Fused heterocyclic benzodiazepine derivatives and uses thereof

Abstract

To provide linkers and cytotoxic agents useful for preparing antibody-drug conjugates.SOLUTION: Provided herein are compounds having the structure of Formula (I) in the figure or pharmaceutically acceptable salts thereof. In the formula: the ring A is a heterocyclic, aryl or heteroaryl ring; the ring B is a heterocyclic ring, preferably a five- or six-membered ring, optionally fused to or substituted with one or more aryl or heteroaryl rings; R1 and R2 are each independently an alkyl group; Z1 is methylene or a linking group; na is an integer equal to 1 or 2; and n1 and n2 are each independently an integer from 1 to 5.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 753,605, filed October 31, 2018, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Antibody-drug conjugates (ADCs) have emerged as a powerful class of antitumor agents with efficacy across a range of cancers. ADCs are commonly composed of three distinct features: a cell-binding or targeting moiety, a linker, and a cytotoxic agent. The linker component of an ADC is a key feature in developing targeted anticancer drugs with the desired target specificity, i.e., high activity in tumor cells but low activity in healthy cells. The combined use of a targeting moiety and a cytotoxic agent that may otherwise harm healthy tissues also alters the calculation of the desired characteristics of such a cytotoxic agent. Thus, there is a need for improved linkers and cytotoxic agents useful in the preparation of ADCs. Summary of the Invention

[0003] In certain embodiments, a compound having the structure of formula (IV): [ka] or a pharma- ceutically acceptable salt thereof is provided herein: During the ceremony, Ring A is a heterocyclic, aryl, or heteroaryl ring; R 1 is alkyl, preferably lower alkyl; W a is H or benzyl, n a is an integer having a value of 1 or 2.

[0004] In certain aspects, provided herein are compounds having the structure of formula (I): [ka] JPEG2025020130000003.jpg8170 or a pharma- ceutically acceptable salt thereof is provided; During the ceremony, Ring A is a heterocyclic, aryl, or heteroaryl ring; Ring B is a heterocyclic ring, preferably a 5- or 6-membered ring, optionally fused to or substituted with one or more aryl or heteroaryl rings; R 1 and R 2 are each independently alkyl, preferably lower alkyl; Z 1 is methylene or a linking group, which may be further conjugated to a cleavable linker and a targeting agent; n a is an integer having a value of 1 or 2, n 1 and n 2 are each independently 1, 2, 3, 4, or 5.

[0005] In certain aspects, provided herein is a conjugate comprising a compound of formula (I), wherein the linking group is a cleavable linker that cleavably links the compound to a targeting agent. In a preferred embodiment, the targeting agent is a cell binding agent.

[0006] In certain embodiments, a compound having the structure of formula (II), (IIa), or (IIb): [ka] or a pharma- ceutically acceptable salt thereof is provided herein: During the ceremony, Ring A is a heterocyclic, aryl, or heteroaryl ring; Ring B is a heterocyclic ring, preferably a 5- or 6-membered ring, optionally fused to or substituted with one or more aryl or heteroaryl rings; R 1 and R 2 are each independently alkyl, preferably lower alkyl; Z 1 is methylene or a linking group, which may be further conjugated to a targeting agent; n a is an integer having a value of 1 or 2, n 1 and n 2 are each independently 1, 2, 3, 4, or 5; Z 2 is absent or a linking group, L 1 is a linking group bonded to SO2 via a heteroatom selected from O, S, and N, preferably O or N; L 1 The bond between L and SO2 is broken. 1 and Z 1 to promote cleavage of the bond between X 1 -O-, -CR a 2-, or -NR'-, preferably -O-; Ar represents a ring such as aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, preferably aryl or heteroaryl; Y 1 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, where X 1 and Y 1 are located on adjacent atoms of Ar, TG is an inducing group that, when activated, reacts with SO2 to produce an N, O, or S atom capable of replacing Z and forming a 5-6 membered ring containing the intervening atoms X-SO2 and Ar; w and x are each independently an integer having a value of 0 or 1; Each R a and R c are independently hydrogen or lower alkyl; Each R b is independently hydrogen or lower alkyl; or The Two R's b form a 3- to 5-membered ring, preferably a 3- to 4-membered ring, together with the carbon atom to which they are attached.

[0007] In some embodiments, the present disclosure provides a compound having a structure of formula (IV): [ka] or a pharma- ceutically acceptable salt thereof, During the ceremony, Ring A is a heterocyclic, aryl, or heteroaryl ring; R 1 is alkyl, W a is H or benzyl, n a is either 1 or 2.

[0008] Additionally, a conjugate having the structure of formula (III): (DL) dl -LG-(CB) cb (III) or a pharma- ceutically acceptable salt thereof is provided herein: During the ceremony, LG is a linking group; CB is a cell binding agent; cb and dl are each independently an integer having a value of 1 to about 20, preferably 1 to about 10; Each DL is independently a group having the structure of a compound of formula (I) or (II).

[0009] The present disclosure also relates to a composition (e.g., a pharmaceutical composition) comprising a compound of Formula (I), (II), (IIa), or (IIb) or a conjugate of Formula (III) and a carrier (e.g., a pharma- ceutically acceptable carrier).

[0010] In certain aspects, the disclosure provides a method of delivering an active agent to a cell, comprising administering a conjugate of formula (III) or a pharmaceutical composition thereof, wherein the targeting moiety is selected to bind to a molecule associated with the target cell. In certain aspects, the present invention provides a conjugate of formula (III) and a pharmaceutical composition thereof for use in a method of delivering an active agent to a cell, wherein the targeting moiety is selected to bind to a molecule associated with the target cell. In particular, the compounds, conjugates, and compositions of the present invention may be useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (e.g., a human), such as when the target cell is a cancer cell and the targeting moiety is selected to bind to a molecule associated with the cancer cell (and not associated with healthy cells, or at least associated preferentially with tumor cells over healthy cells).

[0011] In some embodiments, the present disclosure relates to a method of treating or preventing a disease or disorder, comprising administering a compound of Formula (I) or Formula (II) or a conjugate of Formula (III), or a pharmaceutical composition comprising a compound of Formula (I) or Formula (II) or a conjugate of Formula (III), to a subject in need thereof.

[0012] The conjugates of formula (III) and pharmaceutical compositions thereof may be useful in treating conditions such as cancer, rheumatoid arthritis, multiple sclerosis, graft-versus-host disease (GVHD), transplant rejection, lupus, myositis, infectious diseases, immune disorders such as AIDS, and inflammatory diseases in mammals (e.g., humans). [Brief description of the drawings]

[0013] [Figure 1] 1 shows the in vitro cytotoxic activity of compounds D-101, D-102, D-103, D-105, and D-110 against NCI-N87. [Diagram 2] 1 shows the in vitro cytotoxic activity of compounds D-104, D-106, D-107, D-108, and D-109 against NCI-N87. [Diagram 3] 1 shows the in vitro cytotoxic activity of compounds D-111, D-112, D-113, and D-114 against NCI-N87. [Figure 4] 1 shows the in vitro cytotoxic activity of conjugate T-1-AB against NCI-N87. [Diagram 5] 1 shows the in vitro cytotoxic activity of conjugate T-2-AB against NCI-N87. [Figure 6] 1 shows the in vitro cytotoxic activity of conjugate T-3-AB against NCI-N87. [Figure 7] 1 shows the in vitro cytotoxic activity of conjugate T-4-AB against NCI-N87. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention relates to benzodiazepine (BD) derivatives, such as pyrrolobenzodiazepines (PBD), indolinobenzodiazepines (IBD), tetrahydroisoquinolinobenzodiazepines (TBD), and dimers thereof; compounds and conjugates thereof containing cleavable linkers; and uses thereof. Exemplary compounds and conjugates disclosed herein include an active agent (e.g., a compound of formula (I)) having a desired function or activity, a functional group that undergoes a chemical reaction (e.g., a physicochemical reaction and / or a biological reaction) under predetermined conditions to release a nucleophilic heteroatom, and a SO2 functional group positioned proximal to the nucleophilic heteroatom, thereby reacting with the nucleophilic heteroatom in an intramolecular cyclization reaction to release the active agent. In some embodiments, the compounds and conjugates disclosed herein further include a targeting moiety (e.g., an oligopeptide, a polypeptide, an antibody, etc.) that has binding specificity for a desired target receptor or other molecule associated with a target cell.

[0015] definition Unless otherwise specified herein, scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. In general, the nomenclature 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.

[0016] 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).

[0017] Unless otherwise specified herein, chemical terms used herein are used according to conventional usage in the art, such as in "The McGraw-Hill Dictionary of Chemical Terms", Parker S., Ed., McGraw-Hill, San Francisco, CA (1985).

[0018] 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.

[0019] The term "agent" is used herein to denote a chemical compound (e.g., an organic or inorganic compound, a mixture of compounds, etc.), a biological macromolecule (e.g., nucleic acids, antibodies including portions thereof, as well as humanized, chimeric and human antibodies and monoclonal antibodies, proteins or portions thereof, e.g., peptides, lipids, carbohydrates), or an extract made from biological material such as bacteria, plants, fungi, or animal (especially mammalian) cells or tissues. Agents include, for example, agents of known structure and agents of unknown structure.

[0020] "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).

[0021] "Treating" a condition or patient refers to taking measures to obtain a result, including beneficial or desired clinical results. As used herein and as is well understood in the art, "treatment" is an approach to obtain a result, including beneficial or desired 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 disease state (i.e., not worsening), prevention of disease spread, delay or slowing of disease progression, improvement or palliation of disease state, and remission (whether partial or complete). "Treatment" can also mean prolonging survival compared to expected survival if not receiving treatment.

[0022] The term "prevention" is art-recognized and well understood in the art when used in reference to a condition such as local recurrence (e.g., pain), a disease such as cancer, a complex syndrome such as heart failure, or any other medical condition, 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 not receiving the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving a prophylactic treatment relative to an untreated control population, and / or delaying the onset of detectable cancerous growths in a treated population relative to an untreated control population, for example, by a statistically and / or clinically significant amount.

[0023] "Administering" or "administration" of a substance, compound or agent to a subject can be performed using one of a variety of methods known to those skilled in the art. For example, the compound or agent can be administered intravenously, intraarterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ophthalmically, sublingually, orally (by ingestion), intranasally (by inhalation), intrathecally, intracerebrally, and transdermally (e.g., by absorption through the skin's channels). The compound or agent can also be suitably introduced by rechargeable or biodegradable polymeric or other devices, such as patches and pumps, or formulations that provide extended, slow or controlled release of the compound or agent. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0024] 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.

[0025] As used herein, the phrase "co-administration" refers to any form of administration of two or more different therapeutic agents, where a second agent is administered while a previously administered therapeutic agent is active in the body (e.g., the two agents are active simultaneously in a 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 receiving such treatment may benefit from the combined effect of the different therapeutic agents.

[0026] A "therapeutically effective amount" or "therapeutically effective dose" of a drug or agent is an amount that, when administered to a subject, produces the intended therapeutic effect of the drug or agent. The full therapeutic effect does not necessarily occur in one administration, but may occur only after a series of administrations. Thus, a therapeutically effective amount may be administered in one or more administrations. 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 particular situation by routine experimentation.

[0027] The term "alkenyl" as used herein refers to an aliphatic group containing at least one double bond, and is intended to include both "unsubstituted alkenyl" and "substituted alkenyl", the latter of which refers to an alkenyl moiety having a substituent replacing a hydrogen on one or more carbons of the alkenyl group. Such substitution may occur on one or more carbons that are included or not included in one or more double bonds. Moreover, such substitutions include all of those contemplated for alkyl groups as described below, except where stability precludes such use. For example, substitution of alkenyl groups with one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.

[0028] The term "alkylidenyl" as used herein refers to a = C(R*)(R**), where R* and R** are each independently hydrogen or alkyl. Examples include methylidenyl (=CH2), ethylidenyl (=CHCH3), 1-propylidenyl (=CHCH2CH3), 2-propylidenyl (=C(CH3)2), 1-butylidenyl (=CHCH2CH2CH3), 2-methyl-1-propylidenyl (=CHCH(CH3)2), 2-butylidenyl (=C(CH3)CH2CH3), 1-pentylideneyl (=CHCH2CH2CH2CH3), 2-pentylideneyl (=C(CH3) CH2CH2CH3), 3-pentylidenyl (=C(CH2CH3)2), 3-methyl-2-pentylidenyl (=C(CH3)CH(CH3)2), 3-methyl-1-butylidenyl (=CHCH2CH(CH3)2), 2-methyl-1-butylidenyl (=CHCH(CH3)CH2CH3), 1-hexylidenyl (=CHCH2CH2CH2CH2CH3), 2-hexylidenyl (=C(CH3)CH2CH2CH2CH3), 3-hexylidenyl (=C(CH3)CH2CH2CH2CH3), Alkylidenyl (=C(CH2CH3)(CH2CH2CH3)), 3-methyl-2-pentylidenyl (=C(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentylidenyl (=C(CH3)CH2CH(CH3)2), 2-methyl-3-pentylidenyl (=C(CH2CH3)CH(CH3)2), and 3,3-dimethyl-2-butylidenyl (=C(CH3)C(CH3)3). Alkylidenyl optionally can be one or more of alkyl, alkenyl, alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, imino, alkylamino, acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl, alkylsulfonyl, cyano, NR x R y , and / or COOR y where each R x and R yis independently H, alkyl, alkenyl, aryl, heteroaryl, heterocycle, cycloalkyl, or hydroxyl.

[0029] An "alkyl" group or "alkane" is a fully saturated straight-chain or branched non-aromatic hydrocarbon. Generally, a straight-chain or branched alkyl has from 1 to about 20 carbon atoms, preferably from 1 to about 10, unless otherwise specified. Examples of straight-chain and branched alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl, and octyl. C1-C6 straight-chain or branched alkyl groups are also referred to as "lower alkyl" groups.

[0030] Moreover, the term "alkyl" (or "lower alkyl"), as used throughout the specification, examples, and claims, is intended to include both "unsubstituted alkyl" and "substituted alkyl," the latter of which refers to an alkyl moiety having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents, unless otherwise specified, can include, for example, halogen (e.g., fluoro), 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. In a preferred embodiment, the substituents on the substituted alkyl are C 1-6 Alkyl, C 3-6In a more preferred embodiment, the substituents of the substituted alkyl are selected from fluoro, carbonyl, cyano, or hydroxyl. Those skilled in the art will appreciate that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For example, the substituents of the substituted alkyl can include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonates and phosphinates), sulfonyl (including sulfates, sulfonamides, sulfamoyl, and sulfonates), and silyl groups, as well as ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxylates, and esters), -CF3, and -CN. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, -CF3, -CN, and the like.

[0031] When used in conjunction with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, "C x-y " is meant to include groups containing x to y carbons in the chain. For example, "C x-y The term "alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight chain alkyl and branched alkyl groups, containing x to y carbons in the chain, including haloalkyl groups. Preferred haloalkyl groups include trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, and pentafluoroethyl. CO alkyl refers to hydrogen when the group is in a terminal position and to a bond when internal. 2-y alkenyl" and "C 2-y The term "alkynyl" refers to substituted or unsubstituted unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one double or triple bond respectively.

[0032] As used herein, the term "amide" refers to the group: [ka] where each R A independently represent hydrogen or a hydrocarbyl group, or two R A together with the N atom to which they are attached form a heterocycle having 4 to 8 atoms in the ring structure.

[0033] The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, as well as their salts, e.g., [ka] where each R A independently represent hydrogen or a hydrocarbyl group, or two R A However, together with the N atom to which they are attached, they complete a heterocycle having 4 to 8 atoms in the ring structure.

[0034] The term "aryl" as used herein includes substituted or unsubstituted monocyclic aromatic groups, where each atom of the ring is carbon.Preferably, the ring is a 6-membered or 10-membered ring, more preferably a 6-membered ring.The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, at least one of which is aromatic, and the other cyclic rings can be, for example, cycloalkyl, cycloalkenyl, cycloalkyl, aryl, heteroaryl, and / or heterocyclyl.Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, etc.

[0035] A "cycloalkyl" group is a fully saturated cyclic hydrocarbon. "Cycloalkyl" includes monocyclic and bicyclic rings. Typically, a monocyclic cycloalkyl group has 3 to about 10 carbon atoms, more typically 3 to 8 carbon atoms, unless otherwise specified. The second ring of a bicyclic cycloalkyl may be selected from a saturated ring, an unsaturated ring, and an aromatic ring. Cycloalkyl includes bicyclic molecules in which one, two, or three or more atoms are shared between the two rings. The term "fused cycloalkyl" refers to a bicyclic cycloalkyl in which each of the rings shares two adjacent atoms with the other ring. The second ring of a fused bicyclic cycloalkyl may be selected from a saturated ring, an unsaturated ring, and an aromatic ring. A "cycloalkenyl" group is a cyclic hydrocarbon containing one or more double bonds.

[0036] The terms "halo" and "halogen" as used herein mean halogen and include chloro, fluoro, bromo, and iodo.

[0037] The terms "heteroaryl" and "hetaryl" include substituted or unsubstituted aromatic monocyclic structures, preferably 5-7 membered rings, more preferably 5-6 membered rings, which ring structures contain 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, for example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkyl, aryl, heteroaryl, and / or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.

[0038] The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0039] The terms "heterocyclyl", "heterocycle", and "heterocyclic" refer to substituted or unsubstituted non-aromatic ring structures, preferably 3-10 membered rings, more preferably 3-7 membered rings, which ring structures contain at least one heteroatom, preferably 1-4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heterocyclyl" and "heterocyclic" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, at least one of which is heterocyclic, e.g., the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkyl, aryl, heteroaryl, and / or heterocyclyl. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, tetrahydropyran, tetrahydrofuran, morpholine, lactones, lactams, and the like.

[0040] The term "lower" when used in conjunction with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups in which there are 10 or fewer non-hydrogen atoms in the substituent, preferably 6 or fewer. "Lower alkyl" refers, for example, to alkyl groups containing 10 or fewer carbon atoms, preferably 6 or fewer carbon atoms. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, respectively, whether they appear alone or in combination with other substituents, for example, in the recitation of hydroxyalkyl and aralkyl (where, for example, atoms in an aryl group are not counted when counting the carbon atoms in an alkyl substituent).

[0041] The term "substituted" refers to a moiety having a substituent replacing a hydrogen on one or more carbons of the backbone. It is understood that "substituted" or "substituted with" includes the implicit proviso that such substitution is subject to the permissible valences of the substituted atom and the substituent, as well as the implicit proviso that the substitution results in a stable compound that does not spontaneously undergo transformation, for example, by rearrangement, cyclization, elimination, and the like. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valence of the heteroatom. The substituents may include any of the substituents described herein, such as, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkyl, aralkyl, or aromatic or heteroaromatic moieties. In a preferred embodiment, the substituted alkyl substituent is C 1-6 Alkyl, C 3-6In a more preferred embodiment, the substituent of the substituted alkyl is selected from fluoro, carbonyl, cyano, or hydroxyl. Those skilled in the art will appreciate that the substituents may themselves be substituted, if appropriate. Unless specifically stated as "unsubstituted", references herein to chemical moieties are understood to include substituted variants. For example, references to "aryl" groups or moieties implicitly include both substituted and unsubstituted variants.

[0042] "Protecting group" refers to a group of atoms that, when attached to a reactive functional group in a molecule, masks, reduces or prevents the reactivity of the functional group. Typically, a protecting group can be selectively removed as needed during the synthetic process. Examples of protecting groups can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons, NY, and Harrison et al., Compendium of Synthetic Organic Methods, Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Representative nitrogen protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("CBZ"), tert-butoxycarbonyl ("Boc"), trimethylsilyl ("TMS"), 2-trimethylsilyl-ethanesulfonyl ("TES"), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl ("FMOC"), and nitro-veratryloxycarbonyl ("NVOC"), etc. Representative hydroxyl protecting groups include, but are not limited to, those in which the hydroxyl group is acylated (esterified) or alkylated, such as benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPS groups), glycol ethers, e.g., ethylene glycol and propylene glycol derivatives, and allyl ethers.

[0043] As used herein, the term "modulate" includes the inhibition or suppression of a function or activity (eg, cell proliferation), as well as the enhancement of a function or activity.

[0044] The phrase "pharmacologically acceptable" is art-recognized. In certain embodiments, this term includes compositions, excipients, adjuvants, polymers and other materials and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0045] A "pharmacologically acceptable salt" or "salt" as used herein refers to an acid addition salt or a base addition salt suitable or compatible with the treatment of a patient.

[0046] The term "pharmaceutically acceptable acid addition salts" as used herein refers to any non-toxic organic or inorganic salt of any base compound represented by formula I. Exemplary 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. Exemplary 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. Salts of monobasic and dibasic acids 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 is known to one skilled in the art. Other pharmaceutically unacceptable salts, such as oxalates, may be used, for example, for isolation of compounds of formula I for laboratory use or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

[0047] As used herein, the term "pharmaceutically acceptable base addition salt" refers to any non-toxic organic or inorganic base addition salt of any acid compound represented by formula I or any of its intermediates. Exemplary inorganic bases which form suitable salts include lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or barium hydroxide. Exemplary organic bases which form suitable salts include aliphatic, alicyclic, or aromatic organic amines, such as methylamine, trimethylamine, and picoline or ammonia. The selection of suitable salts is within the skill of the art.

[0048] 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 the R or S configuration, and the R and S designations are used according to the conventions set forth in Pure Appl. Chem. (1976), 45, 11-30. The present disclosure contemplates all stereoisomers of the compounds, including enantiomeric and diastereomeric forms, salts, prodrugs, or mixtures thereof, including mixtures of all possible stereoisomers. See, for example, WO01 / 062726.

[0049] Additionally, certain compounds containing alkenyl groups may exist as Z (zusammen) and E (entgegen) isomers, and in each case the present disclosure includes both mixtures and the individual isomers separately.

[0050] Some of the compounds may 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.

[0051] "Prodrug" or "Pharmaceutically acceptable prodrug" refers to a compound that is metabolized in the host after administration, e.g., hydrolyzed or oxidized to yield a compound of the present disclosure (e.g., a compound of formula I). ​​Representative 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.

[0052] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharma- ceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, useful for formulating a drug for medicinal or therapeutic use.

[0053] The terms "abnormal cell growth" and "proliferative disorder" are used interchangeably in this application. As used herein, "abnormal cell growth" refers to cell growth independent of normal regulatory mechanisms (e.g., loss of contact inhibition), unless otherwise specified. This includes, for example, the abnormal growth of: (1) tumor cells (tumors) that grow due to expression of mutated tyrosine kinases or overexpression of receptor tyrosine kinases, (2) benign and malignant cells of other proliferative diseases where abnormal tyrosine kinase activation occurs, (3) any tumor that grows due to receptor tyrosine kinases, (4) any tumor that grows due to abnormal serine / threonine kinase activation, and (5) benign and malignant cells of other proliferative diseases where abnormal serine / threonine kinase activation occurs.

[0054] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" contains one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung cancer, including adenocarcinoma of the lung and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, acute leukemia, and head / brain and neck cancer.

[0055] Compounds and conjugates of the present invention Compounds of formula (I) The present disclosure provides a compound of formula (I): [ka] or a pharma- ceutically acceptable salt thereof, During the ceremony, Ring A is a heterocyclic, aryl, or heteroaryl ring; Ring B is a heterocyclic ring, preferably a 5- or 6-membered ring, optionally fused to or substituted with one or more aryl or heteroaryl rings; R 1 and R 2 are each independently an alkyl group; Z 1 is methylene or a linking group, which may be further conjugated to a targeting agent; n a is an integer having a value of 1 or 2, n 1 and n 2 are each independently an integer having a value of 1 to 5, In some embodiments, at least one of ring A or ring B is a heteroaryl ring.

[0056] For example, in some embodiments, ring A is [ka] where: A 1 , A 2 , and A 3 are each independently NR a1 , C.R. a2 , O, N, or S; R a1 is H, alkyl, halogen, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R a2 is H or alkyl, preferably lower alkyl.

[0057] In some embodiments, Ring B is a 5- or 6-membered heterocycle, optionally including one or more R selected from alkyl, alkylidenyl, aryl, or heteroaryl. C is substituted with a group.

[0058] In some such embodiments, ring B is: [ka] where: The dotted line indicates the presence of an optional double bond; Each R C is H, OH, alkyl, alkylidenyl, halogen, amino, cyano, or aryl.

[0059] In some such embodiments, ring B is: [ka] where: Each R 1a is H, OH, SH, alkylthiol, -CH2N(CH3)2, alkyl, alkyloxy, alkylidenyl, halogen, amino, cyano, or aryl.

[0060] In some embodiments, ring B is a 5- or 6-membered heterocycle and is fused to ring C' selected from an aryl or heteroaryl ring, preferably a 5- or 6-membered aryl or heteroaryl ring. In some such embodiments, rings B and C together have the following structure: [ka] forming a compound represented by the formula: Ring C' is optionally R 1c is replaced by up to 3 carbon atoms in ring C' may be replaced by nitrogen; Each R 1c is OH, SH, alkylthiol, -CH2N(CH3)2, alkyl, alkyloxy, alkyl, halogen, amino, or cyano; n b is an integer having a value of 1 or 2.

[0061] In some embodiments, ring A and ring C' are independently [ka] is selected from.

[0062] In certain preferred embodiments, ring A is [ka] In certain embodiments, R a1 is C1-C4-alkyl, preferably methyl.

[0063] In certain embodiments, ring C' is [ka] where: B 1 , B 2 , and B 3 are each independently NR a1 , C.R. a2 , O, N, or S; R b1 is H, alkyl, halogen, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R b2 is absent, H, or alkyl.

[0064] In some such embodiments, ring C' is [ka] In certain preferred embodiments, ring C' is selected from: [ka] In certain embodiments, R b1is C1-C4-alkyl, preferably methyl.

[0065] In some embodiments, ring A is [ka] where: A 6 , A 7 , A 8 , and A 9 are each independently a3 or N, R a3 is H, alkyl, halogen, nitro, hydroxy, nitrile, cyano, alkoxy, amino, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, However, A 6 , A 7 , A 8 , and A 9 The condition is that no more than two of are N.

[0066] In certain embodiments, ring C is [ka] where: B 6 , B 7 , B 8 , and B 9 are each independently b3 or N, R b3 is H, alkyl, halogen, nitro, hydroxy, nitrile, cyano, alkoxy, amino, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, However, B 6 , B 7 , B 8 , and B 9 The condition is that no more than two of are N.

[0067] In some embodiments, both ring A and ring B are each independently selected from the rings defined in the embodiments above.

[0068] In certain embodiments, one of ring A or ring C is [ka] and the other of ring A or ring C is selected from the heteroaryl rings defined in any one of the above embodiments.

[0069] In certain embodiments, the compound of formula (I) has the structure of formula (Ia), (Ib-1), (Ib-2), (Ib-3), (Ic-1), (Ic-2), or (Ic-3): [ka] JPEG2025020130000022.jpg188160, or a pharma- ceutically acceptable salt thereof, wherein the variables are defined as above.

[0070] In some such embodiments, R 1 and R 2 For example, in certain embodiments, R 1 and R 2 is C1-C4-alkyl, preferably methyl.

[0071] In some embodiments, Z 1 is methylene.

[0072] In another embodiment, Z 1 teeth, [ka] where: Y 1 CR Y1 or N but one Y 1 Only N is satisfied, R Y1 is H or hydroxyl, amino, amide, or (CH2) y (R Y1a ) yy and R Y1a is amino, aryl, or heteroaryl; y and yy each independently represent an integer having a value of 1 to about 10.

[0073] In a preferred embodiment, Z 1 teeth, [ka] where R Z1 is absent or is hydroxyl, amino, amide, or (CH2) z (R Z1a ) zz and R Z1a is amino, aryl, or heteroaryl, and z is zz, each independently an integer having a value from 1 to about 10. 1 teeth, [ka] It is.

[0074] In certain preferred embodiments, Z 1 provides an amine or phenol for conjugation. Thus, in certain such embodiments, for example, Z 1 may be selected from amino, hydroxy-substituted aryl, or nitrogen-containing heteroaryl, and may be optionally further substituted. In other such embodiments, R Y1a and R Z1a provides an amine or phenol for conjugation. For example, R Z1a may be selected from amino (eg, alkyl-substituted amino), hydroxy-substituted aryl, or nitrogen-containing heteroaryl, and may be optionally further substituted.

[0075] In certain preferred embodiments, Z 1 teeth, [ka] It is.

[0076] In certain embodiments, the compound of formula (I) is [ka] JPEG2025020130000028.jpg73170, or a pharma- ceutically acceptable salt thereof.

[0077] In certain other embodiments, the compound of formula (I) is [ka] JPEG2025020130000030.jpg232170JPEG2025020130000031.jpg90170, or a pharma- ceutically acceptable salt thereof.

[0078] In some embodiments, the present disclosure provides a compound having a structure of formula (IV): [ka] or a pharma- ceutically acceptable salt thereof, During the ceremony, Ring A is a heterocyclic, aryl, or heteroaryl ring; R 1 is alkyl, W a is H or benzyl, n a is either 1 or 2.

[0079] In certain embodiments, ring A is [ka] where: A 1 , A 2 , and A 3 are each independently NR a1 , C.R. a2 , O, N, or S; R a1is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R a2 is H or alkyl, preferably lower alkyl.

[0080] For example, ring A is [ka] may be selected from:

[0081] In some embodiments, ring A is [ka] It is.

[0082] In certain embodiments, R a1 is C1-C4-alkyl, such as methyl.

[0083] In certain embodiments, ring A is [ka] where: A 6 , A 7 , A 8 , and A 9 are each independently a3 or N, R a3 is H, alkyl, halogen, nitro, hydroxy, nitrile, cyano, alkoxy, amino, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, However, A 6 , A 7 , A 8 , and A 9 The condition is that no more than two of are N.

[0084] Conjugates of compounds of formula (I) In some embodiments, provided herein is a conjugate comprising a compound of formula (I), wherein the linking group is a cleavable linker that cleavably links the compound to a targeting agent. In a preferred embodiment, the targeting agent is a cell-binding agent. In some embodiments, the linker comprises a functional group that undergoes a chemical reaction (e.g., a physicochemical reaction and / or a biological reaction) under a predetermined condition to release a nucleophilic heteroatom, and a SO2 functional group located proximal to the nucleophilic heteroatom. In a preferred embodiment, the SO2 functional group reacts with the nucleophilic heteroatom in an intramolecular cyclization reaction to release the active agent. In some embodiments, the conjugate further comprises a targeting moiety (e.g., an oligopeptide, a polypeptide, an antibody, etc.) that has binding specificity for a desired target receptor or other molecule associated with a target cell.

[0085] Compounds of Formulae (II), (IIa), and (IIb) Also, compounds having the structures of formula (II) and (IIa) [ka] or a pharma- ceutically acceptable salt thereof is also provided herein: During the ceremony, Ring A is a heterocyclic, aryl, or heteroaryl ring; Ring B is a heterocyclic ring, preferably a 5- or 6-membered ring, optionally fused to or substituted with one or more aryl or heteroaryl rings; R 1 and R 2 are each independently an alkyl group; Z 1 is methylene or a linking group, n a is an integer having a value of 1 or 2, n 1 and n 2 are each independently an integer having a value of 1 to 5, Z 2 is absent or a linking group, L 1is a linking group bonded to SO2 via a heteroatom selected from O, S, and N, preferably O or N; L 1 The bond between L and SO2 is broken. 1 and Z 1 to promote cleavage of the bond between X 1 -O-, -CR a 2-, or -NR'-, preferably -O-; Ar represents a ring such as aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, preferably aryl or heteroaryl; Y 1 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, where X 1 and Y 1 are located on adjacent atoms of Ar, TG is an inducing group that, when activated, reacts with SO2 to produce an N, O, or S atom capable of replacing Z and forming a 5-6 membered ring containing the intervening atoms X-SO2 and Ar; w and x are each independently an integer having a value of 0 or 1; Each R a and R c are independently hydrogen or lower alkyl; Each R b are independently hydrogen or lower alkyl, or The Two R's b form a 3- to 5-membered ring, preferably a 3- to 4-membered ring, together with the carbon atom to which they are attached.

[0086] In some embodiments, X 1 is -O-.

[0087] In certain embodiments, Ar is aryl. In preferred embodiments, Ar is phenyl or naphthyl.

[0088] In some embodiments, Z 2 are isocyanides, isothiocyanides, 2-pyridyl disulfides, haloacetamides (-NHC(O)CH2-halo), maleimides, dienes, alkenes, halogens, tosylates (TsO - ), aldehydes, sulfonates (R-SO3 - ), [ka] Phosphonic acid (-P(=O)(OH)2), ketone, C8-C 10 Cycloalkynyl, -OH, -NHOH, -NHNH2, -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N3), amino (-NH2), sulfonic acid (-SO3H), alkynone derivatives (-C(O)C≡CR a ), and dihydrogen phosphate (-OP(=O)(OH)2).

[0089] In certain embodiments, x is 0.

[0090] Active agent release As mentioned above, in certain embodiments, the compounds and conjugates disclosed herein are capable of releasing one or more active agents represented by formula (II) through an intramolecular cyclization reaction following a chemical reaction that activates an inducing group. In certain embodiments, the chemical reaction is a physicochemical reaction and / or a biochemical reaction.

[0091] In some embodiments, the compounds and conjugates disclosed herein include a nucleophilic functional group (Y or Y') introduced at the atom adjacent to X (e.g., O) on Ar. Typically, the nucleophilic functional group is masked by a triggering group (TG), as described in further detail below. When activated, the triggering group releases the nucleophilic functional group that reacts with a nearby SO2 moiety in an intramolecular cyclization, ultimately releasing one or more compounds of formula (II), (IIa), or (IIb). In some such embodiments, one or more active agents are released through an intramolecular cyclization reaction after a chemical, physicochemical, and / or biochemical reaction (see, e.g., Reaction Scheme 1), or the active agent is released through 1,6-elimination or 1,4-elimination after an intramolecular cyclization reaction (see, e.g., Reaction Scheme 2).

[0092] For example, Y is -Y'-TG and Q is directly SO 2 When attached to a group, the active agent can be released by the mechanism shown in Reaction Scheme 1.

[0093] [ka] Q is [ka] In this case, Q 1 can be released by the mechanism shown in Reaction Scheme 2.

[0094] [ka] JPEG2025020130000042.jpg51170 In some embodiments, the Q 1 is an activator that includes at least one functional group selected from -OH, -NH-, -SH, and -COOH. According to these embodiments, Q is an activator that includes at least one functional group selected from -OH, -NH-, -SH, and -COOH, as further described herein. 1is conjugated to a compound as described herein through a functional group selected from, for example, ester, amide, thioester, carbamate, urea, oxime, hydrazone, and the like, through -OH, -NH-, -SH, and -COOH.

[0095] In some such embodiments, Q 2 Q 1 is used instead of Q 2 is an amine group-containing drug. 2 is an activator capable of binding to an ammonium unit. In still another embodiment, Q 2 Q 2 Upon release, it is capable of dissociating in its original form bearing the amine groups, where the active agent can be a drug, a toxin, an affinity ligand, a detection probe, or a combination thereof.

[0096] In some embodiments, the compounds and conjugates disclosed herein are chemically and physiologically stable, and in some such embodiments, the compounds and conjugates disclosed herein reach the desired target cells with little dissociation of the active agent in the blood, thereby selectively releasing the drug.

[0097] Inducing group (TG) In some embodiments, the conjugates of the present invention comprise a triggering group (TG). TG is a group that is cleavable, preferably selectively cleavable, by a chemical reaction, such as a biological reaction. In general, the triggering group serves to mask the nucleophilic nature of the Y or Y' group, thereby providing stability to the compounds and conjugates disclosed herein (e.g., by preventing self-immolation or intramolecular cyclization before the conjugate reaches the target location or experiences a predefined triggering condition). When activated, the triggering group releases the nucleophilic Y group, allowing self-immolation or intramolecular cyclization to occur, as described above.

[0098] In some embodiments, the TG comprises a sequence (e.g., a peptide sequence) or moiety recognized by TEV, trypsin, thrombin, cathepsin B, cathespin D, cathepsin K, caspase 1, matrix metalloproteinase (MMP), etc., which may be hydrolyzed by an enzyme (e.g., oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, etc.), and / or may comprise a moiety selected from phosphodiesters, phospholipids, esters, β-galactose, β-glucose, fucose, oligosaccharides, etc.

[0099] 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).

[0100] 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).

[0101] In some embodiments, the TG may contain a reactive chemical moiety or functional group that can be cleaved by irradiation with light (e.g., 2-nitrobenzyl derivatives, phenacyl esters, 8-quinolinylbenzenesulfonates, coumarins, phosphotriesters, bis-arylhydrazones, or bimanbi-thiopropionic acid derivatives).

[0102] In some embodiments, the TG may contain a reactive chemical moiety or functional group that can be cleaved by reducing agent conditions (eg, hydroxylamine, disulfide, levulinate, nitro, or 4-nitrobenzyl derivatives).

[0103] In some embodiments, the TG may contain reactive chemical moieties or functional groups that can be cleaved using acidic conditions (e.g., sugars, tert-butyl carbamate analogs, dialkyl or diaryl dialkoxysilanes, orthoesters, acetals, aconityls, hydrazones, β-thiopropionates, phosphoramidates, imines, trityl, vinyl ethers, polyketals, and alkyl 2-(diphenylphosphino)benzoate derivatives; alkyl esters, 8-hydroxyquinoline esters, and picolinic acid esters).

[0104] 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).

[0105] In certain preferred embodiments, the TG comprises a saccharide, which can be cleaved under acidic or enzymatic conditions. In certain preferred embodiments, the triggering group is -NO2, which can be cleaved under reducing conditions. In certain preferred embodiments, the triggering group is a boronate, which can be cleaved under oxidative conditions. In certain preferred embodiments, the triggering group is an ester, which can be cleaved under acidic, basic, or enzymatic conditions. In certain preferred embodiments, the triggering group is a hydrazone, which can be cleaved under nucleophilic or acidic conditions. In certain preferred embodiments, the triggering group is a hydroxylamine, which can be cleaved under reducing conditions.

[0106] Sugar-derived groups In some embodiments, the compounds and conjugates disclosed herein comprise a saccharide derivative group, for example a derivative group selected from: [ka] In the formula, each R 21 are independently hydrogen or OR 21 is selected to be a hydroxy protecting group (e.g., acetyl), and R 22is hydrogen or lower alkyl (e.g., C1-C6-alkyl). In certain embodiments, hydroxy protecting groups can be used in organic synthesis, including, but not limited to, methyl ether, methoxymethyl ether, methylthiomethyl ether, 2-methoxyethoxymethyl ether, bis(2-chloroethoxy)methyl ether, tetrahydropyranyl ether, tetrahydrothiopyranyl ether, 4-methoxytetrahydropyranyl ether, 4-methoxytetrahydrothiopyranyl ether, tetrahydrofuranyl ether, 1-ethoxyethyl ether, 1-methyl-1-methoxyethyl ether, 2-(phenylselenyl)ethyl ether, t-butyl ether, allyl ether, benzyl ether, o-nitrobenzyl ether, triphenylmethyl ether, α-naphthyldiphenylmethyl ether, p-methoxyphenyldiphenylmethyl ether, 9-(9-phenyl Examples of the silyl ethers include, but are not limited to, -10-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, pivaloate ester, adamantoate 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, nitrate ester, 2,4-dinitrophenylsulfenate ester, and the like.

[0107] Protective groups as triggering groups In some embodiments, TG is a group that can be cleaved by chemical reaction, physicochemical reaction, and / or biological reaction.In certain embodiments, TG is a protecting group.In some such embodiments, the protecting group is an amine protecting group, an alcohol protecting group, or a thiol protecting group.

[0108] Amine Protecting Groups In certain embodiments, the amine protecting group is a general protecting group that can be used in organic synthesis, including, but not limited to, m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, alkyl carbamates, 9-fluorenylmethyl carbamate, 2,2,2-trichloroethyl carbamate, 2-trimethylsilylethyl carbamate (Teoc), t-butyl carbamate (Boc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), 8-quinolyl carbamate, 1-isopropyl aryl carbamate (Ipaoc), ... amine, N-phenylmethylcarbamate, N-hydroxypiperidinylcarbamate, benzyl carbamate, p-methoxybenzyl carbamate, p-nitrobenzyl carbamate, diphenylmethylcarbamate, acetamide, chloroacetamide, trichloroacetamide, phenylacetamide, benzamide, N-phthalimide, N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1-dimethylthiomethyleneamine, N-benzylideneamine, benzenesulfenamide, o-nitrobenzenesulfenamide, triphenylmethylsulfenamide, p-toluenesulfonamide, methanesulfonamide, and the like.

[0109] Alcohol Protecting Groups In certain embodiments, the alcohol protecting group is a general protecting group that can be used in organic synthesis, including, but not limited to, methyl ether, methoxymethyl ether (MOM ether), benzyloxymethyl ether (BOM ether), 2-(trimethylsilyl)ethoxymethyl ether (SEM ether), phenylthiomethyl ether (PTM ether), 2,2-dichloro-1,1-difluoroethyl ether, p-bromophenacyl ether, chloropropyl methyl ether, isopropyl ether, cyclohexyl ether, 4-methoxybenzyl, 2,6-dichlorobenzyl ether, 4-(dimethylaminocarbonyl)benzyl ether, 9-anthrylmethyl ether, 4-picolyl ether, methylthiomethyl ether (MTM ether), 2-methoxyethoxymethyl ether (MEM ether), bis(2-chloroethoxy)methyl ether, tetrahydropyranyl ether (THP ether), tetrahydrothiopyranyl ether, 4-methoxytetrahydropyranyl ether, 4-methoxytetrahydrothio Pyranyl ether, tetrahydrofuranyl ether, 1-ethoxyethyl ether, 1-methyl-1-methoxyethyl ether, 2-(phenylselenyl)ethyl ether, t-butyl ether, allyl ether, benzyl ether, o-nitrobenzyl ether, triphenyl methyl ether, α-naphthyl diphenyl methyl ether, p-methoxyphenyl diphenyl methyl ether, 9-(9-phenyl-10-oxo)anthryl ether, trimethylsilyl ether (TMS ether), isopropyl dimethylsilyl ether silyl ether, t-butyldimethylsilyl ether (TBDMS ether), t-butyldiphenylsilyl ether, tribenzylsilyl ether, triisopropylsilyl ether, formate ester, acetate ester, trichloroacetate ester, phenoxyacetate ester, isobutyrate ester, pivaloate ester, adamantoate ester, benzoate ester, 2,4,6-trimethylbenzoic acid (mesitoate) ester, methyl carbonate, 2,2,Examples of the aryl esters include, but are not limited to, 2-trichloroethyl, allyl carbonate, p-nitrophenyl carbonate, benzyl carbonate, p-nitrobenzyl carbonate, S-benzyl thiocarbonate, N-phenyl carbamate, nitrate ester, 2,4-dinitrophenylsulfenic acid ester, dimethylphosphinyl ester (DMP ester), dimethylthiophosphinyl ester (MPT ester), aryl methanesulfonate, aryl toluenesulfonate, and the like.

[0110] Thiol Protecting Groups In certain embodiments, thiol protecting groups can be used in organic synthesis, including, but not limited to, S-benzyl thioether, Sp-methoxybenzyl thioether, So- or p-hydroxyl or acetoxybenzyl thioether, Sp-nitrobenzyl thioether, S-4-picolyl thioether, S-2-picolyl N-oxide thioether, S-9-anthrylmethyl thioether, S-9-fluorenylmethyl thioether, S-methoxymethyl monothioacetal, A-acetyl derivatives, S-benzoyl derivatives, S-(N-ethylcarbamate), S-(N-methoxymethylcarbamate), and the like.

[0111] linking group In some embodiments, the compounds and conjugates disclosed herein include a linking group that connects each CB and Ar by a covalent bond. Exemplary linking groups are stable, non-hydrolyzable moieties, such as C 10 -C 100 Linear or branched, saturated or unsaturated alkylene, etc. 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 is replaced with (i.e., replaced by) one or more heteroatoms selected from -NH-, -C(=O), -O-, -S-, and -P-; (ii) at least one heteroarylene is contained in an alkylene moiety; (iii) at least one amino acid moiety, glycobond, peptide bond, or amide bond is contained in the alkylene moiety; and (iv) Alkylene is C1-C 20 Alkyl, C6-C 20 Aryl C1-C8 alkyl, -(CH2) s COOH and -(CH2) p NH2, where s is an integer having a value from 0 to 10, and p is an integer having a value from 1 to about 10.

[0112] In certain embodiments, the linking unit comprises at least two, and more preferably at least three, of the following: (i) at least one heteroatom selected from -NH-, -C(=O), -O-, -S-, and -P-; (ii) at least one heteroarylene; (iii) at least one amino acid moiety, glycobond, peptide bond, or amide bond, and (iv) Alkylene is C1-C 20 Alkyl, C6-C 20 Aryl C1-C8 alkyl, -(CH2) s COOH and -(CH2) p NH2, where s is an integer having a value from 0 to 10, and p is an integer having a value from 1 to about 10.

[0113] In other embodiments, the linking group connecting each CB and Ar comprises a functional group produced through a click chemistry reaction.

[0114] In an alternative embodiment, the linking unit comprises a reactive functional group capable of participating in a click chemistry reaction.

[0115] Click chemistry is a reaction that can be carried out under mild conditions and is highly selective for functional groups not commonly found in biomolecules (e.g., azide groups, acetylene groups, etc.). Thus, the reaction can be carried out in the presence of conjugation triggering groups, targeting moieties, etc. Furthermore, click chemistry has high reaction specificity. For example, the click chemistry reaction between azide groups and acetylene groups proceeds selectively without interference from other functional groups present in the molecule. For example, azide-acetylene click chemistry can lead to triazole moieties in high yields.

[0116] Thus, in some embodiments, the linking group connecting each CB and Ar is [ka] and V is a single bond, -O-, -S-, -NR 21 -, -C(O)NR 22 -, -NR 23 C(O)-, -NR 24 SO2-, or -SO2NR 25 - Well, R 21 ~R 25 are each independently hydrogen, (C1-C6) alkyl, (C1-C6) alkyl(C6-C 20 )aryl, or (C1-C6)alkyl (C3-C 20 ) heteroaryl, r can be an integer having a value from 1 to about 10, p can be an integer having a value from 0 to about 10, q can be an integer having a value from 1 to about 10, and L″ can be a single bond.

[0117] In another embodiment, the linking unit connecting each CB and Ar is a linking group represented by formula (A): **-L c -W b1 -(CH2) b -W a3 -(P 1 ) a -Y 2 -W a2 -Y 1 -W a1 -* (A) During the ceremony, * is the point of attachment to CB ** is the point of attachment to Ar W a1 , W a2 , and W a3 are each independently -NH-, -C(=O)-, or (-CH2-) b and W b1 is an amide bond or a triazolylene; P 1 is W a3 and Y 2 a linker which is an amino acid moiety, a peptide bond, or an amide bond; L c is alkylene, Y 2 is a single bond, -W a4 -(CH2) c -W b2 -(CH2) d -W a5 -, or -W a6 -(CH2) e -CR e R f -X-, R e is C1-C8 alkyl or CB-W a7 -Y3-W c1 -(CH2) f - and R f is BW a7 -Y 3 -W c1 -(CH2) f - and X is -NHC(=O)-(CH2) g -W a8 - or -C(=O)NH-(CH2) h -W a9 - and W a4 , W a5 , W a6 , W a7 , W a8 , and W a9 are each independently -NH-, -C(=O)-, or -CH2-; W b2 is an amide bond or a triazolylene; W c1 is -NHC(=O)- or -C(=O)NH-, Y 3 is -(CH2) i -(X'CH2CH2) j -(CH2) k - and X' is -O-, -S-, -NH-, or -CH2-; CB is as defined above, b, c, d, e, f, g, h, i, and j are each independently an integer having a value from 1 to about 10; k and y are each independently an integer having a value from 0 to about 10; Y 1 is -(CH2) q -(CH2CH2X") o -or-(CH2) q -(X”CH2CH2) o - and X″ is —O—, —S—, —NH—, or —CH2—; o and q are integers having a value of 1 to about 10.

[0118] In some embodiments, P 1 comprises at least one unit represented by formula (B) or (C), [ka] During the ceremony, R 12 is hydrogen, C1-C8-alkyl, an amino acid side chain, e.g., a natural amino acid side chain (e.g., H, methyl, isopropyl, isobutyl, sec-butyl, S-methylthioether, benzyl, indole, pyrrolidine, pyrroline, hydroxymethyl, tyrosyl, lysyl, imidazole, glycyl, glutamyl, carbamoylbutanoic acid, carboxamide, aspartic acid, 1-hydroxyethyl, and 2-hydroxyethyl), -(CH2) s COR 13or -(CH2) p NR 14 R 15 and R 13 is OH or -NH(CH2) s' (X”CH2CH2) s” Z, R 14 and R 15 are each independently hydrogen or -(C(O)(CH2) s' (X”CH2CH2) s” Z) m -CB, X″ is —O—, —S—, —NH—, or —CH2—; Z and CB are as defined above; p is an integer having a value from 1 to about 10; s and s″ are integers having a value from 0 to about 10; s' is an integer having a value from 1 to about 10; m is an integer having a value of 0 or 1.

[0119] In some embodiments of formula (B) or (C), R 12 is hydrogen, alkyl, amino acid side chain, -(CH2) s C(O)R 13 , or -(CH2) p NR 14 R 15 and p is an integer having a value from 1 to about 10; s is an integer having a value from 0 to about 10; R 13 is OH or -NH(CH2) s' (X''CH2CH2) s” Z''-(CB) m and R 14 and R 15 are each independently hydrogen or -C(O)(CH2) s' (X''CH2CH2) s” Z''-(CB) m and s″ is an integer having a value from 0 to about 10; s' is an integer having a value from 1 to about 10; m is an integer having a value of 0 or 1; X''' is -O-, -S-, -NH-, or -CH2-; Z'' is CB to R 14 Or R 15 or Z'' is a linking group that includes a reactive group.

[0120] In some such embodiments of formula (B) or (C): R 13 is OH or -NH(CH2) s' (X''CH2CH2) s” Z'', R 14 and R 15 are each independently hydrogen or -C(O)(CH2) s' (X''CH2CH2) s” Z'', Z'' can be any of the following: isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH2-halo), maleimide, diene, alkene, halogen, tosylate (TsO - ), aldehydes, sulfonates (R-SO3 - ), [ka] Phosphonic acid (-P(=O)(OH)2), ketone, C8-C 10 Cycloalkynyl, -OH, -NHOH, -NHNH2, -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N3), amino (-NH2), sulfonic acid (-SO3H), alkynone derivatives (-C(O)C≡CR a , where R a is C1-C 10 -O-P(=O)(OH)2) are reactive precursors of linking units selected from dihydrogen phosphate (-O-P(=O)(OH)2), and dihydrogen phosphate (-O-P(=O)(OH)2).

[0121] In other such embodiments of formula (B) or (C): R 13 is OH or -NH(CH2) s' (X''CH2CH2) s” Z''CB, R 14 and R 15 are each independently hydrogen or -C(O)(CH2) s' (X''CH2CH2) s” Z''CB, Z'' can be any of the following: isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH2-halo), maleimide, diene, alkene, halogen, tosylate (TsO - ), aldehydes, sulfonates (R-SO3 - ), [ka] Phosphonic acid (-P(=O)(OH)2), ketone, C8-C 10 Cycloalkynyl, -OH, -NHOH, -NHNH2, -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N3), amino (-NH2), sulfonic acid (-SO3H), alkynone derivatives (-C(O)C≡CR a , where R a is C1-C 10 R is formed from a precursor selected from dihydrogen phosphate (-OP(=O)(OH)2), 14 or R 15 It is a linking unit that connects CB to the rest of.

[0122] In some embodiments, Y 2 is a single bond or is selected from: [ka] During the ceremony, W b2 is -C(O)NH-, -NHC(O)-, [ka] and R e is C1-C8-alkyl or -(L 1' -Z-) m CB, R f is BW b2' -(CH2) i -(X''CH2CH2) j -NH-C(=O)-(CH2) f - and X a is -NHC(=O)-(CH2) g -NH- or -C(O)NH-(CH2) h -NH-, W b2' is -C(O)NH- or -NHC(=O)-, c, d, e, f, g, h, i, and j are each independently an integer having a value from 1 to about 10; X″ is —O—, —S—, —NH—, or —CH2—; L 1' , Z, m, and B are as defined above.

[0123] In certain embodiments, the linking units connecting each CB and Ar are (CH2) connected to each other by a covalent bond. b , L c , (P 1 ) a , W a1 , W a2 , W a3 , Y 1 , and Y 2 is a linking group comprising a group, W a1 , W a2 , and W a3 are each independently -NH-, -C(O)-, or -CH2-; W b1 is an amide bond or a triazolylene; P 1 is an amide bond, an amino acid residue, or a peptide, L c is alkylene, Y 1is -(CH2) q -(CH2CH2X") o -or-(CH2) q -(X”CH2CH2X”) o - and X″ is —O—, —S—, —NH—, or —CH2—; Y 2 is a single bond or a group selected from the following: [ka] W b2 is an amide bond or a triazolylene; a is 0 to 10; b, c, and d are each independently an integer having a value from 1 to about 10; o and q are each independently an integer having a value of 1 to about 10.

[0124] In some embodiments, R 12 is a naturally occurring amino acid side chain. In another embodiment, R 12 is an unnatural amino acid side chain.

[0125] In some embodiments, the linking unit connecting each CB and Ar is a linking group represented by formula (A): **-L c -W b1 -(CH2) b -W a3 -(P 1 ) a -Y 2 -W a2 -Y 1 -W a1 -* (A) During the ceremony, * is the point of attachment to CB ** is the point of attachment to Ar.

[0126] In some such embodiments, P 1 is as follows: [ka] During the ceremony, R 12 is hydrogen, alkyl, amino acid side chain, -(CH2) s COOH, or -(CH2) p NH2, p is an integer having a value from 1 to about 10; s and s″ are each independently an integer having a value of 0 to about 10.

[0127] In some embodiments, P 1 is as follows: [ka] During the ceremony, R 12 is hydrogen, alkyl, amino acid side chain, -(CH2) s C(O)R 13 , or -(CH2) p NR 14 R 15 and p is an integer having a value from 1 to about 10; s is an integer having a value from 0 to about 10; R 13 is OH or -NH(CH2) s' (X''CH2CH2) s” Z''-(CB) m and R 14 and R 15 are each independently hydrogen or -C(O)(CH2) s' (X''CH2CH2) s” Z''-(CB) m and s″ is an integer having a value from 0 to about 10; s' is an integer having a value from 1 to about 10; m is an integer having a value of 0 or 1; X''' is -O-, -S-, -NH-, or -CH2-; Z'' is CB to R 14 Or R 15or Z'' is a linking group that includes a reactive group.

[0128] P 1 In some such embodiments, R 13 is OH or -NH(CH2) s' (X''CH2CH2) s” Z'', R 14 and R 15 are each independently hydrogen or -C(O)(CH2) s' (X''CH2CH2) s” Z'', Z'' can be any of the following: isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH2-halo), maleimide, diene, alkene, halogen, tosylate (TsO - ), aldehydes, sulfonates (R-SO3 - ), [ka] Phosphonic acid (-P(=O)(OH)2), ketone, C8-C 10 Cycloalkynyl, -OH, -NHOH, -NHNH2, -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N3), amino (-NH2), sulfonic acid (-SO3H), alkynone derivatives (-C(O)C≡CR a , where R a is C1-C 10 -O-P(=O)(OH)2) are reactive precursors of linking units selected from dihydrogen phosphate (-O-P(=O)(OH)2), and dihydrogen phosphate (-O-P(=O)(OH)2).

[0129] P 1 In other such embodiments, R 13 is OH or -NH(CH2) s' (X''CH2CH2) s” Z''CB, R 14 and R 15 are each independently hydrogen or -C(O)(CH2) s'(X''CH2CH2) s” Z''CB, Z'' can be any of the following: isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH2-halo), maleimide, diene, alkene, halogen, tosylate (TsO - ), aldehydes, sulfonates (R-SO3 - ), [ka] Phosphonic acid (-P(=O)(OH)2), ketone, C8-C 10 Cycloalkynyl, -OH, -NHOH, -NHNH2, -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N3), amino (-NH2), sulfonic acid (-SO3H), alkynone derivatives (-C(O)C≡CR a , where R a is C1-C 10 R is formed from a precursor selected from dihydrogen phosphate (-OP(=O)(OH)2), 14 or R 15 It is a linking unit that connects CB to the rest of.

[0130] In an alternative embodiment, the linking unit connecting CB and Ar is a linking group represented by formula (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIh), (IIIh), or (IIIj): [ka] JPEG2025020130000056.jpg172170In formula, R e is alkyl, X 5 is -O-, -S-, -NH-, or -CH2-, W b1 and W b2 each independently represents -C(O)NH-, -NHC(O)-, [ka] and R 12 is hydrogen, alkyl, amino acid side chain, -(CH2) s C(O)R 13 , or -(CH2) p NR 14 R 15 and R 13 is OH or -NH(CH2) s' (X 4 CH2CH2) s” Z''-(CB) m and R 14 and R 15 are each independently hydrogen or -C(O)(CH2) s' (X''CH2CH2) s” Z''-(CB) m and s and s″ are each independently an integer having a value from 0 to about 10; m is an integer having a value of 0 or 1; X 4 and X 5 are each independently -O-, -S-, -NH-, or -CH2-, and b, c, d, e, g, h, o, and q are each independently an integer having a value of 1 to about 10.

[0131] In some such embodiments of formula (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIh), (IIIh), or (IIIj), R 13 is OH or -NH(CH2) s' (X''CH2CH2) s” Z''CB, R 14 and R 15 are each independently hydrogen or -C(O)(CH2) s' (X''CH2CH2) s” Z''CB, Z'' can be any of the following: isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH2-halo), maleimide, diene, alkene, halogen, tosylate (TsO - ), aldehydes, sulfonates (R-SO3 - ), [ka] Phosphonic acid (-P(=O)(OH)2), ketone, C8-C 10 Cycloalkynyl, -OH, -NHOH, -NHNH2, -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N3), amino (-NH2), sulfonic acid (-SO3H), alkynone derivatives (-C(O)C≡CR a , where R a is C1-C 10 R is formed from a precursor selected from dihydrogen phosphate (-OP(=O)(OH)2), 14 or R 15 It is a linking unit that connects CB to the rest of.

[0132] targeting part The compounds and conjugates of the present invention may further comprise a ligand or targeting moiety, CB. In some embodiments, the ligand or targeting moiety is any molecular recognition element that can undergo specific interaction with at least one other molecule by non-covalent binding, such as hydrogen bonding, metal coordination, hydrophobic forces, van der Waals forces, π-π interactions, halogen bonds, electrostatic effects, and / or electromagnetic effects. In certain embodiments, the CB is selected from nanoparticles, immunoglobulins, nucleic acids, proteins, oligopeptides, polypeptides, antibodies, fragments of antigenic polypeptides, repebodies, and the like.

[0133] The compounds and conjugates of the invention may comprise one or more targeting moieties, i.e. the variable cb may have an integer value selected from 1, 2, 3, 4, 5, 1-10, or 1-20.

[0134] In some embodiments, the CB comprises two or more independently selected natural or unnatural amino acids conjugated by a covalent bond (e.g., a peptide bond), and the peptide may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more natural or unnatural amino acids conjugated by peptide bonds. In some embodiments, the ligands include shorter amino acid sequences (e.g., fragments of natural proteins or synthetic polypeptide fragments) as well as full-length proteins (e.g., pre-engineered proteins).

[0135] In some embodiments, the CB is selected from an antibody, hormone, drug, antibody analog (e.g., non-IgG), protein, oligopeptide, polypeptide, etc., that binds to a receptor. In certain embodiments, the CB selectively targets a drug in a specific organ, tissue, or cell. In other embodiments, the CB specifically binds to a receptor that is overexpressed in cancer cells compared to normal cells, and can be classified as a monoclonal antibody (mAb) or antibody fragment, and a small molecule non-antibody. Preferably, the CB is selected from a peptide, a tumor cell-specific peptide, a tumor cell-specific aptamer, a tumor cell-specific carbohydrate, a tumor cell-specific monoclonal antibody, a polyclonal antibody, and an antibody fragment identified in a library screen.

[0136] Exemplary ligands or targeting moieties include carnitine, inositol, lipoic acid, pyridoxal, ascorbic acid, niacin, pantothenic acid, folic acid, riboflavin, thiamine, biotin, vitamin B 12, other water-soluble vitamins (vitamin B), fat-soluble vitamins (vitamins A, D, E, K), RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), transferein, VIP (vasoactive intestinal peptide) receptor, APRPG (Ala-Pro-Arg-Pro-Gly) peptide, TRX-20 (thioredoxin-20), integrins, nucleolin, aminopeptidase N (CD13), endoglin, vascular epidermal 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 (glycoprotein nonmetastatic b), Ley, CA6, CanAng, SLC44A4 (solute carrier family 44 member 4), CEACAM5 (cancer embryonic stem cell lineage) Antigen-related cell adhesion molecule 5), nectin-4, carbonic anhydrase 9, TNNB2, 5T4, CD30, CD37, CD74, CD70, PMEL17, EphA2 (EphrinA2 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, EphrinA, CTLA4 (cytotoxic T-lymphocyte-associated protein 4), FGFR2b (fibroblast growth factor receptor 2b), N-acetylcholine receptor, gonadotropin-releasing hormone receptor, gastrin-releasing peptide receptor, bone morphogenetic protein receptor type 1B (BMPR1B), E16 (LAT1, SLC7A5), STEAP1 (six transmembrane epithelial antigen of prostate), 0772P (CA125, MUC16), MPF (MSLN, mesothelin), Napi3b (SLC34A2), Sema5b (semaphorin 5b), ETBR (endothelin type B receptor), MSG783 (RNF124), STEAP2 (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 (IN20Ra), brevican (BCAN), EphB2R, ASLG659 (B7h), CD276, PSCA (prostate stem cell antigen precursor), GEDA, BAFF-R (BR3), CD22 (BL-CAM), CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2 , SSTR2, SSTR5, SSTR1, SSTR3, SSTR4, ITGAV (integrin, alpha 5), ​​ITGB6 (integrin, beta 6), MET, MUC1, EGFRvIII, CD33, CD19, IL2RA (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, claudin, Her3 (ErbB3), RON (MST1R), CD20, TNC (tenascin-C), FAP, DKK-1, CD52, CS1 (SLAMF7), annexin A1, V-CAM, gp100, MART-1, MAGE-1 (melanoma antigen-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., but are not limited to these.

[0137] target In some embodiments, the target(s) of the molecular recognition element are specifically associated with one or more particular cell types or tissue types. In some embodiments, the target is specifically associated with one or more particular disease states. In some embodiments, the target is specifically associated with one or more particular developmental stages. For example, a cell type specific marker is typically expressed at a level at least 2-fold higher in the cell type than in a reference cell population. In some embodiments, the cell type specific marker is present at a level at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, at least 100-fold, or at least 1,000-fold higher than its average expression in a reference population. Detection or measurement of a cell type specific marker may allow for the identification of a cell type(s) of interest from many, most, or all other types of cells. In some embodiments, the target may include proteins, carbohydrates, lipids, and / or nucleic acids as described herein.

[0138] 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.

[0139] 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.).

[0140] In some embodiments, the conjugates and compounds described herein comprise a targeting moiety that binds to a target specific to one or more particular tissue types (e.g., liver tissue vs. prostate tissue). In some embodiments, the conjugates and compounds described herein comprise a targeting moiety that binds to a target specific to one or more particular cell types (e.g., T cells vs. B cells). In some embodiments, the conjugates and compounds described herein comprise a targeting moiety that binds to a target specific to one or more particular disease states (e.g., tumor cells vs. healthy cells). In some embodiments, the conjugates and compounds described herein comprise a targeting moiety that binds to a target specific to one or more particular developmental stages (e.g., stem cells vs. differentiated cells).

[0141] In some embodiments, a target may be a marker that is exclusively or primarily associated with one or a few cell types, with one or a few diseases, and / or with one or a few developmental stages. A cell type specific marker is typically expressed at a level at least 2-fold higher in that cell type than in a reference cell population, which may consist of, for example, a mixture containing approximately equal amounts of cells from multiple (e.g., 5-10 or more) different tissues or organs. In some embodiments, a cell type specific marker is present at a level at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold, at least 100-fold, or at least 1000-fold higher than its average expression in the reference population. Detection or measurement of a cell type specific marker may enable one to distinguish a cell type(s) of interest from many, most, or all other types of cells.

[0142] In some embodiments, the targets include proteins, carbohydrates, lipids, and / or nucleic acids. In some embodiments, the targets include proteins and / or characteristic portions thereof, such as tumor markers, integrins, cell surface receptors, transmembrane proteins, intracellular proteins, ion channels, membrane transporter proteins, enzymes, antibodies, chimeric proteins, glycoproteins, and the like. In some embodiments, the targets include carbohydrates and / or characteristic portions thereof, such as glycoproteins, sugars (e.g., monosaccharides, disaccharides, polysaccharides), glycocalyx (i.e., the carbohydrate-rich marginal zone on the outer surface of most eukaryotic cells), and the like. In some embodiments, the targets include lipids and / or characteristic portions thereof, such as oils, fatty acids, glycerides, hormones, steroids (e.g., cholesterol, bile acids), vitamins (e.g., vitamin E), phospholipids, sphingolipids, lipoproteins, and the like. In some embodiments, the targets include nucleic acids and / or characteristic portions thereof, such as DNA nucleic acids; RNA nucleic acids; modified DNA nucleic acids; modified RNA nucleic acids; nucleic acids including any combination of DNA, RNA, modified DNA, and modified RNA.

[0143] Many markers are known in the art. Typical markers include cell surface proteins, such as receptors. Exemplary receptors include, but are not limited to, transferrin receptor; LDL receptor; growth factor receptors, such as epidermal growth factor receptor family members (e.g., EGFR, Her2, Her3, Her4) or vascular endothelial growth factor receptors, cytokine receptors, cell adhesion molecules, integrins, selectins, and CD molecules. Markers can be molecules that are present exclusively or in higher amounts on malignant cells, such as tumor antigens.

[0144] Antibody Drug Conjugates (ADCs) In some embodiments, CB is an antibody and Q is a drug. Thus, the compounds and conjugates disclosed herein may be used to conjugate an antibody to a drug moiety to form an antibody-drug conjugate (ADC). Antibody-drug conjugates (ADCs) may increase therapeutic efficacy in treating disease, e.g., cancer, due to the ability of ADCs to selectively deliver one or more drug moieties(s) to target tissues, such as tumor-associated antigens. Thus, in certain embodiments, the present invention provides ADCs for therapeutic use, e.g., for the treatment of cancer.

[0145] The ADC of the present invention comprises an antibody linked to one or more drug moieties. The specificity of the ADC is determined by the specificity of the antibody. In one embodiment, the antibody is linked to one or more cytotoxic drug(s) that are delivered to cancer cells in the body.

[0146] Examples of drugs that can be used in the ADCs of the invention are provided below. The terms "drug," "agent," and "drug moiety" are used interchangeably herein. The terms "linked" and "conjugated" are also used interchangeably herein to indicate that the antibody and the moiety are covalently linked.

[0147] In certain aspects, the present disclosure is directed to ADCs, compositions comprising ADCs, methods of treatment, and methods of formulating ADC compositions. The ADCs comprise an antibody or antibody fragment conjugated 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 linked to the antibody. Types of antibodies, linkers, and cytotoxic compounds encompassed by the present disclosure are described below.

[0148] In some embodiments, the ADC has the following formula (Formula (III)): (DL) dl -LG-(CB) cb (III) or a pharma- ceutically acceptable salt thereof; During the ceremony, LG is a linking group; CB is a cell binding agent; cb and dl are each independently an integer having a value of 1 to about 20, preferably 1 to about 10; Each DL is independently a group having the structure of a compound of formula (I) or (II).

[0149] antibody The antibody of the ADC may be any antibody that typically, but not necessarily, specifically binds to an antigen expressed on the surface of a target cell of interest. The antigen is not required, but in some embodiments, allows for the ADC bound to it to be internalized. The target cell of interest may include a cell in which induction of apoptosis is desired. The target antigen may be any protein, glycoprotein, polysaccharide, lipoprotein, etc., expressed on the target cell of interest, but typically will be a protein that is either uniquely expressed on the target cell and not expressed on normal or healthy cells, or that is overexpressed on the target cell compared to normal or healthy cells, such that the ADC selectively targets the specific cell of interest, e.g., tumor cell. As will be appreciated by those skilled in the art, the specific antigen, and therefore the antibody, selected will depend on the identity of the desired target cell of interest. In a specific embodiment, the antibody of the ADC is an antibody suitable for administration to humans.

[0150] Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins with the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules that lack target specificity. Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end.

[0151] 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.

[0152] The term "antibody" herein is used in the broadest sense to refer to an immunoglobulin molecule that specifically binds to or is immunologically reactive with a particular antigen, including polyclonal, monoclonal, engineered and otherwise modified forms of antibodies, including, but not limited to, murine antibodies, chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies), and antigen-binding fragments of antibodies, including, for example, Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments. The term "scFv" refers to a single chain Fv antibody in which the variable domains of the heavy and light chains from a conventional antibody have been joined to form a single chain.

[0153] Antibodies may be murine, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system that are capable of recognizing and binding to specific antigens (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). Target antigens generally have multiple binding sites, also called epitopes, that are recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen may have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules that contain an antigen binding site or a portion thereof that immunospecifically binds to an antigen of a desired target, such targets including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species. However, in one aspect, the immunoglobulins are of human, murine, or rabbit origin.

[0154] The term "antibody fragment" refers to a portion of a full-length antibody, generally the target binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments. The "Fv" fragment is the smallest antibody fragment that contains a complete target recognition and binding site. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in non-covalent tight association (VH-VL dimer). It is in this arrangement that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. In many cases, six CDRs confer binding specificity to the antibody for a target. However, in some cases, even a single variable domain (or half of an Fv, containing only three target-specific CDRs) can have the ability to recognize and bind to a target. "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains 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 which exhibits sufficient affinity for the target. In a specific embodiment, a single-domain antibody is a camelized antibody (see, e.g., Riechmann, 1999, Journal of Immunological Methods 231:25-38).

[0155] Fab fragments contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. F(ab') fragments are generated by cleavage of the disulfide bond at the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those skilled in the art.

[0156] Both light and heavy chain variable domains have complementarity determining regions (CDRs), also known as hypervariable regions. The more highly conserved parts of the variable domains are called frameworks (FRs). As is known in the art, the amino acid positions / boundaries that define the hypervariable regions of an antibody may vary depending on the context and the various definitions known in the art. Some positions within the variable domain may be considered hybrid hypervariable positions, in that these positions may be considered to be within the hypervariable region under one set of criteria, while being considered to be outside the hypervariable region under a different set of criteria. One or more of these positions may also be found in an extended hypervariable region. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the target binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987)). As used herein, numbering of amino acid residues in immunoglobulins is done according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise specified.

[0157] In certain embodiments, the antibodies of the ADCs of the present disclosure are monoclonal antibodies. The term "monoclonal antibody" (mAb) refers to an antibody derived from a single copy or clone (including, for example, any eukaryotic, prokaryotic, or phage clone), and not the method by which it is produced. Preferably, the monoclonal antibodies of the present disclosure are present in a homogenous or substantially homogenous population. Monoclonal antibodies include both intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) that are capable of specifically binding to a protein. Fab and F(ab')2 fragments lack the Fc fragment of intact antibodies, are cleared more rapidly from the circulatory system of animals, and may have less nonspecific tissue binding than intact antibodies (Wahl et al., 1983, J. Nucl. Med 24:316). Monoclonal antibodies useful in the present disclosure can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. Antibodies of the present disclosure include chimeric, primatized, humanized, or human antibodies.

[0158] In most cases, antibodies are composed only of genetically encoded amino acids, although in some embodiments, non-encoded amino acids may be incorporated at specific positions. Examples of non-encoded amino acids that can be incorporated into antibodies for use in controlling stoichiometry and binding positions, as well as methods for making such modified antibodies, are discussed in Tian et al., 2014, Proc Nat'l Acad Sci USA 111(5):1766-1771 and Axup et al., 2012, Proc Nat'l Acad Sci USA 109(40):16101-16106, the entire contents of which are incorporated herein by reference.

[0159] In certain embodiments, the antibody of the ADC described herein is a chimeric antibody. The term "chimeric" antibody as used herein refers to an antibody having variable sequences derived from a non-human immunoglobulin, such as a rat or mouse antibody, and a human immunoglobulin constant region, typically selected from a human immunoglobulin template. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies et al., 1985, J.Immunol.Methods 125:191-202; U.S. Patent Nos. 5,807,715, 4,816,567, and 4,816,397, which are incorporated herein by reference in their entirety.

[0160] In certain embodiments, the antibodies of the ADCs described herein are humanized antibodies. "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other target-binding subdomains of antibodies) that contain minimal sequence derived from non-human immunoglobulin. In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. A humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc), typically of a human immunoglobulin consensus sequence. Methods for humanizing antibodies are known in the art. See, e.g., Riechmann et al., 1988, Nature 332:323-7; U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,761, and 5,693,762; and U.S. Pat. No. 6,180,370 to Queen et al., EP 239400, PCT Publication No. WO 91 / 09967, U.S. Pat. No. 5,225,539, EP 592106, EP 519596, Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. Natl. Acad Sci. USA 91:969-973, as well as U.S. Pat. No. 5,565,332, all of which are incorporated herein by reference in their entireties.

[0161] In certain embodiments, the antibody of the ADC described herein is a human antibody. Fully "human" antibodies may be desirable for therapeutic treatment of human patients. As used herein, "human antibody" includes antibodies having amino acid sequences of human immunoglobulins, and also includes antibodies isolated from human immunoglobulin libraries, or antibodies isolated from animals transgenic with one or more human immunoglobulins and that do not express endogenous immunoglobulins. Human antibodies can be produced by a variety of methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887, 4,716,111, 6,114,598, 6,207,418, 6,235,883, 7,227,002, 8,809,151, and U.S. Published Application No. 2013 / 189218, the contents of which are incorporated by reference in their entireties. Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. See, e.g., U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, 5,885,793, 5,916,771, 5,939,598, 7,723,270, 8,809,051, and U.S. Published Application No. 2013 / 117871, which are incorporated by reference in their entireties. In addition, companies such as Medarex (Princeton, NJ), Astellas Pharma (Deerfield, Ill.), and Regeneron (Tarrytown, NY) may be engaged to provide human antibodies directed against a selected antigen using techniques similar to those described above. Fully human antibodies that recognize a selected epitope can be generated using a technique referred to as "guided selection."In this approach, a selected non-human monoclonal antibody, such as a murine antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope (Jespers et al., 1988, Biotechnology 12:899-903).

[0162] In certain embodiments, the antibody of the ADC described herein is a primatized antibody. The term "primatized antibody" refers to an antibody that comprises a monkey variable region and a human constant region. Methods for producing primatized antibodies are known in the art. See, for example, U.S. Patent Nos. 5,658,570, 5,681,722, and 5,693,780, which are incorporated herein by reference in their entirety.

[0163] 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.

[0164] In certain embodiments, the antibody of the ADC described herein is a derivatized antibody. For example, but not limited to, a derivatized antibody is typically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to a cellular ligand or other protein, and the like. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Additionally, a derivative can contain one or more unnatural amino acids, for example, using ambrx technology (see, e.g., Wolfson, 2006, Chem. Biol. 13(10):1011-2).

[0165] In certain embodiments, the antibodies of the ADCs described herein have sequences that are modified to alter at least one biological effector function mediated by the constant region, as compared to the corresponding wild-type sequence. For example, in some embodiments, the antibodies can be modified to reduce at least one biological effector function mediated by the constant region, as compared to an unmodified antibody, for example, to reduce binding to Fc receptors (FcRs). Binding to FcRs can be reduced by mutating the immunoglobulin constant region segment of the antibody in specific regions required for interaction with FcRs (see, e.g., Canfield and Morrison, 1991, J. Exp. Med 173:1483-1491, and Lund et al., 1991, J. Immunol. 147:2657-2662).

[0166] In certain embodiments, the antibodies of the ADCs described herein are modified to acquire or improve at least one constant region-mediated biological effector function, e.g., to enhance interaction with FcγR, as compared to an unmodified antibody (see, e.g., US2006 / 0134709). For example, antibodies having constant regions that bind to FcγRIIA, FcγRIIB, and / or FcγRIIIA with higher affinity than the corresponding wild-type constant regions can be produced according to the methods described herein.

[0167] In certain specific embodiments, the antibodies of the ADCs described herein are antibodies that bind to tumor cells, such as antibodies against cell surface receptors or tumor-associated antigens (TAA). In an attempt to discover effective cellular targets for cancer diagnosis and therapy, researchers have attempted to identify transmembrane or otherwise tumor-associated polypeptides that are differentially expressed on the surface of one or more specific types of cancer cells compared to one or more normal non-cancerous cell(s). In many cases, such tumor-associated polypeptides are more abundantly expressed on the surface of cancer cells compared to the surface of non-cancerous cells. Such cell surface receptors and tumor-associated antigens are known in the art and can be prepared for use in generating antibodies using methods and information well known in the art.

[0168] Exemplary Cell Surface Receptors and TAAs Examples of cell surface receptors and TAAs that can be targeted by the antibodies of the ADCs described herein include, but are not limited to, the various receptors and TAAs listed below in Table 1. For convenience, information about these antigens, all of which are known in the art, is listed below, including their names according to the National Center for Biotechnology Information (NCBI) Nucleic Acid and Protein Sequence Identification Conventions, alternative names, Genbank accession numbers, and primary reference(s). Nucleic acid and protein sequences corresponding to the listed cell surface receptors and TAAs are available in public databases, such as GenBank.

[0169] [Table 1] JPEG2025020130000060.jpg252170JPEG2025020130000061.jpg250170JPEG2025020130000062.jpg194170

[0170] Exemplary Antibodies Exemplary antibodies of interest for use in the ADCs of the disclosure include 3F8 (GD2), Abagovomab (CA-125 (mimetic)), adecatumumab (EpCAM), afutuzumab (CD20), alacizumab pegol (VEGFR2), ALD518 (IL-6), alemtuzumab (CD52), altumomab pentetate (CEA), amatuximab (mesothelin), anatumomab mafenatox (Anatumomab mafenatox (TAG-72), apolizumab (HLA-DR), arcitumomab (CEA), bavituximab (phosphatidylserine), bectumomab (CD22), belimumab (BAFF), besilesomab (CEA-related antigen), bevacizumab (VEGF-A), bivatuzumab mertansine (CD44 v6), blinatumomab (CD19), brentuximab vedotin (CD30 (TNFRSF8)), cantuzumab mertansine (mucin CanAg), cantuzumab mertansine (cantuzumab ravtansine (MUC1), capromab pendetide (prostate cancer cells), carlumab (MCP-1), catumaxomab (EpCAM, CD3), CC49 (Tag-72), cBR96-DOX ADC (Lewis Y antigen), cetuximab (EGFR), sitatuzumab vogatoxin (bogatox (EpCAM), cixutumumab (IGF-1 receptor), clivatuzumab tetraxetan (MUC1), conatumumab (TRAIL-E2), dacetuzumab (CD40), darotuzumab (insulin-like growth factor 1 receptor), deratumumab (CD38 (cyclic ADP-ribose hydrolase)), demcizumab (DLL4), denosumab (RANKL), detumomab (B-cell lymphoma) Tumor cells), Drozitumab (DR5), Dusigitumab (ILGF2), Eclomeximab (D3 ganglioside), Eculizumab (C5), Edrecolomab (EpCAM), Elotuzumab (SLAMF7), Elsilimomab (IL-6), Enavatuzumab (TWEAK receptor), Enotikumab (DLL4), Ensituximab (5AC), Epitumomab-siTuxetan (Epitumomab cituxetan (episialin), epratuzumab (CD22), ertumaxomab (HER2 / neu, CD3), etancizumab (integrin αvβ3), farletuzumab (folate receptor 1), FBTA05 (CD20), ficlatuzumab (HGF), figitumumab (IGF-1 receptor), framvotumab (TYRP1 (glycoprotein 75)), fresolimumab (TGF-1), galiximab ( CD80), Ganitumab (IGF-I), Gemtuzumab ozogamicin (CD33), Girentuximab (Carbonic anhydrase 9 (CA-IX)), Glembatumumab vedotin (GPNMB), Ibritumomab tiuxetan (CD20), Icrucumab (VEGFR-1), Igovomab (CA-125), IMAB362 (CLDN18.2), Imgatuzumab (EGFR), Indatuximab ravtansineravtansine (SDC1), intetumumab (CD51), inotuzumab ozogamicin (CD22), ipilimumab (CD152), iratumumab (CD30 (TNFRSF8)), labetuzumab (CEA), lambrolizumab (PDCD1), lexatumumab (TRAIL-R2), lintuzumab (CD33), rolvo Tutumab Mertansine (CD56), Lucatumumab (CD40), Lumiliximab (CD23 (IgE receptor)), Mapatumumab (TRAIL-R1), Margetuximab (ch4DS), Matuzumab (EGFR), Milatuzumab (CD74), Mitumomab (GD3 ganglioside), Mogamulizumab (CCR4), Moxetumomab Passudotoxin (Moxetumomab pasudotox (CD22), Nacolomab tafenatox (C2-42 antigen), Naptumomab estafenatox (5T4), Narutuzumab (RON), Natalizumab (integrin α4), Necitumumab (EGFR), Nesvacumab (angiopoietin 2), Nimotuzumab (EGFR), Nivolumab (IgG4), Ocaratuzumab (CD20), Ofatumumab (CD20), Olaratumab (PDGF-Rα), Onartuzumab (human scatter factor receptor kinase), Ontuxizumab (TEM1), Oportuzumab monat monato (EpCAM), Olefovomab (CA-125), Otlertuzumab (CD37), Panitumumab (EGFR), Pancomab (tumor-specific glycosylation of MUC1), Parsatuzumab (EGFL7), Patritumab (HER3), Pemtumomab (MUC1), Pertuzumab (HER2 / neu), Pidilizumab (PD-1), Pinatuzumab vedotinvedotin (CD22), Pritumumab (Vimentin), Racotumomab (N-Glycolylneuraminic Acid), Radretumab (Fibronectin Extra Domain B), Ramucirumab (VEGFR2), Rilotumumab (HGF), Rituximab (CD20), Lobatumumab (IGF-1 Receptor), Samalizumab (CD200), Satumomab Pendetide (TAG-72), Seriban ERBB3, Sibrotuzumab (FAP), SGN-CD19A (CD19), SGN-CD33A (CD33), Siltuximab (IL-6), Solitomab (EpCAM), Sonepcizumab (Sphingosine-1-phosphate), Tabalumab (BAFF), Tacatuzumab tetraxetan (alpha-fetoprotein), Taplitumomab paptox paptox (CD19), Tenatumomab (tenascin-C), Teprotumumab (CD221), TGN1412 (CD28), Ticilimumab (CTLA-4), Tigatuzumab (TRAIL-R2), TNX-650 (IL-13), Tobetumab (CD40a), Trastuzumab (HER2 / neu), TRBS07 (GD2), Tremelimumab (CTLA-4), Tucotuzumab-cell moleukin (EpCAM), Ublituximab (MS4A), Urelumab (4-1BB), Vandetanib (VEGF), Vanticutumab (Frizzled receptor), Volociximab (integrin α5β1), Vorsetuzumab-mafodotin mafodotin (CD70), votumumab (tumor antigen CTAA16.88), zalutumumab (EGFR), zanolimumab (CD4), and zatuximab (HER1).

[0171] How to generate antibodies The antibodies of the ADCs can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. For example, to express an antibody recombinantly, a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody, thereby allowing the light and heavy chains to be expressed in the host cell and, optionally, secreted into the medium in which the host cell is cultured, from which the antibody can be recovered. Standard recombinant DNA techniques are used to obtain antibody heavy and light chain genes, incorporate these genes into a recombinant expression vector, and introduce the vector into the host cell. This is, for example, as described in Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, NY, 1989), Current Protocols in Molecular Biology (Ausubel, FM et al., eds., Greene Publishing Associates, 1989), and U.S. Patent No. 4,816,397.

[0172] In one embodiment, Fc variant antibodies are similar to their wild-type counterparts except for changes in their Fc domain. To generate nucleic acids encoding such Fc variant antibodies, a DNA fragment encoding the Fc domain or a portion of the Fc domain (referred to as the "wild-type Fc domain") of a wild-type antibody can be synthesized using conventional mutagenesis techniques and used as a template for mutagenesis to generate an antibody as described herein. Alternatively, a DNA fragment encoding the antibody can be directly synthesized.

[0173] Once the DNA fragments encoding the wild-type Fc domain are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the constant region genes into full-length antibody chain genes. In these manipulations, the DNA fragment encoding the CH is operatively linked to another DNA fragment encoding another protein, such as an antibody variable region or a flexible linker. The term "operatively linked" as used in this context is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0174] To express an Fc variant antibody, DNA encoding the partial or full-length light and heavy chains obtained as described above is inserted into an expression vector such that these genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector such that the transcriptional and translational control sequences in the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The variant antibody light chain gene (gne) and the antibody heavy chain gene can be inserted into separate vectors, or more typically, both genes are inserted into the same expression vector.

[0175] The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction enzyme sites on the antibody gene fragment and vector, or blunt end ligation if no restriction enzyme sites are present). Prior to insertion of the variant Fc domain sequence, the expression vector can already carry the variable region sequence of the antibody. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0176] In addition to the antibody chain genes, the recombinant expression vector carries regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif., 1990). It will be appreciated by those of ordinary skill in the art that the design of the expression vector, including the selection of regulatory sequences, can depend on such factors as the choice of the host cell to be transformed, the desired expression level of protein, etc. Suitable regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter / enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and polyoma. For further description of viral regulatory elements and sequences thereof, see, e.g., U.S. Patent No. 5,168,062 by Stinski, U.S. Patent No. 4,510,245 by Bell et al., and U.S. Patent No. 4,968,615 by Schaffner et al.

[0177] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vector can carry additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017, all to Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, puromycin, blasticidin, hygromycin, or methotrexate, on the host cell into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use with methotrexate selection / amplification in DHFR- host cells) and the neo gene (for G418 selection). For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains are transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, and the like.

[0178] Antibodies can be expressed in either prokaryotic or eukaryotic host cells. In certain embodiments, expression of the antibody is carried out in eukaryotic cells, e.g., mammalian host cells, to obtain optimal secretion of a properly folded, immunologically active antibody. Exemplary mammalian host cells for expressing recombinant antibodies include Chinese Hamster Ovary (CHO cells) (including DHFR-CHO cells described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp, 1982, Mol. Biol. 159:601-621), NS0 myeloma cells, COS cells, 293 cells, and SP2 / 0 cells. Once a recombinant expression vector encoding an antibody gene has been introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell or secretion of the antibody into the culture medium in which the host cell is grown. Antibodies can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules.

[0179] In some embodiments, the antibody of the ADC may be a bifunctional antibody. Such an antibody, in which one heavy and one light chain is specific for one antigen and the other heavy and light chains are specific for a second antigen, may be produced by crosslinking the antibody to a second antibody by standard chemical crosslinking methods. Bifunctional antibodies may also be made by expressing a nucleic acid engineered to encode the bifunctional antibody.

[0180] In certain embodiments, bispecific antibodies, i.e., antibodies that use the same binding site to bind one antigen and a second, unrelated antigen, can be produced by mutating amino acid residues in the light and / or heavy chain CDRs. Exemplary second antigens include proinflammatory cytokines, such as lymphotoxin, interferon-γ, or interleukin-1. Bispecific antibodies can be produced, for example, by mutating amino acid residues in the vicinity of the antigen binding site (see, e.g., Bostrom et al., 2009, Science 323:1610-1614). Bifunctional antibodies can be made by expressing a nucleic acid engineered to encode the bispecific antibody.

[0181] Antibodies can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.). Antibodies can also be produced using cell-free platforms (see, e.g., Chu et al., Biochemia No. 2, 2001 (Roche Molecular Biologicals)).

[0182] 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.

[0183] Once an antibody has been produced by recombinant expression, it may be purified by any method known in the art for the purification of immunoglobulin molecules, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography for the antigen, particularly after selection with Protein A or Protein G, affinity chromatography, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

[0184] 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).

[0185] Treatment method targeted therapy The targeting moiety of the conjugate may be recognised by cells, thereby providing a so-called targeted therapy.

[0186] In some embodiments, the present disclosure relates to a method of treating or preventing a disease or disorder, comprising administering a compound of Formula (I) or Formula (II) or a conjugate of Formula (III), or a pharmaceutical composition comprising a compound of Formula (I) or Formula (II) or a conjugate of Formula (III), to a subject in need thereof. In certain such embodiments, the disease or disorder is cancer, an infectious disease, an immune deficiency, an autoimmune disease, or a chronic inflammatory disorder.

[0187] In some embodiments, the present disclosure relates to a method further comprising co-administering a chemotherapeutic agent to the subject, hi certain such embodiments, the chemotherapeutic agent is administered sequentially to the subject with the conjugate as disclosed herein.

[0188] In some embodiments, the conjugates described herein include an active agent as described herein, for example, an active agent of formula (I). In some such embodiments, the active agent is selected from: [ka] JPEG2025020130000064.jpg255170, or a pharma- ceutically acceptable salt thereof.

[0189] In other embodiments, the active agent is selected from: [ka] JPEG2025020130000066.jpg226170JPEG2025020130000067.jpg48170, or a pharma- ceutically acceptable salt thereof.

[0190] Cell proliferation and apoptosis The compounds and conjugates disclosed herein may be used in methods for inducing apoptosis in a cell.

[0191] Dysregulation of apoptosis has been implicated in a variety of diseases, including, for example, autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis, graft-versus-host disease, myasthenia gravis, or Sjogren's syndrome), chronic inflammatory conditions (e.g., psoriasis, asthma, or Crohn's disease), hyperproliferative disorders (e.g., breast cancer, lung cancer), viral infections (e.g., herpes, papilloma, or HIV), and other conditions such as osteoarthritis and atherosclerosis. The compounds, conjugates, and compositions described herein may be used to treat or ameliorate any of these diseases. Such treatment generally involves administering to a subject suffering from the disease, a sufficient amount of the compounds, conjugates, or compositions described herein to provide a therapeutic benefit. The identity of the antibody of the administered compound, conjugate, or composition will depend on the disease being treated, and thus the antibody should bind to a cell surface antigen expressed in a cell type whose inhibition would be beneficial. The therapeutic benefit achieved will also depend on the particular disease being treated. In certain cases, the compounds and compositions disclosed herein may treat or improve the disease itself or symptoms of the disease when administered as a monotherapy. In other cases, the compounds and compositions disclosed herein may be part of an overall treatment regimen that includes other agents that, together with the inhibitors or compounds and compositions disclosed herein, treat or improve the disease or symptoms of the disease being treated. Agents useful for treating or improving a particular disease that can be administered to supplement or in conjunction with the compounds and compositions disclosed herein will be apparent to those skilled in the art.

[0192] Although an absolute cure is always desirable in any therapeutic regimen, achieving a cure is not required to provide a therapeutic benefit. A therapeutic benefit may include halting or slowing the progression of a disease, regressing a disease without a cure, and / or improving or slowing the progression of disease symptoms. Prolonged survival and / or improved quality of life compared to the statistical mean may also be considered a therapeutic benefit.

[0193] One particular class of disease that involves dysregulation of apoptosis and is a significant health burden worldwide is cancer. In a specific embodiment, the compounds and compositions disclosed herein can be used to treat cancer. The cancer can be, for example, a solid tumor or a hematological tumor. Cancers that can be treated with the compounds and compositions disclosed herein include, but are not limited to, bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular carcinoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myeloid leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, myeloma, prostate cancer, small cell lung cancer, and splenic cancer. The compounds and compositions disclosed herein may be particularly beneficial in the treatment of cancer, as they can be used to specifically target tumor cells with antibodies, thereby potentially avoiding or ameliorating the undesirable side effects and / or toxicity that may be associated with systemic administration of unconjugated inhibitors.One embodiment relates to a method for treating a disease associated with dysregulation of intrinsic apoptosis, comprising administering to a subject with a disease associated with dysregulation of apoptosis an amount of the compounds and compositions disclosed herein effective to provide a therapeutic benefit, wherein the ligand of the compounds and compositions disclosed herein binds to a cell surface receptor on cells in which intrinsic apoptosis is dysregulated.One embodiment relates to a method for treating cancer, comprising administering to a subject with cancer an amount of the compounds and compositions disclosed herein effective to provide a therapeutic benefit, wherein the ligand is capable of binding to a cell surface receptor or tumor-associated antigen expressed on the surface of cancer cells.

[0194] In the context of tumorigenic cancers, therapeutic benefit, in addition to including the effects discussed above, may also specifically include halting or slowing the progression of tumor growth, regressing tumor growth, eradicating one or more tumors, and / or increasing patient survival compared to the statistical mean for the type and stage of cancer being treated. In one embodiment, the cancer being treated is a tumorigenic cancer.

[0195] The compounds and conjugates disclosed herein may be administered as monotherapy to provide therapeutic benefit or may be administered to supplement or in conjunction with other chemotherapeutic agents and / or radiotherapy. The chemotherapeutic agents for which the compounds and compositions disclosed herein may be utilized as supplemental therapy may be targeted (e.g., ADCs, protein kinase inhibitors, etc.) or non-targeted (e.g., non-specific cytotoxic agents such as radionucleotides, alkylating agents, and intercalating agents). Non-targeted chemotherapeutic agents with which the compounds and compositions disclosed herein may be administered adjunctively include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbidine, topotecan, nitrogen mustard, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asperaginase, vinblastine, vincristine, vinorelbine, paclitaxel, calicheamicin, and docetaxel.

[0196] The compounds and conjugates disclosed herein, which may not be effective as monotherapy in treating cancer, may be administered to supplement or together with other chemotherapeutic agents or radiotherapy to provide therapeutic benefit. One embodiment relates to a method of administering the compounds or compositions disclosed herein in an amount effective to sensitize tumor cells to standard chemotherapy and / or radiotherapy. Thus, in the context of cancer treatment, "therapeutic benefit" includes administering the compounds and compositions disclosed herein to supplement or together with chemotherapeutic agents and / or radiotherapy as a means of sensitizing tumors to chemotherapy and / or radiotherapy, either in patients who have not yet started chemotherapy and / or radiotherapy or have not yet shown signs of resistance, or in patients who have begun to show signs of resistance.

[0197] Pharmaceutical Compositions and Administration Thereof The compounds and conjugates disclosed herein can be used to treat individuals in need thereof. In certain embodiments, the individual is a mammal, such as a human or a non-human mammal. When administered to an animal, such as a human, the composition or compound is preferably administered as a pharmaceutical composition, for example comprising the disclosed compound and a pharmaceutically acceptable carrier.

[0198] Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiological buffered saline, or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In a preferred embodiment, when such pharmaceutical compositions are for administration to humans, particularly for invasive routes of administration (i.e., routes such as injection or implantation that bypass transport or diffusion through epithelial barriers), the aqueous solutions are pyrogen-free or substantially pyrogen-free. Excipients can be selected, for example, to provide delayed release of the drug or to selectively target one or more cells, tissues, or organs. The pharmaceutical compositions can be in unit dosage forms such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophiles for reconstitution, powders, liquids, syrups, suppositories, injections, and the like. The compositions can also be present in transdermal delivery systems, for example, skin patches. The compositions can also be present in liquids suitable for topical administration, such as ointments or creams.

[0199] A pharma- ceutically acceptable carrier may contain a physiologically acceptable agent that acts, for example, to stabilize, increase the solubility, or increase the absorption of a compound, such as the compound of the present invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose, or dextran, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers or excipients. The choice of a pharma- ceutical acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The pharmaceutical composition preparation may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) may also be, for example, a liposome or other polymer matrix that may have the compound of the present invention incorporated therein. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, metabolizable carriers that are relatively simple to prepare and administer.

[0200] The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of prudent medical judgment, commensurate with a reasonable benefit / risk ratio.

[0201] The phrase "pharmacologically acceptable carrier" as used herein means a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can serve as pharma-ceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository wax; (9) peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and the like. (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar, (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid, (16) pyrogen-free water, (17) isotonic saline, (18) Ringer's solution, (19) ethyl alcohol, (20) phosphate buffers, and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0202] Pharmaceutical compositions (preparations) can be administered to a subject by any of several routes of administration, including, for example, orally (e.g., drenches, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue, such as in aqueous or non-aqueous solutions or suspensions); absorption through the oral mucosa (e.g., sublingually); anally, rectally, or intravaginally (e.g., as a pessary, cream, or foam); parenterally (e.g., as a sterile solution or suspension, including intramuscularly, intravenously, subcutaneously, or intrathecally); intranasally; intraperitoneally; subcutaneously; transdermally (e.g., as a patch applied to the skin); and topically (e.g., as a cream, ointment, or spray applied to the skin, or as eye drops). The compounds may also be formulated for inhalation. In certain embodiments, the compounds may simply be dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable for such may be found, for example, in U.S. Pat. Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896, and patents cited therein.

[0203] The formulations may be conveniently presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of compound that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of the active ingredient, preferably from about 5 percent to about 70 percent, and most preferably from about 10 percent to about 30 percent.

[0204] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the present invention, with the carriers and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0205] Formulations of the present invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (with a flavored base, usually sucrose and acacia or tragacanth), lyophilized powders, powders, granules, or as a solution or suspension in an aqueous liquid or a non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a troche (with an inert base such as gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, each containing a predetermined amount of a compound of the present invention as an active ingredient. The compound, conjugate, or composition may also be administered as a bolus, electuary, or paste.

[0206] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharma- ceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders, such as paraffin. (6) absorption enhancers such as quaternary ammonium compounds; (7) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents such as modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets, and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be employed as fillers in soft-filled and hard-filled gelatin capsules using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

[0207] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactants, or dispersants. Molded tablets can be made by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine.

[0208] Tablets and other solid dosage forms of the pharmaceutical composition, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills, and granules, may be optionally scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may also be formulated to provide slow or controlled release of the active ingredient therein, for example, with hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres in various ratios to provide the desired release profile. They may be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of a sterile solid composition that can be dissolved in sterile water or any other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents, and may belong to compositions that release the active ingredient(s) only, or preferentially, in a certain part of the digestive tract, optionally in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0209] The liquid dosage form useful for oral administration includes pharmaceutically acceptable emulsion, lyophilized agent for reconstitution, microemulsion, solution, suspension, syrup and elixir.In addition to active ingredient, liquid dosage form may contain inert diluent commonly used in the art, such as water or other solvent, solubilizer and emulsifier, such as cyclodextrin and its derivatives, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid ester of sorbitan, and mixtures thereof.

[0210] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents.

[0211] Suspensions may contain, in addition to the active compound, suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, agar-agar, and tragacanth, and mixtures thereof.

[0212] Formulations of the pharmaceutical compositions for rectal, intravaginal, or urethral administration may be presented as suppositories, which can be prepared by mixing one or more active compounds with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, a suppository wax, or a salicylate, which are solid at room temperature, but liquid at body temperature, and therefore will melt in the rectum or vaginal cavity and release the active compounds.

[0213] Formulations of the pharmaceutical composition for administration to the buccal cavity may be presented as a mouthwash, or as an oral spray, or as an oral ointment.

[0214] Alternatively or additionally, the compositions can be formulated for delivery via a catheter, stent, wire, or other intraluminal device, which may be particularly useful for delivery to the bladder, urethra, ureter, rectum, or intestine.

[0215] Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing such carriers as are known in the art to be appropriate.

[0216] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharma- ceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

[0217] The ointments, pastes, creams, and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0218] Powders and sprays may contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0219] Transdermal patches have the added advantage of providing controlled delivery of the compounds of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the active compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate of such flux can be controlled by providing a rate-controlling membrane or dispersing the compound in a polymer matrix or gel.

[0220] Ophthalmic formulations, eye ointments, powders, solutions, and the like are also contemplated within the scope of the present invention. Exemplary ophthalmic formulations are described in U.S. Publication Nos. 2005 / 0080056, 2005 / 0059744, 2005 / 0031697, and 2005 / 004074, and U.S. Patent No. 6,583,124, the contents of which are incorporated herein by reference. If desired, the liquid ophthalmic formulation has properties similar to those of tears, aqueous humor, or vitreous humor, or is compatible with such fluids.

[0221] The phrases "parenteral administration" and "administered parenterally" as used herein mean modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0222] Pharmaceutical compositions suitable for parenteral administration contain one or more active compounds in combination with one or more pharma- ceutically acceptable isotonic aqueous or non-aqueous sterile solutions, dispersions, suspensions, or emulsions, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use.

[0223] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0224] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the incorporation 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, such as sugars, sodium chloride, and the like, in the compositions. In addition, prolonged absorption of the injectable dosage form may be brought about by the incorporation of agents that delay absorption, such as aluminum monostearate and gelatin.

[0225] In some cases, it is desirable to slow down the absorption of drugs from subcutaneous or intramuscular injections in order to prolong the effect of drugs.This can be achieved by using liquid suspensions of crystalline or amorphous materials with low water solubility.The absorption rate of drugs then depends on their dissolution rate, which in turn depends on crystal size and crystalline form.Alternatively, delayed absorption of parenterally administered drugs can be achieved by dissolving or suspending the drug in an oil vehicle.

[0226] Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0227] When used in the methods of the present invention, the active compound may be administered by itself or in combination with a pharma- ceutically acceptable carrier, for example, as a pharmaceutical composition containing 0.1 to about 99.5% (more preferably about 0.5 to about 90.0%) of the active ingredient.

[0228] In some embodiments of the present invention, the compounds of the present invention are co-administered with one or more additional compounds / agents.

[0229] In certain such embodiments, the co-administration is simultaneous. In certain such embodiments, the compound of the present invention is combined with one or more additional compounds. In certain other such embodiments, the compound of the present invention is administered separately but simultaneously with one or more additional compounds. In certain such embodiments, the co-administration is sequential, with the compound of the present invention being administered minutes or hours before or after the administration of one or more additional compounds.

[0230] The method of introducing the compound of the present invention can also be enabled by rechargeable or biodegradable devices.In order to obtain controlled delivery of drugs, including proteinaceous biological agents, various slow release polymeric devices have been developed and tested in vivo in recent years.Various biocompatible polymers, including hydrogels, including both biodegradable and non-degradable polymers, can be used to form implants to obtain sustained release of compounds at specific target sites.

[0231] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0232] The selected dosage level will depend on a variety of factors, including the activity of the particular compound, conjugate, or combination of compounds and / or conjugates, or esters, salts, or amides thereof, used, the route of administration, the time of administration, the excretion rate of the particular compound(s) being used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular compound(s) being used, the age, sex, weight, medical condition, general health, and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0233] A physician or veterinarian of ordinary skill in the art can easily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can begin dosing the pharmaceutical composition or compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. A "therapeutically effective amount" refers to the concentration of the compound sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound varies depending on the subject's weight, sex, age, and medical history. Other factors that affect the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered together with the compound of the present invention. A higher total dose may be delivered by multiple administrations of the agent. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, incorporated herein by reference).

[0234] In general, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors mentioned above.

[0235] If desired, the effective daily dose of the active compound or conjugate can be administered as one, two, three, four, five, six or more sub-doses, which are administered separately at appropriate intervals throughout the day, optionally in unit dosage form.In certain embodiments of the present invention, the active compound can be administered two or three times a day.In preferred embodiments, the active compound will be administered once a day.

[0236] The patient undergoing this treatment may be any animal in need, including primates, particularly humans, as well as other mammals such as horses, cows, pigs, and sheep, as well as poultry and pets in general.

[0237] In certain embodiments, the compounds or conjugates disclosed herein may be used alone or co-administered with another type of therapeutic agent. As used herein, the phrase "co-administration" refers to any form of administration of two or more different therapeutic compounds or conjugates, such that the second compound or conjugate is administered while the previously administered therapeutic compound or conjugate is still effective in the body (e.g., the two compounds or conjugates are effective in the patient at the same time, which may include a synergistic effect of the two compounds or conjugates). For example, the different therapeutic compounds or conjugates may be administered either simultaneously or sequentially, in the same formulation or in separate formulations. In certain embodiments, the different therapeutic compounds or conjugates may be administered within 1 hour, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, 1 week, or longer of each other. Thus, an individual undergoing such treatment may benefit from the combined effect of the different therapeutic compounds or conjugates.

[0238] The present invention includes the use of pharma- ceutically acceptable salts of the compounds or conjugates disclosed herein.In certain embodiments, the contemplated salts of the present invention include, but are not limited to, alkyl salts, dialkyl salts, trialkyl salts, or tetra-alkylammonium salts.In certain embodiments, the contemplated salts of the present invention include, but are not limited to, L-arginine salts, benenthamine salts, benzathine salts, betaine salts, calcium hydroxide salts, choline salts, deanol salts, diethanolamine salts, diethylamine salts, 2-(diethylamino)ethanol salts, ethanolamine salts, ethylenediamine salts, N-methylglucamine salts, hydrabamine salts, 1H-imidazole salts, lithium salts, L-lysine salts, magnesium salts, 4-(2-hydroxyethyl)morpholine salts, piperazine salts, potassium salts, 1-(2-hydroxyethyl)pyrrolidine salts, sodium salts, triethanolamine salts, tromethamine salts, and zinc salts. In certain embodiments, contemplated salts of the present invention include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts.

[0239] Pharmaceutically acceptable acid addition salts may also exist as various solvates, for example with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates may also be prepared. The source of such solvates may be inherent in the preparation or crystallization solvent or from the crystallization solvent adventitious to such solvent.

[0240] Wetting agents, emulsifying agents, and lubricating agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0241] Examples of pharma- ceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium disulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0242] Having now generally described the invention, it will be more readily understood with reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to be limiting of the invention. EXAMPLES

[0243] Synthesis protocol Abbreviation AcO: Acetyl AcOH: acetic acid EA: Ethyl acetate DCM: dichloromethane m-CPBA: meta-chloroperbenzoic acid TBDMSOTf: tert-butyldimethylsilyl triflate TBDMS: tert-butyldimethylsilyl DMF: Dimethylformamide EDCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HOBt: 1-Hydroxybenzotriazole hydrate ACN: Acetonitrile TBDMS-Cl: tert-butyldimethylsilyl chloride DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene THF: tetrahydrofuran DCC: N,N'-dicyclohexylcarbodiimide DMAP: 4-dimethylaminopyridine NHS: N-hydroxysuccinimide DIPEA: Diisopropylethylamine TEA: Triethylamine DEAD: Diethyl azodicarboxylate Boc: tert-butyloxycarbonyl LAH: Lithium aluminum hydride CDI: 1,1'-carbonyldiimidazole BEMP: 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine TPSCl: Triphenylchlorosilane tfa: trifluoroacetyl PyBop: Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate HBTU: N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate TFA: Trifluoroacetic acid DIC: N,N'-diisopropylcarbodiimide DMPA: 2,2-dimethoxy-2-phenylacetophenone TBAF: Tetra-n-butylammonium fluoride AgOTf: Silver trifluoromethanesulfonate (BimC4A)3: 5,5',5''-[2,2',2''-nitrilotris(methylene)tris(1H-benzimidazole-2,1-diyl)]tripentanoic acid tripotassium hydrate

[0244] Example 1: Preparation of compound L-1 [ka] 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 dropwise LAH (3.6 g, 95.15 mmol) under N2 atmosphere at -78 °C. The reaction mixture was stirred at room temperature for 17 h. After the reaction was completed, 15% NaOH solution (4 mL), H2O (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 (400 MHz,DMSO-d6) δ 9.21 (s, 1H), 6.66 (s, 1H), 6.58 (s, 2H), 5.07 (t, J = 6.0 Hz, 2H), 4.38 (d, J = 4.6 Hz, 4H).

[0245] Preparation of compound L-1-2 Compound L-1-1 (2 g, 12.97 mmol) was dissolved in a solution of HBr (5.0 mL, 33% in AcOH) under N2 atmosphere. After stirring at 60 °C for 18 h, the reaction was quenched by adding NaHCO3 solution (pH approx. 8). Distilled water (50 mL) and EA (100 mL x 2) were then added to the reaction mixture. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-1-2 (2.9 g, 80%). 1 H NMR (400 MHz, CDCl3) δ 6.99 (s, 1H), 6.81 (s, 2H), 4.85 (s, 1H), 4.41 (s, 2H).

[0246] Preparation of compound L-1 To a solution of compound L-1-2 (100 mg, 0.36 mmol) in dry DCM (3 mL) was added imidazole (27 mg, 0.39 mmol) and TBDMS-Cl (59 mg, 0.39 mmol) at room temperature under N2 atmosphere. After stirring for 16 h, distilled water (50 mL) and EA (100 mL) were added to the reaction mixture. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-1 (110 mg, 79%). 1 H NMR (400 MHz, CDCl3) δ 7.00 (s, 1H), 6.80 (s, 2H), 4.41 (s, 4H), 0.99 (s, 9H), 0.21 (s, 6H).

[0247] Example 2: Preparation of compound Int-1 [ka] Preparation of compound Int-1-1 To a solution of vanillic acid (50.0 g, 0.30 mol) in MeOH (700 mL) was added SOCl2 (207 mL, 2.85 mol) dropwise at 0 °C under N2 atmosphere. After stirring at room temperature for 15 h, the reaction was adjusted to pH 7-8 with saturated aqueous NaHCO3 solution and then diluted with distilled water (100 mL) and EA (400 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 Int-1-1 (54.2 g, quantitative). 1 H NMR (400 MHz, CDCl3) δ 7.64 (dd, J = 6.4, 1.6 Hz, 1H), 7.55 (s, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.05 (s, 1H), 3.95 (s, 3H), 3.89 (s, 3H).

[0248] Preparation of compound Int-1-2 To a solution of compound Int-1-1 (54.2 g, 0.30 mol) in DMF (200 mL), K2CO3 (61.6 g, 0.45 mol) and benzyl bromide (39.0 mL, 0.33 mol) were added under N2 atmosphere. After stirring at 100 °C for 6 h, the mixture was cooled to room temperature and diluted with distilled water (100 mL) and EA (400 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 Int-1-2 (79.8 g, 98%). 1 H NMR (400 MHz, CDCl3) δ 7.60 (dd, J = 6.4, 2.0 Hz, 1H), 7.56 (d, J = 2.0 Hz, 1H), 7.44 - 7.31 (m, 5H), 6.89 (d, J = 8.4 Hz, 1H), 5.22 (s, 2H), 3.94 (s, 3H), 3.88 (s, 3H).

[0249] Preparation of compound Int-1-3 Compound Int-1-2 (79.8 g, 0.29 mol) was dissolved in acetic anhydride (550 mL) under N2 atmosphere and then cooled to 0° C. Copper(II) nitrate hemi(pentahydrate) (75.0 g, 0.32 mol) was added in portions. After stirring at 0° C. for 6 h, the reaction was quenched with ice water (800 mL). The solid was filtered and washed with distilled water (100 mL) and hexane (400 mL) to give compound Int-1-3 (85.5 g, 92%). 1 H NMR (400 MHz, CDCl3) δ 7.52 (s, 1H), 7.45-7.35 (m, 5H), 7.08 (s, 1H), 5.22 (s, 2H), 3.98 (s, 3H), 3.91 (s, 3H).

[0250] 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 dried over anhydrous Na2SO4, 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 (400 MHz, DMSO-d6) δ 7.69 (s, 1H), 7.47-7.35 (m, 5H), 7.03 (s, 1H), 5.24 (s, 2H), 3.91 (s, 3H).

[0251] 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) and one drop of DMF were slowly added dropwise at 0° C. under N2 atmosphere. After stirring for 30 min, the reaction mixture was concentrated under reduced pressure. Compound Int-1 was used directly in the next step without further purification.

[0252] Example 3: Preparation of compound Int-2 [ka] Preparation of compound Int-2-1 To a brown solution of compound Int-1-3 (24.8 g, 78.2 mmol) in DCM (500 mL) was added 1M BCl3 in DCM (93.8 mL, 93.8 mmol, 1.2 equiv) at -78 °C under N2 atmosphere. After stirring for 3 h, the reaction was quenched by adding MeOH (100 mL). The mixture was allowed to warm to room temperature, and then saturated NaHCO3 (100 mL), brine (100 mL), and DCM (600 mL) were added to the reaction mixture. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Removal of the solvent afforded Int-2-1 (17.5 g, 98%) as a yellow solid. 1 H NMR (400 MHz, CDCl3) δ 7.47 (s, 1H), 7.14 (s, 1H), 6.03 (s, 1H), 4.02 (s, 3H), 3.90 (s, 3H).

[0253] Preparation of compound Int-2-2 A brown solution of compound Int-2-1 (17.5 g, 77.03 mmol) in 1,4-dioxane (250 mL) was treated with 6N NaOH (38.5 mL, 231.0 mmol) at room temperature under N2 atmosphere. After stirring at 40° C. for 5 h, the mixture was allowed to cool to 0° C. and acidified with 2N HCl. The mixture was diluted with H2O (150 mL) and extracted with EA (300 mL×3). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The resulting precipitate was collected by filtration, washed with hexane, and dried under vacuum to give compound Int-2-2 (15.9 g, 97%) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 10.6 (brs, 1H), 7.31 (s, 1H), 7.25 (s, 1H), 3.90 (s, 3H), 3.57 (s, 3H).

[0254] Preparation of compound Int-2-3 A brown solution of compound Int-2-2 (15.9 g, 74.6 mmol) in anhydrous THF (370 mL) was treated with DMAP (1.8 g, 14.92 mmol, 0.2 equiv), acetic anhydride (8.5 mL, 87.5 mmol, 1.2 equiv), and TEA (15.6 mL, 111.9 mmol, 1.5 equiv) at room temperature under N2 atmosphere and stirred for 6 h. The reaction mixture was diluted with water (150 ml) and extracted with EA (300 mL x 2). The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give compound Int-2-3 (18 g, 95%) as a yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 7.99 (s, 1H), 7.46 (s, 1H), 3.94 (s, 3H), 2.30 (s, 3H).

[0255] Preparation of compound Int-2 A brown solution of compound Int-2-3 (14.4 g, 56.43 mmol) in anhydrous THF (15 mL) and anhydrous DCM (40 mL) was treated with oxalyl chloride (7.6 mL, 84.64 mmol, 1.5 equiv.) and DMF (2 drops) under N2 atmosphere at 0° C. and stirred for 6 h. The reaction mixture was concentrated under reduced pressure. Compound Int-2 was used directly in the next step without further purification.

[0256] Example 4: Preparation of compound Mono-1 [ka] Preparation of compound Mono-1-1 To a solution of L-2-thienylalanine (500 mg, 2.92 mmol) in distilled water (5.0 mL), concentrated HCl (206 μL) was added dropwise under N2 atmosphere at 0° C., and then formaldehyde (37%, 261 μL, 3.5 mmol) was added to it. The mixture was refluxed overnight. After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was suspended in IPA (3.0 mL), and 4M HCl (in 1,4-dioxane, 1.0 mL) was added to it. After stirring for 2 hours, the solid was filtered and washed with IPA (5 mL), ether (20 mL) to give compound Mono-1-1 (495.7 mg, 77%). 1 H NMR (400 MHz,DMSO-d6) δ 9.95 (brs, 1H), 7.48 (d, J = 5.2 Hz, 1H), 6.94 (d, J = 5.2 Hz, 1H), 4.48-4.44 (m, 1H), 4.28 (d, J = 15.6 Hz, 1H), 4.18 (d, J = 16.0 Hz, 1H), 3.39 (dd, J = 11.6, 5.2 Hz, 1H), 3.17-3.10 (m, 1H). EI-MS m / z: 184 (M + +1).

[0257] Preparation of compound Mono-1-2 Compound Mono-1-1 (495.7 mg, 2.25 mmol) was dissolved in MeOH (10.0 mL) under N2 atmosphere and then cooled to 0 °C. SOCl2 (491.3 μL, 6.76 mmol) was added dropwise at 0 °C, and the reaction mixture was refluxed for 3 h. After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was washed with diethyl ether (5 mL × 2) to give compound Mono-1-2 (521.5 mg, 99%). 1H 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, EI-MS m / z: 198 (M + +1).

[0258] Preparation of compound Mono-1-3 To a solution of compound Int-1 (856.5 mg, 2.66 mmol) in anhydrous THF (3.0 ml) was added a solution of compound Mono-1-2 (518.5 mg, 2.22 mmol) in DMF (3.0 mL) followed by DIPEA (772.8 μL, 4.44 mmol) at 0° C. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, distilled water (20 mL) and EA (50 mL×2) were added to the reaction mixture. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mono-1-3 (888.5 mg, 89%). EI-MS m / z: 483 (M + +1).

[0259] Preparation of compound Mono-1-4 To a solution of compound Mono-1-3 (880 mg, 1.82 mmol) in anhydrous DCM (5.0 mL) and toluene (15.0 mL), DIBAL (3.6 mL, 3.6 mmol, 1.0 M in toluene) was added dropwise at −78° C. under N2 atmosphere. The reaction mixture was stirred at −78° C. for 3 h. The reaction was quenched with MeOH (5 mL), 2N HCl (20.0 mL) at −78° C., then distilled water (20 mL) and EA (50 mL×2) were added to the reaction mixture. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mono-1-4 (701.9 mg, 85%). EI-MS m / z: 453(M + +1).

[0260] Preparation of compound Mono-1-5 To a solution of compound Mono-1-4 (700 mg, 1.55 mmol) in THF (15.0 mL) and distilled water (3.0 mL) was added Na2S2O4 (2.2 g, 12.4 mmol) at room temperature for 4 h. After the reaction was complete, it was quenched with MeOH (5 mL) and the reaction mixture was concentrated under reduced pressure. The residue was suspended in toluene (20 mL) and evaporated to help remove any residual water. The resulting white solid was further dried completely by leaving it under high vacuum overnight. The residue was suspended in anhydrous MeOH (10 mL) followed by the addition of acetyl chloride (1.1 mL, 15.5 mmol). After 15 min, the cloudy solution was filtered and the solid was washed with anhydrous MeOH (5 mL x 2). The filtrate was stirred for 2 h. After the reaction was completed, the reaction mixture was quenched with NaHCO3 solution pH 7, and distilled water (20 mL) and EA (50 mL x 2) were added to the reaction mixture. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mono-1-5 (701.9 mg, 85%). 1 H NMR (400 MHz,CDCl3) 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).

[0261] Preparation of compound Mono-1 A solution of compound Mono-1-5 (60 mg, 0.15 mmol) in anhydrous DCM (3 mL) was cooled to 0° C., and methanesulfonic acid (700 μL) in DCM (2.0 mL) was added. The mixture was stirred at 0° C. for 2 h. Upon completion of the reaction, the reaction mixture was quenched with NaHCO3 solution (pH approx. 7), and distilled water (5 mL) and EA (20 mL×2) were added to the reaction mixture. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mono-1 (38.3 mg, 82%). 1 H NMR (400 MHz,CDCl3) δ 7.58 (d, J = 5.6 Hz, 1H), 7.54 (s, 1H), 7.23 (d, J = 5.2 Hz, 1H), 6.95 (d, J = 5.2 Hz, 1H), 6.89 (s, 1H), 6.06 (s, 1H), 5.30 (s, 1H), 4.99 (d, J = 16.4 Hz, 1H), 4.44 (d, J = 16.4 Hz, 1H), 4.10-4.04 (m, 1H), 3.99 (s, 3H), 3.32-3.26 (m, 1H). EI-MS m / z: 315(M + +1).

[0262] Example 5: Preparation of compound Mono-2 [ka] Preparation of compound Mono-2-1 N-Boc-L-histidine (900 mg, 3.34 mmol) was dissolved in ACN (5.0 mL) under N2 atmosphere. Cs2CO3 (2.2 g, 6.68 mmol) and iodomethane (208 μL, 3.34 mmol) were added. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, the cloudy solution was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mono-2-1 (549.2 mg, 58%). 1H NMR (400 MHz,CDCl3) δ 7.34 (s, 1H), 6.52 (s, 1H), 5.89 (d, J = 7.2 Hz, 1H), 4.58-4.50 (m, 1H), 3.72 (s, 3H), 3.63 (s, 3H), 3.14-3.08 (m, 2H). EI-MS m / z: 284 (M + +1).

[0263] Preparation of compound Mono-2-2 Compound Mono-2-1 (549.2 mg, 1.94 mmol) was dissolved in DCM (5.0 mL) under N2 atmosphere and cooled to 0° C. 4M HCl (in 1,4-dioxane, 2 mL) was added at 0° C. The reaction was allowed to warm to room temperature and stirred for 5 h. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was washed with ether (5 mL) to give compound Mono-2-2 (429.8 mg, quantitative). 1 H NMR (400 MHz, DMSO-d6) δ 9.00 (s, 1H), 8.80 (brs, 2H), 7.51 (s, 1H), 4.31 (t, J = 6.8 Hz, 1H), 3.81 (s, 3H), 3.74 (s, 3H), 3.27 (t, J = EI-MS m / z: 184 (M + +1).

[0264] Preparation of compound Mono-2-3 Compound Mono-2-2 (425.8 mg, 1.94 mmol) was dissolved in distilled water (4.0 mL) under N2 atmosphere and then cooled to 0 °C. Concentrated HCl (250 μL) was added dropwise at 0 °C, followed by the addition of formaldehyde (37%, 216 μL, 2.9 mmol). The reaction mixture was refluxed overnight. After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was suspended in IPA (3.0 mL), 4M HCl (in 1,4-dioxane, 2.0 mL) was added, and the resulting mixture was stirred for 2 h. The solid was filtered and washed with IPA (5 mL), ether (10 mL × 2) to give compound Mono-2-3 (421.9 mg, quantitative). EI-MS m / z: 182 (M + +1).

[0265] Preparation of compound Mono-2-4 Compound Mono-2-3 (421.9 mg, 1.94 mmol) was dissolved in MeOH (5.0 mL) under N2 atmosphere and then cooled to 0 °C. SOCl2 (423 μL, 5.83 mmol) was added dropwise at 0 °C, and the reaction mixture was refluxed for 5 h. After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was washed with ether (5 mL × 2) to give compound Mono-2-4 (375.2 mg, 84%). 1 H NMR (400 MHz,DMSO-d6) δ 9.02 (s, 2H), 4.66-4.62 (m, 1H), 4.49 (d, J = 15.2 Hz, 1H), 4.33 (d, J = 15.6 Hz, 1H), 3.80 (s, 6H), 3.29 (dd, J = 11.6, 5.2 Hz, 1H), 3.18-3.13, (m, 1H). EI-MS m / z: 196 (M + +1).

[0266] Preparation of compound Mono-2-5 A solution of compound Mono-2-4 (375.2 mg, 1.62 mmol) in DMF (3.0 mL) was added to a solution of compound Int-1 (625.2 mg, 1.94 mmol) in anhydrous THF (3.0 mL), then DIPEA (846.2 μL, 4.86 mmol) was added at 0° C., and the reaction mixture was stirred at room temperature overnight. After the reaction was completed, distilled water (20 mL) and EA (50 mL×2) were added to the reaction mixture. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mono-2-5 (466.8 mg, 60%). EI-MS m / z: 481 (M + +1).

[0267] Preparation of compound Mono-2-6 To a solution of compound Mono-2-5 (100 mg, 0.21 mmol) in anhydrous DCM (0.5 mL) and toluene (1.5 mL), DIBAL (832 μL, 0.832 mmol, 1.0 M in toluene) was added dropwise at −78° C. under N2 atmosphere, and the reaction mixture was stirred at −78° C. for 5 h. The reaction was quenched with MeOH (0.1 mL) and 2N HCl (20 mL) at −78° C., and distilled water (20 mL) and DCM (100 mL) were added to the reaction mixture. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mono-2-6 (43 mg, 46%). EI-MS m / z: 451(M + +1).

[0268] Preparation of compound Mono-2-7 To a solution of compound Mono-2-6 (40 mg, 0.09 mmol) in THF (1.7 mL) and distilled water (1.1 mL) was added Na2S2O4·2H2O (149 mg, 0.71 mmol) at room temperature under N2 atmosphere. After stirring for 4 h, the reaction was quenched with MeOH (2.0 mL). The reaction mixture was concentrated under reduced pressure and the residue was suspended in toluene (20 mL) and evaporated to help remove any residual water. The resulting yellow solid was further dried thoroughly by leaving it under high vacuum overnight. The residue was suspended in anhydrous MeOH (3.0 mL) followed by the addition of acetyl chloride (63 μL, 0.88 mmol). After stirring for 15 min, the cloudy solution was filtered and the solid was washed with anhydrous MeOH (5.0 mL × 2). After stirring for 2 h, the reaction mixture was adjusted to pH 7 with NaHCO3 solution. Distilled water (5.0 mL) and EA (20 mL×2) were added to it. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mono-2-8 (18 mg, 51%). 1H NMR (400 MHz,CDCl3) δ 7.63 (d, J = 5.6 Hz, 1H), 7.51 (s, 1H), 7.46-7.27 (m, 6H), 6.85 (s, 1H), 5.26-5.17 (m, 2H), 4.71 (d, J = 16.4 EI-MS m / z: 403 (M) + +1).

[0269] Preparation of compound Mono-2 To a solution of compound Mono-2-7 (16 mg, 0.04 mmol) in anhydrous DCM (160 μL) was added methanesulfonic acid (80 μL) in DCM (160 μL) at 0° C. The reaction mixture was stirred at the same temperature for 3 h. After the reaction was completed, the mixture was quenched with NaHCO3 solution (pH 8-9). The residue was purified by preparative HPLC to give compound Mono-2 (4.9 mg, 40%). 1 H NMR (400 MHz,CD3OD) δ 8.34 (brs, 1H), 7.68 (s, 1H), 7.28 (s, 1H), 6.42 (s, 1H), 4.83 (d, J = 16.4 Hz, 1H), 4.63 (d, J = 16.4 Hz, 1H), 4.26 (d, J = 8.4 Hz, 1H ), 4.02-3.99 (m, 1H), 3.87 (s, 3H), 3.68 (s, 3H), 2.98-2.86 (m, 2H). EI-MS m / z: 313 (M + +1).

[0270] Example 6: Preparation of compound Mono-3 [ka] Preparation of compound Mono-3-1 L-2-Furyl alanine (2.56 g, 13.35 mmol) was dissolved in MeOH (50.0 mL) under N2 atmosphere and then cooled to 0°C. SOCl2 (1.45 mL, 20.02 mmol) was added dropwise at the same temperature and the reaction mixture was refluxed for 5 h. The mixture was concentrated under reduced pressure. The residue was washed with ether (10 mL) to give compound Mono-3-1 (2.73 g, quantitative). 1 H NMR (400 MHz,DMSO-d6) δ 8.65 (brs, 3H), 7.58 (s, 1H), 6.35 (s, 1H), 6.26 (s, 1H), 4.26 (t, J = 6.4 Hz, 1H), 3.69 (s, 3H), 3.24-3.14 (m, 2H). EI-MS m / z: 170 (M + +1).

[0271] Preparation of compound Mono-3-2 Compound Mono-3-1 (2.7 g, 13.13 mmol) was dissolved in THF (10 mL) and DMF (20.0 mL), then formaldehyde (37%, 2.0 mL, 26.26 mmol) was added under N2 atmosphere. After refluxing the reaction mixture overnight, Na2SO4 (1.0 g) was added to the reaction mixture to remove water, then 4 M HCl (in 1,4-dioxane, 3.5 mL) was added, and the resulting mixture was stirred at room temperature for 3 h. The mixture was quenched with NaHCO3 solution (pH 9) and BOC2O (4.3 g, 19.7 mmol) was added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was filtered, and the filtrate was extracted with EA (50 mL) and distilled water (20 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 Mono-3-2 (906.1 mg, 24.5%). EI-MS m / z: 282 (M + +1).

[0272] Preparation of compound Mono-3-3 To a solution of compound Mono-3-2 (609.1 mg, 3.22 mmol) in DCM (5.0 mL) was added 4M HCl (in 1,4-dioxane, 1.6 mL) at 0° C. under N2 atmosphere. The reaction was allowed to warm to room temperature and stirred for 5 h. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was washed with ether (5 mL) to give compound Mono-3-3 (628.3 mg, 90%). 1 H NMR (400 MHz, DMSO-d6) δ 10.03 (brs, 1H), 7.65 (s, 1H), 6.46 (s, 1H), 4.62-4.58 (m, 1H), 4.11 (q, J = 17.6, 14.8 Hz, 2H), 3.28-3.20 (m, 1H), 3.12-3.00 (m, 3H). EI-MS m / z: 182(M + +1).

[0273] Preparation of compound Mono-3-4 To a solution of compound Int-1 (1.4 g, 4.33 mmol) in anhydrous THF (5.0 ml) was added a solution of compound Mono-3-3 (628.3 mg, 2.88 mmol) in DMF (5.0 mL) followed by DIPEA (846.2 μL, 4.86 mmol) at 0° C. The reaction mixture was stirred at room temperature overnight. After the reaction was completed, distilled water (20 mL) and EA (50 mL×2) were added. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mono-3-4 (1.2 g, 89%). EI-MS m / z: 467 (M + +1).

[0274] Preparation of compound Mono-3-5 To a solution of compound Mono-3-4 (1.2 g, 2.57 mmol) in anhydrous DCM (7.0 mL) and toluene (21.0 mL), 1M DIBAL (5.2 mL, 5.2 mmol, 1.0 M in toluene) was added dropwise at -78 °C. The mixture was stirred at the same temperature for 5 h. After the reaction was completed, the reaction mixture was quenched with MeOH (5 mL), 2N HCl (20.0 mL) at -78 °C, and distilled water (20 mL) and EA (50 mL x 2) were added to the reaction mixture. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mono-3-5 (884 mg, 79%). EI-MS m / z: 436(M + +1).

[0275] Preparation of compound Mono-3-6 To a solution of compound Mono-3-5 (55.4 mg, 0.13 mmol) in THF (3.0 mL) and distilled water (1.0 mL) was added Na2S2O4 (213.4 mg, 1.02 mmol) at room temperature under N2 atmosphere. After stirring for 8 h, the reaction was quenched with MeOH (3.0 mL). The reaction mixture was concentrated under reduced pressure. The residue was suspended in toluene (20 mL) and evaporated to help remove any residual water. The resulting yellow solid was further dried thoroughly by leaving it under high vacuum overnight. The residue was suspended in anhydrous MeOH (2.0 mL) followed by the addition of acetyl chloride (90.2 μL, 1.27 mmol). After stirring for 15 min, the cloudy solution was filtered and the solid was washed with anhydrous MeOH (5.0 mL x 2). After stirring for 2 h, the reaction mixture was adjusted to pH 7 with NaHCO3 solution. Distilled water (5.0 mL) and EA (20 mL×2) were added to it. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mono-3-6 (30.3 mg, 61%). 1H NMR (400 MHz,CDCl3) δ 7.64 (d, J = 5.6 Hz, 1H), 7.51 (s, 1H), 7.45-7.32 (m, 6H), 6.85 (s, 1H), 6.36(s, 1H), 5.24 (q, J = 12.4, 11.2 EI-MS m / z: 389 (M + +1).

[0276] Preparation of compound Mono-3 To a solution of compound Mono-3-6 (100 mg, 0.26 mmol) in anhydrous EtOH (10 mL), 5% Pd / C (273 mg, 0.13 mmol) and 1,4-cyclohexadiene (1.0 mL, 10.30 mmol) were added. The mixture was stirred at room temperature for 8 hours. After the reaction was completed, the solid was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mono-3 (23 mg, 31%). 1 H NMR (400 MHz,CDCl3) δ 7.67 (d, J = 6.0 Hz, 1H), 7.50 (s, 1H), 7.41 (s, 1H), 7.37 (d, J = 3.2 Hz, 1H), 6.89 (s, 1H), 6.37 (s, 1H), 4.58 EI-MS m / z: 299 (M + +1).

[0277] Example 7: Preparation of compound Mono-4 [ka] Preparation of compound Mono-4-1 To a solution of Fmoc-His(Trt)-OH (15.0 g, 24.2 mmol) and HOBT (5.0 g, 24.2 mmol) in anhydrous THF (200 mL) was added DCC (1.15 g, 8 mmol) in THF (40 mL) and MeOH (20 mL) dropwise at -13 °C over 30 min. The reaction mixture was slowly warmed to ambient temperature and stirred for 5 h. After the reaction was completed, distilled water (50 mL) and DCM (200 mL x 2) were added to the reaction mixture. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mono-4-1 (13.0 g, 84%). 1 H NMR (400 MHz,CDCl3) δ 7.75 (d, J = 7.6 Hz, 2H), 7.62 (t, J = 7.6 Hz, 2H), 7.41-7.28 (m, 14H), 7.15-7.06 (m, 7H), 6.54 (s, 1H), 6.52 EI-MS m / z: 634 (M + +1).

[0278] Preparation of compound Mono-4-2 To a solution of compound Mono-4-1 (13 g, 20.51 mmol) in DCM (50 mL) was added methyl iodide (3.8 mL, 61.54 mmol) at 0° C. The reaction mixture was warmed to room temperature and stirred for 5 h. After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mono-4-2 (11 g, 83%). 1H NMR (400 MHz, CDCl3) δ 8.42 (s, 1H), 7.76 (d, J = 7.6 Hz, 2H), 7.70-7.60 (m, 2H), 7.46-7.20 (m, 19h), 6.89 (s, 1H), 6.60 (d, J = 7.2 4.70-4.62 (m, 1H), 4.30-4.12 (m, 3H), 4.01 (s, 3H), 3.67 (s, 3H), 3.50-3.28 (m, 2H). EI-MS m / z: 648 (M + +1).

[0279] Preparation of compound Mono-4-3 To a solution of compound Mono-4-2 (11 g, 16.95 mmol) in DCM (150 mL) was added TFA (40 mL) and triethylsilane (8.12 mL, 50.86 mmol) under N2 atmosphere at 0° C. The reaction was allowed to warm to room temperature and stirred for 6 h. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mono-4-3 (6.25 g, 91%). 1 H NMR (400 MHz, CDCl3) δ 8.87 (s, 1H), 7.78 (d, J = 7.2 Hz, 2H), 7.59 (d, J = 7.6 Hz, 2H),7.45-7.30 (m, 4H), 7.09 (s, 1H), 5.69 (d, J = 6.0 EI-MS m / z: 407 (M) + +1).

[0280] Preparation of compound Mono-4-4 To a solution of compound Mono-4-3 (6.25 g, 15.37 mmol) in DCM (150 mL) was added piperidine (3.0 mL, 30.74 mmol) under N2 atmosphere at 0° C. The reaction mixture was warmed to room temperature and stirred for 7 hours. After the reaction was completed, the mixture was concentrated under reduced pressure to give compound Mono-4-4 (2.65 g, 95%). 1 EI-MS m / z: 184 (M + +1).

[0281] Preparation of compound Mono-4-5 Compound Mono-4-4 (2.65 g, 14.46 mmol) was dissolved in distilled water (100 mL) under N2 atmosphere, then the reaction mixture was cooled to 0 °C. Concentrated HCl (2.5 mL) was added dropwise at 0 °C, followed by the addition of formaldehyde (37%, 2.2 mL, 28.93 mmol). The reaction mixture was refluxed overnight. After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was suspended in IPA (20 mL) and 4M HCl (in 1,4-dioxane, 4.0 mL) was added. The reaction mixture was stirred for 2 hours. The solid was filtered and washed with IPA (5 mL), ether (10 mL x 2) to give compound Mono-4-5 (3.14 g, 99%). EI-MS m / z: 182 (M + +1).

[0282] Preparation of Compound Mono-4-6 To a solution of compound Mono-4-5 (3.14 g, 14.43 mmol) in MeOH (100 mL) was added SOCl2 (2.5 mL, 35.15 mmol) dropwise at -0°C. The reaction mixture was refluxed for 5 h and then concentrated under reduced pressure. The residue was washed with ether (25 mL x 2) to give compound Mono-4-6 (2.18 g, 65%).

[0283] EI-MS m / z: 196 (M+ +1). Preparation of compound Mono-4-7 To a solution of compound Int-1 (3.93 g, 12.23 mmol) and compound Mono-4-6 (2.18 g, 9.41 mmol) in anhydrous THF (30 ml) and DMF (30 ml) was added DIPEA (4.9 mL, 28.22 mmol) at 0° C. After stirring at room temperature for 2 h, the mixture was quenched with distilled water (200 mL) and EA (1000 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 Mono-4-7 (2.81 g, 62%). EI-MS m / z: 481 (M + +1).

[0284] Preparation of compound Mono-4-8 To a solution of compound Mono-4-7 (2.5 g, 5.20 mmol) in anhydrous DCM (12.5 mL) and toluene (37.5 mL), DIBAL (10.4 mL, 10.41 mmol, 1.0 M in toluene) was added dropwise at −78° C. under N2 atmosphere. After stirring at −78° C. for 5 h, the mixture was quenched with MeOH (1.0 mL) and 2N HCl (100 mL) at the same temperature. The mixture was successively diluted with water (100 mL) and DCM (200 mL), then the organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mono-4-8 (1.21 g, 52%). EI-MS m / z: 451 (M + +1).

[0285] Preparation of compound Mono-4-9 To a solution of compound Mono-4-8 (1.2 g, 2.66 mmol) in THF (100 mL) and distilled water (70 mL) was added Na2S2O4 (3.7 g, 21.31 mmol) at room temperature. After stirring for 6 h, the reaction was quenched with MeOH (20 mL) and the mixture was concentrated under reduced pressure three times by using toluene as a co-solvent, thereby removing water. The resulting yellow solid was suspended in anhydrous MeOH (200 mL) to which acetyl chloride (1.9 mL, 26.64 mmol) was added. After stirring for 15 min, the reaction mixture was adjusted to pH 8 by adding saturated NaHCO3 solution and diluted with distilled water (250 mL), MeOH (250 mL), and DCM (200 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 Mono-4-9 (918 mg, 78%). 1 H NMR (400 MHz,CDCl3) δ 7.58 (d, J = 5.6 Hz, 1H), 7.57 (s, 1H), 7.52 (s, 1H), 7.49-7.27 (m, 5H), 6.84 (s, 1H), 5.26-5.15 (m, 2H), 4.66 (s, 2H), 4.16 (t, J = 6.0 Hz, 1H), 3.97 (s, 3H), 3.67 (s, 3H), 3.22-2.94 (m, 2H).EI-MS m / z: 403 (M + +1).

[0286] Preparation of compound Mono-4 To a solution of compound Mono-4-9 (50 mg, 0.12 mmol) in anhydrous DCM (2 mL) was added methanesulfonic acid (0.1 mL) in DCM (0.2 mL) at 0° C. After stirring at room temperature for 1 h, the mixture was adjusted to pH 8 by adding saturated NaHCO3 solution. The residue was purified by preparative HPLC to give compound Mono-4 (27 mg, 71%). 1H NMR (400 MHz, CD3OD) δ 8.34 (brs, 1H), 7.68 (s, 1H), 7.28 (s, 1H), 6.42 (s, 1H), 4.77 (d, J = 16.0 Hz, 1H), 4.56 (d, J = 16.0 Hz, 1H), 4.33 (d, J = 7.6 Hz, 1H), 4.10-4.02 (m, 1H), 3.84 (s, 3H), 3.66 (s, 3H), 3.02-2.82 (m, 2H). EI-MS m / z: 313 (M + +1).

[0287] Example 8: Preparation of compound Mono-5

change

[0288] Example 9: Preparation of compound Mono-6 [ka] Compound Mono-6 was synthesized in a similar manner to that described in Example 4. Compound Mono-6-1 Yield: 95%; 1H NMR (400 MHz, DMSO-d6) δ 10.0 (s, 2H), 7.51 (d, J = 4.8 Hz, 1H), 6.95 (d, J = 5.2 Hz, 1H), 4.46 (d, J = 15.6 Hz, 1H), 4.44-4.40 (m, 1H), 4.34 (d, J = 16.0 Hz, 1H), 3.24 (dd, J = 11.6, 5.2 Hz, 1H), 3.02 - 2.92 (m, 1H); EI-MS m / z: 184 (M + +1). Compound Mono-6-2 Yield: 95%; 1 H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 2H), 7.52 (d, J = 4.8 Hz, 1H), 6.95 (d, J = 5.2 Hz, 1H), 4.62-4.54 (m, 1H), 4.49 (d, J = 16.4 Hz, 1H), 4.34 (d, J = 15.6 Hz, 1H), 3.81 (s, 3H), 3.24 (dd, J = 12.0, 4.8 Hz, 1H), 3.02 - 2.96 (m, 1H); EI-MS m / z: 198 (M + +1). Compound Mono-6-3 Yield: 76%; EI-MS m / z: 483 (M + +1). Compound Mono-6-4 Yield: 74%; EI-MS m / z: 453 (M + +1). Compound Mono-6-5 Yield: 74%; 1H NMR (400 MHz, CDCl3) δ 7.55 (s, 1H), 7.48 (d, J = 6.0 Hz, 1H), 7.46 - 7.28 (m, 5H), 7.25 (s, 1H), 7.01 (d, J = 4.8 Hz, 1H), 6.85 (s, 1H), 5.20 (q, J = 12.0, 11.6 Hz, 2H), 5.08 (d, J = 16.8, 1H), 4.57 (d, J = 16.4, 1H), 4.06 - 4.0 (m, 1H), 3.98 (s, 3H), 3.18 - 3.12 (m, 2H);EI-MS m / z: 405 (M + +1). Compound Mono-6 Yield: 89%; 1 H NMR (400 MHz, CDCl3) δ 7.53 (s, 1H), 7.52 (d, J = 5.2 Hz, 1H), 7.02 (d, J = 5.2 Hz, 1H), 6.89 (s, 1H), 6.06 (s, 1H), 5.09 (d, J = 16.4, EI-MS m / z: 315 (M + +1).

[0289] Example 10: Preparation of compound Mono-8 [ka] Preparation of compound Mono-8-1 To a solution of (s)-(-)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (5.0 g, 28.22 mmol) in MeOH (140 mL) was added SOCl2 (2.30 mL, 31.04 mmol) dropwise under N2 atmosphere at 0°C. After stirring at 40°C for 21 h, the mixture was concentrated under reduced pressure. The residue was washed with ether (50 mL) to give compound Mono-8-1 (6.42 g, 99%). 1H NMR (400 MHz, DMSO-d6) δ 10.02 (s, 2H), 7.27 (s, 4H), 4.60 - 4.56 (m, 1H), 4.39 - 4.29 (m, 2H), 3.82 (s, 3H), 3.19 - 3.12 (m, 2H): EI-MS m / z: 192(M + +1).

[0290] Preparation of compound Mono-8-2 To a solution of compound Mono-8-1 (9.07 g, 28.22 mmol) and compound Int-1 (6.42 g, 28.22 mmol) in anhydrous THF (50 mL) was added TEA (7.9 mL, 56.43 mmol) at 0° C. After stirring at room temperature for 2 h, distilled water (500 mL) and EA (800 mL) were added to it. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Mono-8-2 (12.01 g, 90%). EI-MS m / z: 477 (M + +1).

[0291] Preparation of compound Mono-8-3 To a solution of compound Mono-8-2 (4 g, 8.39 mmol) in anhydrous DCM (18 mL) and toluene (52 mL), DIBAL (16.8 mL, 16.79 mmol, 1.0 M in toluene) was added dropwise at −78° C. under N2 atmosphere. After the reaction mixture was stirred at −78° C. for 4 h, the reaction was quenched at −78° C. with MeOH (0.4 mL), 2N HCl (25 mL), and distilled water (100 mL) and EA (500 mL) were added. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mono-8-3 (3.07 g, 82%). EI-MS m / z: 447 (M + +1).

[0292] Preparation of compound Mono-8-4 To a solution of compound Mono-8-3 (3 g, 6.72 mmol) in THF (130 mL) and distilled water (86 mL) was added Na2S2O4·2H2O (11.3 g, 53.76 mmol) at room temperature under N2 atmosphere. After stirring for 5 h, the reaction was quenched with MeOH (130 mL). The reaction mixture was concentrated under reduced pressure. The residue was suspended in toluene (20 mL) and evaporated to help remove any residual water. The resulting yellow solid was further dried thoroughly by leaving it under high vacuum overnight. The residue was suspended in anhydrous MeOH (220 mL) followed by the addition of acetyl chloride (4.8 mL, 67.19 mmol). After stirring for 15 min, the cloudy solution was filtered and the solid was washed with anhydrous MeOH (50 mL). After stirring for 2 h, the reaction mixture was adjusted to pH 7 with NaHCO3 solution, to which distilled water (100 mL) and EA (500 mL) were added. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Mono-8-4 (2.48 g, 93%). 1 H NMR (400 MHz, CDCl3) δ 7.55 (s, 1H), 7.45 - 7.27 (m, 10H), 6.84 (s, 1H), 5.24 - 5.15 (m, 2H), 5.00 (d, J = 15.2, 1H), 4.56 (d, J = 15.6, 1H), 3.97 (s, 3H), 3.93 - 3.92 (m, 1H), 3.31 - 3.12 (m, 2H). + +1).

[0293] Preparation of compound Mono-8 To a solution of compound Mono-8-4 (1 g, 2.51 mmol) in anhydrous DCM (10 mL) was added methanesulfonic acid (5 mL) in DCM (10 mL) at 0° C. After stirring at 0° C. for 3 h, the mixture was adjusted to pH 7 with NaHCO3 solution, followed by addition of distilled water (100 mL) and EA (400 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 Mono-8 (703 mg, 91%). 1 H NMR (400 MHz, CDCl3) δ 7.54 (s, 1H), 7.48 (d, J = 4.8 Hz, 1H), 7.37 - 7.26 (m, 4H), 6.88 (s, 1H), 6.03 (s, 1H), 5.00 (d, J = 15.6 Hz, EI-MS m / z: 309 (M + +1).

[0294] Example 11: Preparation of compound Mono-9 (IBD monomer) [ka] Mono-9 was synthesized by a route similar to that described in WO2010091150. 1 H NMR (400 MHz,CDCl3) δ 8.27 (d, J = 7.6 Hz, 1H), 7.88 (d, J = 4.4 Hz, 1H), 7.56 (s, 1H), 7.33 - 7.30 (m, 1H), 7.11 (t, J = 7.6 Hz, 1H), 6.92 (s, 1H), 4.50 - 4.46 (m, 1H), 3.99 (s, 3H), 3.78 - 3.67 (m, 1H), 3.53 - 3.50 (m, 1H) ;EI-MS m / z: 295(M + +1).

[0295] Example 12: Preparation of compound Mono-10 [ka] Preparation of compound Mono-10-1 To a solution of trans-4-hydroxy-L-proline (30 g, 230 mmol, CAS 51-35-4) and NaHCO3 (43 g, 570 mmol) in HO (500 mL) in toluene (120 mL) under N2 atmosphere, Cbz-Cl (37.4 mL, 260 mmol) was added. The reaction mixture was stirred at room temperature overnight. The mixture was extracted with EA (500 mL x 3). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain compound Mono-10-1 (57.5 g, 95%) as a light brown oil. 1 H NMR (400 MHz, DMSO-d6) δ 12.7 (brs, 1H), 7.40-7.28 (m, 5H), 5.18-5.02 (m, 2H), 4.31 - 4.24 (m, 2H), 3.51 - 3.35 (m, 2H), 2.23 - 2.10 (m, 1H), 2.00 - 1.87 (m, 1H);EI-MS m / z: 266(M + +1).

[0296] Preparation of compound Mono-10-2 A brown solution of compound Mono-10-1 (57.5 g, 220 mmol) in MeOH (400 mL) was treated with thionyl chloride (45.3 mL, 610 mmol) under N2 atmosphere at 0° C. and stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure to give compound Mono-10-2 (60.5 g, quantitative) as a light brown oil. Compound Mono-10-2 was used directly in the next step without further purification. 1H NMR (400 MHz, CDCl3) δ 7.38-7.28 (m, 5H), 5.23 - 5.09 (m, 2H), 4.56 - 4.47 (m, 2H), 3.80 (s, 1H), 3.76 - 3.66 (m, 2H), 3.58 - 3.52 (m, 1H) 2.38 - 2.26 (m, 1H), 2.16 - 2.08 (m, 1H) ;EI-MS m / z: 270(M + +1).

[0297] Preparation of compound Mono-10-3 To a solution of compound Mono-10-2 (60.5 g, 220 mmol) in anhydrous THF (500 mL) was added LiBH4 (3.9 g, 180 mmol) at 0° C. under N2 atmosphere. After stirring at room temperature for 2 days, the reaction was quenched with water (200 mL) and 2N HCl (100 mL). The mixture was extracted with EA (500 mL×3). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give compound Mono-10-3 (54.6 g, 98%) as a light brown oil. 1 H NMR (400 MHz, CDCl3) δ 7.39-7.30 (m, 5H), 5.21-5.12 (m, 2H), 4.66 (d, J = 7.2 Hz, 1H), 4.39 (s, 1H), 4.21 (q, J = 7.6, 7.2 Hz, 1H), 3.76 (t, J = 9.6 Hz, 2H), 3.66 - 3.58 (m, 1H), 3.50 (dd, J = 8.0, 4.0 Hz, 1H), 2.10 - 2.23 (m, 1H), 1.78 - 1.64 (m, 1H);EI-MS m / z: 252(M) + +1).

[0298] Preparation of compound Mono-10-4 A brown solution of Mono-10-3 (53 g, 210 mmol) in anhydrous DCM (500 mL) was treated with t-butyldimethylsilyl chloride (25.4 g, 170 mmol), TEA (30 mL, 210 mmol), and DBU (6.3 mL, 42.2 mmol) at room temperature under N2 atmosphere and stirred overnight. The mixture was washed with NH4Cl (300 mL) and brine (300 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA=1:1) to give compound Mono-10-4 (48.2 g, 63%) as a light brown oil. 1 H NMR (400 MHz, CDCl3) δ 7.38-7.28 (m, 5H), 5.20-5.08 (m, 2H), 4.50 (s, 1H), 4.12-4.00 (m, 1H), 3.97 (dd, J = 6.4, 4.0 Hz, 1H), 3.71 (dd, J = 5.6, 4.8 Hz, 1H), 3.66 - 3.58 (m, 1H), 3.52-3.48 (m, 1H), 2.28 - 2.18 (m, 1H), 2.02 - 1.92 (m, 1H), 0.10 - -0.08(m, 6H) ;EI-MS m / z: 366(M + +1).

[0299] Preparation of compound Mono-10-5 Palladium on carbon, 5% Pd / C (1.3 g, 1.23 mmol) was added to a stirred solution of Mono-10-4 (15 g, 41.0 mmol) in EA (50 mL) under N2 atmosphere at room temperature. The flask was flushed by bubbling hydrogen gas through the solution at room temperature. The mixture was stirred at the same temperature for 5 h. The mixture was diluted with EA (30 mL), filtered through CELITE®, and the CELITE® plug was washed with EA (50 mL x 2). The filtrate was concentrated under reduced pressure. Compound Mono-10-5 (9.5 g, quant.) as a light brown oil was used directly in the next step without further purification. 1H NMR 400 MHz, CDCl3) δ 4.41 (brs, 1H), 3.60 - 3.44 (m, 3H), 3.12 (dd, J = 7.2 Hz, 4.8 Hz, 1H), 2.89 (d, J = 12 Hz, 1H), 1.84-1.79 (m, 1H), 1.74 - 1.67 (m, 1H), 0.89 (s, 9H), 0.06 (s, 6H) ;EI-MS m / z: 232(M + +1).

[0300] Preparation of compound Mono-10-6 A brown solution of compound Mono-10-5 (11.9 g, 51.42 mmol) and Int-2 (14.4 g, 56.6 mmol) in anhydrous THF (400 ml) was treated with DIPEA (26.9 mL, 154.3 mmol) at 0° C. under N2 atmosphere and stirred for 5 h. The reaction mixture was diluted with distilled water (50 mL) and EA (150 mL×2). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA=2:1 to 2:1) to give compound Mono-10-6 (20 g, 83%) as a yellow foam solid. 1 H NMR (400 MHz, CDCl3) δ 7.95 (s, 1H), 6.87 (s, 1H), 4.60 - 4.51(m, 1H), 4.49-4.41 (m, 1H), 4.24 - 4.08 (m, 1H), 3.91 (s, 3H), 3.80 - 3.68 (m, 1H), 3.37 (dd, J = 7.6 Hz, 4.0 Hz, 1H), 3.14 (d, J = 10.4 Hz, 1H), 2.35 (s, 3H), 2.18-2.08 (m, 1H), 0.91 (s, 9H), 0.1 (s 6H) ;EI-MS m / z: 469(M + +1).

[0301] Preparation of compound Mono-10-7 Palladium on carbon, 5% Pd / C (9.1 g, 4.27 mmol) was added to a stirred solution of Mono-9-6 (20 g, 42.68 mmol) in EA (213 mL) under N2 atmosphere. The flask was flushed by bubbling hydrogen gas through the solution at room temperature. After stirring for 8 h, the mixture was diluted with EA (50 mL), filtered through Celite®, and the Celite® plug was washed with EA (50 mL×2). The filtrate was concentrated under reduced pressure to give compound Mono-10-7 (18.5 g, 99%) as a yellow foamy solid. 1 H NMR (400 MHz,CDCl3) δ 6.81 (s, 1H), 6.44 (s, 1H), 5.79 (brs, 1H), 4.58 - 4.50 (m, 1H), 4.42 - 4.36 (m, 1H), 4.10 (brs, 1H), 3.79 (s, 3H), 3.59 (dd, J = 8.4 Hz, 2.8 Hz, 1H), 3.50 (d, J = 11.2 Hz, 1H), 2.30 - 2.24 (m, 1H), 2.06 - 2.01 (m, 1H), 0.89 (s, 9H), 0.05 (d, J = 1.6 Hz, 6H) ;EI-MS m / z: 439 (M + +1).

[0302] Preparation of compound Mono-10-8 A yellow solution of Mono-10-7 (18.5 g, 42.18 mmol) in anhydrous DCM (210 mL) was treated with 2,2,2-trichloroethyl chloroformate (6.4 mL, 46.4 mmol) and pyridine (6.9 mL, 87.4 mmol) under N2 atmosphere at 0° C. and stirred for 3 h. The reaction mixture was washed with CuSO4 solution (50 mL) and brine (100 mL×2). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA=2:1) ​​to give compound Mono-10-8 (21.2 g, 82%) as a brown foamy solid. 1H NMR (400 MHz,CDCl3) δ 8.87 (brs, 1H), 7.86 (s, 1H), 6.89 (s, 1H), 4.84 (d, J = 12.8 Hz, 1H), 4.71 (d, J = 10.8 Hz, 1H), 4.61 (brs, 1H), 4.45 (s, 1H), 4.20 (brs, 1H), 3.78 (s, 3H), 3.70-3.62 (m, 1H), 3.57 (s, 2H), 2.32 (s, 4H), 2.11 - 2.02 (m, 1H), 1.80 (s, 1H), 0.90 (s, 9H), 0.05 (s, 6H) EI-MS m / z: 615(M + +1).

[0303] Preparation of compound Mono-10-9 A homogeneous solution of oxalyl chloride (21 mL, 24.4 mmol) in anhydrous DCM (50 mL) was treated with DMSO (3.5 mL, 48.9 mmol) in anhydrous DCM (20 mL) at −78° C. under N2 atmosphere and stirred for 1 h. A solution of Mono-10-8 (10 g, 0.33 mmol) in anhydrous DMC (100 mL) was added dropwise to the reaction mixture and stirred for 2 h. The reaction mixture was treated with TEA (22.7 mL, 162.9 mmol) and stirred at room temperature for 1 h. The reaction mixture was extracted with NH4Cl solution (30 mL) and DCM (100 mL×2). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA=4:1 to 1:1) to give compound Mono-10-9 (8.2 g, 83%) as a brown foamy solid. 1H NMR (400 MHz, CDCl3) δ 8.68 (brs, 1H), 7.91 (s, 1H), 6.86 (s, 1H), 5.12 (brs, 1H), 4.80 (s, 2H), 4.13 (brs, 1H), 4.04 (d, J = 17.2 Hz, 1H), 3.98 -3.86 (m, 1H), 3.80 (s, 3H), 3.76-3.62 (m, 1H), 2.84-2.72 (m, 2H), 2.54 (d, J = 17.2 Hz, 1H), 2.32 (s, 3H), 2.08-1.98 (m, 1H), 0.88 (s, 9H), 0.21 (s, 6H); EI-MS m / z: 612(M + +1).

[0304] Preparation of compound Mono-10-10 A yellow solution of Mono-10-9 (3.0 g, 4.9 mmol) and 2,6-lutidine (6.9 mL, 58.8 mmol) in anhydrous DCM (150 mL) was treated with triflic anhydride (8.25 mL, 49 mmol) at −10° C. under N2 atmosphere and stirred for 6 h. The reaction mixture was warmed to room temperature and stirred for 1 h. The reaction mixture was diluted with distilled water (50 mL) and extracted with DCM (100 mL×2). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA=4:1) to give compound Mono-10-10 (1.3 g, 36%) as a yellow oil. 1 H NMR (400 MHz,CDCl3) δ 8.78 (brs, 1H), 7.97 (s, 1H), 6.85 (s, 1H), 6.80 (s, 1H), 4.86 - 4.72 (m, 3H), 4.20 - 4.32 (m, 1H), 3.80 (s, 3H), 3.76 - 3.68 (m, 1H), 3.20 - 3.00 (m, 2H), 2.33 (s, 3H), 0.89 (s, 9H), 0.06 (d, J = 10.6 Hz 6H) ;EI-MS m / z: 745(M + +1).

[0305] Preparation of Compound Mono-10-11 A yellow solution of Mono-10-10 (760 mg, 1.02 mmol) in toluene (8.0 mL), H2O (1.2 mL), and ethanol (8.0 mL) was treated with 4-methoxybenzeneboronic acid, pinacol ester (286.9 mg, 1.22 mmol), Pd(TPP)4 (122 mg, 0.12 mmol), and TEA (185 μL, 2.04 mmol) at room temperature under N2 atmosphere, then stirred for 2 h. The mixture was diluted with distilled water (100 mL) and extracted with EA (100 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA=3:1) to give compound Mono-10-11 (420.9 mg, 59%) as a yellow foam solid. 1 H NMR (400 MHz,CDCl3) δ 8.78 (brs, 1H), 7.95 (s, 1H), 7.22 (d, J = 8.4 Hz, 2H), 6.97 (s, 1H), 6.85 (d, J = 8.4 Hz, 1H), 6.81 (s, 1H), 4.87 - 4.69 (m, 3H), 4.09 - 4.02 (m, 1H), 3.93 - 3.88 (m, 1H), 3.80 (d, J = 8.0 Hz, 6H), 3.20 - 3.12 (m, 1H), 3.05 - 2.97 (m, 1H), 2.34 (s, 3H), 0.85 (s, 9H), 0.60 (d, J = 8.4 Hz, 6H) ;EI-MS m / z: 703(M + +1).

[0306] Preparation of compound Mono-10-12 A yellow solution of Mono-10-11 (420 mg, 0.60 mmol) in THE (4.0 mL) and H2O (2.0 mL) was treated with acetic acid (8.0 mL) at room temperature under N2 atmosphere and stirred overnight. The reaction mixture was diluted with distilled water (10 mL) and extracted with EA (20 mL x 2). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA = 3:1) to give compound Mono-10-12 (279.5 mg, 79%) as a yellow foam solid. 1 H NMR (400 MHz,CDCl3) δ 8.85 (brs, 1H), 7.85 (s, 1H), 7.2 (d, J = 8.8 Hz, 2H), 7.00 (s, 1H), 6.85 (d, J = 7.2 Hz, 1H), 6.72 (s, 1H), 4.95 - 4.87 (m, 1H), 4.74 (d, J = 2.8 Hz, 2H), 4.04 - 3.84 (m, 3H), 3.81 (d, J = 3.6 Hz, 6H), 3.34 - 3.24 (m, 1H), 2.72 (dd, J = 13.2, 3.2 Hz, 1H), 2.34 (s, 3H) ;EI-MS m / z: 588(M + +1).

[0307] Preparation of compound Mono-10-13 A homogeneous solution of oxalyl chloride (52.5 μL, 0.61 mmol) in anhydrous DMC (1.0 mL) was treated with DMSO (86.2 μL, 1.22 mmol) in anhydrous DCM (1.0 mL) at −78° C. under N2 atmosphere and stirred for 15 min. A solution of Mono-10-2 (240 mg, 0.40 mmol) in anhydrous DMC (3.0 mL) was added dropwise to the reaction mixture, which was then stirred for 3 h, followed by the addition of TEA (569 μL, 4.08 mmol). The mixture was stirred at room temperature for 30 min. The reaction mixture was diluted with distilled water (5.0 mL) and extracted with EA (15 mL×2). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA=3:1) to give compound Mono-10-13 (191.4 mg, 80%) as a yellow foam solid. 1 H NMR (400 MHz,CDCl3) δ 7.37 (s, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.11 (s, 1H), 6.90 (d, J = 9.2 Hz, 2H), 5.86 - 5.81 (m, 1H), 5.18 (d, J = 12 Hz, 1H), 4.30 (d, J = 11.6 Hz, 1H), 4.10 - 4.05 (m, 1H), 3.90 (s, 3H), 3.83 (s, 3H), 3.73 (d, J = 4.8 Hz, 1H), 3.44 - 3.35 (m, 1H), 3.12 - 3.05 (m, 1H), 2.37 (s, 3H); EI-MS m / z: 586(M + +1)

[0308] Preparation of compound Mono-10 A yellow solution of Mono-10-13 (150 mg, 0.26 mmol) in MeOH (6.0 mL), H2O (3.0 mL) was treated with K2CO3 (88.5 mg, 0.64 mmol) at room temperature under N2 atmosphere and stirred for 1 h. The reaction mixture was diluted with distilled water (5 mL) and extracted with EA (10 mL x 2). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA = 2:1) to give compound Mono-10 (105 mg, 75%) as a yellow foam solid. 1 H NMR (400 MHz,CDCl3) δ 7.37 (s, 1H), 7.31 (d, J = 8.8 Hz, 2H), 7.30 (s, 1H), 6.94 (s, 1H), 6.89 (d, J = 8.8 Hz, 2H), 5.93 (s, 1H), 5.84 (dd, J = 5.2, 4.4 Hz, 1H), 5.14 (d, J = 11.6 Hz, 1H), 4.32 (d, J = 12 Hz, 1H), 4.07 - 3.99 (m, 1H), 3.97 (s, 3H), 3.83 (s, 1H), 3.64 (d, J = 4.4 Hz, 1H), 3.43 - 3.34 (m, 1H), 3.10 - 3.03 (m, 1H);EI-MS m / z: 544(M + +1)

[0309] Example 13: Preparation of compound D-101 [ka] To a solution of compound Mono-2 (2.0 mg, 0.005 mmol) and compound L-1 (3.3 mg, 0.010 mmol) in DMF (1.0 mL) was added K2CO3 (2.0 mg, 0.012 mmol) at room temperature under N2 atmosphere. After stirring for 3 h, the reaction mixture was purified by preparative HPLC to give compound D-101 (1.2 mg, 34%). EI-MS m / z: 743 (M + +1).

[0310] Example 14: Preparation of compound D-102 [ka] Compound D-102 was synthesized in a manner similar to that described in Example 13. Yield 34%; EI-MS m / z: 743 (M + +1).

[0311] Example 15: Preparation of compound D-103 [ka] To a solution of compound Mono-1 (10.0 mg, 0.03 mmol) and compound L-1 (5.8 mg, 0.015 mmol) in DMF (1.0 mL) was added K2CO3 (9.4 mg, 0.07 mmol) under N2 atmosphere. The mixture was stirred at room temperature for 7 h. After the reaction was completed, the reaction mixture was purified by preparative HPLC to give compound D-103 (5.4 mg, 50%). EI-MS m / z: 747 (M + +1).

[0312] Example 16: Preparation of compound D-104 [ka] Preparation of compound D-104a To a solution of compound Mono-1 (100 mg, 0.318 mmol) and 1,3,5-trisbromomethylbenzene (57 mg, 0.159 mmol) in DMF (3 mL) was added K2CO3 (66 mg, 0.477 mmol) at room temperature under N2 atmosphere. After stirring for 3 h, the reaction mixture was purified by preparative HPLC to give compound D-104a (62 mg, 48%). EI-MS m / z: 824 (M + +1).

[0313] Preparation of compound D-104 To a solution of compound D-104a (62 mg, 0.075 mmol) in DMF (1 mL) was added 1M dimethylamine in THF (0.5 mL) at room temperature under N2 atmosphere. After stirring for 1 h, the reaction mixture was purified by preparative HPLC to give compound D-104 (39 mg, 60%). EI-MS m / z: 788 (M + +1).

[0314] Example 17: Preparation of compound D-105 [ka] Preparation of compound D-105a To a solution of compound Mono-8 (10 mg, 0.03 mmol) and compound Int-1 (25 mg, 0.06 mmol) in DMF (1 mL) was added K2CO3 (4.5 mg, 0.03 mmol) under N2 atmosphere at 40° C. After stirring for 2 h, the reaction mixture was purified by preparative HPLC to give compound D-10a (9 mg, 45%). EI-MS m / z: 622 (M + +1).

[0315] Preparation of compound D-105 To a solution of compound D-105a (9 mg, 0.01 mmol) and compound Mono-4 (2 mg, 0.01 mmol) in DMF (1 mL) was added K2CO3 (2 mg, 0.01 mmol) under N2 atmosphere at 40 ° C. After stirring for 15 h, the reaction mixture was purified by preparative HPLC to give compound D-105 (1.1 mg, 23%). EI-MS m / z: 739 (M + ).

[0316] Example 18: Preparation of compound D-106 [ka] Compound D-106 was synthesized in a manner similar to that described in Example 16. Compound D-106a EI-MS m / z: 820 (M+ +1). Compound D-106 (2 steps, yield 4%). EI-MS m / z: 784 (M + +1)

[0317] Example 19: Preparation of compound D-107 [ka] Compound D-107 was synthesized in a manner similar to that described in Example 16. Compound D-107a EI-MS m / z: 792 (M + +1). Compound D-107 2 steps, 16% yield; EI-MS m / z: 756 (M + +1).

[0318] Example 20: Preparation of compound D-108 [ka] Compound D-108 was synthesized in a manner similar to that described in Example 16. Compound D-108a EI-MS m / z: 824 (M + +1). Compound D-108 2 steps, 16% yield; EI-MS m / z: 788 (M + +1).

[0319] Example 21: Preparation of compound D-109 [ka] Compound D-109 was synthesized in a manner similar to that described in Example 16. Compound D-109a Yield: 20% EI-MS m / z: 982 (M + +1). Compound D-109 Yield: 56% EI-MS m / z: 946 (M + +1).

[0320] Example 22: Preparation of compound D-110 [ka] To a solution of compound D-102 (3.5 mg, 0.005 mmol) in DMF (1 mL) was added iodomethane (0.1 mL) at room temperature under N2 atmosphere. After stirring for 5 h, the reaction mixture was purified by HPLC to give compound D-110 (1.2 mg, 33%). EI-MS m / z: 772 (M + +1).

[0321] Example 23: Preparation of compound D-111 [ka] Compound D-111 was synthesized in a manner similar to that described for compound D-105 in Example 17. Compound D-111a Yield 54%, white solid. EI-MS m / z: 628 (M + +1). Compound D-111 Yield 11%, white solid. EI-MS m / z: 745 (M + +1)

[0322] Example 24: Preparation of compound D-112 [ka] Preparation of compound D-112a A yellow solution of compound Mono-1 (10 mg, 0.032 mmol) and 1,3,5-tris(bromomethyl)benzene (11.35 mg, 0.032 mmol, 1.0 equiv.) in DMF (1 mL) was treated with K2CO3 (4.4 mg, 0.032 mmol, 1.0 equiv.) at room temperature under N2 atmosphere and stirred for 5 h. The reaction mixture was purified by preparative HPLC (column: Innoval ODS-2 10 um, 100 Å, 21.2×250 mm, flow rate: 15 mL / min, A buffer 0.1% formic acid in water / B buffer 0.1% formic acid in ACN, method gradient, Solvent A:Solvent B 95:5 to 5:95, 1 h, wavelength 214 nm) to give compound D-112a (7.5 mg, 40%) as a white solid. EI-MS m / z: 591(M + +1).

[0323] Preparation of compound D-112 A homogeneous solution of compound D-112a (7.5 mg, 0.013 mmol) and Mono-4 (3.9 mg, 0.013 mmol, 1.0 equiv.) in DMF (1 mL) was treated with K2CO3 (1.8 mg, 0.013 mmol, 1.0 equiv.) at room temperature under N2 atmosphere and stirred for 6 h. The reaction mixture was treated with 1 M dimethylamine in THF (0.5 mL) and stirred for 30 min. The reaction mixture was purified by preparative HPLC (column: Innoval ODS-2 10 um, 100 Å, 21.2×250 mm, flow rate: 15 mL / min, A buffer 0.1% formic acid in water / B buffer 0.1% formic acid in ACN, method gradient, Solvent A:Solvent B 95:5 to 5:95, 1 h, wavelength 214 nm) to give compound D-112 (0.9 mg, 9%) as a white solid. EI-MS m / z: 786(M + +1).

[0324] Example 25: Preparation of compound D-113 [ka] Compound D-113 was synthesized in the same manner as in the synthesis of compound D-105 in Example 17. Compound D-113a Yield 32%, white solid. EI-MS m / z: 494 (M + +1). Compound D-113 7% yield, white solid. EI-MS m / z: 725 (M + +1)

[0325] Example 26: Preparation of compound D-114 [ka] Preparation of compound D-114a A homogeneous solution of compound D-112a (20 mg, 0.034 mmol) and Mono-10 (18.4 mg, 0.034 mmol) in DMF (1 mL) was treated with K2CO3 (4.7 mg, 0.034 mmol) at room temperature under N2 atmosphere and stirred for 5 h. The reaction mixture was treated with 1 M dimethylamine in THF (0.5 mL) and stirred for 30 min. The reaction mixture was purified by preparative HPLC (column: Innoval ODS-2 10 um, 100 Å, 21.2×250 mm, flow rate: 15 mL / min, A buffer 0.1% formic acid in water / B buffer 0.1% formic acid in ACN, method gradient, Solvent A:Solvent B 95:5 to 5:95, 1 h, wavelength 214 nm) to give compound D-114a (3.4 mg, 9.8%) as a white solid. EI-MS m / z: 1018(M + +1).

[0326] Preparation of compound D-114 A solution of compound D-114a (3.4 mg, 0.003 mmol) and 10% Cd / Pb (100 mg) in THF (0.5 mL) was treated with 1N NH4OAc (300 μL) at room temperature under N2 atmosphere and stirred for 3 days. The reaction mixture was purified by preparative HPLC (column: Innoval ODS-2 10 um, 100 Å, 21.2×250 mm, flow rate: 15 mL / min, A buffer 0.1% formic acid in water / B buffer 0.1% formic acid in ACN, method gradient, solvent A:solvent B 95:5 to 5:95, 1 h, wavelength 214 nm) to give compound D-114 (0.8 mg, 29%) as a white solid. EI-MS m / z: 824 (M ++1).

[0327] Example 27: Preparation of compound Ref-1 [ka] Preparation of compound Ref-1a Compound Mono-8 (100 mg, 0.32 mmol) and 1,3,5-tris(bromomethyl)benzene (57 mg, 0.16 mmol) were dissolved in DMF (20 mL) at room temperature under N2 atmosphere, and then K2CO3 (45 mg, 0.32 mmol) was added thereto. The mixture was stirred at room temperature for 4 h. After the reaction was completed, EA (100 mL), H2O (50 mL), and 2N HCl aqueous solution (5 mL) were added for extraction, and the obtained organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Ref-1a (54 mg, 42%). EI-MS m / z: 812 (M + +1).

[0328] Preparation of compound Ref-1 Compound Ref-1a (50 mg, 0.01 mmol) was dissolved in dimethylamine (1 mL) at room temperature under N2 atmosphere. After stirring for 1 h, the mixture was purified by preparative HPLC to give compound Ref-1 (2.2 mg, 17%). EI-MS m / z: 776(M + ).

[0329] Example 28: Preparation of compound Ref-2 [ka] To a solution of compound Mono-8 (10.0 mg, 0.03 mmol) and 1,3-bis(bromomethyl)benzene (4.1 mg, 0.015 mmol) in DMF (1.0 mL) was added K2CO3 (4.7 mg, 0.034 mmol) under N2 atmosphere. The mixture was stirred at room temperature overnight. After the reaction was completed, the mixture was purified by preparative HPLC to give compound Ref-2 (8.0 mg, 71%). EI-MS m / z: 719 (M + +1).

[0330] Example 29: Preparation of compound Ref-3 [ka] Compound Ref-3 was synthesized in a similar manner to that described in Example 13. Yield 35%; EI-MS m / z: 734 (M + +1).

[0331] Example 30: Preparation of compound L-2 [ka] Preparation of compound L-2 To a solution of compound L-1-2 (1.0 g, 3.57 mmol) in DCM (35 mL) was added TEA (0.45 mL, 3.21 mmol) at room temperature under N2 atmosphere. SO2F2 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-2 (941.7 mg, 73%). 1 H NMR (400 Hz, CDCl3) δ 7.47 (s, 1H), 7.32 (s, 2H), 4.46 (s, 4H).

[0332] Example 31: Preparation of compound L-3 [ka] Compound L-3 was synthesized in a manner similar to that described in Journal of Polymer Science, Part A: Polymer Chemistry, 2012, 50(19), 3986-3995 (incorporated herein by reference). Preparation of compound L-3-1 Yield 30% 1 H NMR (400 Hz, CDCl3) δ 7.80 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 4.16 (t, J = 4.8 Hz, 2H), 3.74 - 3.58 (m, 14H), 2.45 (s, 3H). Preparation of compound L-3-2 Yield 68% 1 H NMR (400 Hz, CDCl3) δ 3.74 - 3.61 (m, 14H), 3.40 (t, J = 4.8 Hz, 2H), 2.45 (t, J = 6.0Hz, 2H). Preparation of compound L-3-3 Yield 63% 1 H NMR (400 Hz, CDCl3) δ 4.21 (d, J = 2.4 Hz, 2H), 3.72 - 3.67 (m, 14H), 3.39 (t, J = 5.2 Hz, 2H), 2.43 (t, J = 2.4 Hz, 1H). Preparation of compound L-3 Yield: 76% 1 H NMR (400 Hz, CDCl3) δ 4.20 (d, J = 2.4 Hz, 2H), 3.7 - 3.61 (m, 12H), 3.51 (t, J =4.8 Hz, 2H), 2.87 (t, J = 5.6 Hz, 2H), 2.43 (t, J = 2.4 Hz, 1H).

[0333] Example 32: Preparation of compound L-4

change

[0334] Example 33: Preparation of compound L-5 [ka] Preparation of compound L-5-1 To a solution of hexaethylene glycol (5.0 g, 17.71 mmol) in anhydrous DCM (178 mL) was added KI (294 mg, 1.77 mmol), Ag2O (4.92 g, 19.48 mmol), and p-TsCl (3.7 g, 19.48 mmol) under N2 atmosphere. The mixture was stirred at room temperature overnight. After the reaction was completed, the 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-5-1 (5.98 g, 73%). 1 H NMR (400 Hz, CDCl3) δ 7.80 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 4.16 (t, J = 4.8 Hz, 2H), 3.71 - 3.58 (m, 22H), 2.88 (br, 1H), 2.45 (s, 3H).

[0335] Preparation of compound L-5-2 To a solution of compound L-5-1 (5.98 g, 13.7 mmol) in DMF (30 mL) was added NaN3 (1.34 g, 20.55 mmol) under N2 atmosphere. The mixture was stirred at 110 °C for 1 h and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-5-2 (4.1 g, 97%). 1 H NMR (400 Hz, CDCl3) δ 3.72 - 3.60 (m, 22H), 3.39 (t, J = 4.8 Hz, 2H), 2.78 (br, 1H). Preparation of compound L-4-3 Compound L-5-2 (1.9 g, 6.18 mmol) was dissolved in DCM (20 mL) under N2 atmosphere, to which triethylamine (2.0 mL, 14.22 mmol) and p-TsCl (2.4 g, 12.36 mmol) were added, and the mixture was stirred at room temperature overnight. After the reaction was completed, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-5-3 (2.58 g, 91%). 1 H NMR (400 Hz, CDCl3) δ 7.80 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 4.16 (t, J = 4.8 Hz, 2H), 3.70 - 3.61 (m, 16H), 3.56 (s, 1H), 3.39 (t, J = 4.8 Hz, 2H), 2.45 (s, 3H). EI-MS m / z: 462 (M + +1).

[0336] Preparation of compound L-5-4 A homogeneous solution of compound L-4 (1.1 g, 3.4 mmol) in anhydrous THF (30 mL) was treated with NaH (60% dispersion in mineral oil, 135 mg, 3.4 mmol) under N2 atmosphere at 0°C. The mixture was stirred at the same temperature for 20 min, and then compound L-5-3 (1.56 g, 3.4 mmol) was added to it. The reaction was allowed to warm to room temperature and stirred overnight. After the reaction was completed, the reaction mixture 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-5-4 (1.91 g, 93%). EI-MS m / z: 610 (M + +1).

[0337] Preparation of compound L-5 Compound L-5-4 (906.7 mg, 1.49 mmol) was dissolved in EA (4 mL) and ether (4 mL) under N2 atmosphere and then cooled to 0°C. To this was added 5% HCl solution (8 mL), triphenylphosphine (390 mg, 1.49 mmol) was added slowly, and the mixture was diluted with DCM (10 mL). The aqueous layer was extracted with DCM (10 mL x 3). The aqueous layer was concentrated under high vacuum to give compound L-5 (495 mg, 54%). EI-MS m / z: 584 (M + +1).

[0338] Example 34: Preparation of compounds L-6 and L-7 [ka] Preparation of compound L-6-1 To a solution of compound L-5-2 (1.0 g, 3.25 mmol) in EtOH (5 mL) was added 5% Pd / C (1.04 g, 0.49 mmol) under H2 atmosphere. The mixture was stirred at room temperature 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). BOC2O (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-6-1 (330 mg, 28%). 1 H NMR (400 Hz, CDCl3) δ 5.19 (brs, 1H), 3.73 (t, J = 4.8 Hz, 2H), 3.67 (s, 12H), 3.63 - 3.60 (m, 6H), 3.54 (t, J = 5.2 Hz, 2H), 3.34 - 3.27 (m, 1H), 1.44 (s, 9H). EI-MS m / z: 382 (M + +1).

[0339] Preparation of compound L-6-2 A homogeneous solution of compound L-6-1 (450 mg, 1.18 mmol) in anhydrous THF (10 mL) was treated with NaH (60% dispersion in mineral oil, 47.2 mg, 1.18 mmol) under N2 atmosphere at 0°C. The mixture was stirred at 0°C for 20 min, and then L-5-3 (544.5 mg, 1.18 mmol) was added to it. 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-6-2 (582.9 mg, 74%).

[0340] Preparation of compound L-6 To a solution of compound L-6-2 (582.9 mg, 0.87 mmol) in DCM (3 mL) was added 4M-HCl (in 1,4-dioxane, 1 mL) under N2 atmosphere at 0° C. The mixture was stirred at room temperature for 2 h. The mixture was concentrated to give compound L-6 (527.6 mg, quantitative). EI-MS m / z: 571 (M + +1).

[0341] Preparation of compound L-7 Compound L-7 (quantitative, colorless oil) was synthesized in a similar manner to the preparation of compound L-5 in Example 33. EI-MS m / z: 645(M + +1).

[0342] Example 35: Preparation of compound L-8 [ka] Preparation of compound L-8-1 A homogeneous solution of 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 H2O (25 mL) was treated with NaHCO3 (3.8 g, 45.8 mmol, 2.0 equiv.) and BOC2O (6.0 g, 27.5 mmol, 1.2 equiv.) at room temperature under N2 atmosphere 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 dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (1% to 3% MeOH in DCM) to give compound L-8-1 (7.2 g, 99%) as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 5.03 (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).

[0343] Preparation of compound L-8 Compound L-8 (quantitative, colorless oil) was synthesized in a similar manner to the preparation of compound L-5 in Example 33. Yield 95%, colourless oil. EI-MS m / z: 293 (M + +1)

[0344] Example 36: Preparation of compound L-9 [ka] Preparation of compound L-9-1 A homogeneous solution of Boc-L-serine methyl ester (5.0 g, 22.8 mmol) in DCM (30 mL) was treated with pyridine (8 mL), para-toluenesulfonyl chloride (5.22 g, 27.4 mmol) at room temperature under N2 atmosphere and stirred overnight. The reaction was quenched by adding water (50 mL) and extracted with EA (100 mL x 3). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA = 9:1 to 2:1) to give compound L-9-1 (7.0 g, 82%) as a white solid. 1 H NMR (600 MHz, CDCl3) δ 7.76 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 7.8 Hz, 2H), 5.29 (S, 1H), 4.53 - 4.47 (m, 1H), 4.39 (dd, J = 2.4, 7.8 Hz, 1H), 4.29 (d, J = 7.2, 2.4 Hz, 1H), 3.69 (s, 3H), 2.45 (s, 3H).

[0345] Preparation of compound L-9-2 A suspension of CsCO3 (1.05 g, 3.21 mmol, 0.6 equiv) in DMF (12 mL) was treated with a solution of thioacetic acid (498 μL, 6.96 mmol) and L-9-1 (2.0 g, 5.36 mmol) in DMF (8 mL) at room temperature under N2 atmosphere and stirred overnight. The mixture was quenched by adding water (50 mL) and extracted with EA (100 mL × 3). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA = 5:1) to give compound L-9-2 (1.4 g, 95%) as a white solid. 1 H NMR (600 MHz, CDCl3) δ 5.24 (s, 1H), 4.53-4.49 (m, 1H), 3.75 (s, 3H), 2.45 (s, 3H), 4.41-4.31 (m, 2H).

[0346] Preparation of compound L-9-3 To a solution of compound L-9-2 (1.2 g, 4.33 mmol) in AcOH (10 mL) was added 35% hydrogen peroxide (4 mL) at room temperature under N2 atmosphere. The mixture was stirred for 7 h and then concentrated under reduced pressure. The residue was diluted with water (5 mL) and basified to pH 9 with saturated aqueous NaHCO3 at 0 °C. Boc2O (1.4 g, 6.49 mmol, 1.5 equiv) was added and the resulting mixture was stirred overnight. The mixture was neutralized with 2N HCl at 0 °C and concentrated under reduced pressure. The residue was purified by column chromatography (DCM:MeOH:AcOH = 8:1:0.01 to 5:1:0.01) to give compound L-9-3 (521.5 mg, 42%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 6.96 (d, J = 7.2 Hz, 1H), 4.20 (q, J = 6.8, 4.8 Hz, 1H), 3.58 (s, 3H), 2.84 (dd, J = 14, 6.4 Hz, 1H), 2.76 (dd, J = 9.2, 4.4 Hz, 1H), 1.37 (s, 9H).

[0347] Preparation of compound L-9-4 A homogeneous solution of L-9-3 (71 mg, 0.25 mmol) in THF / HO (2.0 mL / 4.0 mL) was treated with LiOH (17.3 mg, 0.41) at room temperature under N2 atmosphere and stirred for 3 h. The mixture was neutralized with 2N HCl at 0° C. and concentrated under reduced pressure to give compound L-9-4 (67 mg, 99%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 6.40 (d, J = 7.2 Hz, 1H), 3.96 (q, J = 6.4, 5.6 Hz, 1H), 2.88 - 2.78 (n, 2H), 1.36 (s, 9H).

[0348] Preparation of compound L-9 L-8-4 (35 mg, 0.13 mmol), N-hydroxysuccinimide (22.4 mg, 0.19 mmol), and EDCI-HCl (50 mg, 0.26 mmol) were dissolved in DMF (2 mL) at room temperature under N2 atmosphere. The mixture was stirred overnight, after which compound L-9 was used directly in the next step without further purification. EI-MS m / z: 367 (M + +1).

[0349] Example 37: Preparation of BGal-Br (hereinafter "Int-TG") [ka] 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 N2 atmosphere. The mixture was allowed to warm to room temperature. After stirring at room temperature for 4 h, the mixture was concentrated under reduced pressure, then EA (1000 mL) and saturated aqueous sodium bicarbonate (1000 mL) were added. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG (5.2 g, 99%). 1 H NMR (400 Hz, CDCl3) δ 6.70 (d, J = 4.0 Hz, 1H), 5.52 (d, J = 2.4 Hz, 1H), 5.41 (dd, J = 7.6, 2.8 Hz, 1H), 5.05 (dd, J = 6.4, 4.0 Hz, 1H), 4.49 (t, J = 6.4 Hz, 1H), 4.22 - 4.09 (m, 2H), 2.16 - 2.01 (m, 12H).

[0350] Example 38: Preparation of compounds Int-TG1 and Int-TG2 [ka] Preparation of compound Int-TG1-1 To a solution of 3-formyl-4-hydroxybenzoic acid (5 g, 43.06 mmol) in DMF (100 mL) was added benzyl bromide (5.1 mL, 43.06 mmol) and NaHCO3 (2.53 g, 43.06 mmol) at room temperature under N2 atmosphere. The mixture was stirred at room temperature under N2 atmosphere overnight. The reaction was extracted with EA (200 mL x 2) and distilled water (100 mL). The obtained organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-TG1-1 (2.56 g, 39%). 1 H NMR (400 Hz, CDCl3) δ 11.41 (s, 1H), 9.95 (s, 1H), 8.34 (d, J = 2.0 Hz, 1H), 8.23 ​​(dd, J = 6.4 Hz, 2.4 Hz, 1H), 7.46 - 7.35 (m, 5H), 7.04 (d, J = 9.2 Hz, 1H), 5.37 (s, 2H).

[0351] Preparation of compound Int-TG1-2 To a solution of compound Int-TG1-1 (1.0 g, 3.90 mmol) and compound Int-TG (1.6 g, 3.90 mmol) in anhydrous ACN (30 mL), molecular sieves (8 g) and Ag2O (3.62 g, 15.61 mmol) were added at room temperature under N2 atmosphere. The mixture was stirred at room temperature for 1 hour and then filtered through CELITE®. The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG1-2 (2.1 g, 92%). 1H NMR (400 Hz, CDCl3) δ 10.34 (s, 1H), 8.55 (d, J = 2.0 Hz, 1H), 8.26 (dd, J = 6.8, 2.0 Hz, 1H), 7.45 - 7.35 (m, 5H), 7.17 (d, J = 8.8 Hz, 1H), 5.63 - 5.60 (m, 1H), 5.50 (d, J = 3.6 Hz, 1H), 5.37 (s, 2H), 5.23 (d, J = 8.0 Hz, 1H), 5.16 (dd, J = 7.2, 3.6 Hz, 1H) 4.24 - 4.10 (m, 4H), 2.20 (s, 3H), 2.10 - 2.03 (m, 9H).

[0352] Preparation of compound Int-TG1-3 To a solution of compound Int-TG1-2 (2.1 g, 3.58 mmol) in DCM (30 mL) was added m-CPBA (2.65 g, 10.74 mmol) at 0° C. under N2 atmosphere. After stirring at 0° C. for 7 h, the mixture was quenched by adding saturated sodium bicarbonate (40 mL×2). The mixture was separated and the organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was dissolved in DCM (5 mL) and hydrazine hydrate (261 μL, 5.37 mmol) was added to the solution at 0° C. under N2 atmosphere. After stirring at 0° C. for 1 h, EA (30 mL×2) and 1M HCl aqueous solution (10 mL) were added. The resulting organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give compound Int-TG1-3 (1.1 g, 55%). EI-MS m / z: 574 (M + +Na)

[0353] Preparation of compound Int-TG1-4 To a solution of compound Int-TG1-3 (280 mg, 0.49 mmol) in DCM (5 mL) was added TBDMS-OTf (224 μL, 0.97 mmol) and Et3N (207 μL, 1.46 mmol) at 0° C. under N2 atmosphere. The mixture was stirred at room temperature for 1.5 h and then quenched by adding citric acid (20 mL). The organic layer was washed with brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG1-4 (246.3 mg, 68%). 1 H NMR (400 Hz, CDCl3) δ 7.67 (d, J = 8.4 Hz, 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 = 7.6 Hz, 1H), 5.10 (dd, J = 6.8, 3.2 Hz, 1H) 4.20 - 4.11 (m, 2H), 4.05 (t, J = 6.8 Hz, 2H), 2.19 (s, 3H), 2.04(s, 3H), 2.01 (d, J = 6.0 Hz, 6H), 1.02 (s, 9H), 0.20 (d, J = 15.6 Hz, 6H).

[0354] Preparation of compound Int-TG1-5 To a solution of compound Int-TG1-4 (283.2 mg, 0.41 mmol) in EA (5 mL) was added Pd / C (5%, 87.5 mg, 0.04 mmol) at room temperature under H2. The mixture was stirred for 1 h, filtered through CELITE®, and then concentrated under reduced pressure. Compound Int-TG1-5 was used directly in the next step without further purification (246 mg, quantitative). 1H NMR (400 Hz, CDCl3) δ 7.67 (d, J = 8.8 Hz, 1H), 7.57 (s, 1H), 7.05 (d, J = 8.4 Hz, 1H), 5.49 - 5.45 (m, 2H), 5.22 (d, J = 7.6 Hz, 1H), 5.12 (dd, J = 7.2, 3.6 Hz, 1H) 4.20 - 4.06 (m, 4H), 2.19 (s, 3H), 2.05(s, 3H), 2.02 (d, J = 7.6 Hz, 6H), 1.01 (s, 9H), 0.21 (d, J = 15.2 Hz, 6H).

[0355] Preparation of compound Int-TG1 To a solution of compound Int-TG1-5 (243.2 mg, 0.41 mmol) and 11-azido-3,6,9-trioxaundecan-1-amine (Aldrich, CAS 134179-38-7, 89.5 mg, 0.41 mmol) in DMF (5 mL), PyBOP (275 mg, 0.53 mmol) and DIPEA (176 μL, 1.02 mmol) were added at room temperature under N2 atmosphere. The mixture was stirred at room temperature for 2 h under N2 atmosphere. The reaction was extracted with EA (30 mL x 2) and distilled water (10 mL). The obtained organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG1 (272.8 mg, 84%). 1H NMR (400 Hz, CDCl3) δ 7.34(s, 1H), 7.31 (d, J = 9.2 Hz, 1H), 7.02 (d, J= 8.0 Hz, 1H), 6.73(s, 1H), 5.48 - 5.44 (m, 2H), 5.19 (d, J = 7.6 Hz, 1H), 5.10 (dd, J = 6.4, 3.6 Hz, 1H), 4.20 - 4.10 (m, 2H), 4.06 (t, J = 6.4 Hz, 2H), 3.66 (s, 14H), 3.38 (t, J = 4.4 Hz, 2H), 2.19 (s, 3H), 2.02 (t, J = 8.4 Hz, 9H), 1.00 (s, 9H), 0.20 (d, J = 14.4 Hz, 6H). EI-MS m / z: 799 (M + +1).

[0356] Preparation of compound Int-TG2 To a solution of compounds Int-TG1-5 (246 mg, 0.41 mmol) and L-6 (249.5 mg, 0.41 mmol) in DMF (3 mL), PyBOP (278 mg, 0.53 mmol) and DIPEA (179 uL, 1.02 mmol) were added at room temperature under N2 atmosphere. After stirring the mixture for 2 h, the reaction mixture was subjected to preparative HPLC to obtain compound Int-TG2 (384.6 mg, 81%). EI-MS m / z: 1152 (M + +1).

[0357] Example 39: Preparation of compounds Int-TG3 and Int-TG4 [ka] Preparation of compound Int-TG3-1 A homogeneous solution of compound Int-TG1-5 (1.0 g, 0.26 mmol) and L-8 (586 mg, 2.0 mmol, 1.2 equiv) in DMF (10 mL) was treated with PyBOP (1.13 g, 2.17 mmol, 1.3 equiv) and DIPEA (873 uL, 5.01 mmol, 3.0 equiv) at room temperature under N2 atmosphere and stirred for 4 h. The reaction was quenched with water (20 mL) and extracted with EA (30 mL x 2). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (Hex:EA = 1:1 to 1:3) to give compound Int-TG3-1 (1.05 g, 72%) as a white foam solid. EI-MS m / z: 874 (M + +1).

[0358] Preparation of compound Int-TG3a To a solution of 4-hydroxybenzaldehyde (1 g, 8.19 mmol) in DCM (3 mL) was added Et3N (2.28 mL, 16.38 mmol) at room temperature under N2 atmosphere. SO2F2 gas was introduced via 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 dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG3a (790 mg, 63%). 1 H NMR (400 Hz, CDCl3) δ 10.06 (s, 1H), 8.05 (d, J = 8.0 Hz, 2H), 7.55 (d, J = 8.8 Hz, 2H).

[0359] Preparation of compound Int-TG3-2 A homogeneous solution of compound Int-TG3-1 (500 mg, 0.57 mmol) and compound Int-TG3a (140 mg, 0.69 mmol, 1.2 equiv) in anhydrous ACN (10 mL) was treated with BEMP (66.3 μL, 0.23 mmol, 0.4 equiv) at room temperature under N2 atmosphere and stirred for 4 h. The reaction was quenched with water (20 mL) and extracted with EA (30 mL×2). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (4% MeOH in DCM) to give compound Int-TG3-2 (495 mg, 85%) as a white foam solid. EI-MS m / z: 869 (M + +1).

[0360] Preparation of compound Int-TG3-3 To a solution of compound Int-TG3-2 (495 mg, 0.52 mmol) in anhydrous THF (5.0 mL) at 0° C. under N2 atmosphere, NaBH4 (39.7 mg, 1.05 mmol, 2.0 equiv) was added and the resulting mixture was stirred for 2 h. The reaction was quenched with water (20 mL) and extracted with EA (30 mL×2). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (2%-3% MeOH in DCM) to give compound Int-TG3-3 (418 mg, 91%) as a white foam solid. EI-MS m / z: 945 (M + +1).

[0361] Preparation of compound Int-TG3-4 To a solution of compound Int-TG3-3 (214.2 mg, 0.23 mmol) in anhydrous THF (5.0 mL) at 0° C. under N2 atmosphere, methanesulfonyl chloride (24.6 μL, 0.32 mmol, 1.4 equiv.) and TEA (79.2 μL, 0.57 mmol, 1.5 equiv.) were added and the resulting mixture was stirred at room temperature overnight. The reaction was quenched with water (10 mL) and extracted with DCM (20 mL×2). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (100% DCM to 5% MeOH in DCM) to give compound Int-TG3-4 (164 mg, 70%) as a white foam solid. EI-MS m / z: 1024 (M + +1).

[0362] Preparation of compound Int-TG3 To a solution of compound Int-TG3-4 (164 mg, 0.16 mmol) in anhydrous THF (10 mL) at room temperature under N2 atmosphere, LiBr (69.6 mg, 0.80 mmol, 5.0 equiv.) was added and the resulting mixture was stirred for 3 h. The reaction was diluted with water (10 mL) and extracted with DCM (20 mL x 2). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (3%-5% MeOH in DCM) to give compound Int-TG3 (161 mg, 99%) as a white foam solid. EI-MS m / z: 1008 (M + +1).

[0363] Compound Int-TG4 was synthesized in the same manner as in the preparation of compound Int-TG3. Preparation of compound Int-TG4-1 Yield 72%, colourless oil. EI-MS m / z: 1226 (M + +1). Preparation of compound Int-TG4-2 Yield 82%, colourless oil. EI-MS m / z: 1296 (M + +1). Preparation of compound Int-TG4-3 Yield 75%, colourless oil. EI-MS m / z: 1298 (M + +1). Preparation of compound Int-TG4-4 Yield 82%, colourless oil. EI-MS m / z: 1376 (M + +1). Preparation of compound Int-TG4 Yield 82%, colourless oil. EI-MS m / z: 1361 (M + +1).

[0364] Example 40: Preparation of compound Int-TG5 [ka] Preparation of compound Int-TG5-1 To a solution of compound Int-TG1 (100 mg, 0.13 mmol) and compound Int-TG3a (26 mg, 0.13 mmol) in anhydrous ACN (3 mL) was added DBU (4 μL, 25 μmol). The mixture was stirred at room temperature for 1 h, washed with distilled water (10 mL), and extracted with EA (15 mL×2). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG5-1 (103 mg, 94%). EI-MS m / z: 869 (M + +1).

[0365] Preparation of compound Int-TG5-2 To a solution of compound Int-TG5-1 (103 mg, 0.12 mmol) in THF (8 mL) was added NaBH4 (9 mg, 0.24 mmol) at 0° C. under N2 atmosphere. After stirring at room temperature for 2 h, distilled water (10 mL) and EA (10 mL×2) were added. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give compound Int-TG5-2 (101 mg, 98%). EI-MS m / z: 871 (M + +1).

[0366] Preparation of compound Int-TG5-3 To a solution of compound Int-TG5-2 (320.5 mg, 0.0.37 mmol) in DCM (3 ml) was added 1M PBr3 in DCM (165 μL, 0.19 mmol) under N2 atmosphere at 0° C. After stirring at 0° C. for 2 h, the mixture was quenched by adding saturated sodium bicarbonate (8 mL×2). The mixture was separated and the organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG5-3 (202.6 mg, 59%). EI-MS m / z: 934 (M + +1).

[0367] Preparation of compound Int-TG5 To a solution of compound Int-TG5-3 (10 mg, 0.01 mmol) in DMF (1 mL) was added dimethylamine (0.1 mL) at room temperature under N2 atmosphere. After stirring at room temperature for 10 min, the reaction mixture was purified by preparative HPLC to give compound Int-TG5 (6 mg, 63%). EI-MS m / z: 898 (M + +1).

[0368] Example 41: Preparation of compound MPS-D1 [ka] Preparation of compound MPS-D1-1 To a solution of 4-acetylbenzoic acid (9 g, 54.82 mmol) in EtOH (50 mL) was added piperidine hydrochloride (6.66 g, 54.82 mmol), paraformaldehyde (4.95 g, 164.5 mmol), and concentrated HCl (0.6 mL) at room temperature under N2 atmosphere. The mixture was stirred at 100° C. for 16 h, then cooled to room temperature, to which acetone (90 mL) was added dropwise. The mixture was stirred at 0° C. for 1 h. The solid was filtered and washed with diethyl ether (30 mL×2) to obtain compound MPS-D1-1 (6.11 g, 38%). 1H NMR (400 Hz, DMSO-d6) δ 8.08 (s, 4H), 5.73 (s, 1H), 3.65 (t, J = 7.2 Hz, 2H), 3.35 (t, J = 7.2 Hz, 2H), 3.31 (m, 6H), 1.74 (s, 4H).

[0369] Preparation of compound MPS-D1-2 To a solution of MPS-D1-1 (6.11 g, 20.52 mmol) in EtOH (40 mL) and MeOH (26 mL) was added 4-methoxybenzenethiol (2.55 g, 20.52 mmol) and piperidine (0.3 mL, 3.08 mmol) at room temperature. The mixture was stirred at 100° C. for 16 h, cooled to 0° C., and stirred for another 1 h. The solid was filtered and washed with ether (30 mL×2) to give compound MPS-D1-2 (5.56 g, 90%). 1 H NMR (400 Hz, CDCl3) δ 8.04 - 7.99 (m, 4H), 7.27 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 7.6 Hz, 2H), 3.39-3.36 (m, 2H), 3.25-3.21 (m, 2H), 2.27 (s, 3H).

[0370] Preparation of compound MPS-D1 To a solution of MPS-D1b (5.56 g, 18.51 mmol) in MeOH (90 mL) and distilled water (90 mL) was added oxone (25.03 g, 40.72 mmol) at 0° C. under N2 atmosphere. After stirring at room temperature for 14 h, the mixture was quenched with distilled water (100 mL) and chloroform (150 mL×3). The organic layer was washed with brine (200 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain compound MPS-D1 (5.29 g, 86%). 1H NMR (400 Hz, CDCl3) δ 8.04 - 7.99 (m, 4H), 7.81 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 3.63 (t, J = 7.2 Hz, 2H), 3.41 (t, J = 7.2 Hz, 2H), 2.44 (s, 3H). EI-MS m / z: 333 (M + +1).

[0371] Example 42: Preparation of compound mMPS-D1 [ka] Compound mMPS-D1 was synthesized in a manner similar to that described in Example 41. Preparation of compound mMPS-D1-1 Yield 21%, white solid. EI-MS m / z: 262 (M + +1). Preparation of compound mMPS-D1-2 Yield 72%, white solid. 1 H NMR (600 Hz, DMSO-d6) δ 8.40 (s, 1H), 8.18 - 8.15 (m, 2H), 7.66 - 7.63 (m, 1H), 7.26 (d, J = 7.8 Hz, 2H), 7.14 (d, J = 7.8 Hz, 2H), 3.40 - 3.37 (m, 2H), 3.26 - 3.23 (m, 2H), 2.27 (s, 3H). EI-MS m / z: 301 (M + +1). Preparation of compound mMPS-D1 Yield 47%, yellowish solid. 1H NMR (600 Hz, DMSO-d6) δ 8.37 - 8.36 (m, 1H), 8.20 - 8.16 (m, 2H), 7.81 (d, J = 7.8 Hz, 2H), 7.68 - 7.65 (m, 1H), 7.46 (d, J = 8.4 Hz, 2H), 3.66 - 3.63 (m, 2H), 3.44 - 3.41 (m, 2H), 2.41 (s, 3H). EI-MS m / z: 333 (M + +1).

[0372] Example 43: Preparation of compound pyrMPS-D1 [ka] Preparation of compound pyrMPS-D4-1 A solution of methyl 5-bromonicotinate (3 g, 13.89 mmol), PdCl2(PPh3)2 (487 mg, 0.96 mmol), and tributyl(1-ethoxyvinyl)tin (5.86 mL, 17.36 mmol) in anhydrous toluene (60 mL) was heated to reflux at room temperature under N2 atmosphere for 3 h. The mixture was cooled to 0 °C, filtered through CELITE®, and washed with MeOH (100 mL). The filtrate was concentrated under reduced pressure. The residue was dissolved in MeOH (30 mL) and 10 M HCl (30 mL) at room temperature under N2 atmosphere and allowed to stand for 2 h. The reaction was quenched with saturated Na2CO3 (120 mL) and extracted with EA (150 mL x 3). The organic layer was washed with brine (250 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (EA:HEX=1:2) to give compound pyrMPS-D1-1 (2.22 g, 89%) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 9.37 (s, 1H), 9.31 (s, 1H), 8.79 (s, 1H), 4.00 (s, 3H), 2.69 (s, 3H).

[0373] Preparation of compound pyrMPS-D1-2 A homogeneous solution of pyrMPS-D1-1 (636 mg, 3.55 mmol) in MeOH (11 mL) was treated with 1N NaOH (10.64 mL) at room temperature under N2 atmosphere and stirred for 1 h. The mixture was concentrated under reduced pressure, and the reaction was quenched with 1N HCl (pH 2) and extracted with EA (80 mL x 3). The organic layer was washed with brine (150 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to give compound pyrMPS-D1-2 (crude) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.31 (s, 1H), 9.25 (s, 1H), 8.64 (s, 1H), 2.69 (s, 3H).

[0374] Compounds pyrMPS-D1-3, pyrMPS-D1-4, and pyrMPS-D1 were synthesized by a method similar to the preparation method of compound MPS-D1 in Example 41. Preparation of compound pyrMPS-D1-3 Yield 30%, white solid. EI-MS m / z: 264 (M + +1). Preparation of compound pyrMPS-D1-4 Yield 11%, white solid. 1 H NMR (400 Hz, DMSO-d6) δ 9.26 (d, J = 2 Hz, 1H), 9.23 (d, J = 2 Hz, 1H), 8.58 (m, 1H), 7.26 (d, J = 8 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H), 3.46 (t, J = 7.2 Hz, 2H), 3.24 (d, J = 7.2 Hz, 2H), 2.26 (s, 3H);EI-MS m / z: 302 (M + +1) Preparation of compound pyrMPS-D1 Yield 43%, white solid. EI-MS m / z: 334 (M + +1).

[0375] Example 44: Preparation of compounds MPS-D2, mMPS-D2, and PyrMPS-D2 [ka] Preparation of compound MPS-D2 Compound MPS-D1 (652.4 mg, 1.96 mmol) and compound L-3 (500 mg, 1.96 mmol) were dissolved in DMF (5 mL) under N2 atmosphere. HBTU (893.3 mg, 2.36 mmol) and DIPEA (0.684 mL, 3.93 mmol) were added to it, and the mixture was stirred at room temperature for 3 hours. After the reaction was completed, EA (30 mL x 2) and H2O (20 mL) were added to extract the organic layer. The obtained organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound MPS-D2 (854.1 mg, 80%). 1 H NMR (400 Hz, CDCl3) δ 8.11 - 7.94 (m, 4H), 7.83 (d, J = 7.6 Hz, 2H), 7.44 (brs, 1H), 7.38 (d, J = 8.0 Hz, 2H), 4.15 (s, 2H), 3.69 - 3.65 (m, 14H), 3.58 - 3.48(m, 4H), 2.80 (s, 1H), 2.46(s, 3H). EI-MS m / z: 546 (M + +1).

[0376] Compounds mMPS-D2 and pyrMPS-D2 were synthesized in a manner similar to that of compound MPS-D2. Preparation of compound mMPS-D2 Yield 60%, white solid. EI-MS m / z: 546 (M + +1). Preparation of compound pyrMPS-D2 Yield 22%, yellowish oil. EI-MS m / z: 547 (M + +1).

[0377] Example 45: Preparation of compound MPS-D3 [ka] Compounds MPS-D3 and mMPS-D3 were synthesized by a synthetic route similar to that described in Example 44. Preparation of compound MPS-D3 Yield: 72% EI-MS m / z: 899 (M + +1). Preparation of compound mMPS-D3 Yield 32%, white solid. EI-MS m / z: 899 (M + +1).

[0378] Example 46: Preparation of compound Mal-1 [ka] A homogeneous solution of N-succinimidyl 4-(N-maleimidomethyl)cyclohexanecarboxylate (85.5 mg, 0.26 mmol) and L-3 (75.3 mg, 0.28 mmol, 1.1 equiv) in anhydrous DMC was treated with DIPEA (44.5 μL, 0.26 mmol) at room temperature under N2 atmosphere and stirred at room temperature for 45 h. The reaction mixture was diluted with DCM (32 mL), washed with 1N HCl (30 mL), brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC to give the title compound Mal-1 (70.8 mg, 61%) as a white gum. EI-MS m / z: 451 (M + +1).

[0379] Example 47: Preparation of compound BCN-PNP [ka] (1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethanol (800 mg, 5.3 mmol) was dissolved in DCM (125 mL) at room temperature under N2 atmosphere. To this was added pyridine (1.22 mL, 15.9 mmol) and 4-nitrophenyl chloroformate (1.75 g, 8.74 mmol). After the mixture was stirred at the same temperature for 4 h, the reaction was quenched by adding saturated NH4Cl solution (100 mL) and extracted with EA (100 mL x 4). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography (Hex:EA = 10:1) to give compound BCN-PNP (1.34 g, 84%) as a white solid. 1 H NMR (600 MHz, CDCl3) δ 8.29 (d, J = 9 Hz, 2H), 7.39 (d, J = 9 Hz, 2H), 4.41 (d, J = 8.4 Hz, 2H), 2.36 - 2.24 (m, 6H), 1.62 - 1.55 (m, 2H), 1.53 - 1.49 (m, 1H), 1.07 (t, J = 10.2 Hz, 2H).

[0380] Example 48: Preparation of compound T-Int-1 [ka] Preparation of compound T-Int-1-1 To a solution of compound Mono-4 (14 mg, 0.04 mmol) and compound L-2 (8.0 mg, 0.02 mmol) in DMF (0.6 mL) was added K2CO3 (9.3 mg, 0.07 mmol) under N2 atmosphere at 30 °C. After stirring for 3 h, the reaction mixture was purified by preparative PLC to give compound T-Int-1-1 (5.4 mg, 29%). EI-MS m / z: 825 (M + +1).

[0381] Preparation of compound T-Int-1-2 To a solution of compound T-Int-1-1 (7.5 mg, 0.01 mmol) and compound Int-TG1 (14 mg, 0.02 mmol) in ACN (0.5 mL) and DMF (0.5 mL) was added BEMP (1 μL, 0.004 mmol) at room temperature under N2 atmosphere. After stirring at room temperature for 5 h, the reaction mixture was purified by HPLC to give compound T-Int-1-2 (67 mg, 83%). EI-MS m / z: 1490 (M + +1).

[0382] Preparation of compound T-Int-1 To a solution of compound T-Int-1-2 (8.1 mg, 0.01 mmol) in MeOH (1 mL) and MC (0.1 mL) was added K2CO3 (5.6 mg, 0.04 mmol) under N2 atmosphere. After stirring at 0 °C for 1 h, the reaction mixture was purified by HPLC to give compound T-Int-1 (5.5 mg, 76%). EI-MS m / z: 1322 (M + +1).

[0383] Example 49: Preparation of compound T-Int-2 [ka] Preparation of compound T-Int-2-1 To a solution of compound D-109 (22.8 mg, 0.03 mmol) and compound Int-TG3 (32 mg, 0.03 mmol) in DMF (2 mL), DIPEA (12 μL, 0.07 mmol) was added under N2 atmosphere at 40° C. After stirring at room temperature for 5 h, the reaction mixture was purified by preparative HPLC to give compound T-Int-2-1 (28.9 mg, 61%). EI-MS m / z: 1641 (M + +1).

[0384] Preparation of compound T-Int-2 To a solution of compound T-Int-2-1 (28.9 mg, 0.02 mmol) in MeOH (2 mL) was added K2CO3 (12.2 mg, 0.09 mmol) under N2 atmosphere. After stirring at 0 °C under N2 atmosphere for 1 h, the reaction mixture was purified by preparative HPLC to give compound T-Int-2 (18.4 mg, 71%). EI-MS m / z: 1473 (M + +1).

[0385] Example 50: Preparation of compound T-Int-3 [ka] Preparation of compound T-Int-3-1 To a solution of compound D-106a (3.0 mg, 0.004 mmol) and compound Int-TG5 (6.0 mg, 0.008 mmol) in DMF (1 mL) was added DIPEA (3 μL, 0.018 mmol) at room temperature under N2 atmosphere. After stirring at room temperature for 5 h, the reaction mixture was purified by preparative HPLC to give compound T-Int-3-1 (3.1 mg, 52%). EI-MS m / z: 1637 (M + +1).

[0386] Preparation of compound T-Int-3 To a solution of compound T-Int-3-1 (3.1 mg, 0.002 mmol) in MeOH (1 mL) was added K2CO3 (1.2 mg, 0.009 mmol) under N2 atmosphere. After stirring at 0 °C under N2 atmosphere for 1 h, the reaction mixture was purified by preparative HPLC to give compound T-Int-3 (2.0 mg, 72%). EI-MS m / z: 1469 (M + +1).

[0387] Example 51: Preparation of compound T-Int-4 [ka] Compound T-Int-4 was synthesized in a manner similar to that described in Example 49. Preparation of compound T-Int-4-1 Yield: 50% EI-MS m / z: 1609 (M + +1). Preparation of compound T-Int-4 Yield: 55% EI-MS m / z: 1441 (M + +1).

[0388] Example 52: Preparation of compound T-Int-5 [ka] Compound T-Int-5 was synthesized in a manner similar to that described in Example 48. Preparation of compound T-Int-5-1 Yield: 61% EI-MS m / z: 1842 (M + +1). Preparation of compound T-Int-5 Yield: 62% EI-MS m / z: 1674 (M + +1).

[0389] Example 53: Preparation of compounds T-Int-6 and T-Int-7 [ka] Compound T-Int-6-2 was synthesized in the same manner as in Example 49, compound T-Int-2. Preparation of compound T-Int-6-1 Yield: 61%, ivory solid. EI-MS m / z: 1716(M + +1), 858(M + / 2+1). Preparation of compound T-Int-6-2 Yield 67%: ivory solid. EI-MS m / z: 1548(M + +1).

[0390] Preparation of compound T-Int-6-3 A homogeneous solution of compound T-Int-6-2 (92 mg, 0.059 mmol) in anhydrous DCM (4.0 mL) was treated with TFA / DCM (0.6 mL / 2 mL) and stirred for 2 h under N2 atmosphere at 0° C. The reaction mixture was purified by preparative HPLC (0.1% formic acid in water / 0.1% formic acid in ACN) to give compound T-Int-6-3 (72.5 mg, 84%) as a yellow solid. EI-MS m / z: 1448(M + +1).

[0391] Preparation of compound T-Int-6 A homogeneous solution of compound T-Int-6-3 (18.6 mg, 0.013 mmol) and BCN-PNP (4.0 mg, 0.013 mmol) in DMF (1.5 mL) was treated with DIPEA (4.4 μL, 0.026 mmol) at room temperature under N2 atmosphere and stirred for 2 h. The reaction mixture was purified by preparative HPLC (0.1% formic acid in water / 0.1% formic acid in ACN) to give compound T-Int-6 (12.9 mg, 62%) as a pale yellow solid. EI-MS m / z: 1624(M + +1), 812(M + / 2+1).

[0392] Compound T-Int-7 was synthesized in the same manner as compound T-Int-6-3. Preparation of compound T-Int-7-1 Yield 73%, ivory solid EI-MS m / z: 2069 (M + +1), 1035(M + / 2+1). Preparation of compound T-Int-7-2 72% yield, ivory solid EI-MS m / z: 1901 (M + +1), 951(M + / 2+1). Preparation of compound T-Int-7 65% yield, yellow solid EI-MS m / z: 1801 (M + +1), 901(M + / 2+1)

[0393] Example 54: Preparation of compound T-Int-8 [ka] Preparation of compound T-Int-8-1 A clear solution of T-Int-6-3 (20 mg, 0.014 mmol) and compound L-9 (5.1 mg, 0.014 mmol) in DMF (1.0 mL) was treated with DIPEA (7.3 μL, 0.042 mmol) at room temperature under N2 atmosphere and stirred for 2 h. The reaction mixture was purified by preparative HPLC (0.1% formic acid in water / 0.1% formic acid in ACN) to give compound T-Int-8-1 (19.9 mg, 85%) as a yellow solid. EI-MS m / z: 1699(M + +1), 850(M + / 2+1).

[0394] Preparation of compound T-Int-8-2 A clear solution of compound T-Int-8-1 (19.9 mg, 0.012 mmol) in anhydrous DCM (1.0 mL) was treated with TFA / DCM (0.3 mL / 3.0 mL) and stirred for 4 h under N2 atmosphere at 0° C. The reaction mixture was purified by preparative HPLC (0.1% formic acid in water / 0.1% formic acid in ACN) to give compound T-Int-8-2 (13.2 mg, 71%) as an ivory solid. EI-MS m / z: 1599(M + +1), 800(M + / 2+1).

[0395] Preparation of compound T-Int-8 A clear solution of compound T-Int-8-2 (13.2 mg, 0.00083 mmol) and BCN-PNP (1.8 mg, 0.0058 mmol) in DMF (1.0 mL) was treated with DIPEA (2.8 μL, 0.017 mmol) at room temperature under N2 atmosphere and stirred for 28 h. The reaction mixture was purified by preparative HPLC (0.1% formic acid in water / 0.1% formic acid in ACN) to give compound T-Int-8 (3.6 mg, 35%) as a pale yellow solid. EI-MS m / z: 1776(M + +1), 888(M + / 2+1).

[0396] Example 55: Preparation of compound T-Int-9 [ka] Compound T-Int-9 was synthesized in a manner similar to that used for preparing compound T-Int-8 in Example 54. Preparation of compound T-Int-9-1 Yield 56%, ivory solid. EI-MS m / z: 2052 (M + +1), 1026(M + / 2+1) Preparation of compound T-Int-9-2 Yield 71%, ivory solid. EI-MS m / z: 1952 (M + +1), 976(M + / 2+1) Preparation of compound T-Int-9 Yield 31%, white solid. EI-MS m / z: 2128(M + +1), 1064(M + / 2+1)

[0397] Example 56: Preparation of compound T-Int-10 [ka] Preparation of compound T-Int-10-1 A yellow solution of compound Mono-8 (100 mg, 0.324 mmol) and L-2 (352.2 mg, 0.972 mmol) in DMF (2.0 mL) was treated with K2CO3 (45 mg, 0.347 mmol) at room temperature under N2 atmosphere and stirred for 7 h. The reaction was diluted with water (10 mL) and extracted with EA (30 mL x 2). The organic layer was washed with 2N HCl (5 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (MeOH / DCM = 0%-3%) to give compound T-Int-10-1 (136.7 mg, 71%) as a yellow solid. EI-MS m / z: 590(M + +1).

[0398] Preparation of compound T-Int-10-2 A clear solution of compound T-Int-10-1 (25 mg, 0.042 mmol) and Mono-4 (13.2 mg, 0.042 mmol) in DMF (2 mL) was treated with K2CO3 (11.7 mg, 0.085 mmol) at room temperature under N2 atmosphere and stirred for 7 h. The reaction mixture was purified by preparative HPLC (0.1% formic acid in water / 0.1% formic acid in ACN) to give compound T-Int-10-2 (11.1 mg, 32%) as a white solid. EI-MS m / z: 821(M + +1).

[0399] Preparation of compound T-Int-10-3 A clear solution of compound T-Int-10-2 (18.1 mg, 0.022 mmol) and compound Int-TG1 (21.1 mg, 0.026 mmol) in ACN (3 mL) was treated with BEMP (2.6 μL, 0.009 mmol) at room temperature under N2 atmosphere and stirred for 6 h. The reaction mixture was purified by preparative HPLC (0.1% formic acid in water / 0.1% formic acid in ACN) to give compound T-Int-10-3 (15 mg, 46%) as an ivory solid. EI-MS m / z: 1486(M + +1).

[0400] Preparation of compound T-Int-10 A cloudy solution of compound T-Int-10-3 (13.2 mg, 0.009 mmol) in MeOH (2 mL) was treated with KCO (8.6 mg, 0.063 mmol) and stirred for 30 min under N2 atmosphere at 0° C. The reaction mixture was purified by preparative HPLC (0.1% formic acid in water / 0.1% formic acid in ACN) to give compound T-Int-10 (7.4 mg, 63%) as a white solid. EI-MS m / z: 1318(M + +1).

[0401] Example 57: Preparation of compound T-Int-11 [ka] Compound T-Int-11 was synthesized in a manner similar to that of compound T-Int-10 in Example 56. Preparation of compound T-Int-11-1 Yield 52%, white solid. EI-MS m / z: 576 (M + +1). Preparation of compound T-Int-11-2 29% yield, yellow solid. EI-MS m / z: 807 (M + +1). Preparation of compound T-Int-11-3 Yield 66%, yellow solid. EI-MS m / z: 1472 (M + +1). Preparation of compound T-Int-11 Yield 57%, yellow solid. EI-MS m / z: 1304(M + +1).

[0402] Example 58: Preparation of compound T-Int-12 [ka] Compound T-Int-12 was synthesized in the same manner as in Example 48 for the preparation of compound T-Int-1. Preparation of compound T-Int-12-1 Yield 66%, ivory solid. EI-MS m / z: 829 (M + +1). Preparation of compound T-Int-12-2 Yield 64%, yellow solid. EI-MS m / z: 1494 (M + +1). Preparation of compound T-Int-12 Yield 71%, yellow solid. EI-MS m / z: 1326 (M + +1).

[0403] Example 59: Preparation of Compound T-1 [ka] To a solution of T-Int-1 (2.3 mg, 0.002 mmol) in DMSO (2 mL), (BimC4A)3 (1348 μL, 5 mM) was added, followed by a solution of CuBr (189 μL, 100 mM). The mixture was stirred for 2 min. To this was added compound MPS-D2 (3.7 mg, 0.007 mmol) in DMSO (674 μL), followed by stirring for 10 min. After the reaction was completed, the mixture was separated and purified by preparative HPLC to obtain compound T-1 (1.0 mg, 32%). EI-MS m / z: 1868(M + +1).

[0404] Example 60: Preparation of compound T-2 [ka] To a solution of compound T-Int-2 (2.4 mg, 0.002 mmol) in DMSO (337 μL), (BimC4A)3 (1348 μL, 5 mM) was added, followed by a solution of CuBr (189 μL, 100 mM). The mixture was stirred for 2 min. To this was added compound MPS-D3 (5.7 mg, 0.006 mmol) in DMSO (673 μL), followed by stirring for 10 min. After the reaction was completed, the mixture was separated and purified by preparative HPLC to obtain compound T-2 (1.3 mg, 34%). EI-MS m / z: 2371(M + +1).

[0405] Example 61: Preparation of compound T-3 [ka] Compound T-3 was synthesized in the same manner as in Example 60 for preparing compound T-2. Yield: 34%. EI-MS m / z: 2371(M + +1).

[0406] Example 62: Preparation of compound T-4 [ka] Compound T-4 was synthesized in a similar manner to that described in Example 60. Yield: 24% EI-MS m / z: 2339 (M + +1).

[0407] Example 63: Preparation of compound T-5 [ka] Compound T-5 was synthesized in a similar manner to that described in Example 60. Yield 15%, white solid. EI-MS m / z: 1869(M + +1).

[0408] Example 64: Preparation of compound T-6 [ka] Compound T-6 was synthesized in the same manner as in Example 60 for preparing compound T-2. Yield 79%, white solid. EI-MS m / z: 2372(M + +1), 1186(M + / 2+1).

[0409] Example 65: Preparation of compound T-7 [ka] Compound T-7 was synthesized in the same manner as in Example 59 for preparing compound T-1. Yield 66%, white solid EI-MS m / z: 2224(M + +1), 1112(M + / 2+1).

[0410] Example 66: Preparation of compound T-8 [ka] Compound T-8 was synthesized in the same manner as in Example 59 for preparing compound T-1. 65% yield, white solid EI-MS m / z: 2224(M + +1), 1112(M + / 2+1).

[0411] Example 67: Preparation of compound T-9 [ka] A solution of compound T-2 (1.3 mg, 0.0005 mmol), HO (25 μL), and sodium bisulfite (65.7%, 0.16 mg, 0.0010 mmol) in IPA (50 μL) was stirred at room temperature under N for 6 h. The mixture was lyophilized to give compound T-9 (1.3 mg, 92%) as a yellow solid. EI-MS m / z: 2580(M + +1)

[0412] Example 68: Preparation of compound T-10 [ka] Compound T-10 was synthesized in the same manner as in Example 59 for preparing compound T-1. Yield 59%, white solid EI-MS m / z: 1864(M + +1), 932(M + / 2+1).

[0413] Example 69: Preparation of Compounds T-11 and T-12 [ka] Compounds T-11 and T-12 were synthesized in the same manner as in Example 59 for preparing compound T-1. Preparation of compound T-11 32% yield, white solid EI-MS m / z: 1864(M + +1), 932(M + / 2+1). Preparation of compound T-12 50% yield, white solid EI-MS m / z: 2216(M + +1), 1108(M + / 2+1).

[0414] Example 70: Preparation of compound T-13 [ka] Compound T-13 was synthesized in the same manner as in Example 59 for preparing compound T-1. 75% yield, white solid EI-MS m / z: 1849(M + +1), 925(M + / 2+1).

[0415] Example 71: Preparation of compound T-14 [ka] Compound T-14 was synthesized in the same manner as in Example 61 for preparing compound T-3. 65% yield, white solid EI-MS m / z: 1925(M + +1), 963(M + / 2+1).

[0416] Example 72: Preparation of compound T-Int-13 [ka] Compound T-Int-13 was synthesized in a manner similar to that used for preparing compound T-Int-6 in Example 53. Preparation of compound T-Int-13-1 70% yield, white solid EI-MS m / z: 1704 (M + +1). Preparation of compound T-Int-13-2 Yield 81%, white solid EI-MS m / z: 1536 (M + +1). Preparation of compound T-Int-13-3 Yield 81%, ivory solid EI-MS m / z: 1436 (M + +1). Preparation of compound T-Int-13 81% yield, beige solid EI-MS m / z: 1612 (M + +1).

[0417] Example 73: Preparation of compound T-16 [ka] Compound T-16-2 was synthesized in the same manner as in Example 49, compound T-Int-2. Preparation of compound T-16-1 Yield: 71%. EI-MS m / z: 1630 (M +1 ). Preparation of compound T-16-2 Yield: 60%. EI-MS m / z: 1462 (M +1 ).

[0418] Compound T-Int-16 was synthesized in the same manner as in Example 59, compound T-1. Preparation of compound T-16 Yield 69%, pale yellow solid. EI-MS m / z: 2360 (M + +1), 1180 (M / 2 + +1).

[0419] Example 74: Preparation of compound L-10 [ka] Compound L-10 was synthesized in the same manner as in Example 33 for preparing compound L-5. Preparation of compound L-10-1 Yield 96%, colorless oil. 1 H NMR (600 MHz, CDCl3) δ 3.67 - 3.63 (m, 32H), 3.37 (t, J = 5.2 Hz, 4H);EI-MS m / z: 465 (M + +1). Preparation of compound L-10 Yield 97%, colourless oil. EI-MS m / z: 439 (M + +1).

[0420] Example 75: Preparation of compound MPS-D4 [ka] Compound MPS-D4 was synthesized in a manner similar to that described in Example 44. Preparation of compound MPS-D4 Yield 53%, yellow oil. EI-MS m / z: 753 (M + +1).

[0421] Biological testing Example 76: In vitro analysis of benzodiazepine dimer derivatives NCI-N87 cancer cells were cultured in a 24-well plate at 25 × 10 3 Cells were seeded at 1 mL medium per well. The plates were incubated at 37°C for 24 hours in a humidified 5% CO2 air atmosphere. Benzodiazepine dimer derivatives were first dissolved in DMSO at a concentration of 10 mM. Serial dilutions were made in DMSO. This series of DMSO dilutions of compounds were added to triplicate wells of a 24-well plate at 5 μL per well. Three wells of each plate received 5 μL of DMSO without compound as a control. The final concentration of DMSO per well was 0.5%. The plates were incubated at 37°C for 6 days in a humidified 5% CO2 air atmosphere. Cell viability was quantified by MTT assay and IC was calculated using sigmoidal dose-response nonlinear regression curve fitting (GraphPad Software Inc.). 50 The results are shown in Figures 1, 2, and 3, and in Table 2. [Table 2]

[0422] Example 77: Preparation of conjugates Reduction / oxidation of antibodies for conjugation: Cysteine ​​engineered monoclonal antibodies were reduced with approximately 20-50 fold excess of TCEP (tris(2-carboxyethyl)phosphine hydrochloride) or DTT (dithiothreitol) in 4 mM Tris pH 7.3 containing 1 mM EDTA for 1 hour at 37°C. The reduced thiomab was diluted and loaded onto a PD-10 column in PBS. The column was eluted with 10 mM PBS pH 7.3. The eluted reduced thiomab was re-established by air oxidation. The thiol / Ab value was confirmed by quantifying the reduced antibody concentration from the absorbance of the solution at 280 nm and the thiol concentration by reaction with DTNB (Aldrich, CAS number D8130) and quantification of the absorbance at 412 nm.

[0423] Conjugation method: After reduction and reoxidation reaction, the antibody was dissolved in PBS. A solution of compound T-1 (5.62 μL, 3.0 mmol, as linker-toxin intermediate) obtained in Example 59 in DMSO was treated with reduced and reoxidized antibody (44 μL, 0.080 mmol) and gently stirred at room temperature for 3 hours. Hydroxylamine (5.62 μL, 1,500 mmol) was added to the reaction mixture solution and incubated at 37° C. for 8 hours to block the reversible deconjugation reaction. The conjugation mixture was loaded and eluted through a PD-10 column to remove excess drug-linker intermediate and other impurities. The mixture was concentrated by centrifugal ultrafiltration, and the conjugate was purified on a HIC NPR column (TOSOH #0007656 TSKgel Phenyl-5PW, 21.5 × 150 mm, 13 μm) and eluted with a 0.8 ml / min linear gradient of 40 to 100% B (A buffer was 1.5 M ammonium sulfate in 50 mM sodium phosphate, pH 7.0; B buffer was 20% acetonitrile in 50 mM sodium phosphate, pH 7.0).

[0424] Compounds T-1, T-2, T-3, and T-4 obtained in Examples 59, 60, 61, and 62 were used to carry out conjugation reactions to the thiol groups of engineered cysteines of trastuzumab (anti-HER-2) with reference to the methods shown in the literature, thereby preparing T-1-AB, T-2-AB, T-3-AB, and T-4-AB as thiomab drug conjugates (TDCs), respectively. [See Nature Biotechnology, 2008, 26, 925-932, Bioconjugate Chem., 2013, 24, 1256-1263, Bioconjugate Chem., 2016, 27, 1324-1331, Bioconjugate Chem. 2014, 25, 460-469]. The DAR (drug-to-antibody ratio) of the conjugated antibodies was analyzed by HIC, and the analysis results are shown in Table 3. [Table 3]

[0425] Example 78: In vitro analysis of protein drug conjugates NCI-N87 cancer cells were seeded in 96-well plates at a density of 5000 cells per well in 100 μL of medium and cultured for 24 hours. The four compound ADCs of Example 77 were treated at 1:4 serial dilutions from 50 nM to 0.0003 nM, and the antibody drug conjugate T-DM1 was treated at 1:4 serial dilutions from 50 nM to 0.0007 nM. DMSO dilutions of this series of compounds were added to triplicate wells of a 24-well plate at 5 μL per well. Three wells of each plate received 5 μL of DMSO without compound as a control. The final concentration of DMSO per well was 0.5%. The plates were incubated at 37°C for 6 days in a humidified 5% CO2 air atmosphere. Cell viability was quantified by MTT assay and IC was calculated using a sigmoidal dose-response nonlinear regression curve fit (GraphPad software Inc.). 50 was generated.

[0426] The results of the in vitro analysis of compounds T-1-AB, T-2-AB, T-3-AB, and T-4-AB are shown in Figures 4, 5, 6, 7, and Table 4. [Table 4]

[0427] Example 79: Solubility Test Solubility tests were performed to confirm the relative solubility of D-102, D-103, D-104, D-105, D-106, and D-107 in terms of retention time using analytical HPLC. The retention times of these compounds were analyzed by HPLC and the analytical results are shown in Table 5. [Table 5]

[0428] Incorporation by Reference All publications and patents mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication and patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present specification, including any definitions herein, will control.

[0429] Equivalent While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not limiting. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims that follow. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

1. Formula (I): 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof, During the ceremony, Ring A is a heterocyclic, aryl, or heteroaryl ring; Ring B is a heterocyclic ring, preferably a 5- or 6-membered ring, optionally fused to or substituted with one or more aryl or heteroaryl rings; R １ and R ２ are each independently an alkyl group; Z １ is methylene or a linking group, n ａ is an integer having a value of 1 or 2, n １ and n ２ are each independently an integer having a value of 1 to 5, or a pharma- ceutically acceptable salt thereof.

2. The compound of claim 1 , wherein Ring A is a heteroaryl ring.

3. Ring A is 【Chemistry 2】 where: A １ , A ２ , and A ３ Each independently represents NR ａ１ , C.R. ａ２ , O, N, or S; R ａ１ is H, alkyl, halogen, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R ａ２ 3. The compound of claim 1 or 2, wherein is H or alkyl, preferably lower alkyl.

4. Ring A is 【Chemistry 3】 【change】 The compound of claim 3 selected from:

5. Ring A is 【Chemistry 4】 5. The compound of claim 4, selected from:

6. R ａ１ C １ -C ４ -alkyl.

7. R ａ１ The compound of claim 6, wherein is methyl.

8. Ring A is 【Chemistry 5】 where: A ６ , A ７ , A ８ , and A ９ However, each independently, CR ａ３ or N, R ａ３ is H, alkyl, halogen, nitro, hydroxy, nitrile, cyano, alkoxy, amino, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; However, A ６ , A ７ , A ８ , and A ９ 3. The compound of claim 1 or 2, provided that no more than two of are N.

9. Ring B is a 5- or 6-membered heterocycle, optionally containing one or more R selected from alkyl, alkylidenyl, aryl, or heteroaryl. Ｃ The compound according to any one of claims 1 to 8, which is substituted with a group.

10. Ring B is 【Chemistry 6】 and The dotted line indicates the presence of an optional double bond; Each R Ｃ 10. The compound of claim 9, wherein is independently selected from H, OH, alkyl, alkylidenyl, halogen, amino, cyano, or aryl.

11. Ring B is 【Chemistry 7】 and Ring C' is optionally represented by R １ａ and is substituted by 1 to 4 substituents independently selected from Each R １ａ are independently H, OH, SH, alkylthiol, -CH ２ N (CH ３ ) ２ 11. The compound of claim 10, wherein the aryl group is selected from alkyl, alkyloxy, alkyl, alkylidenyl, halogen, amino, cyano, aryl, or alkyl.

12. The compound according to any one of claims 1 to 8, wherein ring B is a 5- or 6-membered heterocyclic ring and is fused to ring C' selected from an aryl or heteroaryl ring, preferably a 5- or 6-membered aryl or heteroaryl ring.

13. Rings B and C together form the following structure: 【Chemistry 8】 Forming Ring C' is optionally R １ｃ is an aryl or heteroaryl substituted by 1 to 4 substituents independently selected from Each R １ｃ are independently OH, SH, alkylthiol, or -CH ２ N (CH ３ ) ２ , alkyloxy, alkyl, halogen, amino, or cyano; n ｂ 13. The compound of claim 12, wherein is an integer having a value of 1 or 2.

14. Ring C is 【Chemistry 9】 and B １ , B ２ , and B ３ Each independently represents NR ａ１ , C.R. ａ２ , O, N, or S; R ｂ１ is H, alkyl, halogen, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R ｂ２ The compound of any one of claims 9 to 13, wherein is absent, H, or alkyl.

15. Ring C' is 【Chemistry 10】 15. The compound of claim 14, selected from:

16. Ring C' is 【Chemistry 11】 16. The compound of claim 15, selected from:

17. Ring A is 【Chemistry 12】 Ring C' is selected from 【Chemistry 13】 17. The compound of claim 16, selected from:

18. R ｂ１ C １ -C ４ -alkyl.

19. R ｂ１ The compound of claim 18 , wherein is methyl.

20. Ring C' is 【Chemistry 14】 where: B ６ , B ７ , B ８ , and B ９ However, each independently, CR ｂ３ or N, R ｂ３ is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; However, B ６ , B ７ , B ８ , and B ９ The compound of any one of claims 9 to 13, provided that no more than two of are N.

21. One of ring A and ring C' is 【Chemistry 15】 and the other of ring A or ring C' is heteroaryl.

22. A compound according to any one of the preceding claims having the structure of formula (Ia), (Ib-1), (1b-2), (1b-3), (1c-1), (1c-2), or (1c-3); 【Chemistry 16】 【change】 or a pharma- ceutically acceptable salt thereof.

23. R １ and R ２ 4. A compound according to any one of the preceding claims, wherein

24. R １ and R ２ But, C １ -C ４ 2. A compound according to any one of the preceding claims, wherein - alkyl.

25. R １ and R ２ 4. A compound according to any one of the preceding claims, wherein is methyl.

26. Z １ The compound according to any one of claims 1 to 25, wherein is methylene.

27. Z １ but, 【Chemistry 17】 where: Y １ But, CR Ｙ１ or N, R Ｙ１ is H or alkyl, preferably lower alkyl; R Ｚ１ is absent or is hydroxyl, amino, amide, or (CH ２ ) ｚ R Ｚ１ａ and R Ｚ１ａ is amino (preferably tertiary amino), aryl, or heteroaryl; The compound of any one of claims 1 to 25, wherein z is an integer having a value from 1 to about 10.

28. Z １ but, 【Chemistry 18】 28. The compound of claim 27,

29. Z １ but, 【Chemistry 19】 where: Y ２ 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, or heteroaryl; The compound of any one of claims 1 to 25, wherein z is an integer having a value from 0 to about 10.

30. Z, 【Chemistry 20】 30. The compound of claim 29,

31. The compound is 【Chemistry 21】 【change】 The compound according to claim 1, selected from or a pharma- ceutically acceptable salt thereof.

32. The compound is 【Chemical 22】 【change】 【change】 The compound according to claim 1, selected from or a pharma- ceutically acceptable salt thereof.

33. 33. A conjugate comprising the compound of any one of claims 1 to 32, a cleavable linker, and a targeting agent, wherein the cleavable linker cleavably links the compound to the targeting agent.

34. 34. The conjugate of claim 33, wherein the targeting agent is a cell binding agent.

35. The linker comprises a functional group that undergoes a chemical reaction (e.g., a physicochemical reaction and / or a biological reaction) under defined conditions to release a nucleophilic heteroatom, and a -S(O) functional group located proximal to the nucleophilic heteroatom. ２ - a functional group.

36. The -S(O) ２ 36. The conjugate of claim 35, wherein the functional group reacts with the nucleophilic heteroatom in an intramolecular cyclization reaction to release the active agent.

37. The conjugate of any one of claims 33 to 36, further comprising a targeting moiety (e.g., an oligopeptide, polypeptide, antibody, etc.) having binding specificity for a desired target receptor or other molecule associated with a target cell.

38. having the structure of formula (II), (IIa), or (IIb): 【Chemistry 23】 During the ceremony, Ring A is a heterocyclic, aryl, or heteroaryl ring; Ring B is a heterocyclic ring, preferably a 5- or 6-membered ring, optionally fused to or substituted with one or more aryl or heteroaryl rings; R １ and R ２ are each independently an alkyl group; Z １ is methylene or a linking group, n ａ is an integer having a value of 1 or 2, n １ and n ２ are each independently an integer having a value of 1 to 5, Z ２ is absent or is a linking group; L １ is a heteroatom selected from O, S, and N, preferably O or N, connected to said SO ２ is a linking group bonded to L １ and S.O. ２ The cleavage of the bond between L １ and Z １ to promote cleavage of the bond between the X １ However, -O-, -CR ａ ２ - or -NR'-, preferably -O-; Ar represents a ring such as aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, preferably aryl or heteroaryl; Y １ But - (CR ｂ ２ ) ｙ N (R ａ ) -, - (CR ｂ ２ ) ｙ O- or -(CR ｂ ２ ) ｙ S-, and when y is 1, the N, O, or S atom is positioned to bond to TG, where X １ and Y １ are positioned on adjacent atoms of Ar; When the TG is activated, the SO ２ and reacts with Z to replace X-SO ２ and an inducing group that produces an N, O, or S atom capable of forming a 5- or 6-membered ring with the intervening atom of Ar; w and x are each independently an integer having a value of 0 or 1; Each R ａ and R ｃ is independently hydrogen or lower alkyl; Each R ｂ is independently hydrogen or lower alkyl; or Two R's ｂ together with the carbon atom to which they are attached form a 3-5 membered ring, preferably a 3-4 membered ring, or a pharma- ceutically acceptable salt thereof.

39. X １ The conjugate of claim 38, wherein is -O-.

40. 40. The conjugate of claim 38 or 39, wherein Ar is aryl.

41. 41. The conjugate of claim 40, wherein Ar is phenyl or naphthyl.

42. Z ２ However, isocyanides, isothiocyanides, 2-pyridyl disulfides, haloacetamides (-NHC(O)CH ２ -halo), maleimide, diene, alkene, halogen, tosylate (TsO-), aldehyde, sulfonate (R-SO ３ － ), 【Chemistry 24】 Phosphonic acid (-P(=O)(OH) ２ ), ketone, C ８ -C １０ Cycloalkynyl, -OH, -NHOH, -NHNH ２ , -SH, carboxylic acid (-COOH), acetylene (-C≡CH), azide (-N ３ ), amino (-NH ２ ), sulfonic acid (-SO ３ H), alkynone derivatives (-C(O)C≡C-R ａ ), and dihydrogen phosphate (-OP(=O)(OH) ２ 41. The conjugate of any one of claims 38 to 40, wherein the linking group comprises one or more groups selected from:

43. The conjugate of any one of claims 38 to 42, wherein x is 0.

44. TGが、-NO ２ 、-OC(O)-(CH ２ ) r C(O)R １ 、 -NHNH ２ , -BR ２ R ３ ,or 【Chemistry 25】 where: R １ But, C １ -C ６ is alkyl, R ２ and R ３ each independently represents hydrogen, C １ -C ６ Alkyl, C １ -C ６ alkoxy, or hydroxy; R ４ , R ５ , R ６ , and R ７ are each independently hydrogen or C １ -C ６ 44. The conjugate of any one of claims 38 to 43, wherein r is alkyl and r is an integer having a value of 1, 2, 3, 4, or 5.

45. T.G. 【Chemistry 26】 is selected from During the ceremony, Each R ２１ is independently hydrogen or acetyl; R ２２ The conjugate of any one of claims 38 to 43, wherein is hydrogen or lower alkyl.

46. TG is -NO ２ , -C(O)-(CH ２ ) ２ The conjugate of any one of claims 38 to 43, wherein the alkyl group is selected from C(O)-alkyl and nitrobenzyl.

47. A conjugate having the structure of formula (III): (D-L) ｄｌ (CB) ｃｂ (III) or a pharma- ceutically acceptable salt thereof, During the ceremony, LG is a linking group; CB is a cell binding agent; cb and dl are each independently an integer having a value from 1 to about 20, preferably from 1 to about 10; The conjugate, or a pharma- ceutically acceptable salt thereof, wherein each DL is independently a group having the structure of a compound according to any one of claims 1 to 28 and 38 to 46.

48. LG comprises at least two of the following: １０ -C １００ 48. The conjugate of claim 47, which is a linear or branched, saturated or unsaturated alkylene moiety: (i) at least one heteroatom selected from -NH-, -C(=O), -O-, -S-, and -P-; (ii) at least one heteroarylene; (iii) at least one amino acid moiety, glycobond, peptide bond, or amide bond, and (iv) C １ -C ２０ Alkyl, C ６ -C ２０ Aryl C １ -C ８ Alkyl, -(CH ２ ) ｓ COOH, and -(CH ２ ) ｐ N.H. ２ is an integer having a value from 0 to 10, and p is an integer having a value from 1 to about 10.

49. 49. The conjugate of claim 47 or 48, wherein LG comprises a functional group that can be generated through a click chemistry reaction, such as a triazole.

50. L.G. 【Chemical 27】 and During the ceremony, ＊ However, (D-L) ｄｌ represents the attachment point to ＊＊ represents the point of attachment to CB, Each V is independently a single bond, —O—, —S—, or —NR ２１ --, --C(O)NR ２２ --, --NR ２３ C(O)-, -NR ２４ SO ２ - or -SO ２ N.R. ２５ - and R ２１ , R ２２ , R ２３ , R ２４ , and R ２５ each independently represents hydrogen, (C １ -C ６ ) alkyl, (C １ -C ６ ) alkyl(C ６ -C ２０ ) aryl, or (C １ -C ６ ) alkyl(C ３ -C ２０ ) heteroaryl; r is an integer having a value from 1 to about 10; p is an integer having a value from 0 to about 10; q is an integer having a value from 1 to about 10; L ３ The conjugate according to any one of claims 47 to 49, wherein is a single bond.

51. LG are connected to each other in a linear chain by a covalent bond (CH ２ ) ｂ , L ｃ , (P １ ) ａ , W ａ１ , W ａ２ , W ａ３ , Y ３ , and Y ４ group, where W ａ１ , W ａ２ , and W ａ３ each independently represents -NH-, -C(O)-, or -CH ２ - and W ｂ１ is an amide bond or a triazolylene; P １ is an amide bond, an amino acid residue, or a peptide; L ｃ is alkylene, Y １ But -(CH ２ ) ｑ - (CH ２ CH ２ X ４ ) ｏ - or - (CH ２ ) ｑ - (X ４ CH ２ CH ２ X ３ ) ｏ - and X ４ is -O-, -S-, -NH- or -CH ２ - and Y ２ is a single bond or a group selected from the following: 【Chemistry 28】 【change】 W ｂ２ is an amide bond or a triazolylene; a is 0 to 10; b, c, and d are each independently an integer having a value from 1 to about 10; 50. The conjugate of any one of claims 47 to 49, wherein o and q are each independently an integer having a value from 1 to about 10.

52. LG is a linking group of formula (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIh), (IIIh), or (IIIj); 【Chemical 29】 【change】 During the ceremony, R ｅ is alkyl, X ５ is -O-, -S-, -NH-, or -CH ２ - and W ｂ１ and W ｂ２ each independently represents -C(O)NH-, -NHC(O)-, 【Chemistry 30】 and R １２ is hydrogen, alkyl, amino acid side chain, -(CH ２ ) ｓ C(O)R １３ , or -(CH ２ ) ｐ N.R. １４ R １５ and R １３ is OH or -NH(CH ２ ) ｓ' (X ４ CH ２ CH ２ ) ｓ” Z''-(CB) ｍ and R １４ and R １５ each independently represents hydrogen or -C(O)(CH ２ ) ｓ' (X''CH ２ CH ２ ) ｓ” Z''-(CB) ｍ and s and s″ are each independently an integer having a value from 0 to about 10; m is an integer having a value of 0 or 1; X ４ and X ５ each independently represents -O-, -S-, -NH-, or -CH ２ - and b, c, d, e, g, h, o, and q are each independently an integer having a value from 1 to about 10.

53. 53. The conjugate of any one of claims 47 to 52, wherein the cell binding agent binds to a target cell selected from a tumor cell, a virus-infected cell, a microbial cell, an activated cell, a bone marrow cell, an activated T cell, a B cell, or a melanocyte; a cell expressing CD4, CD6, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40, CD44, CD56, EpCAM, CanAg, CALLA, or Her-2 antigen; a cell expressing a Her-3 antigen or an insulin growth factor receptor, an epidermal growth factor receptor, and a folate receptor.

54. 54. The conjugate of claim 53, wherein the tumor cell is selected from a breast cancer cell, a prostate cancer cell, an ovarian cancer cell, a colorectal cancer cell, a gastric cancer cell, a squamous cell carcinoma cell, a small cell lung cancer cell, and a testicular cancer cell.

55. The conjugate of claim 53 or 54, wherein the cell binding agent is an antibody, a single chain antibody, an antibody fragment, a monoclonal antibody, a single chain monoclonal antibody or a monoclonal antibody fragment that specifically binds to the target cell, a chimeric antibody, a chimeric antibody fragment, a domain antibody, a domain antibody fragment that specifically binds to the target cell, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, or a nutrient transport molecule.

56. 56. The conjugate of any one of claims 53 to 55, wherein the antibody is a resurfaced antibody, a resurfaced single chain antibody or a resurfaced antibody fragment, a monoclonal antibody, a single chain monoclonal antibody or a monoclonal antibody fragment thereof, a chimeric antibody fragment, a domain antibody or a domain antibody fragment, a humanized antibody, a humanized single chain antibody or a humanized antibody fragment.

57. A pharmaceutical composition comprising a compound according to any one of claims 1 to 46 or a conjugate according to any one of claims 47 to 56, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

58. 48. The pharmaceutical composition of claim 47, further comprising a chemotherapeutic agent.

59. 57. A method for treating or preventing a disease or disorder, comprising administering to a subject in need thereof a compound according to any one of claims 1 to 46, a conjugate according to any one of claims 47 to 56, or a pharmaceutical composition according to claim 57 or 58.

60. 60. The method of claim 59, wherein the disease or disorder is cancer, an infectious disease, an immunodeficiency, an autoimmune disease, or a chronic inflammatory disorder.

61. 61. The method of claim 59 or 60, further comprising administering to the subject a chemotherapeutic agent.

62. 62. The method of claim 61 , wherein the chemotherapeutic agents are administered to the subject sequentially.

63. A compound having the structure of formula (IV): 【Chemistry 31】 or a pharma- ceutically acceptable salt thereof, During the ceremony, Ring A is a heterocyclic, aryl, or heteroaryl ring; R １ is alkyl, W ａ is H or benzyl, n ａ is 1 or 2, or a pharma- ceutically acceptable salt thereof.

64. Ring A is 【Chemistry 32】 where: A １ , A ２ , and A ３ Each independently represents NR ａ１ , C.R. ａ２ , O, N, or S; R ａ１ is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; R ａ２ 64. The compound of claim 63, wherein is H or alkyl, preferably lower alkyl.

65. Ring A is 【Chemical 33】 65. The compound of claim 63 or 64, selected from:

66. Ring A is 【Chemical 34】 66. The compound of claim 65, selected from:

67. R ａ１ C １ -C ４ -alkyl.

68. R ａ１ The compound according to any one of claims 64 to 67, wherein is methyl.

69. Ring A is 【Chemistry 35】 where: A ６ , A ７ , A ８ , and A ９ However, each independently, CR ａ３ or N, R ａ３ is H, alkyl, halogen, nitro, hydroxy, nitrile, cyano, alkoxy, amino, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; However, A ６ , A ７ , A ８ , and A ９ 64. The compound of claim 63, with the proviso that no more than two of are N.

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