After stirring for 1 h, the reaction mixture was extracted with EA (30 mL×2) and 1 M aqueous HCl (10 mL) was added. The resulting organic layer was washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered, and concentrated under reduced pressure to give compound Int-TG6-3 (1.1 g, 55%). EI-MS m / z: 574(M + +Na)

[0441] Preparation of compound Int-TG6-4 A solution of compound Int-TG6-3 (280 mg, 0.49 mmol) in DCM (5 mL) was diluted with TBDMS-OTf (224 μL, 0.97 mmol), Et 3 N (207 μL, 1.46 mmol) was used at 0° C. 2The mixture was treated under atmospheric pressure and stirred for 1.5 h. The reaction mixture was quenched by the addition of citric acid (20 ml). The organic layer was washed with brine (20 mL) and diluted with Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure, and the residue was purified by column chromatography to give compound Int-TG6-4 (246.3 mg, 68%). 1 H NMR (400Hz, CDCl 3 )δ7.67(d,J=8.4Hz,1H),7.57(s,1H),7.44-7.34(m,5H),7.02(d,J=8.4 Hz,1H),5.49-5.44(m,2H),5.30(s,2H),5.19(d,J=7.6Hz,1H),5.10(dd, J=6.8,3.2Hz,1H)4.20-4.11(m,2H),4.05(t,J=6.8Hz,2H),2.19(s,3H), 2.04(s,3H),2.01(d,J=6.0Hz,6H),1.02(s,9H),0.20(d,J=15.6Hz,6H).

[0442] Preparation of compound Int-TG6-5 To a solution of compound Int-TG6-4 (283.2 mg, 0.41 mmol) in EA (5 mL), 2 At room temperature under reduced pressure, pd / C (5%, 87.5 mg, 0.04 mmol) was added. The mixture was stirred for 1 h, filtered through CELITE®, and then concentrated under reduced pressure. The compound Int-TG6-5 was used directly in the next step without further purification (246 mg, quantitative). 1 H NMR (400Hz, CDCl 3 )δ7.67(d,J=8.8Hz,1H),7.57(s,1H),7.05(d,J=8.4Hz,1H),5.49-5.45(m,2H),5.22(d,J=7.6Hz,1H),5.12(dd,J=7. 2,3.6Hz,1H)4.20-4.06(m,4H),2.19(s,3H),2.05(s,3H),2.02(d,J=7.6Hz,6H),1.01(s,9H),0.21(d,J=15.2Hz,6H).

[0443] Preparation of compound Int-TG6 A solution of compounds Int-TG6-5 (243.2 mg, 0.41 mmol) and 11-azido-3,6,9-trioxaundecan-1-amine (Aldrich, CAS134179-38-7, 89.5 mg, 0.41 mmol) in DMF (5 mL) was reacted with PyBOP (275 mg, 0.53 mmol) and DIPEA (176 μL, 1.02 mmol) at room temperature under N 2 The mixture was treated under atmospheric pressure and stirred for 2 h. The reaction mixture was extracted with EA (30 mL x 2) and distilled water (10 mL). The resulting organic layer was washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure, and the residue was purified by column chromatography to give compound Int-TG6 (272.8 mg, 84%). 1 H NMR (400Hz, CDCl 3 )δ7.34(s,1H),7.31(d,J=9.2Hz,1H),7.02(d, J=8.0Hz,1H),6.73(s,1H),5.48-5.44(m,2H),5.19(d,J=7.6Hz,1H),5.10(dd,J=6.4,3.6Hz,1H),4.20-4.10(m,2H),4.06(t ,J=6.4Hz,2H),3.66(s,14H),3.38(t,J=4.4Hz,2H),2.19(s,3H),2.02(t,J=8.4Hz,9H),1.00(s,9H),0.20(d,J=14.4Hz,6H). EI-MS m / z: 799(M + +1).

[0444] Preparation of compound Int-TG7 A solution of compound Int-TG1-6 (1.05 g, 1.75 mmol) and L-12 (565 mg, 2.1 mmol) in DMF (10 mL) was heated at 0 °C under N 2 The reaction was treated with DIPEA (0.77 mL, 4.38 mmol), PyBOP (1.09 g, 2.1 mmol) under atmospheric pressure and stirred at room temperature for 2 h. 2O (250 mL) was added and extracted with EA (250 mL × 3). 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure, and the residue was purified by column chromatography to give compound Int-TG7 (1.17 g, 83%). 1 H NMR (400 MHz, CDCl 3 )δ7.00-6.96(m,2H),6.90(s,1H),5.48-5.43(m,2H),5.16(d,J=8.0Hz,1H) ,5.10(dd,J=3.2,10.4Hz,1H),4.20-4.11(m,2H),4.05(t,J=7.2Hz,1H),3.7 6-3.49(m,14H),3.46-3.39(m,2H),3.10-3.04(m,3H),2.19(s,3H),2.04(s, EI-MS m / z:813(M + +1).

[0445] Example 12 Preparation of Compounds Int-TG8 and Int-TG8a [ka]

[0446] Compound Int-TG8 was synthesized by a method similar to that described in Example 11. Compound Int-TG8-1 Yield: 65% 1 H NMR (400 MHz, CDCl 3 )δ10.32(s,1H),8.54(d,J=2.4Hz,1H),8.28(dd,J=8.8Hz,1H),7.45-7.35(m,5H),7.16 (d,J=8.8Hz,1H),5.39-5.34(m,6H),4.28-4.26(m,1H),3.72(s,3H),2.11-2.06(m,9H).

[0447] Compound Int-TG8-2 Yield: 63% 1 H NMR (400 MHz, CDCl 3 )δ7.66(d,J=2Hz,1H),7.60(dd,J=8.4Hz,1H),7.43-7.31(m,5H),7.00(d,J=8.4Hz,1H),6.13(s,1H),5.41- 5.28(m,5H),5.12(d,J=7.2Hz,1H),4.23(d,J=9.2Hz,1H),3.76(s,3H),2.09(s,3H),2.06(d,J=3.6Hz,6H).

[0448] Compound Int-TG8-3 Yield 70% 1 H NMR (400 MHz, CDCl 3 )δ7.60(dd,J=2.0,2.0Hz,1H),7.43(d,J=0.8Hz,1H),7.48-7.32(m,5H),7.01(d,J=8 .4Hz,1H),5.40-5.26(m,6H),4.18(d,J=9.2Hz,1H),3.72(s,3H),2.09-2.04(m,9H). 0.99(s,9H),0.18(d,J=12.8Hz,1H).

[0449] Compound Int-TG8-4 Harvest EI-MS m / z:607(M + +Na)

[0450] Compound Int-TG8-5 Yield 96% 1 H NMR(400Hz,DMSO-d6)δ9.73(brs,1H),7.44(d,J=2.0Hz,1H),7.37(dd,J=2.4,6.4Hz,1H),7.08(d,J=8.4Hz,1H),5. 61(d,J=7.6Hz,2H),5.45(t,J=9.6Hz,1H),5.15-5.02(m,2H),4.67(d,J=10Hz,1H)3.63(s,3H),2.04-1.98(m,9H). EI-MS m / z:785(M + +1)

[0451] Compound Int-TG8-6 Yield: 78% EI-MS m / z: 1097(M + +1)

[0452] Compound Int-TG8 Yield: 85% EI-MS m / z: 785(M + +1)

[0453] Compound Int-TG8a Yield: 70% EI-MS m / z: 971(M + +1)

[0454] Example 13 Preparation of compound Int-TG9 [ka]

[0455] Preparation of compound Int-TG9 To a solution of 4-hydroxybenzaldehyde (1 g, 8.19 mmol) in DCM (3 mL), 3 N (2.28 mL, 16.38 mmol) at room temperature 2 The mixture was added under atmospheric pressure. 2 F 2 Gas was introduced via a balloon and the mixture was stirred at room temperature for 2 h. The mixture was then washed with DCM (30 mL x 3) and brine (30 mL), and the organic layer was washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure, and the residue was purified by column chromatography to give compound Int-TG9 (790 mg, 63%). 1 H NMR (400Hz, CDCl 3 )δ10.06(s,1H),8.05(d,J=8.0Hz,2H),7.55(d,J=8.8Hz,2H).

[0456] Example 14 Preparation of Compounds Int-TG10 and Int-TG11 [ka]

[0457] Preparation of compound Int-TG10-1 Compounds Int-TG6 (2.0 g, 2.5 mmol), Int-TG9 (560 mg, 2.75 mmol), and a solution of Int-TG9 in anhydrous ACN (25 mL) were dissolved in N 2 The mixture was treated with BEMP (292 μl, 1.0 mmol) at room temperature under atmospheric pressure and stirred for 4 h. The reaction was quenched with water (20 mL) and extracted with EA (30 mL×2). 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-TG10-1 (1.7 g, 81%) as a white foamy solid. EI-MS m / z: 869(M + +1).

[0458] Preparation of compound Int-TG10-2 A solution of compound Int-TG10-1 (1.7 g, 1.96 mmol) in anhydrous THF (45 mL) was heated at 0° C. with N 2 Under atmosphere, NaBH 4 (150 mg, 3.91 mmol) and stirred for 2 h. The reaction was quenched with water (30 mL) and extracted with EA (50 mL×2). The organic layer was 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 Int-TG10-2 (1.17 g, 69%) as a white foamy solid. EI-MS m / z: 871(M + +1).

[0459] Preparation of compound Int-TG10-3 A solution of compound Int-TG10-2 (1.17 g, 1.34 mmol) in anhydrous THF (40 mL) was heated at 0 °C and cooled to 20 °C. 2The mixture was treated with methanesulfonyl chloride (312 μl, 4.0 mmol), TEA (940 μl, 6.72 mmol) under atmospheric pressure and stirred overnight. The reaction was quenched with water (20 mL) and extracted with DCM (60 mL×2). 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-TG10-3 (1.25 g, 98%) as a white foamy solid. EI-MS m / z: 949(M + +1).

[0460] Preparation of compound Int-TG10 A solution of compound Int-TG10-3 (1.25 g, 1.32 mmol) in anhydrous THF (40 mL) was stirred at room temperature under N 2 The reaction was treated with LiBr (570 mg, 6.58 mmol) under atmospheric pressure and stirred for 3 h. The reaction was diluted with water (30 mL) and extracted with DCM (50 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, and the residue was purified by column chromatography to give compound Int-TG10 (1.2 g, 98%) as a white foamy solid. EI-MS m / z: 933(M + ),935(M + +2).

[0461] Compound Int-TG11 was synthesized by a synthetic route similar to that used to prepare compound Int-TG10. Preparation of compound Int-TG11-1 Yield: 80% 1 H NMR (400 MHz, CDCl 3) δ 10.04 (s, 1H), 8.00 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.4 Hz, 2H), 7.44 - 7.27 (m, 3H), 5.57 - 5.51 (m, 1H), 5.47 (d, J = 3.2 Hz, 1H), 5.14 - 5.10 (m, 2H), 4.27 - 4.09 (m, 3H), 3.76 - 3.53 (m, 14H), 3.42 - 3.36 (m, 2H), 3.12 - 3.04 (m, 3H), 2.19 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.02 (s, 3H); EI-MS m / z: 883 (M + +1).

[0462] Preparation of Compound Int-TG11-2 Yield 81% 1 H NMR (400 MHz, CDCl 3 ) δ 7.47 - 7.42 (m, 2H), 7.40 - 7.31 (m, 3H), 7.24 - 7.21 (m, 2H), 5.54 - 5.45 (m, 2H), 5.11 - 5.07 (m, 2H), 4.74 - 4.70 (m, 2H), 4.25 - 4.21 (m, 1H), 4.17 - 4.12 (m, 1H), 4.06 (t, J = 7.2 Hz, 1H), 3.74 - 3.44 (m, 12H), 3.37 (t, J = 4.8 Hz, 2H), 3.07 - 3.04 (s, 3H), 2.20 (s, 3H), 2.06 (s, 6H), 2.02 (s, 3H).

[0463] Preparation of Compound Int-TG11-3 Yield 98% EI-MS m / z: 963 (M + +1).

[0464] Preparation of Compound Int-TG11 Yield 90% 1 H NMR (400 MHz, CDCl 3)δ7.53-7.41(m,4H),7.37-7.33(m,2H),7.29-7.28(m,1H),5.59-5.55(m,1H),5.47(d,J=3.2Hz,1H),5.13-5.09(m,2H),4.26-4.22(m,1H), EI-MS m / z:948(M + +1).

[0465] Example 15 Preparation of compound Int-TG12 [ka]

[0466] Preparation of compound Int-TG12-1 A solution of compound Int-TG8 (100 mg, 0.13 mmol) and compound Int-TG9 (31.8 mg, 0.16 mmol) in anhydrous ACN (2.5 mL) was stirred at room temperature under N 2 The reaction mixture was treated with DBU (7.6 μl, 0.052 mmol) and stirred for 5 h under atmospheric conditions. 2 The mixture was extracted with O (10 mL) and EA (15 mL × 2). The organic layer was washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure, and the residue was purified by column chromatography to give compound Int-TG12-1 (54.5 mg, 50%). EI-MS m / z: 855(M + +1).

[0467] Preparation of compound Int-TG12a [ka] Compound Int-TG11a was synthesized by a synthetic route similar to that used to prepare compound Int-TG12.

[0468] Compound Int-TG12 was synthesized by a synthetic route similar to that described in Example 14. Compound Int-TG12-2 Yield: 77% EI-MS m / z: 857(M + +1).

[0469] Compound Int-TG12-3 Yield: 98% EI-MS m / z: 935(M + +1).

[0470] Compound Int-T12 Yield: 81% EI-MS m / z: 920(M + +1).

[0471] Example 16 Preparation of compound Int-TG13 [ka] Compound Int-TG13 was synthesized by a synthetic route similar to that described in Example 11 and Example 14.

[0472] Preparation of compound Int-TG13-1 Yield 72%, colorless oil EI-MS m / z: 1226(M + +1).

[0473] Preparation of compound Int-TG13-2 Yield 82%, colorless oil EI-MS m / z: 1296(M + +1).

[0474] Preparation of compound Int-TG13-3 Yield 75%, colorless oil EI-MS m / z: 1298(M + +1).

[0475] Preparation of compound Int-TG13-4 Yield 82%, colorless oil EI-MS m / z: 1376(M + +1).

[0476] Preparation of compound Int-TG13 Yield 82%, colorless oil EI-MS m / z: 1361(M + +1).

[0477] Example 17 Preparation of Compounds Int-TG14 and Int-TG15 [ka]

[0478] Preparation of compound Int-TG14-1 Compound Int-TG14-1 was synthesized by a method similar to the preparation of Int-TG6-5 in Example 11. Yield: 99% 1 H NMR (400Hz, CDCl 3 )δ7.67(s,1H),7.62(dd,J=6.4,2.0Hz,1H),7.02(d,J=8.4Hz,1H),5.51-5.45(m,2H),5.16(dd,J=7.2,3 .6Hz,1H),5.04(d,J=8.0Hz,1H),4.21-4.09(m,4H),2.20(s,3H),2.12(s,3H),2.01(d,J=7.6Hz,8.4H).

[0479] Preparation of compound Int-TG14-2 A solution of compound Int-TG14-1 (578 mg, 1.19 mmol) and compound L-2 (384.8 mg, 1.43 mmol) in DMF (12 mL) was heated at room temperature with N 2 The mixture was treated with PyBOP (807.2 mg, 1.55 mmol) and DIPEA (520 μL, 2.98 mmol) under atmospheric pressure and stirred for 1 h. The reaction mixture was extracted with EA (40 mL×2) and distilled water (25 ml). The resulting organic layer was washed 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 Int-TG14-2 (660 mg, 80%). EI-MS m / z: 699(M + +1).

[0480] Preparation of compound Int-TG15-1 Compound Int-TG14-1 was synthesized via a similar method to prepare compound Int-TG14-2. Yield: 76% 1 H NMR (400 MHz, CDCl 3 )δ7.36-7.31(m,2H),7.01(d,J=8.0Hz,1H),6.64-6.58(m,1H),6.06(brs,1H),5.50-5.46(m,2H),5.14(dd,J=7. 6,3.2Hz,1H),4.99(d,J=8.0Hz,1H),4.28-4.08(m,3H),3.70-3.64(m,14H),3.46(t,J=5.2Hz,2H),2.18(s,3H). ,2.12(s,3H),2.09(s,3H),2.04(s,3H). EI-MS m / z: 685(M + +1).

[0481] Preparation of compound Int-TG14 To a solution of compound Int-TG14-2 (480 mg, 0.69 mmol) in DCM (10 mL), Et 3 N (335uL, 2.4mmol) at room temperature, 2 The mixture was added under atmospheric pressure. 2 F 2 Gas was introduced via a balloon and the mixture was stirred at room temperature for 3 h. The mixture was then washed with DCM (30 mL x 3) and brine (30 mL), and the organic layer was diluted with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure, and the residue was purified by column chromatography to give compound Int-TG14 (430 mg, 80%). EI-MS m / z: 781(M + +1).

[0482] Preparation of compound Int-TG15 Compound Int-TG15 was synthesized via a similar method to prepare compound Int-TG14. Yield: 86% 1 H NMR (400 MHz, CDCl 3 )δ7.86(s,1H),7.83(dd,J=6.4,2.0Hz,1H),7.31(d,J=8.4Hz 1H),6.89-6.83(m,1H),5.61-5.56(m,1H),5.50-5.48(m,1H),5.18(d,J=8.0Hz,1H),5.13(dd,J=7.2,3.2 Hz 1H),4.27-4.10(m,4H),3.70-3.62(m,14H),3.37(t,J=5.2 Hz 2H),2.20(s,3H),2.08(s,6H),2.02(s,3H). EI-MS m / z: 767(M + +1).

[0483] Example 18 Preparation of Compound Mono-1 [ka] Mono-1 was obtained by carrying out the reaction in a similar manner as described in the document WO2020 / 089687.

[0484] Preparation of compound Mono-1-1 Yield: 77% 1 H NMR(400MHz,DMSO-d6)δ9.95(brs,1H),7.48(d,J=5.2Hz,1H),6.94(d,J=5.2Hz,1H),4.48-4.44(m,1 H),4.28(d,J=15.6Hz,1H),4.18(d,J=16.0Hz,1H),3.39(dd,J=11.6,5.2Hz,1H),3.17-3.10(m,1H). EI-MS m / z:184(M + +1).

[0485] Preparation of compound Mono-1-2 Yield: 99% 1 H NMR (400 MHz, DMSO-d6) δ 10.22 (brs, 2H), 7.49 (d, J = 5.2 Hz, 1H), 6.94 (d, J = 5.2 Hz, 1H), 4.65 - 4.61 (m, 1H), 4.30 (d, J = 15.6 Hz, 1H), 4.19 (d, J = 15.6 Hz, 1H), 3.80 (s, 3H), 3.60 (dd, J = 11.6, 5.2 Hz, 1H), 3.21 - 3.14, (m, 1H). EI-MS m / z: 198 (M + +1).

[0486] Preparation of Compound Mono-1-3 Yield: 89% EI-MS m / z: 483 (M + +1).

[0487] Preparation of Compound Mono-1-4 Yield: 85% EI-MS m / z: 453 (M + +1).

[0488] Preparation of Compound Mono-1-5 Yield: 85% 1 H NMR (400 MHz, CDCl 3 ) δ 7.55 (d, J = 5.6 Hz, 1H), 7.47 (m, 5H), 7.22 (d, J = 5.2 Hz, 1H), 6.95 (d, J = 5.2 Hz, 1H), 6.85 (s, 1H), 5.26 - 5.14 (m, 2H), 4.98 (d, J = 16.4 Hz, 1H), 4.44 (d, J = 16.8 Hz, 1H), 4.08 - 4.02 (m, 1H), 3.98 (s, 3H), 3.32 - 3.26 (m, 1H). EI-MS m / z: 453 (M + +1).

[0489] Preparation of Compound Mono-1 Yield: 82% 1 H NMR (400 MHz, CDCl 3)δ7.58(d,J=5.6Hz,1H),7.54(s,1H),7.23(d,J=5.2Hz,1H),6.95(d,J=5.2Hz,1H),6.89(s,1H),6.06(s,1H),5 .30(s,1H),4.99(d,J=16.4Hz,1H),4.44(d,J=16.4Hz,1H),4.10-4.04(m,1H),3.99(s,3H),3.32-3.26(m,1H). EI-MS m / z: 315(M + +1).

[0490] Example 19 Preparation of Compound Mono-2 [ka] Preparation of compound Mono-2-1 A solution of compound Mono-1-1 (1 g, 4.28 mmol) in 20 mL of dry THF was heated at 0 °C under N 2 The mixture was treated with a 1M solution of LAH in THF (5.31 mL, 5.31 mmol) under atmospheric pressure and stirred for 15 h. The reaction mixture was diluted with water (5.3 mL), 15% NaOH (5.3 mL), H 2 The mixture was quenched with 200 mL of HO (16.0 mL) and stirred for 30 min. The inorganic solid was filtered and washed with EA. The organic layer was washed with Na 2 SO 4 The mixture was dried at rt, filtered and concentrated under reduced pressure to provide compound Mono-2-1 (652 mg, 3.85 mmol, 90%) as a red solid which was used without further purification. 1 H NMR (400 MHz, CDCl 3 )δ7.08(d,J=4.8,1H),6.73(d,J=5.2Hz,1H),4.01-3.88(m,2H),3.80(dd,J=11.2Hz,1H) ,3.55(dd,J=8.4Hz,1H),3.13-3.07(m,1H),2.78-2.74(m,1H),2.60-2.51(m,1H);ET-MS m / z:170.0(M +1 +1).

[0491] Preparation of compound Mono-2-2 A solution of compound Mono-2-1 (700 mg, 4.14 mmol) in anhydrous DCM (20 mL) was stirred at 0 °C for 2 h at 25 °C for 3 min. 2 The reaction mixture was treated with imidazole (844 mg, 12.41 mmol), TBDMS-Cl (686 mg, 4.55 mmol) under atmospheric pressure and stirred at room temperature for 4 h. 2 The mixture was extracted with 100 mL of HO and 300 mL of DCM. 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure, and the residue was purified by column chromatography to give compound 2-2 (792 mg, 67%). 1 H NMR (400 MHz, CDCl 3 )δ7.07(d,J=5.2Hz,1H),6.75(d,J=5.2Hz,1H),4.04-3.92(m,2H),3.77(dd,J=9.6Hz,1H ),3.65(dd,J=9.6Hz,1H),3.05-3.00(m,1H),2.75-2.71(m,1H),2.65-2.59(m,1H);ET-MS m / z:284.1(M +1 +1).

[0492] Preparation of compound Mono-2-3 A solution of compound Int-2 (536 mg, 1.96 mmol) and compound Mono-2-2 (666 mg, 2.35 mmol) in anhydrous DMF (1.8 mL) was treated with DIPEA (0.85 mL, 4.89 mmol) at 0° C. under N2 atmosphere and stirred at room temperature for 3 h. The reaction mixture was extracted with EA / H2O. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The reaction mixture was purified by column chromatography (EA / HEX: 1 / 1) to give a yellow solid Mono-2-3 (758.5 ​​mg, 76%); EI-MS m / z: 521 (M + +1).

[0493] Preparation of compound Mono-2 A solution of compound Mono-2-3 (200 mg, 0.384 mmol) in MeOH (4.5 mL) was heated at 0 °C under a N2 atmosphere. 2 CO 3(63.7 mg, 0.461 mmol) and stirred for 20 min. The reaction mixture was extracted with EA / H2O. The organic layer was washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered, concentrated under reduced pressure, and dried in vacuum to give a yellow solid Mono-2 (189.8 mg quantitative); EI-MS m / z: 479 (M + +1).

[0494] Example 20 Preparation of Compound Mono-4 [ka]

[0495] Compound Mono-4 was synthesized by a method similar to that described in Example 19. Preparation of compound Mono-4 Yield: 93% 1 H NMR(400MHz,CDCl3)δ8.04-7.98(m,1H),7.26-7.19(m,3H),7.00-6.75(m,2H),4.41-4.22(m,2H),3.92(s,3H),3. 86(s,1H),3.76-3.66(m,1H),3.51-3.42(m,1H),3.23-2.68(m,2H),0.89-0.73(m,9H),0.05--0.08(m,6H);EI-MS m / z:473(M + +1).

[0496] Example 21 Preparation of Compound Mono-5 [ka] Compound Mono-5 was obtained by carrying out the reaction in a similar manner as described in Journal of Medicinal Chemistry, 2001, Vol. 44, No. 5, 737.

[0497] Preparation of compound Mono-5 Yield: 67% 1 H NMR (400Hz, CDCl 3)δ4.95-4.90(m,2H),3.69-3.54(m,4H),3.35-3.27(m,1H),2.46-2.44(m,1H),2.27-2.25(m,1H),0.89(s,9H),0.05(s,6H).

[0498] Example 22 Preparation of Compound D-1 [ka]

[0499] Preparation of compound D-1 A solution of compound Mono-1 (100 mg, 0.32 mmol) and 1,3,5-tris(bromomethyl)benzene (56.6 mg, 0.16 mmol) in DMF (2.0 mL) was subjected to K 2 CO 3 (44.2 mg, 0.32 mmol) at room temperature. 2 The mixture was treated with dimethylamine (0.5 mL) and stirred for 30 min under atmospheric pressure, and the mixture was purified by preparative HPLC to give compound D-1 (29 mg, 23%). EI-MS m / z: 788(M + +1).

[0500] Example 23 Preparation of Compound D-2 [ka]

[0501] Preparation of Compound D-2-1 A solution of 1,3,5-tris(bromomethyl)benzene (3.9 g, 11.0 mmol) of compound Int-2 (4.96 g, 21.9 mmol) in DMF (10.0 mL) was added to the 2 At room temperature under atmospheric conditions, 2 CO 3(44.2 mg, 0.32 mmol, 1.0 equiv) and stirred for 6 h. The reaction mixture was treated with dimethylamine (5.0 mL) and stirred for 30 min. The reaction mixture was diluted with distilled water (50 mL) and extracted with DCM (100 mL x 2). The organic layer was washed with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure, and the residue was purified by column chromatography to give compound D-2-1 (2.74 g, 41%). 1 H NMR (400 MHz, CDCl 3 )δ7.50(s,2H), 7.41(d,J=12.0Hz,3H),7.08(s,2H),5.20(s,4H),3.97(s,6H),3.91(s,6H),3.47(s,2H),2.25(s 6H);EI-MS m / z:614(M + +1).

[0502] Preparation of Compound D-2-2 Compound D-2-1 (2.74 g, 4.46 mol) in THF (75 mL) and H 2 To a solution in 20O (50 mL) was added LiOH (937 mg, 22.33 mol). After stirring for 5 h, the reaction mixture was concentrated under reduced pressure. The residue was cooled to 0° C. and adjusted to pH 2 by addition of 2N HCl solution, then the solid was filtered and purified with H 2 O (30 mL) and EA (100 mL) to obtain compound D-2-2 (2.5 g, 96%). 1 H NMR(400MHz,DMSO-d6)δ7.71(s,2H),7.60(d,J=17.6Hz,3H),7.32(s,2H),5.30(s,4H),3.91(s,6H),2.67(s,6H);EI-MS m / z:586(M + +1).

[0503] Preparation of compound D-2-3 A solution of compound D-2-2 (1.5 g, 2.56 mmol) and compound Mono-2 (1.52 g, 5.38 mmol) in DMF (50.0 mL) was stirred at room temperature under N 2The mixture was treated with PyBOP (3.5 g, 6.40 mmol) and DIPEA (2.2 mL, 12.8 mmol) under atmospheric pressure and stirred for 2 h. The reaction mixture was diluted with distilled water (100 mL) and extracted with EA (100 mL x 2). 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 D-2-3 (2.7 g, 94%); EI-MS m / z: 1116 (M + +1).

[0504] Preparation of compound M-2-4 A solution of compound D-2-3 (2.7 g, 2.42 mmol) in EA (50.0 mL) was heated at room temperature with H 2 The mixture was treated with 5% Pd / C (5.1 g, 2.42 mmol) under reduced pressure and stirred for 1 h. The reaction mixture was filtered through CELITE® and then concentrated under reduced pressure to give compound D-2-4 (1.87 g, 93%); EI-MS m / z: 1056 (M + ).

[0505] Preparation of compound D-2-5 A solution of compound D-2-4 (100 mg, 0.095 mmol) and Int-TG3 (189 mg, 0.28 mmol) in anhydrous THF (3.0 mL) was added to a flask at room temperature under N 2 The mixture was treated with HOBT (13.0 mg, 0.095 mmol) and DIPEA (36 μl, 0.208 mmol) under atmospheric pressure and stirred for 44 h. The reaction mixture was extracted with distilled water (10 mL) and EA (20 mL × 2), and the organic layer was washed with saturated NH 4 The organic layer was washed with anhydrous NaCl (50 mL). 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 D-2-5 (76 mg, 40%); EI-MS m / z: 2017 (M + ).

[0506] Preparation of compound D-2-6 Compound D-2-5 (116.7 mg, 0.06 mmol) in ACN (2.0 mL), H 2 The solution in 200 µL was incubated at 0 °C for 2 h at 4 °C for 1 h in H2O (800 µL). 2 The mixture was treated with TFA / ACN (1.0 mL) under atmospheric pressure and stirred for 2 h. The residue was purified by preparative HPLC to give compound D-2-6 (83.3 mg, 80%); EI-MS m / z: 1788 (M + ).

[0507] Preparation of compound D-2 A solution of compound D-2-6 (83.3 mg, 0.046 mmol) in anhydrous DCM (3.0 mL) was incubated at 0 °C for 2 h at 25 °C for 3 h. 2 The mixture was treated with Dess-Martin periodinane (45.4 mg, 0.11 mmol) under atmospheric pressure and stirred for 4 h. The reaction mixture was diluted with distilled water (10 mL) and extracted with EA (30 mL × 2). The organic layer was 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 preparative HPLC to give compound D-2 (59.3 mg, 71%); EI-MS m / z: 1784 (M + ).

[0508] Example 24 Preparation of Compound D-3 [ka] Compound D-3 was synthesized by a method similar to that described in Example 23.

[0509] Compound D-3-1 Yield: 9% EI-MS m / z: 1989(M + ).

[0510] Compound D-3-2 Yield: 65% EI-MS m / z: 1760(M + ).

[0511] Compound D-3 Yield: 48% EI-MS m / z: 1756(M+ ).

[0512] Example 25 Preparation of Compound D-4 [ka] Compound D-4 was synthesized by a synthetic route similar to that described in Example 23.

[0513] Compound D-4-1 Yield: 56% EI-MS m / z: 1054.42(M + / 2), 2107.27(M + ).

[0514] Compound D-4-2 Yield: 77% EI-MS m / z: 1879.18 (M+1).

[0515] Compound D-4 Yield: 74% EI-MS m / z: 1874.36(M + ).

[0516] Example 26 Preparation of Compound D-5 [ka] Preparation of Compound D-5-1 A solution of compound L-1 (189 mg, 0.48 mmol) and compound Int-2 (239.6 mg, 1.05 mmol) in DMF (5.0 mL) was diluted with N 2 At room temperature under atmospheric pressure 2 CO 3 (166 mg, 1.20 mmol) and stirred for 6 h. The reaction mixture was diluted with EA (30 mL x 2), H 2 The mixture was extracted with 2,4-dichloromethane (15 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound D-5-1 (227.8 mg, 83%). 1 H NMR (400 MHz, CDCl 3)δ7...

Claims

1. A drug conjugate comprising a compound of formula (VII) or (VIII) and a linker group: 【Chemistry 1】 Or a pharmaceutically acceptable salt thereof; During the ceremony: A is a heterocycle; Each R ａ ' and R ｂ ' is independently halogen, amino, hydroxyl, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; Two Germinal R ｂ ' are optionally taken together to form oxo or =CH ２ or two R ｂ ', together with the intervening atoms, optionally completes a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R ｃ ' is sulfonate, sulfate, hydroxyl, amino, or thiol; R ｄ ' is -L"-Gly, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; provided that at least one Rc' is a sulfonate or sulfate, or at least one R ｄ’ Ga-L ’’ -Gly; R ｅ ' is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; m is an integer selected from 0 to 3; n is an integer selected from 0 to 8 depending on the valence; Ring Cy is selected from aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; 【Chemistry 2】 is a single or double bond; X' is a halogen; X″ is —NR—, —S—, or —O—; R is hydrogen or alkyl; Each R ａ " and R ｂ " is independently halogen, amino, hydroxyl, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, alkenyl, alkynyl, ═O, carboxyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L''') ｒ -X″-Gly; d is an integer selected from 0 to 4; r is an integer from 0 to 1; each L''' is independently a bond or a linker; R ｅ " is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; p is an integer selected from 0 to 4; DBD is the DNA binding domain; L" is a bond or a linker; and Gly is a monosaccharide, disaccharide, or oligosaccharide.

2. each L''' optionally contains one or more double and / or triple bonds; １０ -C １００ 2. The drug conjugate of claim 1, wherein the alkylene moiety is a straight or branched chain, saturated or unsaturated alkylene moiety of the formula:

3. 2. The drug conjugate of claim 1, wherein the compound is represented by formula (VII): 【Transformation 3】 Or a pharmaceutically acceptable salt thereof.

4. 4. The drug conjugate of claim 3, wherein the compound is: 【Chemistry 4】 or a pharmaceutically acceptable salt thereof; During the ceremony 【Transformation 5】 is a single bond or a double bond.

5. Two R's ｂ 10. The drug conjugate of claim 1, wherein ' together with the intervening atom completes a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

6. 4. The drug conjugate of claim 3, wherein the compound is: 【Transformation 6】 or a pharmaceutically acceptable salt thereof.

7. 10. The drug conjugate of claim 1, wherein the compound is represented by formula (VIII): 【Transformation 7】 Alternatively, the drug conjugate is represented by a pharmaceutically acceptable salt thereof.

8. 8. The drug conjugate of claim 7, wherein the compound has formula (VIIIa) or (VIIIb): 【Transformation 8】 Alternatively, the drug conjugate is represented by a pharmaceutically acceptable salt thereof.

9. 8. The drug conjugate of claim 7, wherein the DBD-(L''') ｒ The -X″-Gly unit is: 【Chemistry 9】 or a pharmaceutically acceptable salt thereof; During the ceremony, Y″ is C or N; X″ is selected from —NR—, —S—, or —O—; R is hydrogen or alkyl; r is an integer from 0 to 1; Each R ｂ " are independently halogen, amino, hydroxyl, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L''') ｒ -X″-Gly; R ｋ is alkyl, preferably C １ -C ３ is alkyl; q is an integer selected from 0 to 3; and 【Chemistry 10】 is a single bond or a double bond.

10. 8. The drug conjugate of claim 7, wherein the compound is represented by formula (VIIIc), (VIIId), (VIIIe), or (VIIIf): 【Chemistry 11】 Alternatively, the drug conjugate is represented by a pharmaceutically acceptable salt thereof.

11. 8. The drug conjugate of claim 7, wherein: Each R ａ " is independently halogen, amino, hydroxyl, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, =0, carboxyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and Each R b ″ is independently selected from halogen, amino, hydroxyl, alkoxy, cyano, nitro, C １－６ Alkyl, heteroalkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C ３－１０ cycloalkyl, 4- to 10-membered heterocycloalkyl, C ６－１０ The drug conjugate is aryl, or 5-10 membered heteroaryl.

12. 8. The drug conjugate of claim 7, wherein the compound is: 【Chemistry 12-1】 【Chemistry 12-2】 or a pharmaceutically acceptable salt thereof.

13. 8. The drug conjugate of claim 7, wherein L''' is 【Chemistry 13】 is a linker selected from: During the ceremony, R ａ "''' is hydrogen, halogen, amino, hydroxyl, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, =0, carboxyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and Each R ｂ "''' is independently hydrogen, halogen, amino, hydroxyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and The drug conjugate wherein h is an integer selected from 0 to 4 depending on the valence.

14. 14. The drug conjugate of claim 13, wherein L''' is 【Chemistry 14】 The drug conjugate,

15. 2. The drug conjugate of claim 1, wherein Gly is a monosaccharide or a disaccharide.

16. The drug conjugate of claim 15, wherein Gly is: i) glucose, glucuronic acid, fucose, or galactose; ii) 【Chemistry 15】 wherein optionally one or more —OH groups are masked by a protecting group; iii) 【Chemistry 16】 ； iv) 【Chemistry 17】 wherein optionally one or more —OH groups are masked by a protecting group; and v) [Chemistry 18] The drug conjugate is selected from:

17. 10. The drug conjugate of claim 1, having formula (IX), (X), or (XI): 【Chemistry 19】 or a pharmaceutically acceptable salt thereof; During the ceremony: Z' is a coupling group; Ar is aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; Y' is -(CR ｂ ２ ) ｙ N (R ａ ) -, -(CR ｂ ２ ) ｙ O- or -(CR ｂ ２ ) ｙ S- and when y is 1, an N, O, or S atom is positioned to bond to TG; When TG is activated, SO ２ In response to (Q) ｑ -(L') ｗ Replace X-SO ２ and a trigger group that generates an N, O, or S atom that can form a 5- to 6-membered ring containing the intervening atom of Ar; X is —O—, —C(R ｂ ) ２ -, or -N(R ｃ )- and; L', when present, is bonded to SO via a heteroatom selected from O, S, and N. ２ and L' and SO ２ is selected such that cleavage of the bond between facilitates release of the active agent; w is an integer selected from 0 to 1; r is an integer from 0 to 1; Z ２ is the linking group; Z ３ is the linking group; R ａ , R ｂ and R ｃ are each independently hydrogen or lower alkyl; y is an integer selected from 0 to 1; t is an integer from 1 to 5; and The drug conjugate, wherein e is an integer of 1 to 5.

18. 18. The drug conjugate of claim 17, wherein Z ３ but below: 【Chemistry 20】 Selected from: During the ceremony, X ５ is —O— or —NR ｘ - and; Y １ But, CR ｙ or N; R ｘ and R ｙ are each independently hydrogen or C １－６ is alkyl; each b is independently an integer from 1 to 3; and The drug conjugate, wherein c is an integer of 1 to 5.

19. 19. The drug conjugate of claim 18, wherein Z ３ but below: 【Chemistry 21】 The drug conjugate is selected from:

20. 18. The drug conjugate of claim 17, wherein Z ２ but, 【Chemistry 22】 and During the ceremony: Y ５ But, CR Ｙ１ or N, with the proviso that Y ５ and only one of is N; R Ｙ１ is H, hydroxyl, amino, amide, or (CH ２ ) ｙ (R Ｙ１ａ ) and R Ｙ１ａ is amino (e.g., secondary or tertiary amino), aryl (e.g., phenyl), or heteroaryl; and The drug conjugate wherein y is an integer having a value from 1 to about 10.

21. 21. The drug conjugate of claim 20, wherein Z ２ but 【Chemistry 23】 The drug conjugate,

22. 18. The drug conjugate of claim 17, wherein Z ２ but, 【Chemistry 24】 and During the ceremony, Y ６ But, CR Ｙ２ or N; R Ｙ２ is H or alkyl, preferably lower alkyl; R Ｚ２ However, (CH ２ ) ｚ R Ｚ２ａ and R Ｚ２ａ is amino (preferably tertiary amino), aryl (e.g., phenyl), or heteroaryl; and The drug conjugate wherein z is an integer having a value from 0 to about 10.

23. Y' is -(CR ｂ ２ ) ｙ N (R ａ )-or-(CR ｂ ２ ) ｙ The drug conjugate of claim 17, which is O-.

24. 18. The drug conjugate of claim 17, wherein L' is present and forms an -O-, -OC(O)-, -OC(O)O-, -NHC(O)O-, or -OC(O)NH- bond (including a heteroatom of the active drug).

25. 18. The drug conjugate of claim 17, wherein TG is: i) monosaccharides containing glucose, glucuronic acid, fucose, or galactose; ii) 【Chemistry 25】 wherein optionally one or more —OH groups are masked by a protecting group; iii) a disaccharide containing glucose, glucuronic acid, fucose, galactose, or a combination thereof; or iv) 【Chemistry 26】 wherein optionally one or more —OH groups are masked by a protecting group; The drug conjugate,

26. 18. The conjugate of claim 17, wherein Z' is 【Chemistry 27-1】 【Chemistry 27-2】 【Chemistry 27-3】 【Chemistry 27-4】 is selected from During the ceremony: R ｚａ is H or methyl; R ｚｂ is —OH, ═O, or ═NHOH; m and n are each independently an integer from 1 to 10; x is an integer selected from 1 to 2; 【Chemistry 28】 is a single bond or a double bond; and Z” is below 【Chemistry 29】 The conjugate is selected from:

27. A targeted drug conjugate comprising the drug conjugate of claim 1 and a targeting moiety (TM).

28. 28. The targeted drug conjugate of claim 27, having formula (XII), (XIII) or (XIV): 【Transformation 30】 or a pharmaceutically acceptable salt thereof; wherein TM is a targeting moiety.

29. 29. The targeted drug conjugate of claim 28, wherein Z' is 【Chemistry 31-1】 【Chemistry 31-2】 【Chemistry 31-3】 【Chemistry 31-4】 is selected from During the ceremony, R ｚａ is H or methyl; R ｚｂ is —OH, ═O, or ═NHOH; n and m are each independently an integer from 1 to 10; x is an integer selected from 1 to 2; 【Chemistry 32】 is a single bond or a double bond; a" represents the bond between Z' and the drug conjugate; b" represents the bond between Z' and TM; and Z” is below 【Transformation 33】 The targeted drug conjugate is selected from:

30. 28. The targeted drug conjugate of claim 27, wherein the TM is a nanoparticle, an immunoglobulin, a nucleic acid, a protein, an oligopeptide, a polypeptide, an antibody, a fragment of an antigenic polypeptide, or a lipebody.

31. 31. The targeted drug conjugate of claim 30, wherein the TM is an antibody selected from an intact polyclonal antibody, an intact monoclonal antibody, an antibody fragment, a single-chain Fv (scFv) variant, a multispecific antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, a fusion protein comprising an antigenic determinant of an antibody, and other modified immunoglobulin molecules comprising an antigen recognition site.

32. The antibody is selected from the group consisting of muromonab-CD3, abciximab, rituximab, daclizumab, palivizumab, infliximab, trastuzumab (Herceptin), etanercept, basiliximab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, alefacept, omalizumab, efalizumab, and tositumomab-I １３１ , cetuximab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, rilonacept, certolizumab 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 31. The targeted drug conjugate of claim 30, selected from EGFR, aflibercept, HuMax-CD4, Ala-Ala, ChAglyCD3, TRX4, catumaxomab, IGN101, MT-201, pregovomab, CH-14.18, WX-G250, AMG-162, AAB-001, motavizumab, MEDI-524, efumugumab, aurograb, raxibacumab, third generation anti-CD20, LY2469298, and veltuzumab.

33. 18. The drug conjugate of claim 17, wherein the drug conjugate comprises: 【Chemistry 34-1】 【Chemistry 34-2】 【Chemistry 34-3】 【Chemistry 34-4】 【Chemistry 34-5】 【Chemistry 34-6】 【Chemistry 34-7】 【Chemistry 34-8】 【Chemistry 34-9】 or a pharmaceutically acceptable salt thereof.

34. A pharmaceutical composition comprising the drug conjugate of claim 1.

35. Compounds represented by formula (VII) or (VIII): 【Chemistry 35】 Or a pharmaceutically acceptable salt thereof; During the ceremony: A is a heterocycle; Each R ａ ' and R ｂ ' is independently halogen, amino, hydroxyl, acetyl, hydroxyalkyl, alkoxy, cyano, nitro, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; Two Germinal R ｂ ' optionally taken together to form oxo or =CH ２ or two R ｂ ', together with the intervening atoms, optionally completes a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; Rc' is sulfonate, sulfate, hydroxyl, amino, or thiol; R ｄ ' is -L"-Gly, hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; However, at least one R ｃ ' is a sulfonate or sulfate, or at least one R ｄ ' is -L ” -Gly; R ｅ ' is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; m is an integer selected from 0 to 3; n is an integer selected from 0 to 8 depending on valence; Ring Cy is selected from aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; 【Transformation 36】 is a single bond or a double bond; X' is a halogen; X″ is —NR—, —S—, or —O—; Each R ａ " and R ｂ " are independently halogen, amino, hydroxyl, alkoxy, acetyl, hydroxyalkyl, cyano, nitro, alkyl, alkenyl, alkynyl, ═O, carboxyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or -(L''') ｒ -X″-Gly; d is an integer selected from 0 to 4; r is an integer selected from 0 to 1; each L''' is a bond or a linker; R ｅ " is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; p is an integer selected from 0 to 4; DBD is a DNA binding domain; L" is a bond or a linker; and Gly is a monosaccharide, disaccharide, or oligosaccharide.

36. Use of a compound, drug conjugate, targeted drug conjugate, or pharmaceutical composition according to any one of claims 1 to 35 in the manufacture of a medicament for the treatment of cancer in a subject in need thereof.

37. 37. The use of claim 36, wherein the cancer is selected from leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung cancer, bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, bladder cancer, kidney cancer, renal pelvis cancer, oral cancer, pharyngeal cancer, endometrial cancer, or melanoma.

38. Use of a compound, drug conjugate, targeted drug conjugate, or pharmaceutical composition according to any one of claims 1 to 35 in the manufacture of a medicament for the treatment of an autoimmune or inflammatory disease in a subject in need thereof.

39. 39. The use of claim 38, wherein the autoimmune or inflammatory disease is selected from a B-cell mediated autoimmune or inflammatory disease, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), idiopathic thrombocytopenic purpura (ITP), Waldenstrom's hypergammaglobulinemia, Sjogren's syndrome, multiple sclerosis (MS), or lupus nephritis.

Images