Fused heterocyclic benzodiazepine derivatives and their uses

Abstract

The present disclosure provides compounds and compositions capable of extending lifespan, and methods of using the same.

Description

Technology Field

[0001] Related applications

[0002] 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

[0003] Background Technology

[0004] Antibody-drug conjugates (ADCs) are emerging as a class of potent anti-tumor agents with efficacy across various cancers. ADCs typically comprise three distinct characteristics: a cell-binding agent or targeting moiety; a linker; and a cytotoxic agent. The linker component of an ADC is a critical feature in the development of targeted anticancer agents that possess desirable target specificity—that is, high activity in tumor cells but low activity in healthy cells. The use of a targeting moiety combined with a cytotoxic agent, which can be harmful to healthy tissues if untargeted, also alters the calculation of the desirable characteristics of such cytotoxic agents. Therefore, there is a need for improved linkers and cytotoxic agents useful for the manufacture of ADCs.

[0005] Summary of the Invention

[0006] In a specific embodiment, a compound having the structure of chemical formula (IV):

[0007]

[0008] Or its pharmaceutically acceptable salt is provided herein,

[0009] Here:

[0010] Ring A is a heterocyclic, aryl, or heteroaryl ring;

[0011] R 1 It is an alkyl, preferably a lower alkyl;

[0012] W ais H or benzyl;

[0013] n a is an integer with a value of 1 or 2.

[0014] In a specific embodiment, a compound having the structure of chemical formula (I):

[0015]

[0016] Or its pharmaceutically acceptable salt is provided herein,

[0017] Here:

[0018] Ring A is a heterocyclic, aryl, or heteroaryl ring;

[0019] Ring B is a heterocyclic ring, preferably a 5- or 6-membered ring, fused to or substituted with one or more optional aryl or heteroaryl rings;

[0020] R 1 and R 2 Each is independently an alkyl, preferably a lower alkyl;

[0021] Z 1 is a methylene or linking group that can be additionally conjugated to a cleavable linker and a targeting agent;

[0022] n a is an integer with a value of 1 or 2;

[0023] n 1 and n 2 Each is independently 1, 2, 3, 4, or 5.

[0024] In a specific embodiment, a conjugate comprising a compound of formula (I) is provided herein, 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.

[0025] In a specific embodiment, a compound having the structure of formula (II), (IIa), or (IIb):

[0026]

[0027]

[0028] Or its pharmaceutically acceptable salt is provided herein,

[0029] Here:

[0030] Ring A is a heterocyclic, aryl, or heteroaryl ring;

[0031] Ring B is a heterocyclic ring, preferably a 5- or 6-membered ring, fused to or substituted with one or more optional aryl or heteroaryl rings;

[0032] R 1 and R 2 Each is independently an alkyl, preferably a lower alkyl;

[0033] Z 1 is a methylene or linking group that can be additionally conjugated to a targeting agent;

[0034] n a is an integer with a value of 1 or 2;

[0035] n 1 and n 2 are each independently 1, 2, 3, 4, or 5;

[0036] Z 2 is absent or a connecting device;

[0037] L 1 is a linker attached to SO2 through a heteroatom selected from O, S, and N, preferably O or N, and L 1 L that releases an active agent upon the cleavage of the bond between and SO2 1 and Z 1 Selected to facilitate the severance of the bond between;

[0038] X 1 -O-, -CR a 2-, or -NR'-, preferably -O- and;

[0039] Ar represents a ring, e.g., aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, preferably aryl or heteroaryl;

[0040] Y 1 Silver, arranged so that N, O, or S atoms are attached to TG when y is 1, -(CR b 2) y N(R a )-, -(CR b 2) y O-, or -(CR b 2) y S- and; here X 1 and Y 1 Silver is placed on an adjacent atom of Ar;

[0041] TG is a triggering group that, upon activation, reacts with SO2 to produce an N, O, or S atom capable of replacing Z and forming a 5-6 member ring containing an intervening atom of X-SO2 and Ar;

[0042] w and x are integers that each independently have a value of 0 or 1;

[0043] Each R a and R c is independently hydrogen or lower alkyl; and

[0044] Each R b is independently hydrogen or lower alkyl; or

[0045] 2 Rs b Together with the carbon atoms to which they are attached, they form a 3-5 member ring, preferably a 3-4 member ring.

[0046] In some embodiments, the present disclosure describes a compound having the structure of formula (IV):

[0047]

[0048] or it concerns his pharmaceutically acceptable salt,

[0049] Here:

[0050] Ring A is a heterocyclic, aryl, or heteroaryl ring;

[0051] R 1 is alkyl;

[0052] W a is H or benzyl; and

[0053] n a is 1 or 2.

[0054] Also, a conjugate having the structure of chemical formula (III):

[0055] (DL) dl -LG-(CB) cb

[0056] (III)

[0057] Or his pharmaceutically acceptable salt is provided,

[0058] Here:

[0059] LG is a connector;

[0060] CB is a cell-binding agent;

[0061] cb and dl are each independently integers having a value of 1 to about 20, preferably 1 to about 10;

[0062] Each DL is a group that independently has the structure of a compound of formula (I) or (II).

[0063] 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 pharmaceutically acceptable carrier).

[0064] In a specific embodiment, the present disclosure provides a method for 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 a specific embodiment, the present invention provides a conjugate of formula (III) and a pharmaceutical composition thereof for use in a method for delivering an active agent to a cell, wherein the targeting moiety is selected to bind to a molecule associated with the target cell. In particular, the compounds, conjugates, and compositions of the present invention may be useful for inhibiting abnormal cell growth or treating proliferative disorders in mammals (e.g., humans), such as when the target cell is a cancer cell and the targeting moiety is selected to bind to a molecule associated with the cancer cell (also not associated with healthy cells, or at least preferentially associated with tumor cells rather than healthy cells).

[0065] In some embodiments, the present disclosure relates to a method for treating or preventing a disease or disorder, comprising administering to a subject in need a pharmaceutical composition comprising a compound of formula (I) or formula (II), a conjugate of formula (III), or a compound of formula (I) or formula (II) or a conjugate of formula (III).

[0066] The conjugate of formula (III) and its pharmaceutical composition may be useful for the treatment of pathological conditions in mammals (e.g., humans), such as cancer, rheumatoid arthritis, multiple sclerosis, graft-versus-host disease (GVHD), graft rejection, lupus, myositis, infection, immunodeficiency, such as AIDS, and inflammatory diseases. Brief explanation of the drawing

[0067] Figure 1 shows the in vitro cytotoxic activity of compounds D-101, D-102, D-103, D-105, and D-110 against NCI-N87. Figure 2 shows the in vitro cytotoxic activity of compounds D-104, D-106, D-107, D-108, and D-109 against NCI-N87. Figure 3 shows the in vitro cytotoxic activity of compounds D-111, D-112, D-113, and D-114 against NCI-N87. Figure 4 shows the in vitro cytotoxic activity of conjugate T-1-AB against NCI-N87. Figure 5 shows the in vitro cytotoxic activity of conjugate T-2-AB against NCI-N87. Figure 6 shows the in vitro cytotoxic activity of conjugate T-3-AB against NCI-N87. Figure 7 shows the in vitro cytotoxic activity of conjugate T-4-AB against NCI-N87. Specific details for implementing the invention

[0068] Detailed description of the invention

[0069] The present disclosure relates to benzodiazepine (BD) derivatives, such as pyrrolobenzodiazepine (PBD), indolinobenzodiazepine (IBD), tetrahydroisoquinolinobenzodiazepine (TBD), and dimers thereof; compounds and conjugates thereof comprising a cleavable linker; and uses thereof. Representative compounds and conjugates disclosed herein include a functional group having a desired function or activity that releases a nucleophilic heteroatom when subjected to a chemical reaction (e.g., physicochemical and / or biological reaction) under predetermined conditions, and an active agent (e.g., a compound of formula (I)) having an SO2 functional group positioned proximal to the nucleophilic heteroatom so as to react with the nucleophilic heteroatom in intramolecular cyclization to release the active agent. In some embodiments, the compounds and conjugates disclosed herein further comprise a targeting moiety (e.g., oligopeptide, polypeptide, antibody, etc.) having binding specificity to a desired target receptor or other molecule associated with a target cell.

[0070] definition

[0071] Unless otherwise defined herein, scientific and technical terms used in this application have the meanings commonly understood by those skilled in the art. Generally, the nomenclature and techniques used in connection with 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 widely known and commonly used in the art.

[0072] The methods and techniques of the present disclosure are generally, unless otherwise indicated, carried out in accordance with prior methods widely known in the art and as described in the various general and more specific references cited and discussed throughout this specification. For example, see below: "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).

[0073] Chemical terms used herein, unless otherwise defined herein, are used in accordance with conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, CA (1985).

[0074] All of the foregoing mentioned in this application, as well as any other published literature, patents, and published patent applications, are specifically incorporated herein by reference. In the event of a conflict, this specification shall prevail, including its specific definitions.

[0075] The term "agent" is used herein to refer to chemical compounds (e.g., organic or inorganic compounds, mixtures of chemical compounds), biological macromolecules (e.g., nucleic acids, antibodies, including parts thereof as well as humanized, chimeric, human, and monoclonal antibodies, proteins or parts thereof, e.g., peptides, lipids, carbohydrates), or extracts made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents include, for example, agents of known structure and those of unknown structure.

[0076] "Patient," "Subject," or "Individual" are used interchangeably and refer to human or non-human animals. These terms include mammals, e.g., humans, primates, domestic animals (including cattle, pigs, etc.), companion animals (e.g., dogs, cats, etc.), and rodents (e.g., mice and rats).

[0077] "Treatment" of a condition or patient refers to the performance of steps to obtain beneficial or desired outcomes, including clinical outcomes. As used herein and as understood in the art, "treatment" is an approach to obtain beneficial or desired outcomes, including clinical outcomes. Beneficial or desired clinical outcomes may include, but are not limited to, the alleviation or improvement of one or more detectable or indetectable symptoms or conditions; the reduction of the severity of the disease; a stabilized (i.e., non-deterioration) state of the disease; prevention of disease spread; delay or deceleration of disease progression; improvement or alleviation of the disease state; and remission (partial or total). "Treatment" may also mean an extension of survival compared to the survival expected without treatment.

[0078] The term "prevention" is recognized in the art and is well understood in the art when used in relation to conditions such as local recurrence (e.g., pain), diseases such as cancer, syndrome complexes such as heart failure, or any other medical condition, and involves the administration of a composition that reduces the frequency of the medical condition in a subject or delays the onset of its symptoms compared to a subject who does not accept the composition. Thus, prevention of cancer includes, for example, a reduction in the number of detectable cancer growths in a group of patients who received prophylactic treatment compared to an untreated control group by a statistically and / or clinically significant amount, and / or a delay in the appearance of detectable cancer growths in a treated group compared to an untreated control group.

[0079] "Administering" or the "administration of" a substance, compound, or agent to a subject may be performed using any of the various methods known to those skilled in the art. For example, a compound or agent may be administered intravenously, arterially, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, orally (by ingestion), intranasally (by inhalation), intrathecally, intracerebrally, and transdermally (e.g., through a skin canal, by absorption). A compound or agent may also be appropriately introduced by a rechargeable or biodegradable polymer device or other device, e.g., a patch and a pump, or by a formulation that provides an extended, slow, or controlled release of the compound or agent. Administration may also be performed, for example, once, multiple times, and / or over one or more extended periods.

[0080] The appropriate method of administration of a substance, compound, or agent to a subject will also depend, for example, on the subject's age and / or physical condition 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 orally to the subject, for example, by ingestion. In some embodiments, the orally administered compound or agent is in an extended-release or slow-release formulation, or is administered using a device for such slow or extended release.

[0081] As used herein, the term “combined administration” refers to any form of administration of two or more different therapeutic agents such that a second agent is administered while a previously administered therapeutic agent is still effective in the body (e.g., both agents are simultaneously effective in the patient, which may include a synergistic effect of the two agents). For example, different therapeutic compounds may be administered simultaneously or sequentially, either within the same formulation or within separate formulations. Thus, an individual receiving such treatment may benefit from the combined effect of different therapeutic agents.

[0082] The "therapeutic effective dose" or "therapeutic effective dosage" of a drug or agent is the amount of drug or agent that produces the intended therapeutic effect when administered to a subject. A full therapeutic effect does not necessarily occur with a single dose, but may occur only after a series of doses. Therefore, the therapeutic effective dose may be administered in one or more doses. The exact effective dose required for a subject will depend, for example, on the subject's size, health, and age, and on the nature and severity of the condition being treated, such as cancer or MDS. A person skilled in the art can easily determine the effective dose for a given situation through routine experimentation.

[0083] 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 that replaces a hydrogen on one or more carbons of the alkenyl group. Such substituents may appear on one or more carbons that are included in or not included in one or more double bonds. Additionally, such substituents include all that are considered for the alkyl groups discussed below, except where stability is prohibited. For example, substitution of the alkenyl group by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is considered.

[0084] The term "alkylidenyl" refers to =C(R*)(R**) as used herein, 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-pentylidenyl (=CHCH2CH2CH2CH3), 2-pentylidenyl (=C(CH3)CH2CH2CH3), 3-pentylidenyl (=C(CH2CH3)2), 3-methyl-2-pentylidenyl (=C(CH3)CH(CH3)2), 3-methyl-1-butylidenyl (=CHCH2CH(CH3)2), and 2-methyl-1-butylidenyl (=CHCH(CH3)CH2CH3), 1-hexylidenyl (=CHCH2CH2CH2CH2CH3), 2-hexylidenyl (=C(CH3)CH2CH2CH2CH3), 3-hexylidenyl (=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 is optionally one or more 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 It can be substituted as, where each R x and R yis independently H, alkyl, alkenyl, aryl, heteroaryl, heterocyclic, cycloalkyl, or hydroxyl.

[0085] An "alkyl" group or "alkane" is a fully saturated straight-chain or branched-chain non-aromatic hydrocarbon. Typically, a straight-chain or branched-chain alkyl group has 1 to about 20, preferably 1 to about 10, carbon atoms unless otherwise defined. Examples of straight-chain and branched-chain 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-chain alkyl groups are also referred to as "lower alkyl" groups.

[0086] Additionally, 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 a substituent that replaces a hydrogen on one or more carbons of a hydrocarbon backbone. These substituents may comprise, for example, halogens (e.g., fluoro), hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidin, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moiety, unless otherwise specified. In a preferred embodiment, the substituent on the substituted alkyl phase is C 1-6 Alkyl, C 3-6It is selected from cycloalkyl, halogen, carbonyl, cyano, or hydroxyl. In a more preferred embodiment, the substituent on the substituted alkyl is selected from fluoro, carbonyl, cyano, or hydroxyl. It will be understood by those skilled in the art that the substituted moiety on the hydrocarbon chain may itself be substituted, where appropriate. For example, the substituent on the substituted alkyl may include amino, azido, imino, amido, phosphoryl (including phosphonates and phosphinates), sulfonyl (including sulfates, sulfonamido, sulfamoyl and sulfonates), and silyl groups, as well as substituted and unsubstituted forms such as ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN, etc. Exemplary substituted alkyls are described below. Cycloalkyl can be further substituted with alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl-substituted alkyl, -CF3, -CN, etc.

[0087] Term "C x-y " is intended to contain a group having x to y carbons within the chain when used with chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy. For example, the term "C x-y "alkyl" refers to a substituted or unsubstituted saturated hydrocarbon group, including a haloalkyl group, a straight-chain alkyl group containing x to y carbons in the chain, and a branched-chain alkyl group. Preferred haloalkyl groups include trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, and pentafluoroethyl. CO alkyl indicates a hydrogen at a terminal position of the group, and indicates a bond when internal. The term "C 2-y "Alkenyl" and "C" 2-y "Alkynyl" refers to a substituted or unsubstituted unsaturated aliphatic group similar in length and possible substitution to the alkyl described above, but each containing at least one double or triple bond.

[0088] The term "amide", as used herein, refers to the following group:

[0089]

[0090] Here, each R A represents independently a hydrogen or hydrocarbyl group, or two Rs A It completes a heterocycle having 4 to 8 atoms within a ring structure together with the N atoms to which they are attached.

[0091] The terms "amine" and "amino" are recognized in the art and refer to unsubstituted and substituted amines and their salts, moietyes that can be represented, for example:

[0092] or

[0093] Here, each R A represents independently a hydrogen or hydrocarbyl group, or two Rs A It completes a heterocycle having 4 to 8 atoms within a ring structure together with the N atoms to which they are attached.

[0094] The term "aryl" comprises a substituted or unsubstituted single-ring aromatic group in which each atom of the ring is a carbon, as used herein. Preferably, the ring is a 6- or 10-membered ring, more preferably a 6-membered ring. The term "aryl" also comprises a polycyclic ring system having two or more cyclic rings in which two or more carbons are common to two adjacent rings, wherein at least one of the rings is aromatic, and, for example, the other cyclic ring may be a cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. The aryl group includes benzene, naphthalene, phenanthrene, phenol, aniline, etc.

[0095] The "cycloalkyl" group is a fully saturated cyclic hydrocarbon. "Cycloalkyl" includes monocyclic and bicyclic rings. Typically, unless otherwise defined, a monocyclic cycloalkyl group has 3 to about 10 carbon atoms, more typically 3 to 8 carbon atoms. The second ring of a bicyclic cycloalkyl may be selected from saturated, unsaturated, and aromatic rings. Cycloalkyl includes bicyclic molecules in which 1, 2, or 3 or more atoms are shared between two rings. The term "fused cycloalkyl" refers to a bicyclic cycloalkyl in which each ring shares two adjacent atoms with the other ring. The second ring of a fused bicyclic cycloalkyl may be selected from saturated, unsaturated, and aromatic rings. The "cycloalkenyl" group is a cyclic hydrocarbon containing one or more double bonds.

[0096] The terms “halo” and “halogen” mean halogens as used herein, and include chloro, fluoro, bromo, and iodo.

[0097] The terms “heteroaryl” and “hetaryl” comprise a substituted or unsubstituted aromatic single ring structure, preferably a 5- to 7-membered ring, more preferably a 5- to 6-membered ring, wherein the ring structure comprises at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms “heteroaryl” and “hetaryl” also comprise a polycyclic ring system having two or more cyclic rings in which two or more carbons are common to two adjacent rings, wherein at least one of the rings is heteroaromatic, and, for example, the other cyclic ring may be a cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0098] The term "heteroatom" means an atom of any element other than carbon or hydrogen, as used herein. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0099] The terms “heterocyclil,” “heterocycle,” and “heterocyclic” refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, wherein the ring structure comprises at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms “heterocyclil” and “heterocyclic” also comprise a polycyclic ring system having two or more cyclic rings in which two or more carbons are common to two adjacent rings, wherein at least one of the rings is heterocyclic, and, for example, the other cyclic ring may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclil. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, tetrahydropyran, tetrahydrofuran, morpholine, lactone, lactam, etc.

[0100] The term “lower” is intended to comprise a group having 10 or fewer, preferably 6 or fewer, non-hydrogen atoms within the substituent when used with chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy. “Lower alkyl” refers to, for example, an alkyl group containing 10 or fewer, preferably 6 or fewer carbon atoms. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are each a lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, whether they appear alone or in combination with other substituents as in the mention of hydroxyalkyl and aralkyl (wherein, for example, atoms in the aryl group are not counted when counting carbon atoms in the alkyl substituent).

[0101] The term "substituted" refers to a moiety having a substituent that replaces a hydrogen on one or more carbons of the main chain. The implied condition that "substituted" or "substituted" includes that such substitutions are subject to the allowed valence of the substituted atoms and substituents, and that said substitutions, for exampleIt will be understood that conversion results in a stable compound that does not spontaneously undergo, for example, rearrangement, cyclization, elimination, etc. As used herein, the term “substituted” is considered to include all acceptable substituents of an organic compound. In a broad sense, acceptable substituents include acyclic and cyclic, branched and unbranched, carbon-cyclic and heterocyclic, aromatic and non-aromatic substituents of the organic compound. There may be one or more acceptable substituents and may be the same or different for a suitable organic compound. For the purposes of the present invention, a heteroatom, e.g., nitrogen, may have any acceptable substituent and / or hydrogen substituent of the organic compound described herein that satisfies the valence of the heteroatom. The substituent may comprise any substituent described herein, e.g., halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidin, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclil, aralkyl, or aromatic or heteroaromatic moiety. In a preferred embodiment, the substituent on the substituted alkyl phase is C 1-6 Alkyl, C 3-6It is selected from cycloalkyl, halogen, carbonyl, cyano, or hydroxyl. In a more preferred embodiment, the substituent on the substituted alkyl phase is selected from fluoro, carbonyl, cyano, or hydroxyl. It will be understood by those skilled in the art that the substituent itself may be substituted where appropriate. Unless specifically stated as "unsubstituted," references to chemical moiety herein are understood to include substituted variants. For example, references to an "aryl" group or moiety implicitly include both substituted and unsubstituted variants.

[0102] A "protecting group" refers to a group of atoms that, when attached to a reactive functional group within a molecule, masks, reduces, or prevents the reactivity of the functional group. Typically, protecting groups can be selectively removed as desired during the synthesis process. Examples of protecting groups can be found in the following literature: Greene and Wuts, Protective Groups in Organic Chemistry, 3 rd Ed., 1999, John Wiley & Sons, NY and Harrison et al. Compendium of Synthetic Organic MethodsRepresentative 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"), nitro-veratryloxycarbonyl ("NVOC"), etc. Representative hydroxyl protecting groups include, but are not limited to, hydroxyl groups that are 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, such as ethylene glycol and propylene glycol derivatives, and allyl ethers.

[0103] The term "modulation" as used herein includes not only the inhibition or suppression of a function or activity (e.g., cell proliferation) but also the enhancement of a function or activity.

[0104] The phrase "pharmaceuticalally acceptable" is recognized in the art. In certain embodiments, the term includes compositions, excipients, ajuvants, polymers, and other substances and / or dosage forms suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within the scope of sound medical judgment and in accordance with a reasonable benefit / risk ratio.

[0105] "Pharmaceuticalally acceptable salt" or "salt" is used herein to refer to an acid addition salt or a basic addition salt that is suitable or compatible with the treatment of a patient.

[0106] The term “pharmaceutically acceptable acid addition salt” means any non-toxic organic or inorganic salt of any base compound represented by Formula I as used herein. Exemplary inorganic acids that form suitable salts include hydrochloric acid, hydrobromide, 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, pyruvate, 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. Monovalent or divalent acid salts may be formed, and these salts may exist in hydrated, solvated, or substantially anhydrous forms. Generally, the acid addition salt of the compound of Formula I is more soluble in water and various hydrophilic organic solvents and generally exhibits a higher melting point compared to its free base form. The selection of a suitable salt will be known to those skilled in the art. Other non-pharmaceutical acceptable salts, e.g., oxalates, may be used in the isolation of the compound of Formula I, e.g., for laboratory use or for subsequent conversion into a pharmaceutically acceptable acid addition salt.

[0107] The term “pharmaceutically acceptable basic addition salt” means any non-toxic organic or inorganic basic addition salt of any acid compound represented by Formula I or any intermediate thereof, as used herein. Exemplary inorganic bases that form a suitable salt include lithium, sodium, potassium, calcium, magnesium, or barium hydroxide. Exemplary organic bases that form a suitable salt include aliphatic, alicyclic, or aromatic organic amines, such as methylamine, trimethylamine, picoline, or ammonia. The selection of a suitable salt will be known to those skilled in the art.

[0108] Many compounds useful in the methods and compositions of the present disclosure have at least one stereogenic center within their structure. This stereogenic center may exist in an R or S arrangement, and the R and S notations are used in accordance with the rules set forth in Pure Appl. Chem. (1976), 45, 11-30. The present disclosure considers all stereoisomer forms of compounds, salts, prodrugs, or mixtures thereof, such as enantiomers and diastereomers (including mixtures of all possible stereoisomers). See, for example, WO 01 / 062726.

[0109] Additionally, certain compounds containing alkenyl groups may exist as Z (same-sided) or E (opposite-sided) isomers. In each case, the present disclosure includes both the mixture and the separate individual isomers.

[0110] Some of the compounds may also exist in tautomeric forms. These forms are intended to be included within the scope of this disclosure, even if they are not explicitly indicated in the formulas described herein.

[0111] “Prodrug” or “pharmaceutically acceptable prodrug” refers to a compound that is metabolized within a host after administration, for example, by hydrolysis or oxidation, to form the compound of the present disclosure (e.g., a compound of Formula I). ​​Typical examples of a prodrug include a compound having a biologically labile or cleavable (protecting) group on a functional moiety of the active compound. A prodrug includes a compound capable of being oxidized, reduced, aminated, deaminated, hydroxylated, dihydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, or dephosphorylated to produce an active compound. Examples of prodrugs using an ester or phosphoramidate as the biologically labile or cleavable (protecting) group are disclosed in U.S. Patents 6,875,751, 7,585,851, and 7,964,580, the contents of which are incorporated herein by reference. The prodrug of the present disclosure is metabolized to produce a compound of Formula I. The present disclosure includes, within its scope, a prodrug of the compound described herein. Conventional procedures for selecting and preparing a suitable prodrug are described, for example, in “Design of Prodrugs” Ed. H. Bundgaard, Elsevier, 1985.

[0112] The phrase "pharmaceuticalally acceptable carrier" means a pharmaceutically acceptable substance, composition, or vehicle useful for the formulation of a drug for medicinal or therapeutic use as used herein, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material.

[0113] The terms “abnormal cell growth” and “proliferative disorder” are used interchangeably herein. “Abnormal cell growth” as used herein refers to cell growth independent of normal regulatory mechanisms, unless otherwise indicated ( for example, loss of contact inhibition). This includes, for example, the following abnormal growths: (1) tumor cells (tumors) that proliferate by expressing a mutated tyrosine kinase or by overexpression of a receptor tyrosine kinase; (2) benign and malignant cells of other proliferative diseases in which abnormal tyrosine kinase activation occurs; (3) any tumor proliferated by a receptor tyrosine kinase; (4) any tumor proliferated by abnormal serine / threonine kinase activation; and (5) benign and malignant cells of other proliferative diseases in which abnormal serine / threonine kinase activation occurs.

[0114] The terms "cancer" and "cancerous" refer to or describe physiological pathologies in mammals typically characterized by uncontrolled cell growth. "Tumor" contains one or more cancerous cells. Examples of cancer include, but are not limited to, the following: carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma ( for example , epithelial squamous cell carcinoma), 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 carcinoma, gastrointestinal cancer, etc. Stomach or stomach cancer ( It includes gastric or stomach cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, acute leukemia, as well as head / brain and neck cancer.

[0115] Compounds and conjugates of the present invention

[0116] Compound of chemical formula (I)

[0117] The present disclosure describes a compound having the structure of chemical formula (I):

[0118]

[0119] or provides his pharmaceutically acceptable salt,

[0120] Here:

[0121] Ring A is a heterocyclic, aryl, or heteroaryl ring;

[0122] Ring B is a heterocyclic ring, preferably a 5- or 6-membered ring, fused to or substituted with one or more optional aryl or heteroaryl rings;

[0123] R 1 and R 2 are each independently alkyl;

[0124] Z 1 is a methylene or linking group that can be additionally conjugated to a targeting agent;

[0125] n a is an integer with a value of 1 or 2;

[0126] n 1 and n 2 Each is an integer that independently has a value from 1 to 5.

[0127] In some embodiments, at least one of ring A or ring B is a heteroaryl ring.

[0128] For example, in some embodiments, ring A is and, here:

[0129] A 1 , A 2 , and A 3 NR each independently a1 , CR a2 , O, N, or S and;

[0130] R a1 is H, alkyl, halogen, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;

[0131] R a2 is H, or alkyl, preferably lower alkyl.

[0132] In some embodiments, ring B is a 5- or 6-membered heterocyclic ring, and one or more R's selected optionally from alkyl, alkylidenyl, aryl, or heteroaryl. C It is replaced by gi.

[0133] In some of these embodiments, ring B is or and, here:

[0134] The dotted line indicates that double bonds exist arbitrarily;

[0135] R C is H, OH, alkyl, alkylidenyl, halogen, amino, cyano, or aryl.

[0136] In some of these embodiments, ring B is and, here

[0137] R 1a is H, OH, SH, alkylthiol, -CH2N(CH3)2, alkyl, alkyloxy, alkylidenyl, halogen, amino, cyano, or aryl.

[0138] In some embodiments, 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. In some of these embodiments, rings B and C together form the following structure:

[0139] , Here:

[0140] Ring C' is arbitrarily R 1c Substituted by;

[0141] Three or fewer carbon atoms within ring C' can be replaced by nitrogen;

[0142] R 1c is OH, SH, alkylthiol, -CH2N(CH3)2, alkyl, alkyloxy, alkyl, halogen, amino, or cyano;

[0143] n b is an integer with a value of 1 or 2.

[0144] In some embodiments, ring A and ring C' are independently , , , , , and Selected from.

[0145] In a specific preferred embodiment, ring A is Selected from. In a specific implementation, R a1 It is a C1-C4-alkyl, preferably methyl.

[0146] In a specific embodiment, ring C' is and, here:

[0147] B 1 , B 2 , and B 3 NR each independently a1 , CR a2 , O, N, or S and;

[0148] R b1 is H, alkyl, halogen, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl;

[0149] R b2 is absent, H, or alkyl.

[0150] In some of these implementations, ring C' is

[0151] , and It is selected from. In a specific preferred embodiment, ring C' is Selected from. In a specific implementation, R b1 It is a C1-C4-alkyl, preferably methyl.

[0152] In some embodiments, ring A is and, here:

[0153] A 6 , A7 , A 8 , and A 9 CR each independently a3 or N and;

[0154] R a3 It is H, alkyl, halogen, nitro, hydroxy, nitrile, cyano, alkoxy, amino, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, but,

[0155] Only 2 or fewer A's 6 , A 7 , A 8 , and A 9 is N.

[0156] In a specific embodiment, ring C is and, here:

[0157] B 6 , B 7 , B 8 , and B 9 CR each independently b3 or N and;

[0158] R b3 It is H, alkyl, halogen, nitro, hydroxy, nitrile, cyano, alkoxy, amino, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, but,

[0159] Only 2 or fewer B 6 , B 7 , B 8 , and B 9 is N.

[0160] In some embodiments, ring A and ring B are each independently selected from rings as defined in the above embodiments.

[0161] In a specific embodiment, either ring A or ring C is And, the other of ring A or ring C is selected from a heteroaryl ring as defined in any one of the above embodiments.

[0162] In a specific embodiment, the compound of formula (I) has the structure of formula (Ia), (Ib-1), (Ib-2), (Ib-3), (Ic-1), (Ic-2), or (Ic-3):

[0163]

[0164]

[0165] It is his pharmaceutically acceptable salt, where the variable is defined as described above.

[0166] In some of these implementations, R 1 and R 2 is identical. For example, in a specific implementation, R 1 and R 2 is a C1-C4-alkyl, preferably methyl.

[0167] In some implementations, Z 1 It is methylene.

[0168] In another embodiment, Z 1 silver and, here:

[0169] Y 1 is CR Y1 or N, provided there is only one Y 1 This is N and;

[0170] R Y1 It is H or hydroxyl, amino, amido, or (CH2) y (R Y1a ) yy And;

[0171] R Y1a is an amino, aryl, or heteroaryl;

[0172] y and yy are each independent integers with values ​​ranging from 1 to about 10.

[0173] In a specific preferred embodiment, Z 1 silver and, here R Z1 is absent, or hydroxyl, amino, amido, or (CH2) z (RZ1a ) zz and; R Z1a is an amino, aryl, or heteroaryl; z and zz are each independently integers having a value from 1 to about 10. In a more preferred embodiment, Z 1 silver ; , or is. In a specific preferred embodiment, Z 1 silver and, here, Y 2 is CR Y2 or N and; R Y2 is H or alkyl, preferably a lower alkyl; R Z2 is (CH2) z R Z2a and; R Z2a is an amino (preferably a tertian amino), aryl, or heteroaryl; and z is an integer having a value of 0 to about 10.

[0174] In a specific preferred embodiment, Z 1 ... provides an amine or phenol for conjugation. Accordingly, in certain such embodiments, for example, Z 1 It can be selected from amino, hydroxy-substituted aryl, or nitrogen-containing heteroaryl, and may optionally be further substituted. In other such embodiments, R Y1a and R Z1a provides an amine or phenol for conjugation. For example, R Z1a can be selected from amino (e.g., alkyl-substituted amino), hydroxy-substituted aryl, or nitrogen-containing heteroaryl, and optionally can be further substituted.

[0175] In a specific preferred embodiment, Z 1 silver or am.

[0176] In a specific embodiment, the compound of formula (I) is selected from the following:

[0177]

[0178]

[0179] or his pharmaceutically acceptable salt.

[0180] In a specific other embodiment, the compound of formula (I) is selected from the following:

[0181]

[0182]

[0183]

[0184] or his pharmaceutically acceptable salt.

[0185] In some embodiments, the present disclosure describes a compound having the structure of formula (IV):

[0186]

[0187] or it concerns his pharmaceutically acceptable salt,

[0188] Here:

[0189] Ring A is a heterocyclic, aryl, or heteroaryl ring;

[0190] R 1 is alkyl;

[0191] W a is H or benzyl; and

[0192] n a is 1 or 2.

[0193] In a specific embodiment, ring A is and, here:

[0194] A 1 , A 2 , and A 3 NR each independently a1 , CR a2 , O, N, or S and;

[0195] R a1 is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and

[0196] R a2 is H, or alkyl, preferably lower alkyl.

[0197] For example, ring A is

[0198] and It can be selected from.

[0199] In some embodiments, ring A is am.

[0200] In a specific implementation, R a1 It is a C1-C4-alkyl, for example, methyl.

[0201] In a specific embodiment, ring A is and, here:

[0202] A 6 , A 7 , A 8 , and A 9 CR each independently a3 or N and;

[0203] R a3 It is H, alkyl, halogen, nitro, hydroxy, nitrile, cyano, alkoxy, amino, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, but,

[0204] Only 2 or fewer A's 6 , A 7 , A 8 , and A 9 is N.

[0205] Conjugate of the compound of chemical formula (I)

[0206] In some embodiments, a conjugate comprising a compound of formula (I) is provided herein, 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 releases a nucleophilic heteroatom when subjected to a chemical reaction (e.g., physicochemical reaction and / or biological reaction) under predetermined conditions, and an 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 an active agent. In some embodiments, the conjugate further comprises a targeting moiety (e.g., oligopeptide, polypeptide, antibody, etc.) having binding specificity to a desired target receptor or other molecule associated with the target cell.

[0207] Compounds of chemical formulas (II), (IIa), and (IIb)

[0208] In addition, compounds having the structures of chemical formulas (II) and (IIa):

[0209]

[0210] Or its pharmaceutically acceptable salt is provided herein,

[0211] Here:

[0212] Ring A is a heterocyclic, aryl, or heteroaryl ring;

[0213] Ring B is a heterocyclic ring, preferably a 5- or 6-membered ring, fused to or substituted with one or more optional aryl or heteroaryl rings;

[0214] R 1 and R 2 Each is independently an alkyl group;

[0215] Z 1 is a methylene or linker;

[0216] n a is an integer with a value of 1 or 2;

[0217] n 1 and n 2 Each is an integer that independently has a value from 1 to 5.

[0218] Z 2 is absent or a connecting device;

[0219] L 1 is a linker attached to SO2 through a heteroatom selected from O, S, and N, preferably O or N, and L 1 L that releases an active agent upon the cleavage of the bond between and SO2 1 and Z 1 Selected to facilitate the severance of the bond between;

[0220] X 1 -O-, -CR a 2-, or -NR'-, preferably -O- and;

[0221] Ar represents a ring, e.g., aryl, heteroaryl, cycloalkyl, or heterocycloalkyl, preferably aryl or heteroaryl;

[0222] Y 1 Silver, arranged so that N, O, or S atoms are attached to TG when y is 1, -(CR b 2) y N(R a )-, -(CR b 2) y O-, or -(CR b 2) y S- and; here X 1 and Y 1 Silver is placed on an adjacent atom of Ar;

[0223] TG is a triggering group that, upon activation, reacts with SO2 to produce an N, O, or S atom capable of replacing Z and forming a 5-6 member ring containing an intervening atom of X-SO2 and Ar;

[0224] w and x are integers that each independently have a value of 0 or 1;

[0225] Each R a and R c is independently hydrogen or lower alkyl; and

[0226] Each R b is independently hydrogen or lower alkyl; or

[0227] 2 Rs b Together with the carbon atoms to which they are attached, they form a 3-5 member ring, preferably a 3-4 member ring.

[0228] In some implementations, X 1 It is -O-.

[0229] In a specific embodiment, Ar is an aryl. In a preferred embodiment, it is a phenyl or naphthyl.

[0230] In some implementations, Z 2 isocyanid, isothiocyanid, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH2-halo), maleimide, diene, alkene, halogen, tosylate (TsO - ), aldehyde, sulfonate (R-SO3 - ), , , 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 derivative (-C(O)C≡CR a It is a linking group comprising one or more groups selected from ), and dihydrogen phosphate (-OP(=O)(OH)2).

[0231] In a specific implementation, x is 0.

[0232] Release of active agent

[0233] As described above, in certain embodiments, the compound and conjugate disclosed herein may dissociate one or more activators represented by Formula (II) through an intramolecular cyclization reaction following a chemical reaction that activates the inducing group. In certain embodiments, the chemical reaction is a physicochemical reaction and / or a biochemical reaction.

[0234] In some embodiments, the compound and conjugate disclosed herein are X ( for example It includes a nucleophilic functional group (Y or Y') introduced from an adjacent atom on Ar for (, O). Typically, the nucleophilic functional group is masked by a causative group (TG) as further described below. Upon activation, the causative group releases the nucleophilic functional group to react with an adjacent SO2 moiety in intramolecular cyclization, ultimately releasing one or more compounds of formula (II), (IIa), or (IIb). In some such embodiments, one or more activators are released via an intramolecular cyclization reaction following a chemical reaction, a physicochemical reaction, and / or a biochemical reaction (see, e.g., Reaction Scheme 1), or the activators are released via 1,6-elimination or 1,4-elimination following an intramolecular cyclization reaction (see, e.g., Reaction Scheme 2).

[0235] As an example, Y is -Y'-TG and Q is SO 2 When directly conjugated to the gi, the active agent can be released by the mechanism shown in Reaction Scheme 1:

[0236] Reaction Equation 1:

[0237]

[0238] Q is In the case of Q 1 It can be released by the mechanism shown in Reaction Scheme 2:

[0239]

[0240] In some embodiments, Q 1is an activator comprising at least one functional group selected from -OH, -NH-, -SH, and -COOH when released. According to these embodiments, as further described herein, Q 1 is conjugated to compounds as described herein by -OH, -NH-, -SH, and -COOH through functional groups selected from, for example, esters, amides, thioesters, carbamates, ureas, oximes, hydrazones, etc. In some such embodiments, Q 2 is Q 1 Used instead, and Q 2 is an amine group-containing drug. In another embodiment, Q 2 is an activator capable of binding to ammonium units. In another embodiment, Q 2 is Q 2 Upon release, it can dissociate into its initial form having an amide group, and the activator may be a drug, a toxin, an affinity ligand, a probe for detection, or a combination thereof.

[0241] In some embodiments, the compounds and conjugates disclosed herein are chemically and physiologically stable. In some such embodiments, the compounds and conjugates disclosed herein selectively release a drug by reaching a desired target cell with minimal dissociation of the activator in the blood.

[0242] Inducing agent (TG)

[0243] In some embodiments, the conjugate of the present invention comprises a causative group (TG). The TG is a group that can be cleaved by a chemical reaction, e.g., a biological reaction, preferably optionally cleaved. Generally, the causative group serves to block the nucleophilic nature of the Y or Y' group, thereby thereby improving stability ( for example, by preventing self-sacrificial or intramolecular cyclization before the conjugate reaches a target site or experiences a predetermined trigger condition) is provided to the compounds and conjugates disclosed herein. Upon activation, the trigger group releases a nucleophilic Y group and causes self-sacrificial or intramolecular cyclization to occur as described above.

[0244] In some embodiments, TG comprises a sequence (e.g., a peptide sequence) or a moiety recognized by TEV, trypsin, thrombin, cathepsin B, cathepsin D, cathepsin K, caspase 1, matrix metalloproteinase (MMP), etc., and this includes an enzyme ( for example , oxidoreductase, transferase, hydrolase, degrading enzyme, isomerase, ligase, etc. Can be hydrolyzed by ) or phosphodiesters, phospholipids, esters, β-galactose, β-glucose, fucose, oligosaccharides, etc. It may include a moiety selected from.

[0245] In some embodiments, TG comprises a reactive chemical moiety or functional group, wherein it is a nucleophilic reagent condition ( for example It can be cleaved under silyl ether, 2-N-acyl nitrobenzenesulfonamide, unsaturated vinyl sulfide, sulfonamide after activation, malon dialdehyde-indole derivative, levulinoy ester, hydrazone, or acyl hydrazone.

[0246] In some embodiments, TG may include a reactive chemical moiety or functional group, provided that it is under basic reagent conditions ( for example It can be cleaved under , 2-cyanoethyl ester, ethylene glycol disuccinate, 2-sulfonylethyl ester, alkyl thioester, or thiophenyl ester.

[0247] In some embodiments, TG may include a reactive chemical moiety or functional group that can be cleaved by photo-irradiation ( for example, 2-nitrobenzyl derivatives, phenacyl esters, 8-quinolinylbenzenesulfonate, coumarin, phosphotriesters, bis-arylhydrazone, or bi-thiophionic acid derivatives).

[0248] In some embodiments, TG may include a reactive chemical moiety or functional group, which may be cleaved under reducing agent conditions ( for example , hydroxylamine, disulfide, revelinate, nitro, or 4-nitrobenzyl derivative).

[0249] In some embodiments, TG may include a reactive chemical moiety or functional group, which may be cleaved under acidic conditions ( for example , sugars, tert-butylcarbamate analogs, dialkyl or diaryl dialkoxysilanes, orthoesters, acetals, aconityl, hydrazone, β-thiopropionate, phosphoramidate, imines, trityl, vinyl ethers, polyketals, and alkyl 2-(diphenylphosphino)benzoate derivatives; alkyl esters, 8-hydroxyquinoline esters, and picolinate esters).

[0250] In some embodiments, TG may include a reactive chemical moiety or functional group, provided that it is subject to oxidation conditions ( for example , boronate, proximity diol, paramethoxybenzyl derivative, or selenium compound) It can be cut off under.

[0251] In a specific preferred embodiment, TG comprises a sugar, which can be cleaved under acidic or enzymatic conditions. In a specific preferred embodiment, the initiating group is -NO2, which can be cleaved under reducing conditions. In a specific preferred embodiment, the initiating group is boronate, which can be cleaved under oxidizing conditions. In a specific preferred embodiment, the initiating group is an ester, which can be cleaved under acidic, basic, or enzymatic conditions. In a specific preferred embodiment, the initiating group is a hydrazone, which can be cleaved under nucleophilic or acidic conditions. In a specific preferred embodiment, the initiating group is hydroxylamine, which can be cleaved under reducing conditions.

[0252] Sugar-inducing agent

[0253] In some embodiments, the compounds and conjugates disclosed herein comprise a sugar-inducing group, for example, a group selected from the following:

[0254] and

[0255] Among the formulas, each R 21 is independently hydrogen or OR 21 hydroxy protector ( for example , selected to be acetyl); R 22 is hydrogen or lower alkyl (for example, C1-C6-alkyl) In certain embodiments, the hydroxy protecting group may be used in organic synthesis and includes, without limitation, the following: 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, triphenyl methyl ether, α-naphthyldiphenyl methyl ether, p-Methoxyphenyldiphenylmethyl ether, 9-(9-phenyl-10-oxo)anthyl 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, adamanthoate 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, etc. However, it is not limited to these.

[0256] Protector as an inducing agent

[0257] In some embodiments, TG is a group that can be cleaved by a chemical reaction, a physicochemical reaction, and / or a biological reaction. In certain embodiments, TG is a protecting group. In some such embodiments, the protecting group is an amine group protecting group, an alcohol protecting group, or a thiol protecting group.

[0258] Amine protector

[0259] In certain embodiments, the amine protecting group is a general protecting group that can be used in organic synthesis and includes, without limitation, the following: m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, alkyl carbamate, 9-fluorenylmethyl carbamate, 2,2,2-trichloroethyl carbamate, 2-trimethylsilylethyl carbamate (Teoc), t-butyl carbamate (Boc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, benzyl carbamate, p-methoxybenzyl carbamate, p-nitrobenzyl carbamate, diphenyl methyl carbamate, acetamide, chloroacetamide, trichloroacetamide, Phenylacetamide, Benzamide, N-Phthalimide, N-2,3-Diphenylmaleimide, N-2,5-Dimethylpyrrole, N-1,1-Dimethylthiomethyleneamine, N-Benzylideneamine, Benzenesulfenamide, o-Nitrobenzenesulfenamide, Triphenylmethylsulfenamide, p-Toluenesulfonamide, Methanesulfonamide, etc. However, it is not limited to these.

[0260] alcohol protector

[0261] In certain embodiments, the alcohol protecting group is a general protecting group that can be used in organic synthesis and includes, without limitation, the following: 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, chloropropylmethyl 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-methoxytetrahydrothiopyranyl ether, tetrahydrofuranyl ether, 1-ethoxyethyl ether, 1-methyl-1-methoxyethyl ether, 2-(phenylselenyl)ethyl ether), t-butyl ether, allyl ether, benzyl ether, o-nitrobenzyl ether, triphenylmethyl ether, α-naphthyldiphenylmethyl ether, p-methoxyphenyldiphenylmethyl ether, 9-(9-phenyl-10-oxo)anthyl ether, trimethylsilyl ether (TMS ether), isopropyldimethylsilyl ether, t-butyldimethylsilyl ether (TBDMS ether), t-butyldiphenylsilyl ether, tribenzylsilyl ether, triisopropylsilyl ether, formate ester, acetate ester, trichloroacetate ester, phenoxyacetate ester, isobutyrate ester, pivaloate ester, adamanthoate ester, benzoate ester, 2,4,6-trimethylbenzoate (methitoate) ester, methyl carbonate, 2,2,2-trichloroethyl carbonate, allyl carbonate, p-nitrophenyl carbonate, benzyl carbonate, p-nitrobenzyl carbonate, S-benzyl thiocarbonate, N-phenylcarbamate,Nitrate esters, 2,4-dinitrophenylsulfenate esters, dimethylphosphinyl esters (DMP esters), dimethylthiophosphinyl esters (MPT esters), aryl methanesulfonate, aryl toluenesulfonate, etc. However, it is not limited to these.

[0262] Thiol protector

[0263] In certain embodiments, thiol protecting groups may be used in organic synthesis and include, without limitation, the following: 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-anthylmethyl thioether, S-9-fluorenylmethyl thioether, S-methoxymethyl monothioacetal, A-acetyl derivative, S-benzoyl derivative, S-(N-ethyl carbamate), S-(N-methoxymethyl carbamate), etc. However, it is not limited to these.

[0264] Connector

[0265] In some embodiments, the compounds and conjugates disclosed herein include a linking group connecting each CB and Ar through a covalent bond. A typical linking group is a stable, non-hydrolyzable moiety, e.g., C 10 -C 100 It is a linear or branched, saturated or unsaturated alkylene. In a specific embodiment, the linking unit satisfies at least two of the following four criteria, and more preferably at least three:

[0266] (i) At least one -CH2- in the alkylene moiety is substituted (i.e. replaced by) one or more heteroatoms selected from -NH-, -C(=O), -O-, -S- and -P-;

[0267] (ii) At least one heteroarylene is contained within an alkylene moiety;

[0268] (iii) at least one amino acid moiety, sugar bond, peptide bond, or amide bond is contained within an alkylene moiety; and

[0269] (iv) The above alkylene is C1-C 20 Alkyl, C6-C 20 Aryl C1-C8 alkyl, -(CH2) s COOH, and -(CH2) p It may be further substituted with one or more substituents selected from the group consisting of NH2, wherein s is an integer having a value from 0 to 10, and p is an integer having a value from 1 to about 10.

[0270] In a specific embodiment, the connection unit comprises at least two of the following, and more preferably at least three:

[0271] (i) at least one heteroatom selected from -NH-, -C(=O), -O-, -S- and -P-;

[0272] (ii) at least one heteroarylene;

[0273] (iii) at least one amino acid moiety, sugar bond, peptide bond, or amide bond; and

[0274] (iv) The above alkylene is C1-C 20 Alkyl, C6-C 20 Aryl C1-C8 alkyl, -(CH2) s COOH, and -(CH2) p It may be further substituted with one or more substituents selected from the group consisting of NH2, wherein s is an integer having a value from 0 to 10, and p is an integer having a value from 1 to about 10.

[0275] In another embodiment, the linker connecting each CB and AR includes a functional group generated through a click chemical reaction.

[0276] In an alternative embodiment, the linking unit includes a reaction functional group capable of participating in a click chemical reaction.

[0277] Click chemical reactions can be carried out under mild conditions, and functional groups not typically found in biological molecules ( for example It is an extremely selective reaction for azide groups, acetylene groups, etc. Therefore, such reactions can be carried out in the presence of complex inducing groups, targeted moiety, etc. Furthermore, click chemistry possesses high reaction specificity. For example, the click chemical reaction between an azide group and an acetylene group proceeds selectively without interference from other functional groups present in the molecule. For example, azide-acetylene click chemistry can yield triazole moiety in high yield.

[0278] Therefore, in some embodiments, the connector connecting each CB and Ar is or Includes, where V is a single bond, -O-, -S-, -NR 21 -, -C(O)NR 22 -, -NR 23 C(O)-, -NR 24 SO2-, or -SO2NR 25 -It could be, R 21 to R 25 Each is independently hydrogen, (C1-C6)alkyl, (C1-C6)alkyl(C6-C 20 )aryl, or (C1-C6)alkyl(C3-C 20 ) It may be a heteroaryl, r may be an integer having a value from 1 to about 10, p may be an integer having a value from 0 to about 10, q may be an integer having a value from 1 to about 10, and L" may be a single bond.

[0279] In another embodiment, the connection unit connecting each CB and Ar is Equation ( A )to It is a connector presented by:

[0280]

[0281] During the meal:

[0282] * is the attachment point for CB;

[0283] ** is the attachment point for Ar;

[0284] W a1 , W a2 , and W a3 Each independently -NH-, -C(=O)-, or (-CH2-) b And;

[0285] W b1 is an amide bond or triazolilene;

[0286] P 1 is W a3 and Y 2 It is a linker connecting, and is an amino acid moiety, a peptide bond, or an amide bond;

[0287] L c is an alkylene;

[0288] 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- and;

[0289] R e is a C1-C8 alkyl or CB-W a7 -Y3-W c1 -(CH2) f - and;

[0290] R f is BW a7 -Y 3 -W c1 -(CH2) f - and;

[0291] X is -NHC(=O)-(CH2) g -W a8 - or -C(=O)NH-(CH2)h -W a9 - and;

[0292] W a4 , W a5 , W a6 , W a7 , W a8 , and W a9 Each is independently -NH-, -C(=O)-, or -CH2-;

[0293] W b2 is an amide bond or triazolilene;

[0294] W c1 is -NHC(=O)- or -C(=O)NH- and;

[0295] Y 3 -(CH2) i -(X'CH2CH2) j -(CH2) k - and;

[0296] X' is -O-, -S-, -NH-, or -CH2-;

[0297] CB is the same as defined above;

[0298] b, c, d, e, f, g, h, i, and j are each independent integers having a value from 1 to about 10;

[0299] k and y are each independently integers having a value from 0 to about 10;

[0300] Y 1 -(CH2) q -(CH2CH2X") o - or -(CH2) q -(X"CH2CH2) o - and;

[0301] X" is -O-, -S-, -NH-, or -CH2-; and

[0302] o and q are integers with values ​​ranging from 1 to about 10.

[0303] In some embodiments, P 1 is the formula ( B) or ( C Includes at least one unit presented by ):

[0304]

[0305] During the meal:

[0306] R 12 is hydrogen, C1-C8-alkyl, amino acid side chain, e.g., natural amino acid side chain ( for example , H, methyl, isopropyl, isobutyl, sec-butyl, S-methyl thioether, benzyl, indole, pyrrolidine, pariroline, hydroxymethyl, tyrosyl, lysyl, imidazole, glycyl, glutamyl, carbamoylbutanoic acid, carboxamide, aspartic acid, 1-hydroxyethyl, and 2-hydroxyethyl), -(CH2) s COR 13 or -(CH2) p NR 14 R 15 And;

[0307] R 13 is OH or -NH(CH2) s' (X"CH2CH2) s" It is Z;

[0308] R 14 and R 15 Each independently hydrogen or -(C(O)(CH2) s' (X"CH2CH2) s" Z) m -CB and;

[0309] X" is -O-, -S-, -NH-, or -CH2-;

[0310] Z and CB are the same as defined above;

[0311] p is an integer having a value from 1 to about 10;

[0312] s and s" are integers having values ​​from 0 to about 10;

[0313] s' is an integer having a value from 1 to about 10;

[0314] m is an integer with a value of 0 or 1.

[0315] In some implementations of expression (B) or (C):

[0316] R 12 is hydrogen, alkyl, amino acid side chain, -(CH2) s C(O)R 13 or -(CH2) p NR 14 R 15 And;

[0317] p is an integer having a value from 1 to about 10;

[0318] s is an integer having a value from 0 to about 10;

[0319] R 13 is OH or -NH(CH2) s' (X"'CH2CH2) s" Z"-(CB) m And;

[0320] R 14 and R 15 Each independently hydrogen or -C(O)(CH2) s' (X"'CH2CH2) s" Z"-(CB) m And;

[0321] s" is an integer with a value from 0 to about 10;

[0322] s' is an integer having a value from 1 to about 10;

[0323] m is an integer with a value of 0 or 1;

[0324] X"' is -O-, -S-, -NH-, or -CH2-; and

[0325] Z" is CB R 14 or R 15 It is a linker that connects to the rest of; or Z" is a linker containing a reactive group.

[0326] In a part of such an implementation of formula (B) or (C):

[0327] R 13 is OH or -NH(CH2)s' (X"'CH2CH2) s" Z" and;

[0328] R 14 and R 15 Each independently hydrogen or -C(O)(CH2) s' (X"'CH2CH2) s" Z" and; and

[0329] Z" is isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH2-hal), maleimide, diene, alkene, halogen, tosylate (TsO - ), aldehyde, sulfonate (R-SO3 - ), 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 derivative (-C(O)C≡CR a It is a reaction precursor of a linkage unit selected from, and R a is C1-C 10 -alkyl), and dihydrogen phosphate (-OP(=O)(OH)2).

[0330] In other such implementations of expression (B) or (C):

[0331] R 13 is OH or -NH(CH2) s' (X"'CH2CH2) s" Z"CB and;

[0332] R 14 and R 15 Each independently hydrogen or -C(O)(CH2) s' (X"'CH2CH2) s" Z"CB and; and

[0333] Z" is CB, R formed from a precursor selected from the following. 14 or R 15It is a linking unit that connects to the remainder of: isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH2-hal), maleimide, diene, alkene, halogen, tosylate (TsO - ), aldehyde, sulfonate (R-SO3 - ), 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 derivative (-C(O)C≡CR a , R a is C1-C 10 -alkyl), and dihydrogen phosphate (-OP(=O)(OH)2).

[0334] In some implementations, Y 2 is selected from a single combination or the following:

[0335] and

[0336]

[0337] During the meal:

[0338] W b2 -C(O)NH-, -NHC(O)-, or And;

[0339] R e is a C1-C8-alkyl or -(L 1' -Z-) m It is CB;

[0340] R f is BW b2' -(CH2) i -(X"CH2CH2) j -NH-C(=O)-(CH2) f - and;

[0341] X a -NHC(=O)-(CH2) g-NH- or -C(O)NH-(CH2) h -NH- and;

[0342] W b2' is -C(O)NH- or -NHC(=O)- and;

[0343] Each of c, d, e, f, g, h, i, and j is an integer having a value from 1 to about 10 independently;

[0344] X" is -O-, -S-, -NH-, or -CH2-; and

[0345] L 1' , Z, m, and B are equal as defined above.

[0346] In a specific embodiment, the connecting unit connecting each CB and Ar is (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 It is a connecting unit including a unit, and among the formulas:

[0347] W a1 , W a2 , and W a3 Each is independently -NH-, -C(O)-, or -CH2-;

[0348] W b1 is an amide bond or triazolilene;

[0349] P 1 is an amide bond, an amino acid residue, or a peptide;

[0350] L c is an alkylene;

[0351] Y 1 -(CH2) q -(CH2CH2X") o - or -(CH2) q -(X"CH2CH2X") o - and;

[0352] X" is -O-, -S-, -NH- or -CH2- and;

[0353] Y 2 is a single combination or a group selected from the following:

[0354]

[0355] and

[0356] W b2 is an amide bond or triazolilene;

[0357] a is 0 to 10;

[0358] b, c, and d are each independently integers having a value from 1 to about 10; and

[0359] o and q are each integers with values ​​ranging from 1 to about 10 independently.

[0360] In some implementations, R 12 is a natural amino acid side chain. In another embodiment, R 12 is a non-natural amino acid side chain.

[0361] In some embodiments, the connection unit connecting each CB and Ar is Equation ( A )to It is a connector presented by:

[0362]

[0363] During the meal:

[0364] * is the attachment point for CB;

[0365] ** is the attachment point for Ar.

[0366] In some such implementations, P 1 Is

[0367] or Igo

[0368] During the meal:

[0369] R 12is hydrogen, alkyl, amino acid side chain, -(CH2) s COOH or -(CH2) p NH2 and;

[0370] p is an integer having a value from 1 to about 10;

[0371] s and s" are each integers that independently have a value from 0 to about 10.

[0372] In some implementations, P 1 Is

[0373] or Igo

[0374] During the meal:

[0375] R 12 is hydrogen, alkyl, amino acid side chain, -(CH2) s C(O)R 13 or -(CH2) p NR 14 R 15 And;

[0376] p is an integer having a value from 1 to about 10;

[0377] s is an integer having a value from 0 to about 10;

[0378] R 13 is OH or -NH(CH2) s' (X"'CH2CH2) s" Z"-(CB) m And;

[0379] R 14 and R 15 Each independently hydrogen or -C(O)(CH2) s' (X"'CH2CH2) s" Z"-(CB) m And;

[0380] s" is an integer with a value from 0 to about 10;

[0381] s' is an integer having a value from 1 to about 10;

[0382] m is an integer with a value of 0 or 1;

[0383] X"' is -O-, -S-, -NH-, or -CH2-; and

[0384] Z" is CB R 14 or R 15 It is a linker that connects to the rest of; or Z" is a linker containing a reactive group.

[0385] P 1 In some of such implementation examples:

[0386] R 13 is OH or -NH(CH2) s' (X"'CH2CH2) s" Z" and;

[0387] R 14 and R 15 Each independently hydrogen or -C(O)(CH2) s' (X"'CH2CH2) s" Z" and; and

[0388] Z" is isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH2-hal), maleimide, diene, alkene, halogen, tosylate (TsO - ), aldehyde, sulfonate (R-SO3 - ), 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 derivative (-C(O)C≡CR a It is a reaction precursor of a linkage unit selected from, and R a is C1-C 10 -alkyl), and dihydrogen phosphate (-OP(=O)(OH)2).

[0389] P 1 In other such implementations of:

[0390] R 13is OH or -NH(CH2) s' (X"'CH2CH2) s" Z"CB and;

[0391] R 14 and R 15 Each independently hydrogen or -C(O)(CH2) s' (X"'CH2CH2) s" Z"CB and; and

[0392] Z" is CB, R formed from a precursor selected from the following. 14 or R 15 It is a linking unit that connects to the remainder of: isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH2-hal), maleimide, diene, alkene, halogen, tosylate (TsO - ), aldehyde, sulfonate (R-SO3 - ), 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 derivative (-C(O)C≡CR a , R a is C1-C 10 -alkyl), and dihydrogen phosphate (-OP(=O)(OH)2).

[0393] In an alternative embodiment, the linking unit connecting CB and Ar is a linker represented by the formulas (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIh), (IIIh), or (IIIj):

[0394]

[0395]

[0396] Here:

[0397] R eis alkyl and;

[0398] X 5 is -O-, -S-, -NH-, or -CH2-;

[0399] W b1 and W b2 -C(O)NH-, -NHC(O)-, respectively, independently , or And;

[0400] R 12 is hydrogen, alkyl, amino acid side chain, -(CH2) s C(O)R 13 or -(CH2) p NR 14 R 15 And;

[0401] R 13 OH or -NH(CH2) s' (X 4 CH2CH2) s" Z"-(CB) m And;

[0402] R 14 and R 15 are independently hydrogen or -C(O)(CH2) s' (X"'CH2CH2) s" Z"-(CB) m And;

[0403] s and s" are each independently integers having values ​​from 0 to about 10;

[0404] m is an integer with a value of 0 or 1;

[0405] X 4 and X 5 Each is independently -O-, -S-, -NH-, or -CH2-;

[0406] b, c, d, e, g, h, o, and q are each independent integers with values ​​ranging from 1 to about 10.

[0407] In these embodiments of some of the chemical formulas (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIh), (IIIh), or (IIIj):

[0408] R 13 OH or -NH(CH2) s' (X"'CH2CH2) s" Z"CB and;

[0409] R 14 and R 15 are independently hydrogen or -C(O)(CH2) s' (X"'CH2CH2) s" Z"CB and; and

[0410] Z" is isocyanid, isothiocyanid, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH2-hal), maleimide, diene, alkene, halogen, tosylate (TsO - ), aldehyde, sulfonate (R-SO3 - ), , , 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 derivative (-C(O)C≡CR a , here R a is C1-C 10 R formed from a precursor selected from -alkyl), and dihydrogen phosphate (-OP(=O)(OH)2). 14 or R 15 It is a connection unit that connects CB to the remainder of.

[0411] Targeting Moity

[0412] The compounds and conjugates of the present invention may additionally comprise a ligand or targeting moiety, CB. In some embodiments, the ligand or targeting moiety is any molecular recognition element, which may undergo specific interactions with at least one other molecule through, for example, non-covalent bonds such as hydrogen bonds, metal coordination, hydrophobic forces, van der Waals forces, π-π interactions, halogen bonds, electrostatics, 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 other homologous materials.

[0413] The compound and conjugate of the present invention may include one or more targeting moiety. That is, the variable cb may have an integer value selected from 1, 2, 3, 4, 5, 1-10, or 1-20.

[0414] In some embodiments, CB is a covalent bond ( for example Comprising two or more independently selected natural or non-natural amino acids joined by peptide bonds go 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 non-natural amino acids joined by peptide bonds. In some embodiments, the ligand has a shorter amino acid sequence ( for example , fragments of natural proteins or synthetic polypeptide fragments) as well as whole proteins ( for example , includes pre-engineered protein.

[0415] In some embodiments, CB is an antibody, hormone, drug, or antibody analog that binds to a receptor ( for example , non-IgG), protein, oligopeptide, polypeptide, etc.It is selected from. 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 overexpressed in cancer cells compared to normal cells and may be classified as a monoclonal antibody (mAb) or antibody fragment and a small-molecule non-antibody. Preferably, the CB is selected from peptides identified in a library screen, tumor cell-specific peptides, tumor cell-specific aptamers, tumor cell-specific carbohydrates, tumor cell-specific monoclonal antibodies, polyclonal antibodies, and antibody fragments.

[0416] Exemplary ligands or targeting moietyes include, but are not limited to, carnitine, inositol, lipoic acid, pyridoxal, ascorbic acid, niacin, pantothenic acid, folic acid, riboflavin, thiamine, biotin, vitamin B 12, other water-soluble vitamins (Vitamin B), fat-soluble vitamins (Vitamins A, D, E, K), RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), Transferin, VIP (Vasoactive Enteric Peptide) receptor, APRPG (Ala-Pro-Arg-Pro-Gly) peptide, TRX-20 (Thioredoxin-20), Integrin, Nucleolin, Aminopeptidase N (CD13), Endoglin, Vascular Epithelial Growth Factor Receptor, Low-Density Lipoprotein Receptor, Transferin Receptor, Somatostatin Receptor, Bombesin, Neuropeptide Y, Luteinizing Hormone-Lung Hormone Receptor, Folic Acid Receptor, Epidermal Growth Factor Receptor, Transforming Growth Factor, Fibroblast Growth Factor Receptor, Asialoglycoprotein Receptor, Galectin-3 Receptor, E-Selectin Receptor, Hyaluronic Acid Receptor, Prostate-Specific Membrane Antigen (PSMA), Cholecystokinin A receptor, Cholecystokinin B receptor, Discoydin 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, Tetanustoxin Tet1, Tetanustoxin G23, RVG (Rabies virus glycoprotein) peptide, HER2 (Human epidermal growth factor receptor 2), GPNMB (Glycoprotein non-transferable b), Ley, CA6, CanAng, SLC44A4 (Solute carrier family 44 member 4), CEACAM5 (Carcinoma embryonic antigen-associated cell adhesion molecule 5), Nectin-4, Carbonic anhydrous enzyme 9, TNNB2, 5T4, CD30, CD37, CD74, CD70, PMEL17, EphA2 (Ephrin A2 receptor), Trop-2, SC-16, Tissue factor, ENPP-3 (AGS-16),SLITRK6 (SLIT and NTRK-like family member 6), CD27, Lewis Y antigen, LIV1, GPR161 (G protein-coupled receptor 161), PBR (peripheral-type benzodiazeoine receptor), MERTK (Mer receptor tyrosine kinase) receptor, CD71, LLT1 (lectin-like transcript 1 or CLED2D), interleukin-22 receptor, sigma 1 receptor, peroxisome proliferator-activated receptor, DLL3, C4.4a, cKIT, ephrin A, CTLA4 (cytotoxic T-lymphocyte-associated protein 4), FGFR2b (fibroblast growth factor receptor 2b), N-acetylcholine receptor, gonadotropin-releasing hormone receptor, gastrin-releasing peptide receptor, bone morphogenetic protein receptor-type 1B (BMPR1B), E16 (LAT1, SLC7A5), STEAP1 (6 transmembrane epithelial antigens of the prostate), 0772P (CA125, MUC16), MPF (MSLN, mesothelin), Napi3b (SLC34A2), Sema5b (semaphorin 5b), ETBR (endothelin type B receptor), MSG783 (RNF124), STEAP2 (6 transmembrane epithelial antigens of prostate II), TrpM4 (transient receptor translocation cation 5 channel, subfamily M, member 4), CRIPTO (teratoma-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 II 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 Cyclasase 2C), Liv-1 (SLC39A6), CD56, CanAg, TIM-1, RG-1, B7-H4, PTK7, CD138, Claudin, Her3 (ErbB3), RON (MST1R), CD20, TNC (Thenassin 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), Gangliosides GD2, GD3, GM2, GM3, VEP8, VEP9, My1, VIM-D5, D156-22, OX40, RNAK, PD-L1, TNFR1, TNFR2, etc.

[0417] Target

[0418] In some embodiments, the targets or targets of the molecular recognition element are specifically associated with one or more specific cell or tissue types. In some embodiments, the targets are specifically associated with one or more specific disease states. In some embodiments, the targets are specifically associated with one or more specific developmental stages. For example, cell type-specific markers are typically expressed at a level at least 2 times greater in the corresponding cell type than in a reference population of cells. In some embodiments, cell type-specific markers are present at a level at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 50 times, at least 100 times, or at least 1,000 times greater than their average expression in the reference population. Detection or measurement of cell type-specific markers enables distinguishing cell types or types of interest from many, most, or all other types of cells. In some embodiments, the targets may include proteins, carbohydrates, lipids, and / or nucleic acids as described herein.

[0419] In some embodiments, a substance is considered "targeted" when it specifically binds to a targeting moiety, e.g., a nucleic acid targeting moiety. In some embodiments, the targeting moiety, e.g., a nucleic acid targeting moiety, specifically binds to a target under strict conditions.

[0420] In specific embodiments, the conjugates and compounds described herein are one or more targets associated with an organ, tissue, cell, extracellular matrix component, and / or intracellular compartment ( for exampleIt includes a targeting moiety that specifically binds to an antigen. In some embodiments, the conjugates and compounds described herein include a targeting moiety that specifically binds to a target associated with a specific organ or organ system. In some embodiments, the conjugates and compounds described herein include 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 include 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 include a specific cell type ( for example , endothelial cell, cancer cell, malignant cell, prostate cancer cell, etc. It includes a targeting moiety that specifically binds to a target associated with ).

[0421] In some embodiments, the conjugates and compounds described herein are one or more specific tissue types ( for example , liver tissue vs. It includes a targeting moiety that binds to a specific target for prostate tissue. In some embodiments, the conjugate and compound described herein are one or more specific cell types ( for example , T cells vs. It includes a targeting moiety that binds to a specific target for B cells. In some embodiments, the conjugate and compound described herein are for one or more specific disease conditions ( for example , tumor cells vs. It includes a targeting moiety that binds to a target specific to healthy cells. In some embodiments, the conjugate and compound described herein comprise one or more specific developmental stages ( for example , stem cells vs. It includes a targeting moiety that binds to a specific target for differentiated cells.

[0422] In some embodiments, the target may be a marker exclusively or primarily associated with one or several cell types, one or several diseases, and / or one or several developmental stages. Cell type-specific markers are typically, for example, a plurality of ( for example (5-10 or greater) expressed at a level at least 2 times greater in the corresponding cell type than in a reference population of cells that may consist of a mixture containing cells from different tissues or organs. In some embodiments, the cell type-specific marker is present at a level at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 50 times, at least 100 times, or at least 1000 times greater than its average expression in the reference population. Detection or measurement of the cell type-specific marker enables distinguishing the cell type of interest or types from many, most, or all other types of cells.

[0423] In some embodiments, the target comprises proteins, carbohydrates, lipids, and / or nucleic acids. In some embodiments, the target comprises proteins and / or characteristic portions thereof, e.g., tumor markers, integrins, cell surface receptors, transmembrane proteins, intercellular proteins, ion channels, membrane transport proteins, enzymes, antibodies, chimeric proteins, glycoproteins, etc. In some embodiments, the target comprises carbohydrates and / or characteristic portions thereof, e.g., glycoproteins, sugars ( for example , monosaccharides, disaccharides, polysaccharides), glycocalyx ( in other words , carbohydrate-rich peripheral zones on the outer surface of most eukaryotic cells), etc. ...includes. In some embodiments, the target is a lipid and / or a characteristic part thereof, e.g., oil, fatty acid, glyceride, hormone, steroid ( for example , cholesterol, bile acid), vitamin ( for example , Vitamin E), phospholipids, sphingolipids, lipoproteins, etc. Includes. In some embodiments, the target comprises a nucleic acid and / or a characteristic portion thereof, e.g., DNA nucleic acid; RNA nucleic acid; modified DNA nucleic acid; modified RNA nucleic acid; any combination of DNA, RNA, modified DNA, and modified RNA.

[0424] Numerous markers are known in the art. Typical markers include cell surface proteins, e.g., receptors. Exemplary receptors include, but are not limited to, the following: transferrin receptors; LDL receptors; growth factor receptors, such as members of the epidermal growth factor receptor family (e.g., EGFR, Her2, Her3, Her4) or vascular endothelial growth factor receptors, cytokine receptors, cell adhesion molecules, integrins, selectins, and CD molecules. The marker may be a molecule present exclusively or in higher amounts on malignant cells, e.g., tumor antigens.

[0425] Antibody-drug conjugate (ADC)

[0426] In some embodiments, CB is an antibody, and Q is a drug. Accordingly, the compounds and conjugates disclosed herein can form antibody-drug conjugates (ADCs) by conjugating an antibody to a drug moiety. Antibody-drug conjugates (ADCs) are capable of selectively delivering one or more drug moiety(s) to target tissues, e.g., tumor-associated antigens, due to the ADC's ability to deliver these to diseases, for example , can increase the therapeutic efficacy for treating cancer. Therefore, in certain embodiments, the present invention is for therapeutic use, for example , provides ADCs for the treatment of cancer.

[0427] The ADC of the present invention comprises an antibody connected to one or more drug moieties. The specificity of the ADC is defined by the specificity of the antibody. In one embodiment, the antibody is connected to one or more cytotoxic drug(s) that are delivered internally to cancer cells.

[0428] Examples of drugs that may be used in the ADC of the present invention are provided below. The terms "drug," "formulation," and "drug moiety" are used interchangeably herein. The terms "linked" and "conjugated" are also used interchangeably herein and indicate that the antibody and the moiety are covalently bonded.

[0429] In certain embodiments, the present disclosure relates to an ADC, a composition comprising an ADC, a therapeutic method, and a method for formulating an ADC composition. The ADC comprises 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. The types of antibodies, linkers, and cytotoxic compounds included in the present disclosure are described below.

[0430] In some embodiments, the ADC has the following chemical formula (chemical formula (III)):

[0431] (DL) dl -LG-(CB) cb

[0432] (III)

[0433] or his pharmaceutically acceptable salt,

[0434] Here:

[0435] LG is a connector;

[0436] CB is a cell-binding agent;

[0437] cb and dl are each independently integers having a value of 1 to about 20, preferably 1 to about 10; and

[0438] Each DL is a group that independently has the structure of a compound of formula (I) or (II).

[0439] antibodies

[0440] The antibody of the ADC may be any antibody that binds typically but not necessarily specifically to an antigen expressed on the surface of a target cell of interest. In some embodiments, the antigen may, although not necessarily, internalize the ADC bound thereto into the cell. 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, expressed on the target cell of interest. etc. It may be, but typically not a normal or healthy cell, a protein that is uniquely expressed on the target cell, or is overexpressed on the target cell compared to a normal or healthy cell, thereby allowing the ADC to selectively target specific cells of interest, e.g., tumor cells. As is recognized by the skilled, the specific antigen, and thus the selected antibody, 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.

[0441] Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins with identical structural characteristics. While antibodies exhibit binding specificity to specific targets, immunoglobulins include both antibodies and other antibody-like molecules that lack target specificity. Natural antibodies and immunoglobulins are generally heterotemeric glycoproteins of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has a variable domain (VH) at one end followed by numerous constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end.

[0442] The reference "VH" refers to a variable region of the immunoglobulin heavy chain of an antibody, comprising the heavy chain of Fv, scFv, or Fab. The reference "VL" refers to a variable region of the immunoglobulin light chain, comprising the light chain of Fv, scFv, dsFv, or Fab.

[0443] In this specification, the term "antibody" is used in the broadest sense and refers to an immunoglobulin molecule that specifically binds to a specific antigen or is immunologically reactive with said antigen, and is not limited to, chimeric antibodies, humanized antibodies, heteroconjugate antibodies ( for example Includes polyclonal, monoclonal, genetically engineered, and otherwise modified forms of antibodies, including dispecific antibodies, diabadies, triabadies, and tetraabadies), and antigen-binding fragments of antibodies, such as 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 chain and light chain from a traditional antibody are linked to form a single chain.

[0444] Antibodies can be rodents, humans, humanized, chimeras, or derived from other species. Antibodies are proteins produced by the immune system 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 numerous binding sites, also known as epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, a single antigen can have more than one corresponding antibody. Antibodies are the full-length immunoglobulin molecule or the immunologically active portion of the full-length immunoglobulin molecule, in other words, comprising a molecule containing an antigen-binding site that immunospecifically binds to an antigen or a part thereof of a target of interest, such target comprises, without limitation, cells or cancer cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein are of any type ( for example , IgG, IgE, IgM, IgD, and IgA), class ( for example It may be a subclass of immunoglobulin molecules (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). Immunoglobulins may be derived from any species. In one embodiment, however, immunoglobulins are of human, rodent, or rabbit origin.

[0445] The term "antibody fragment" refers to a portion of a full-length antibody, generally the target binding or variable region. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments. The "Fv" fragment is a minimal antibody fragment containing a complete target recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain (VH-VL dimer) bound by strong, non-covalent bonds. In this configuration, three CDRs of each variable domain interact to define the target binding site on the surface of the VH-VL dimer. Often, six CDRs confer target binding specificity to the antibody. However, in some cases, even a single variable domain (or half of the Fv containing only three CDRs specific to the target) may possess the ability to recognize and bind to the target. "Single-chain Fv" or "scFv" antibody fragments contain the antibody's VH and VL domains in a single polypeptide chain. Generally, the Fv polypeptide comprises a polypeptide linker between the VH and VL domains that enables the scFv to form a desired structure for additional target binding. The "single-domain antibody" consists of a single VH or VL domain that exhibits sufficient affinity for the target. In a specific embodiment, the single-domain antibody is a camelized antibody (see, for example , Riechmann, 1999, Journal of Immunological Methods 231:25-38).

[0446] The Fab fragment contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab′ fragment differs from the Fab fragment by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cystes from the antibody hinge region. The F(ab′) fragment is produced by the cleavage of a disulfide bond at the hinge cysteine ​​of the F(ab′)2 pepsin digestion product. Further chemical coupling of antibody fragments is known to those skilled in the art.

[0447] Both the light and heavy chain variable domains possess a complementary determining region (CDR), also known as the supervariability region. The more highly conserved portion of the variable domain is called the framework (FR). As is known in the art, the amino acid positions / boundaries marking the supervariability region of an antibody can vary depending on the context and various definitions known in the art. Within the variable domain, some positions may be viewed as hybrid supervariability regions in that they are considered outside the supervariability region under a different set of criteria, while being considered within the supervariability region under one set of criteria. One or more of these positions may also be found in the extended supervariability region. In each chain, the CDR is held together in very close proximity by the FR region and contributes to the formation of the antibody's target binding site along with the CDR from the other chain (see Kabat etc. , Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987). As used herein, the numbering of immunoglobulin amino acid residues is, unless otherwise indicated, Kabat etc. It is carried out according to the immunoglobulin amino acid residue numbering system.

[0448] In certain embodiments, the antibody of the ADC of the present disclosure is a monoclonal antibody. The term "monoclonal antibody" (mAb) is not produced by a method, for example ...refers to antibodies derived from a single copy or clone, including any eukaryotic, prokaryotic, or phage clone. Preferably, the monoclonal antibodies of this disclosure exist in a homogeneous or substantially homogeneous population. Monoclonal antibodies comprise both the intact molecule capable of specifically binding to a protein, as well as antibody fragments (e.g., Fab and F(ab')2 fragments). The Fab and F(ab')2 fragments lack the Fc fragment of the intact antibody, are cleared more rapidly from animal circulation, and may have less non-specific tissue binding than the intact antibody (Wahl etc. , 1983, J. Nucl.Med 24:316). Monoclonal antibodies useful for the present disclosure may be produced using various techniques known in the art, including the use of hybridoma, recombinant, and phage display techniques, or combinations thereof. The antibodies of the present disclosure include chimeric, primate-modified, humanized, or human antibodies.

[0449] In most cases, antibodies consist solely of genetically encoded amino acids, whereas in some embodiments, non-encoded amino acids may be specifically incorporated. Examples of non-encoded amino acids that may be incorporated into antibodies for use in stoichiometry and attachment site control, as well as methods for preparing such modified antibodies, are discussed below: Tian etc. , 2014, Proc Nat'l Acad Sci USA 111(5):1766-1771 and Axup etc. , 2012, Proc Nat'l Acad Sci USA 109(40):16101-16106 The entire contents thereof are incorporated herein by reference.

[0450] In certain embodiments, the antibody of the ADC described herein is a chimeric antibody. As used herein, the term “chimeric” antibody refers to a non-human immunoglobulin, such as a rat or mouse antibody, typically selected from a human immunoglobulin template, and an antibody having a variable sequence derived from a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. reference , for example , Morrison, 1985, Science 229(4719):1202-7; Oi etc. , 1986, BioTechniques 4:214-221; Gillies etc. , 1985, J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816,397, the entirety of which is incorporated herein by reference.

[0451] In certain embodiments, the antibody of the ADC described herein is a humanized antibody. Non-human ( for example The "humanized" form of the antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., the Fv, Fab, Fab', F(ab')2, or other target-binding subdomains of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. Generally, the humanized antibody will comprise at least one, and typically two, variable domains substantially all, wherein all or substantially all CDR regions correspond to those of the non-human immunoglobulin and all or substantially all FR regions correspond to those of the human immunoglobulin sequence. The humanized antibody may also comprise at least a portion of the immunoglobulin constant region (Fc), typically of the human immunoglobulin common sequence. Methods of antibody humanization are known in the art. reference , for example , Riechmann etc. , 1988, Nature 332:323-7; U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and U.S. Patent No. 6,180,370, Queen etc. ;EP239400; PCT Publication WO 91 / 09967; U.S. Patent No. 5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol. , 28:489-498; Studnicka etc. , 1994, Prot.Eng. 7:805-814; Roguska etc. , 1994, Proc. Natl. Acad Sci. USA 91:969-973; and U.S. Patent No. 5,565,332, all of which are incorporated herein by reference in their entirety.

[0452] In certain embodiments, the antibody of the ADC described herein is a human antibody. An entirely “human” antibody may be desirable for the therapeutic treatment of human patients. As used herein, “human antibody” comprises an antibody having the amino acid sequence of a human immunoglobulin, and comprises an antibody isolated from an animal transgenic to one or more human immunoglobulins or from a human immunoglobulin library and not expressing the endogenous immunoglobulin. The human antibody may be implemented by various methods known in the art, including a phage display method using an antibody library derived from the human immunoglobulin sequence. 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. Publication No. 2013 / 189218, the contents of which are incorporated herein by reference in their entirety. Human antibodies may also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins but can express human immunoglobulin genes. Note, for example, U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; 5,939,598; 7,723,270; 8,809,051 and U.S. Publication Application No. 2013 / 117871, the whole of which is incorporated herein by reference. Additionally, companies such as Medarex (Princeton, NJ), Astellas Pharma (Deerfield, Ill.), and Regeneron (Tarrytown, NY) may be involved in providing human antibodies to selected antigens using technology similar to that described above. Fully human antibodies recognizing selected epitopes can be generated using a technique referred to as "induced selection." Non-human monoclonal antibodies selected in this approach, for example Mouse antibodies are used to guide the selection of fully human antibodies that recognize the same epitope (Jespers etc. , 1988, Biotechnology 12:899-903).

[0453] In certain embodiments, the antibody of the ADC described herein is a primate-modified antibody. The term "primate-modified antibody" refers to an antibody comprising a monkey variable region and a human constant region. Methods for producing primate-modified antibodies are known in the art. reference , for example , U.S. Patent Nos. 5,658,570; 5,681,722; and 5,693,780, the entirety of which is incorporated herein by reference.

[0454] In certain embodiments, the antibodies of the ADCs described herein are bispecific antibodies or bivariate domain antibodies (DVDs). Bispecific and DVD antibodies are monoclonal, often human or humanized, antibodies having binding specificity to at least two different antigens. DVDs are, for example, described below: U.S. Patent No. 7,612,181, the disclosure thereof of which is incorporated herein by reference.

[0455] In certain embodiments, the antibodies of the ADCs described herein are derived antibodies. For example, without limitation, derived antibodies are typically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protective / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. Any numerous chemical modifications, without limitation, include specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. It can be performed by known techniques including. Additionally, derivatives, for example , using Ambrix technology, it may contain one or more non-natural amino acids ( reference , for example , Wolfson, 2006, Chem.Biol. 13(10):1011-2).

[0456] In certain embodiments, the antibody of the ADC described herein has a modified sequence to alter at least one constant region-mediated biological effector function relative to the corresponding wild-type sequence. For example, in some embodiments, the antibody has at least one constant region-mediated biological effector function relative to the unmodified antibody, for example , can be modified to reduce binding to the Fc receptor (FcR). FcR binding can be reduced by mutating the immunoglobulin constant region segment of the antibody in the specific region required for FcR interaction ( reference , for example, Canfield and Morrison, 1991, J. Exp. Med 173:1483-1491; and Lund etc. , 1991, J. Immunol. 147:2657-2662).

[0457] In a specific embodiment, the antibody of the ADC described herein acquires or improves at least one invariant region-mediated biological effector function compared to an unmodified antibody, for example , is modified to enhance FcγR interaction ( reference , for example , US 2006 / 0134709). For example, antibodies having a constant region that binds to FcγRIIA, FcγRIIB and / or FcγRIIIA with a greater affinity than the corresponding wild-type constant region can be produced according to the methods described herein.

[0458] In certain embodiments, the antibodies of the ADCs described herein are antibodies that bind to tumor cells, such as cell surface receptors or tumor-associated antigens (TAAs). To discover effective cell targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or other tumor-associated polypeptides that are specifically expressed on the surface of one or more specific types of cancer cells compared to one or more normal non-cancerous cell(s). Often, such tumor-associated polypeptides are expressed more abundantly 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 antibody generation using methods and information known in the art.

[0459] Exemplary cell surface receptors and TAA

[0460] Examples of cell surface receptors and TAAs to which the antibodies of the ADCs described herein may be targeted include, but are not limited to, the various receptors and TAAs listed in Table 1 below. For convenience, information regarding all of these antigens known in the art is listed below and includes names, alternative names, gene bank deposit numbers, and primary reference(s) in accordance with the nucleic acid and protein sequence identification protocols of the National Center for Biotechnology Information (NCBI). Nucleic acid and protein sequences corresponding to the listed cell surface receptors and TAAs are available in public databases, such as gene banks.

[0461]

[0462]

[0463]

[0464]

[0465]

[0466] Exemplary antibody

[0467] Exemplary antibodies to be used with the ADC of the present disclosure include, but are not limited to: 3F8 (GD2), avagovomab (CA-125 (misc)), adecatumumab (EpCAM), aputuzumab (CD20), alacizumab pegol (VEGFR2), ALD518 (IL-6), alemtuzumab (CD52), altumomab pentetate (CEA), amatouximab (mesothelin), anatumonab maphenatox (TAG-72), apolizumab (HLA-DR), arcitumomab (CEA), babituximab (phosphatidylserine), vectumumab (CD22), belimumab (BAFF), becilesomab (CEA-associated antigen), bevacizumab (VEGF-A), vibatuzumab mertansine (CD44 v6), blinatumomab (CD19), brentuximab vedotin ((CD30 (TNFRSF8)), cantuzumab mertansine (mucin CanAg), cantuzumab ravtansine (MUC1), capromab pendetide (prostate carcinoma cells), charlumab (MCP-1), catumaxomab (EpCAM, CD3), CC49 (Tag-72), cBR96-DOX ADC (Lewis-Y antigen), cetuximab (EGFR), citatuzumab vogatox (EpCAM), sixutumab (IGF-1 receptor), clivatuzumab tetraxetane (MUC1), conatumumab (TRAIL-E2), dasetuzumab (CD40), dalotuzumab (insulin-like growth factor 1 receptor), daratumumab ((CD38 (cyclic ADP ribose hydrolase)), dempizumab (DLL4), denosumab (RANKL), detumomab (B-lymphoma Cell), drogitumab (DR5), ducigitumab (ILGF2), ecromeximab (D3 ganglioside), eculizumab (C5), edrecolomab (EpCAM), elotuzumab (SLAMF7), elcilimomab (IL-6), enavatuzumab (TWEAK receptor), enoticumab (DLL4), encituximab (5AC), epitumomab situxetane (episialin), efratuzumab (CD22), ertumaxomab ((HER2 / neu, CD3)), etansizumab (integrin αvβ3), parletuzumab (folic acid receptor 1),FBTA05 (CD20), piclatuzumab (HGF), pizitumab (IGF-1 receptor), flambotumab (TYRP1 (glycoprotein 75)), presolimumab (TGF-1), galiximab (CD80), ganitumab (IGF-I), gemtuzumab ozogamicin (CD33), gilentuximab (carbonic anhydrous enzyme 9 (CA-IX)), glembatumab vedotin (GPNMB), ibritumomab tiuxetane (CD20), icrucumab (VEGFR-1), igobomab (CA-125), IMAB362 (CLDN18.2), imgatuzumab (EGFR), indatuximab lavatansine (SDC1), intertumumab (CD51), inotuzumab ozogamicin (CD22), ipilimumab (CD152), iratumumab ((CD30 (TNFRSF8)), Labetuzumab (CEA), Lambrolizumab (PDCD1), Lexatumumab (TRAIL-R2), Lintuzumab (CD33), Lorvotuzumab Mertansine (CD56), Lucatumumab (CD40), Lumiliximab ((CD23 (IgE receptor)), Mapatumumab (TRAIL-R1), Margetuximab (ch4DS), Matuzumab (EGFR), Milatuzumab (CD74), Mitumomab (GD3 ganglioside), Mogamulizumab (CCR4), Moxetumumab Fasudox (CD22), Nacolomab Tafenatox (C2-42 antigen), Naptumomab Estafenatox (5T4), Narnatumab (RON), Natalizumab (Integrin α4), Necitumumab (EGFR), Nesvacumab (Angiopoietin 2), nimotuzumab (EGFR), nivolumab (IgG4), ocaratuzumab (CD20), ofatumumab (CD20), olaratumab (PDGF-R α), onatuzumab (human scattering factor receptor kinase), ontuxizumab (TEM1), ofportuzumab monato (EpCAM), oregovomab (CA-125), othletuzumab (CD37), panitumumab (EGFR), pancomab (tumor-specific glycosylation of MUC1), parsatuzumab (EGFL7), patritumab (HER3), femtumumab (MUC1), pertuzumab (HER2 / neu), fidilizumab (PD-1), finatuzumab vedotin (CD22),Pritumumab (Vimentin), Lacotumumab (N-glycolylneuraminic acid), Radretumab (Fibronectin additional domain-B), Ramucirumab (VEGFR2), Rilotumumab (HGF), Rituximab (CD20), Lovatumumab (IGF-1 receptor), Samalizumab (CD200), Satumomab pendetide (TAG-72), Serivantumab (ERBB3), Sibrotuzumab (FAP), SGN-CD19A (CD19), SGN-CD33A (CD33), Siltuximab (IL-6), Solitomab (EpCAM), Sonefizumab (Sphingosine-1-phosphate), Tavalumab (BAFF), Tacatuzumab tetraxetane (alpha-fetoprotein), Taplitumomab poptox (CD19), Tenatumomab (Thenassine C), Teprotumumab (CD221), TGN1412 (CD28), tilcilimumab (CTLA-4), tigatuzumab (TRAIL-R2), TNX-650 (IL-13), tovetumab (CD40a), trastuzumab (HER2 / neu), TRBS07 (GD2), tremelimumab (CTLA-4), tucotuzumab selmolukin (EpCAM), ublituximab (MS4A), urelumab (4-1BB), vandetanib (VEGF), vantictumab (Prizzled receptor), voloxiximab (integrin α5β1), borsetuzumab mafodotin (CD70), botumumab (tumor antigen CTAA16.88), zalutumumab (EGFR), zanolimumab (CD4), and zatuximab (HER1).

[0468] Method for manufacturing antibodies

[0469] Antibodies of ADCs can be produced by the recombinant expression of immunoglobulin light and heavy chain genes within host cells. For example, to recombinantly express antibodies, host cells are transfected with one or more recombinant expression vectors carrying DNA fragments encoding the antibody's immunoglobulin light and heavy chains, thereby expressing the light and heavy chains in the host cells and, optionally, secreting them into the culture medium in which the host cells are cultured, from which the antibodies can be recovered. Standard recombinant DNA methodologies are used to obtain antibody heavy and light chain genes, to incorporate these genes into a recombinant expression vector, and to introduce the vector into host cells, such as those described below: Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, NY, 1989), Current Protocols in Molecular Biology (Ausubel, FM etc. , eds., Greene Publishing Associates, 1989) and U.S. Patent No. 4,816,397.

[0470] In one embodiment, the Fc variant antibody is similar to its wild-type equivalent except for a change in its Fc domain. To produce a nucleic acid encoding such an Fc variant antibody, a DNA fragment encoding the Fc domain or a portion of the Fc domain of the wild-type antibody (referred to as the “wild-type Fc domain”) can be synthesized and used as a mutagenic template to produce the antibody as described herein using routine mutagenic techniques; alternatively, the DNA fragment encoding the antibody can be synthesized directly.

[0471] Once DNA fragments encoding a wild-type Fc domain are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert a constant region gene into a full-length antibody chain gene. In these manipulations, the CH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody variable region or a flexible linker. As used in this context, the term "operably linked" is intended to mean that the two DNA fragments are linked so that the amino acid sequence encoded by the two DNA fragments remains in-frame.

[0472] To express Fc variant antibodies, DNAs encoding partial or full-length light and heavy chains obtained as described above are inserted into an expression vector, thereby operably linking the gene to transcription and translation control sequences. In this context, the term " "Operatorily ligated" means that the antibody gene is ligated into a vector, thereby allowing the transcription and translation control sequences within the vector to perform their intended functions of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The variant antibody light chain gene and antibody heavy chain gene may be inserted into separate vectors, or, more typically, both genes are inserted into the same expression vector.

[0473] Antibody genes are placed in the expression vector using the standard method ( for example, is inserted via ligation of a complementary restriction site on the antibody gene fragment and vector, or via smooth end ligation if no restriction site is present. Prior to the insertion of the variant Fc domain sequence, the expression vector may already carry the antibody variable region sequence. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that promotes the secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into the vector so that the signal peptide is inframe-linked to the amino terminus of the antibody chain gene. The signal peptide is an immunoglobulin signal peptide or a heterogeneous signal peptide ( in other words , it can be a signal peptide from a non-immunoglobulin protein.

[0474] In addition to the antibody chain gene, the recombinant expression vector carries a regulatory sequence that controls the expression of the antibody chain gene in the host cell. The term "regulatory sequence" refers to promoters, enhancers, and other expression control elements that control the transcription or translation of the antibody chain gene ( for example It is intended to include polyadenylation signals). Such regulatory sequences are, for example, described below: Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif., 1990). It will be acknowledged by those skilled in the art that the design of an expression vector, including the selection of regulatory sequences, may depend on factors such as the selection of the host cell to which it is to be converted, the expression level of the desired protein, etc. Regulatory sequences suitable for mammalian host cell expression are promoters and / or enhancers derived from viral elements that induce high levels of protein expression within mammalian cells, such as cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), primate virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus, ( for example, includes the adenovirus major late promoter (AdMLP) and polyoma. For further description of viral regulatory factors and their sequences, refer to, for example , U.S. Patent No. 5,168,062 by Stinski, U.S. Patent No. 4,510,245 Bell etc. Author, and U.S. Patent No. 4,968,615 Schaffner etc. I.

[0475] In addition to the antibody chain gene and regulatory sequence, the recombinant expression vector has additional sequences, such as a sequence that regulates the replication of the vector in a host cell ( for example It can carry , origin of replication) and selectable marker genes. Selectable marker genes facilitate the selection of host cells into which the vector is introduced ( reference , for example , U.S. Patent Nos. 4,399,216, 4,634,665 and 5,179,017, all Axel etc. (Belonging to ). For example, typically selectable marker genes confer resistance to drugs, such as G418, puromycin, blasticidin, hygromycin, or methotrexate, on host cells into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR host cells with methotrexate selection / amplification) and the neo gene (for G418 selection). For the expression of light and heavy chains, the expression vector(s) encoding the heavy and light chains are transfected into host cells using standard techniques. Various forms of terms " "Transfection" refers to various techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, for example It is intended to cover electroporation, lipofection, calcium-phosphate precipitation, DEAE-dextran transfection, and other homologous methods.

[0476] It is possible to express antibodies in either a prokaryotic or eukaryotic host cell. In a specific embodiment, antibody expression is performed in a eukaryotic cell, for optimal secretion of a properly folded and immunologically active antibody. for example , is performed in mammalian host cells. Exemplary mammalian host cells for recombinant antibody expression include (DHFR CHO cells, Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA Described in 77:4216-4220, used with DHFR screening markers, for example As described in Kaufman and Sharp, 1982, Mol. Biol. 159:601-621, the host cells include Chinese hamster ovaries (CHO cells), NS0 myeloma cells, COS cells, 293 cells, and SP2 / 0 cells. When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period sufficient to allow for antibody expression in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. The antibody can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to produce intact antibodies, such as Fab fragments or parts of scFv molecules.

[0477] In some embodiments, the antibody of the ADC may be a bifunctional antibody. Such an antibody, in which one heavy chain and one light chain are specific to one antigen and the other heavy chain and light chain are specific to a second antigen, may be produced by cross-linking the antibody to the second antibody using a standard chemical cross-linking method. A bifunctional antibody may also be produced by expressing a nucleic acid engineered to encode the bifunctional antibody.

[0478] In a specific embodiment, a dual-specific antibody, in other wordsAntibodies that bind one antigen and a second, unrelated antigen using the same binding site can be produced by mutating amino acid residues in the light chain and / or heavy chain CDR. An exemplary second antigen includes a pro-inflammatory cytokine (e.g., lymphotoxin, interferon-γ, or interleukin-1). The bispecific antibody is, for example , can be produced by mutating amino acid residues around the antigen binding site ( reference , for example , Bostrom etc. , 2009, Science 323:1610-1614). Bifunctional antibodies can be produced by expressing nucleic acids engineered to encode bispecific antibodies.

[0479] Antibodies are also produced by chemical synthesis ( for example , Solid Phase Peptide Synthesis It can be produced by the method described in The Pierce Chemical Co., Rockford, Ill., , 2nd ed., 1984. Antibodies can also be produced using a cell-free platform ( reference , for example , Chu etc. , Biochemia No. 2, 2001 (Roche Molecular Biologicals).

[0480] The method for recombinant expression of the Fc fusion protein is described below: Flanagan etc. , Methods in Molecular Biolog , vol. 378: Monoclonal Antibodies:Methods and Protocols.

[0481] Once the antibody has been produced by recombinant expression, for the purification of the immunoglobulin molecule, any method known in the art, for example, chromatography ( for exampleProteins can be purified by ion exchange, affinity, particularly by affinity for antigens after selection of protein A or protein G, and by sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification.

[0482] Once isolated, the antibody, if desired, for example , high-performance liquid chromatography ( reference , for example , Fisher, Laboratory Techniques In Biochemistry And Molecular Biology It can be further purified by gel filtration chromatography on a Superdex™ 75 column (Pharmacia Biotech AB, Uppsala, Sweden) (Work and Burdon, eds., Elsevier, 1980).

[0483] Treatment methods

[0484] Target-oriented therapy

[0485] The targeting moiety of the conjugate can provide so-called targeted-oriented therapy by being recognized by cells.

[0486] In some embodiments, the present disclosure relates to a method for treating or preventing a disease or disorder, comprising administering to a subject in need a pharmaceutical composition comprising a compound of formula (I) or formula (II), or a conjugate of formula (III), a compound of formula (I) or formula (II), or a conjugate of formula (III). In certain such embodiments, the disease or disorder is cancer, infection, immunodeficiency, autoimmune disease, or chronic inflammatory disorder.

[0487] In some embodiments, the present disclosure relates to a method further comprising co-administering a chemotherapy agent to a subject. In certain such embodiments, the chemotherapy agent is administered to the subject sequentially with a conjugate as disclosed herein.

[0488] In some embodiments, the conjugate described herein comprises an active agent as described herein, for example, an active agent of formula (I). In some such embodiments, the active agent is selected from the following:

[0489]

[0490]

[0491] or his pharmaceutically acceptable salt.

[0492] In another embodiment, the active agent is selected from the following:

[0493] *280

[0494]

[0495] or his pharmaceutically acceptable salt.

[0496] Cell proliferation and apoptosis

[0497] The compounds and conjugates disclosed in this specification may be used in a method for inducing apoptosis in cells.

[0498] Dysregulated apoptosis has been implicated in various diseases, including, for example: autoimmune disorders ( for example , systemic lupus erythematosus, rheumatoid arthritis, graft-versus-host disease, myasthenia gravis, or Sjögren's syndrome), chronic inflammatory conditions ( for example , psoriasis, asthma, or Crohn's disease), hyperproliferative disorders ( for example , breast cancer, lung cancer), viral infection ( for example, 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 improve any of these diseases. Such treatment generally involves administering an amount of the compounds, conjugates, or compositions described herein sufficient to provide therapeutic benefit to a subject suffering from the disease. The identity of the antibodies of the administered compounds, conjugates, or compositions will depend on the disease being treated—and thus said antibodies must bind to cell-surface antigens expressed on cell types where inhibition is beneficial. The therapeutic benefit achieved will also depend on the specific disease being treated. In certain cases, the compounds and compositions disclosed herein may treat or improve the disease itself or the symptoms of the disease when administered as monotherapy. In other instances, the compounds and compositions disclosed herein may be part of an overall therapeutic regimen comprising an inhibitor or other agents that treat or improve the disease being treated or the symptoms of the disease in conjunction with the compounds and compositions disclosed herein. Agents useful for treating or improving specific diseases that may be attached to or administered together with the compounds and compositions disclosed herein will be apparent to those skilled in the art.

[0499] While absolute cure is always desirable in any treatment regimen, achieving a cure is not required to provide therapeutic benefit. Therapeutic benefit may include halting or slowing disease progression, regressing the disease without cure, or improving or slowing the progression of disease symptoms. Extended survival and / or improved quality of life compared to the statistical mean may also be considered therapeutic benefit.

[0500] Cancer is a specific class of diseases that involve abnormally regulated apoptosis and constitute a significant global health burden. In certain embodiments, the compounds and compositions disclosed herein may be used to treat cancer. Cancer may be, for example, a solid tumor or a hematological tumor. Cancers that may 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, multiple myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, multiple 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 because antibodies can be used to specifically target tumor cells, thereby avoiding or improving undesirable side effects and / or toxicity that may be associated with the systemic administration of potentially unconjugated inhibitors. One embodiment relates to a method for treating a disease involving abnormally controlled apoptosis, said method comprising administering an amount of the compounds and compositions disclosed herein effective for providing therapeutic benefit to a subject having a disease involving abnormally controlled apoptosis, wherein the ligands of the compounds and compositions disclosed herein bind to cell surface receptors on cells having abnormally controlled apoptosis. One embodiment relates to a method for treating cancer comprising the step of administering the compounds and compositions disclosed herein to a subject having cancer, said method, wherein the ligands may bind to cell surface receptors or tumor-associated antigens expressed on the surface of cancer cells in an amount effective for providing therapeutic benefit.

[0501] In the context of tumorigenic cancer, therapeutic benefits, in addition to the effects discussed above, may also specifically include halting or slowing the progression of tumor growth, regression of tumor growth, eradication of one or more tumors, and / or increased patient survival compared to the statistical mean for the type and stage of the cancer being treated. In one embodiment, the cancer being treated is tumorigenic cancer.

[0502] The compounds and conjugates disclosed herein may be administered as monotherapy to provide therapeutic benefits, or may be administered adjunctively to or together with other chemotherapy agents and / or radiotherapy. Chemotherapy agents to which the compounds and compositions disclosed herein may be used as adjunctive therapy may be targeted (e.g., ADCs, protein kinase inhibitors, etc. ) or can be non-targeted (e.g., non-specific cytotoxic drugs such as radionucleotides, alkylating agents, and intermediaries). Non-targeted chemotherapeutic agents to which the compounds and compositions disclosed herein may additionally be administered include, but are not limited to, the following: methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustard, cyclosan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecin, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, calichiamycin, and Docetaxel.

[0503] Compounds and conjugates disclosed herein, which may not be effective as monotherapy for treating cancer, may be administered in addition to or together with other chemotherapy agents or radiotherapy to provide therapeutic benefits. One embodiment relates to a method in which a compound or composition disclosed herein is administered in an amount effective for sensitizing tumor cells to standard chemotherapy and / or radiotherapy. Accordingly, in the context of cancer treatment, "therapeutic benefits" include the administration of the compounds and compositions disclosed herein in addition to or together with chemotherapy agents and / or radiotherapy as a means of sensitizing tumors to either chemotherapy and / or radiotherapy, in patients who have not yet started such therapy or have not yet shown signs of resistance, or in patients who have begun to show signs of resistance.

[0504] Pharmaceutical composition and administration thereof

[0505] The compounds and conjugates disclosed herein may be used to treat an individual in need. In certain embodiments, the individual is a mammal, e.g., a human, or a non-human mammal. When administered to an animal, e.g., a human, the composition or compound is administered, preferably as a pharmaceutical composition comprising, for example, the disclosed compound and a pharmaceutically acceptable carrier.

[0506] Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycol, glycerol, oils such as olive oil, or injectable organic esters. In a preferred embodiment, where such a pharmaceutical composition is for human administration, particularly for an invasive route of administration (i.e., a route that avoids transport or diffusion through the epithelial barrier, such as injection or implantation), the aqueous solution is non-pyrogenous or substantially non-pyrogenous. Excipients may be selected, for example, to enable delayed release of the formulation or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition may be in dosing unit forms such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, reconstitution lyophiles, powders, solutions, syrups, suppositories, injections, etc. The composition may also be present in a transdermal delivery system, for example, a skin patch. The composition may also be present in a solution suitable for topical administration, such as an ointment or cream.

[0507] A pharmaceutically acceptable carrier may contain, for example, a physiologically acceptable agent that acts to increase the absorption of a compound, such as the compound of the present invention, or to stabilize or increase its solubility. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose, or dextran; antioxidants, such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; or other stabilizers or excipients. The selection of a pharmaceutically acceptable carrier containing a physiologically acceptable agent depends, for example, on the route of administration of the composition. The preparation of the pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) may also be a liposome or other polymer matrix, for example, a compound of the present invention, which may be incorporated therein. For example, liposomes containing phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively simple to prepare and administer.

[0508] The phrase “pharmaceuticalally acceptable” is used herein to refer to compounds, substances, compositions, and / or dosage forms suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, corresponding to a reasonable benefit / harm ratio within the scope of sound medical judgment.

[0509] As used herein, the phrase “pharmaceuticalally acceptable carrier” means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be “acceptable” in the sense that it is compatible with other components of the formulation and is not harmful to the patient. Some examples of substances that may serve as pharmaceutically acceptable carriers include the following: (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 carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycol, e.g., propylene glycol; (11) polyol, e.g., glycerin, sorbitol, mannitol, and polyethylene glycol; (12) ester, e.g., ethyl oleate and ethyl laurate; (13) agar; (14) buffer, e.g., magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) non-pyrogenous water; (17) isotonic saline solution; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solution; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0510] The pharmaceutical composition (formulation) may be administered to a subject by any number of routes of administration, for example, including: orally (e.g., drenches, aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), bolus, powder, granules, paste for application to the tongue); absorption via oral mucosa (e.g., sublingually); anally, rectally, or vaginally (e.g., as a pessary, cream, or foam); parenterally (e.g., as a sterile solution or suspension, including intramuscularly, intravenously, subcutaneously, or intrathecally); nasally; intraperitoneally; subcutaneously; transdermally (e.g., as a patch applied to the skin); and topically (e.g., as a cream, ointment, or spray applied to the skin, or as eye drops). The compound may also be formulated for inhalation. In certain embodiments, the compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for such purposes may be found below: e.g., U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and 4,172,896, as well as the patents cited herein.

[0511] The formulation can be conveniently presented in a unit dosage form and can be manufactured by any method well known in the field of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific method 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 the compound producing a therapeutic effect. Generally, out of 100 percent, this amount will be in the range of about 1 percent to about 99 percent, preferably about 5 percent to about 70 percent, most preferably about 10 percent to about 30 percent of the active ingredient.

[0512] A method for preparing these formulations or compositions comprises the step of associating an active compound, such as a compound of the present invention, with a carrier and, optionally, one or more auxiliary components. Generally, a formulation is prepared by uniformly and intimately associating a compound of the present invention with a liquid carrier, or a finely ground solid carrier, or both, and then, if necessary, shaping the product.

[0513] Formulations of the present invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cassettes, pills, tablets, lozenges (flavoring bases, generally using sucrose and acacia or tragacanth), hydrophilic substances, powders, granules, or solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as candy-type pills (using inert bases, e.g., gelatin and glycerin, or sucrose and acacia) and / or as mouthwashes and other similar forms, each containing a predetermined amount of the compound of the present invention as an active ingredient. The compound, conjugate, or compositions thereof may also be administered as boluses, softs, or pastes.

[0514] To manufacture solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, coated tablets, powders, granules, and other similar forms), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retardants, such as paraffin; and (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, e.g., cetyl alcohol and glycerol monostearate; (8) absorbents, e.g., kaolin and bentonite clay; (9) lubricants, e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, e.g., modified and unmodified cyclodextrin; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical composition may also include a buffer. A similar type of solid composition may also be used as a filler in soft and hard-filled gelatin capsules using excipients such as lactose or lactose, as well as high molecular weight polyethylene glycol and other similar ones.

[0515] Tablets may be made by compression or molding, optionally with one or more auxiliary components. Compressed tablets may be manufactured using a binder (e.g., gelatin or hydroxypropylmethylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), a surfactant, or a dispersant. Molded tablets may be made by molding from a suitable mechanical mixture of powdered compounds moistened with an inert liquid diluent.

[0516] Tablets and other solid dosage forms of the pharmaceutical composition, such as coated tablets, capsules (including sprinkle capsules and gelatin capsules), pills, and granules, may optionally be obtained or manufactured with coatings and shells, such as intestinal coatings and other coatings well known in pharmaceutical formulation technology. They may also be formulated to provide slow or controlled release of the active ingredient within them using, for example, hydroxypropylmethylcellulose in variable proportions to provide a desired release profile, different polymer matrices, liposomes, and / or microspheres. They may be sterilized, for example, by filtration through a bacteria-fixing filter, or by incorporation of a sterile agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions may also optionally contain an opacifying agent and may be compositions that release the active ingredient(s) alone, or optionally, preferentially in a delayed manner in a specific part of the gastrointestinal tract. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient may also be in a micro-encapsulated form with one or more of the excipients described above, if appropriate.

[0517] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, hydrophilic substances for reconstitution, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage form may contain the following: inert diluents commonly used in the art, e.g., water or other solvents, cyclodextrins and derivatives thereof, solubilizers and emulsifiers, e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (particularly cottonseed, peanut, corn, germ, olive, castor, and sesame oils), fatty acid esters of glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and sorbitan, and mixtures thereof.

[0518] In addition to inert diluents, the oral composition may also include adjuvants such as humectants, emulsifiers and suspending agents, sweeteners, flavoring agents, coloring agents, fragrances and preservatives.

[0519] In addition to the active compound, the suspension may contain a suspending agent, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof.

[0520] A formulation of a pharmaceutical composition for rectal, vaginal, or urethral administration may be presented as a suppository, which may be prepared by mixing one or more active compounds with one or more suitable non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol, suppository wax, or salicylate, which is solid at room temperature but liquid at body temperature, and thus will melt in the rectal or vaginal cavity and release the active compound.

[0521] A formulation of a pharmaceutical composition for oral administration may be presented as a mouthwash, an oral spray, or an oral ointment.

[0522] Alternatively or additionally, the composition may be formulated for delivery via a catheter, stent, wire, or other lumenical device. Delivery via such devices may be particularly useful for delivery to the bladder, urethra, ureter, rectum, or intestine.

[0523] Formulations suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing such carriers as known in the art, where appropriate.

[0524] 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 with a pharmaceutically acceptable carrier under sterile conditions and with any preservatives, buffers, or propellants that may be required.

[0525] 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 glycol, silicone, bentonite, silica, talc, and zinc oxide, or mixtures thereof.

[0526] The powder and spray 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 materials. The spray may additionally contain conventional propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

[0527] Transdermal patches have the added advantage of providing controlled delivery of the compounds of the present invention to the body. Such a dosage form can be made by dissolving or dispersing the active compound in a suitable medium. Absorption enhancers can also be used to increase the flow of the compound through the skin. The rate of such flow can be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0528] Ophthalmic formulations, eye ointments, powders, solutions, and other like products are also considered to be within the scope of the present invention. Exemplary ophthalmic formulations are described below: 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, liquid ophthalmic formulations have properties similar to or compatible with fluids such as tear fluids, aqueous humor, or vitreous fluids.

[0529] As used herein, the phrases "parenteral administration" and "parenterally administered" " Modes of administration other than intestinal and local administration, generally meaning by injection, and include, without limitation, the following: intravenous, intramuscular, intra-arterial, intra-spinal, intraorbital, intracardiac, intradermal, intraperitoneal, transtubercular, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injections and infusions.

[0530] A pharmaceutical composition suitable for parenteral administration comprises one or more active compounds in combination with sterile powders, which can be reconstituted immediately before use into one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that may contain antioxidants, buffers, bacteriostatic agents, and solutes or suspensions or thickeners that make the formulation isotonic with the blood of the intended recipient.

[0531] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical composition of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and other similar ones), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Suitable fluidity can be maintained, for example, by the use of a coating material, such as lecithin, by maintaining the particle size required in the case of a dispersion, and by the use of a surfactant.

[0532] These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of microbial action can be ensured by the encapsulation of various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, and other similar substances. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and other similar substances, in the composition. Additionally, long-term absorption in the injectable pharmaceutical form may be achieved by the encapsulation of absorption-delaying agents, such as aluminum monostearate and gelatin.

[0533] In some cases, to prolong the effect of the drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous material with poor water solubility. The absorption rate of the drug then depends, in turn, on its dissolution rate, which can depend on crystal size and crystal form. Alternatively, delayed absorption of parenterally administered drug forms is achieved by dissolving or suspending the drug in an oil vehicle.

[0534] Injectable depot formulations are created by forming a microencapsulated matrix of the compound in a biodegradable polymer, such as polylactide-polyglycolide. The rate of drug release can be controlled depending on the drug-to-polymer ratio and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by drug capture in liposomes or microemulsions compatible with body tissues.

[0535] For use in the method of the present invention, the active compound may be provided in combination with a pharmaceutically acceptable carrier, for example, as a pharmaceutical composition containing 0.1 to about 99.5% (more preferably about 0.5 to about 90.0%) of the active ingredient, or as itself.

[0536] In some embodiments of the present invention, the compound of the present invention is administered in combination with one or more additional compounds / preparations.

[0537] In certain such embodiments, co-administration is simultaneous. In certain such embodiments, the compound of the present invention is co-formulated 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, co-administration is sequential, with the administration of the compound of the present invention before or after the administration of one or more additional compounds by minutes or hours.

[0538] The method of introducing the compound of the present invention may also be provided by a rechargeable or biodegradable device. Various sustained-release polymer devices have been developed and have recently been tested in vivo for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. Various biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, may be used to form implants for sustained release of the compound at specific target sites.

[0539] The actual dosage level of the active ingredient in a pharmaceutical composition can be varied to obtain an amount of the active ingredient effective in achieving a desired therapeutic response for a specific patient, composition, and mode of administration without toxicity to the patient.

[0540] The selected dosage level will depend on various factors including the following: the activity of the specific compound, conjugate, or combination of compound and / or conjugate, or its ester, salt, or amide; the route of administration of the specific compound(s) used, the time of administration, the elimination rate, the duration of treatment; other drugs, compounds, and / or substances used in combination with the specific compound(s) used; the age, sex, weight, health status, general health and prior medical history of the patient being treated; and similar factors well known in the medical field.

[0541] A physician or veterinarian with ordinary skills in the art can easily determine and prescribe the therapeutic effective dose of the required pharmaceutical composition. For example, a physician or veterinarian may start a dose of the pharmaceutical composition or compound at a lower level than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. "Therapeutic effective dose" refers to a concentration of the compound sufficient to induce the desired therapeutic effect. It is generally understood that the effective dose of the compound will vary depending on the subject's weight, sex, age, and medical history. Other factors affecting the effective dose may, without limitation, include: 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 with the compound of the present invention. A larger total dose may be delivered by multiple administrations of the formulation. 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).

[0542] Generally, a suitable daily dose of the active compound used in the composition and method of the present invention will be the amount of the compound that is the lowest effective dose for producing a therapeutic effect. Such an effective dose will generally depend on the factors described above.

[0543] If desired, the effective daily dose of the active compound or conjugate may be administered, optionally, as 1, 2, 3, 4, 5, 6, or greater sub-dose, administered separately at appropriate intervals throughout the day in the form of unit doses. In a specific embodiment of the invention, the active compound may be administered two or three times daily. In a preferred embodiment, the active compound will be administered once daily.

[0544] Patients receiving such treatment are primates, particularly humans, and other mammals such as horses, cattle, pigs, and sheep; and any animals in need, generally including poultry and pets.

[0545] In certain embodiments, the compounds or conjugates disclosed herein may be used alone or administered co-administered with another type of therapeutic agent. As used herein, the phrase “co-administered” refers to any form of administration of two or more different therapeutic compounds or conjugates such that a second compound or conjugate is administered while the previously administered therapeutic compound or conjugate is still effective in the body (e.g., two compounds or conjugates are simultaneously effective in the patient, which may include a synergistic effect of the two compounds or conjugates). For example, different therapeutic compounds or conjugates may be administered simultaneously or sequentially within the same formulation or within separate formulations. In certain embodiments, different therapeutic compounds or conjugates may be administered within 1 hour, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, weeks, or longer than that. Thus, an individual receiving such treatment may benefit from the combined effect of different therapeutic compounds or conjugates.

[0546] The present invention comprises the use of pharmaceutically acceptable salts of the compounds or conjugates disclosed herein. In certain embodiments, the salts considered of the present invention include, but are not limited to: alkyl, dialkyl, trialkyl, or tetraalkyl ammonium salts. In certain embodiments, the salts considered of the present invention include, but are not limited to: L-arginine, benetamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucarmine, hydravamin, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, the salts considered of the present invention include, but are not limited to, the following: Na, Ca, K, Mg, Zn, or other metal salts.

[0547] Pharmaceutically acceptable acid addition salts may also exist as various solvates with, for example, water, methanol, ethanol, dimethylformamide, and other similar substances. Mixtures of such solvates may also be prepared. The source of such solvates is derived from the solvent of crystallization and may be inherent to the solvent of preparation or crystallization, or may be incidental to such solvent.

[0548] Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavoring agents and fragrances, preservatives, and antioxidants may also be present in the composition.

[0549] Examples of pharmaceutically acceptable antioxidants include the following: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and other similar types; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and other similar types; and (3) metal-chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and other similar types.

[0550] The present invention will be more easily understood by reference to the following examples, which are included for the purpose of illustrative examples of specific aspects and embodiments of the invention and are not intended to limit the invention.

[0551] Experimental Example

[0552] Synthesis protocol

[0553] abbreviation

[0554] AcO : Acetyl

[0555] AcOH: Acetic acid

[0556] EA: Ethyl acetate

[0557] DCM: Dichloromethane

[0558] m-CPBA: Meta-chloroperoxybenzoic acid

[0559] TBDMSOTf : tert-butyldimethylsilyl trilate

[0560] TBDMS: tert-butyldimethylsilyl

[0561] DMF: Dimethylformamide

[0562] EDCI : 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0563] HOBt: 1-Hydroxybenzotriazole hydrate

[0564] ACN: Acetonitrile

[0565] TBDMS-Cl : tert-butyldimethylsilyl chloride

[0566] DBU : 1,8-Diazabicyclo[5.4.0]Undek-7-en

[0567] THF: Tetrahydrofuran

[0568] DCC : N,N'-Dicyclohexylcarbodiimide

[0569] DMAP: 4-Dimethylaminopyridine

[0570] NHS: N-hydroxysuccinimide

[0571] DIPEA: Diisopropylethylamine

[0572] TEA: Triethylamine

[0573] DEAD: Diethyl azodicarboxylate

[0574] Boc: tert-butyloxycarbonyl

[0575] LAH: Lithium Aluminum Hydrate

[0576] CDI: 1,1'-Carbonyldiimidazole

[0577] BEMP: 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorin

[0578] TPSCl : Triphenylchlorosilane

[0579] tfa: trifluoroacetyl

[0580] PyBop : Benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate

[0581] HBTU : N,N,N′,N′-tetramethyl-O-(1H-benzotriazole-1-yl)uronium hexafluorophosphate

[0582] TFA: Trifluoroacetic acid

[0583] DIC : N,N'-Diisopropylcarbodiimide

[0584] DMPA: 2,2-Dimethoxy-2-phenylacetophenone

[0585] TBAF: Tetra-n-butylammonium fluoride

[0586] AgOTf: Silver trifluoromethanesulfonate

[0587] (BimC4A)3: Tripotassium 5,5′,5′′-[2,2′,2′′-Nitrilotris(methylene)tris(1 H -Benzimidazole-2,1-diyl)]Tripentanoate hydrate

[0588] Example 1: Preparation of Compound L-1

[0589]

[0590] Preparation of Compound L-1-1

[0591] LAH (3.6 g, 95.15 mmol) was added dropwise to a solution of dimethyl 5-hydroxyisophthalate (5 g, 23.79 mmol) in dry THF (300 mL) under an N2 environment at -78°C. The reaction mixture was stirred at room temperature for 17 hours. After the reaction was complete, 15% NaOH solution (4 mL), H2O (8 mL), and EA (100 mL) were added, and the reaction mixture was then stirred for 1 hour. The mixture was filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-1-1 (3.02 g, 82%).

[0592] 1 ¹H 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).

[0593] Preparation of Compound L-1-2

[0594] Compound L-1-1 (2 g, 12.97 mmol) was dissolved in HBr (5.0 mL, 33% in AcOH) under an N2 environment. After stirring at 60°C for 18 hours, the reaction was quenched by adding a NaHCO3 solution (pH ~8). Subsequently, distilled water (50 mL) and EA (100 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 obtain compound L-1-2 (2.9 g, 80%).

[0595] 1 ¹H NMR (400 MHz, CDCl₃) δ 6.99 (s, 1H), 6.81 (s, 2H), 4.85 (s, 1H), 4.41 (s, 2H).

[0596] Preparation of Compound L-1

[0597] Imidazole (27 mg, 0.39 mmol) and TBDMS-Cl (59 mg, 0.39 mmol) were added to a solution of compound L-1-2 (100 mg, 0.36 mmol) in dry DCM (3 mL) under an N2 environment at room temperature. After stirring for 16 hours, 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 obtain compound L-1 (110 mg, 79%).

[0598] 1 ¹H 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).

[0599] Example 2: Preparation of Compound Int-1

[0600]

[0601] Preparation of Compound Int-1-1

[0602] SOCl2 (207 mL, 2.85 mol) was added dropwise to a solution of vanillic acid (50.0 g, 0.30 mol) in MeOH (700 mL) under an N2 environment at 0°C. After stirring for 15 hours at room temperature, the reaction was adjusted to a pH of 7 to 8 with a 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 obtain compound Int-1-1 (54.2 g, quantitative).

[0603] 1 ¹H 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).

[0604] Preparation of Compound Int-1-2

[0605] K2CO3 (61.6 g, 0.45 mol) and benzyl bromide (39.0 mL, 0.33 mol) were added to a solution of compound Int-1-1 (54.2 g, 0.30 mol) in DMF (200 mL) under an N2 environment. After stirring at 100°C for 6 hours, 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 obtain compound Int-1-2 (79.8 g, 98%).

[0606] 1 ¹H 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).

[0607] Preparation of Compound Int-1-3

[0608] Compound Int-1-2 (79.8 g, 0.29 mol) was dissolved in acetic anhydride (550 mL) under an N2 environment and then cooled to 0°C. Copper(II) nitrate half-(pentahydrate) (75.0 g, 0.32 mol) was added in portions. After stirring for 6 hours at 0°C, 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 obtain compound Int-1-3 (85.5 g, 92%).

[0609] 1 ¹H 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).

[0610] Preparation of Compound Int-1-4

[0611] 2N NaOH (404 mL, 0.81 mol) was added to a solution of compound Int-1-3 (85.5 g, 0.27 mol) in THF (800 mL) and MeOH (300 mL). After stirring at 65°C for 5 hours, the reaction was cooled to room temperature, adjusted to pH 2 by adding 2N HCl solution, and then extracted with distilled water (100 mL) and EA (300 mL x 2). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue solid was collected and washed with hexane to obtain compound Int-1-4 (79.2 g, 97%).

[0612] 1 ¹H 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).

[0613] Preparation of Compound Int-1

[0614] Oxalyl chloride (42.4 μL) and 1 drop of DMF were slowly added dropwise to a solution of compound Int-1-4 (100 mg, 0.33 mmol) in anhydrous THF (500 μL) and anhydrous DCM (1.5 mL) at 0°C under an N2 environment. After stirring for 30 minutes, the reaction mixture was concentrated under reduced pressure. Compound Int-1 was used directly in the next step without further purification.

[0615] Example 3: Preparation of Compound Int-2

[0616]

[0617] Preparation of Compound Int-2-1

[0618] To a brown solution of compound Int-1-3 (24.8 g, 78.2 mmol) in DCM, 1M BCl3 (93.8 mL, 93.8 mmol, 1.2 equivalents) was added at -78°C under an N2 environment. After stirring for 3 hours, the reaction was quenched by the addition of MeOH (100 mL). The mixture was heated 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. Upon removal of the solvent, Int-2-1 (17.5 g, 98%) was obtained as a yellow solid.

[0619] 1 ¹H 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).

[0620] Preparation of Compound Int-2-2

[0621] 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 an N2 environment. After stirring at 40°C for 5 hours, the mixture was cooled to 0°C and acidified with 2N HCl. The mixture was diluted with H2O (150 mL) and extracted with EA (300 mL x 3). The combined organic layer was 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 obtain compound Int-2-2 (15.9 g, 97%) as a yellow solid.

[0622] 1 ¹H NMR (400 MHz, DMSO- d6 ) δ10.6 (brs, 1H), 7.31 (s, 1H), 7.25 (s, 1H), 3.90 (s, 3H), 3.57 (s, 3H).

[0623] Preparation of Compound Int-2-3

[0624] 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 equivalents), acetic anhydride (8.5 mL, 87.5 mmol, 1.2 eq), and TEA (15.6 mL, 111.9 mmol, 1.5 eq) at room temperature under an N2 environment and stirred for 6 hours. 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 obtain compound Int-2-3 (18 g, 95%) as a yellow solid.

[0625] 1 ¹H NMR (400 MHz, DMSO- d6 ) δ 7.99 (s, 1H), 7.46 (s, 1H), 3.94 (s, 3H), 2.30 (s, 3H).

[0626] Preparation of Compound Int-2

[0627] A brown solution of compound Int-2-3 (14.4 g, 56.43 mmol) in anhydrous THF (15 mL) and anhydrous DCM (40 mL) at 0 °C under an N2 environment was treated with oxalyl chloride (7.6 mL, 84.64 mmol, 1.5 eq) and DMF (2 drops) and stirred for 6 hours. The reaction mixture was concentrated under reduced pressure. Compound Int-2 was used directly in the next step without further purification.

[0628] Example 4: Preparation of compound Mono-1

[0629]

[0630] Preparation of compound Mono-1-1

[0631] 206 μL of conc. HCl was added dropwise to a solution of L-2-thienylalanine (500 mg, 2.92 mmol) in distilled water (5.0 mL) and stirred under an N2 environment at 0°C, followed by the addition of formaldehyde (37%, 261 μL, 3.5 mmol). The mixture was refluxed overnight. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was suspended in IPA (3.0 mL), and 1.0 mL of 4M HCl (in 1,4-dioxane) was added to it. After stirring for 2 hours, the solid was filtered and washed with IPA (5 mL) and ether (20 mL) to obtain compound Mono-1-1 (495.7 mg, 77%).

[0632] 1 ¹H 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).

[0633] Preparation of compound Mono-1-2

[0634] Compound Mono-1-1 (495.7 mg, 2.25 mmol) was dissolved in MeOH (10.0 mL) under an N2 environment 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 hours. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was washed with diethyl ether (5 mL x 2) to obtain compound Mono-1-2 (521.5 mg, 99%).

[0635] 1 H NMR (400 MHz, DMSO-d6) δ 10.22 (brs, 2H), 7.49 (d, J = 5.2 Hz, 1H), 6.94 (d, J = 5.2 Hz, 1H), 4.65-4.61 (m, 1H), 4.30 (d, J = 15.6 Hz, 1H), 4.19 (d, J = 15.6 Hz, 1H), 3.80 (s, 3H), 3.60 (dd, J = 11.6, 5.2 Hz, 1H), 3.21-3.14, (m, 1H). EI-MS m / z: 198 (M + +1).

[0636] Preparation of compound Mono-1-3

[0637] A solution of compound Mono-1-2 (518.5 mg, 2.22 mmol) in DMF (3.0 mL) was added to a solution of compound Int-1 (856.5 mg, 2.66 mmol) in anhydrous THF (3.0 mL), followed by the addition of DIPEA (772.8 μL, 4.44 mmol) at 0°C. The reaction mixture was stirred overnight at room temperature. After the reaction was complete, 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 obtain compound Mono-1-3 (888.5 mg, 89%).

[0638] EI-MS m / z: 483 (M + +1).

[0639] Preparation of compound Mono-1-4

[0640] DIBAL (3.6 mL, 3.6 mmol, 1.0 M in toluene) was added dropwise to a solution of compound Mono-1-3 (880 mg, 1.82 mmol) in anhydrous DCM (5.0 mL) and toluene (15.0 mL) under an N2 environment at -78°C. The reaction mixture was stirred at -78°C for 3 hours. The reaction was quenched with MeOH (5 mL) and 2N HCl (20.0 mL) at -78°C, followed by the addition of distilled water (20 mL) and EA (50 mL x 2) 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 obtain compound Mono-1-4 (701.9 mg, 85%).

[0641] EI-MS m / z: 453(M + +1).

[0642] Preparation of compound Mono-1-5

[0643] Na2S2O4 (2.2 g, 12.4 mmol) was added to a solution of compound Mono-1-4 (700 mg, 1.55 mmol) in THF (15.0 mL) and distilled water (3.0 mL) at room temperature for 4 hours. After the reaction was complete, the mixture 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 remove any residual water. The obtained white solid was left overnight under high vacuum to dry completely. The residue was suspended in anhydrous MeOH (10 mL), and acetyl chloride (1.1 mL, 15.5 mmol) was added. After 15 minutes, the turbid solution was filtered, and the solid was washed with anhydrous MeOH (5 mL x 2). The filtrate was stirred for 2 hours. When the reaction was complete, the reaction mixture was quenched to a pH of 7 in a NaHCO3 solution, 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 obtain compound Mono-1-5 (701.9 mg, 85%).

[0644] 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).

[0645] EI-MS m / z: 453 (M + +1).

[0646] Preparation of compound Mono-1

[0647] 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 hours. After the reaction was complete, the reaction was quenched with a NaHCO3 solution (pH ~7), and distilled water (5 mL) and EA (20 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 obtain compound Mono-1 (38.3 mg, 82%).

[0648] 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).

[0649] EI-MS m / z: 315(M + +1).

[0650] Example 5: Preparation of compound Mono-2

[0651]

[0652] Preparation of compound Mono-2-1

[0653] N-Boc-L-histidine (900 mg, 3.34 mmol) was dissolved in ACN (5.0 mL) under an N2 environment. Cs2CO3 (2.2 g, 6.68 mmol) and iodomethane (208 μL, 3.34 mmol) were added. The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the turbid solution was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Mono-2-1 (549.2 mg, 58%).

[0654] 1 H 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).

[0655] Preparation of compound Mono-2-2

[0656] Compound Mono-2-1 (549.2 mg, 1.94 mmol) was dissolved in DCM (5.0 mL) under an N2 environment and cooled to 0°C. 4M HCl (2 mL in 1,4-dioxane) was added at 0°C. The reaction was heated to room temperature and stirred for 5 hours. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was washed with ether (5 mL) to obtain compound Mono-2-2 (429.8 mg, quantitative).

[0657] 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= 6.0 Hz, 2H). EI-MS m / z: 184 (M + +1).

[0658] Preparation of compound Mono-2-3

[0659] Compound Mono-2-2 (425.8 mg, 1.94 mmol) was dissolved in distilled water (4.0 mL) under an N2 environment and then cooled to 0°C. After adding conc. HCl (250 μL) dropwise at 0°C, formaldehyde (37%, 216 μL, 2.9 mmol) was added. The reaction mixture was refluxed overnight. After the reaction was complete, 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 hours. The solid was filtered and washed with IPA (5 mL) and ether (10 mL x 2) to obtain compound Mono-2-3 (421.9 mg, quant.).

[0660] EI-MS m / z: 182 (M + +1).

[0661] Preparation of compound Mono-2-4

[0662] Compound Mono-2-3 (421.9 mg, 1.94 mmol) was dissolved in MeOH (5.0 mL) under an N2 environment 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 hours. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was washed with ether (5 mL x 2) to obtain compound Mono-2-4 (375.2 mg, 84%).

[0663] 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).

[0664] Preparation of compound Mono-2-5

[0665] 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), after which DIPEA (846.2 μL, 4.86 mmol) was added at 0°C, and the reaction mixture was stirred overnight at room temperature. After the reaction was complete, 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 obtain compound Mono-2-5 (466.8 mg, 60%).

[0666] EI-MS m / z: 481 (M + +1).

[0667] Preparation of compound Mono-2-6

[0668] DIBAL (832 μL, 0.832 mmol, 1.0 M in toluene) was added dropwise to a solution of compound Mono-2-5 (100 mg, 0.21 mmol) in anhydrous DCM (0.5 mL) and toluene (1.5 mL) at -78°C under an N2 environment, and the reaction mixture was stirred at -78°C for 5 hours. 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 obtain compound Mono-2-6 (43 mg, 46%).

[0669] EI-MS m / z: 451(M + +1).

[0670] Preparation of compound Mono-2-7

[0671] Na2S2O4·2H2O (149 mg, 0.71 mmol) was added to a solution of compound Mono-2-6 (40 mg, 0.09 mmol) in THF (1.7 mL) and distilled water (1.1 mL) at room temperature under an N2 environment. After stirring for 4 hours, the reaction was quenched with MeOH (2.0 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 obtained yellow solid was left overnight under high vacuum to dry completely. The residue was suspended in anhydrous MeOH (3.0 mL), and acetyl chloride (63 μL, 0.88 mmol) was added. After stirring for 15 minutes, the turbid solution was filtered, and the solid was washed with anhydrous MeOH (5.0 mL x 2). After stirring for 2 hours, the reaction mixture was adjusted to pH 7 with a NaHCO3 solution. Distilled water (5.0 mL) and EA (20 mL x 2) were added thereto. 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-2-8 (18 mg, 51%).

[0672] 1 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 Hz, 1H), 4.58 (d, J = 16.8 Hz, 1H), 4.15-4.05 (m, 1H), 3.99 (s, 3H), 3.68 (s, 3H), 3.22-3.08 (m, 2H). EI-MS m / z: 403 (M + +1).

[0673] Preparation of compound Mono-2

[0674] 80 μL of methanesulfonic acid in 160 μL of DCM was added to a solution of compound Mono-2-7 (16 mg, 0.04 mmol) in anhydrous DCM (160 μL) at 0°C. The reaction mixture was stirred at the same temperature for 3 hours. After the reaction was complete, the mixture was quenched with a NaHCO3 solution (pH 8–9). The residue was purified by preparative HPLC to obtain compound Mono-2 (4.9 mg, 40%).

[0675] 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).

[0676] Example 6: Preparation of compound Mono-3

[0677]

[0678] Preparation of compound Mono-3-1

[0679] L-2-furylalanine (2.56 g, 13.35 mmol) was dissolved in MeOH (50.0 mL) under an N2 environment 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 hours. The mixture was concentrated under reduced pressure. The residue was washed with ether (10 mL) to obtain compound Mono-3-1 (2.73 g, quant.).

[0680] 1H 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).

[0681] Preparation of compound Mono-3-2

[0682] Compound Mono-3-1 (2.7 g, 13.13 mmol) was dissolved in THF (10 mL) and DMF (20.0 mL), and then formaldehyde (37%, 2.0 mL, 26.26 mmol) was added under an N2 environment. After refluxing the reaction mixture overnight, Na2SO4 (1.0 g) was added to the reaction mixture to remove water, followed by the addition of 4M HCl (3.5 mL in 1,4-dioxane), and the resulting mixture was stirred at room temperature for 3 hours. The mixture was quenched with a 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 obtain compound Mono-3-2 (906.1 mg, 24.5%).

[0683] EI-MS m / z: 282 (M + +1).

[0684] Preparation of compound Mono-3-3

[0685] 4M HCl (in 1,4-dioxane, 1.6 mL) was added to a solution of compound Mono-3-2 (609.1 mg, 3.22 mmol) in DCM (5.0 mL) at 0°C under an N2 environment. The reaction was heated to room temperature and stirred for 5 hours. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was washed with ether (5 mL) to obtain compound Mono-3-3 (628.3 mg, 90%).

[0686] 1 ¹H 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).

[0687] Preparation of compound Mono-3-4

[0688] A solution of compound Mono-3-3 (628.3 mg, 2.88 mmol) in DMF (5.0 mL) was added to a solution of compound Int-1 (1.4 g, 4.33 mmol) in anhydrous THF (5.0 mL), followed by the addition of DIPEA (846.2 μL, 4.86 mmol) at 0°C. The reaction mixture was stirred overnight at room temperature. After the reaction was complete, distilled water (20 mL) and EA (50 mL x 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 obtain compound Mono-3-4 (1.2 g, 89%).

[0689] EI-MS m / z: 467 (M + +1).

[0690] Preparation of compound Mono-3-5

[0691] 1 M DIBAL (5.2 mL, 5.2 mmol, 1.0 M in toluene) was added dropwise at -78°C 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). The mixture was stirred at the same temperature for 5 hours. After the reaction was complete, the reaction mixture was quenched at -78°C with MeOH (5 mL) and 2N HCl (20.0 mL), 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 obtain compound Mono-3-5 (884 mg, 79%).

[0692] EI-MS m / z: 436(M + +1).

[0693] Preparation of compound Mono-3-6

[0694] Na2S2O4 (213.4 mg, 1.02 mmol) was added 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) at room temperature under an N2 environment. After stirring for 8 hours, 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 obtained yellow solid was left overnight under high vacuum to dry completely. The residue was suspended in anhydrous MeOH (2.0 mL), and then acetyl chloride (90.2 μL, 1.27 mmol) was added. After stirring for 15 minutes, the turbid solution was filtered, and the solid was washed with anhydrous MeOH (5.0 mL x 2). After stirring for 2 hours, the reaction mixture was adjusted to pH 7 with a NaHCO3 solution. Distilled water (5.0 mL) and EA (20 mL x 2) were added thereto. 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-3-6 (30.3 mg, 61%).

[0695] 1 H 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 Hz, 2H), 4.59 (s, 2H), 4.13 (t, J = 6.4 Hz, 1H), 3.23 (dd, J = 9.2, 6.8 Hz, 1H), 3.10 (d, J = 16.8 Hz, 1H). EI-MS m / z: 389 (M + +1).

[0696] Preparation of compound Mono-3

[0697] 5% Pd / C (273 mg, 0.13 mmol) and 1,4-cyclohexadiene (1.0 mL, 10.30 mmol) were added to a solution of compound Mono-3-6 (100 mg, 0.26 mmol) in anhydrous EtOH (10 mL). The mixture was stirred at room temperature for 8 hours. After the reaction was complete, the solid was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Mono-3 (23 mg, 31%).

[0698] 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 (s, 2H), 4.15 (t, J = 6.8 Hz, 1H), 3.91 (s, 3H), 3.24 (dd, J = 9.6, 6.8 Hz, 1H), 3.12 (d, J = 16.8 Hz, 1H); EI-MS m / z: 299 (M + +1).

[0699] Example 7: Preparation of compound Mono-4

[0700]

[0701] Preparation of compound Mono-4-1

[0702] DCC (1.15 g, 8 mmol) in THF (40 mL) and MeOH (20 mL) was added dropwise over 30 minutes at -13°C 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). The reaction mixture was slowly heated to room temperature while stirring for 5 hours. After the reaction was complete, 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 obtain the compound Mono-4-1 (13.0 g, 84%).

[0703] 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 (d, J = 7.6 Hz, 1H), 4.66-4.59 (m, 1H), 4.38-4.22 (m, 2H), 3.63 (s, 3H), 3.07 (t, J = 6.4 Hz, 1H). EI-MS m / z: 634 (M + +1).

[0704] Preparation of compound Mono-4-2

[0705] Methyl iodide (3.8 mL, 61.54 mmol) was added to a solution of compound Mono-4-1 (13 g, 20.51 mmol) in DMF (50 mL) at 0°C. The reaction mixture was heated to room temperature and stirred for 5 hours. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Mono-4-2 (11 g, 83%).

[0706] 1 H 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 Hz, 1H), 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).

[0707] Preparation of compound Mono-4-3

[0708] TFA (40 mL) and triethylsilane (8.12 mL, 50.86 mmol) were added to a solution of compound Mono-4-2 (11 g, 16.95 mmol) in DCM (150 mL) at 0°C under an N2 environment. The reaction was heated to room temperature and stirred for 6 hours. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Mono-4-3 (6.25 g, 91%).

[0709] 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 Hz, 1H), 4.64-4.50 (m, 2H), 4.48-4.38 (m, 1H), 3.79 (s, 6H), 3.51-3.44 (m, 1H), 3.29-3.10 (m, 2H). EI-MS m / z: 407 (M + +1).

[0710] Preparation of compound Mono-4-4

[0711] Piperidine (3.0 mL, 30.74 mmol) was added to a solution of compound Mono-4-3 (6.25 g, 15.37 mmol) in DCM (150 mL) at 0°C under an N2 environment. The reaction mixture was heated to room temperature and stirred for 7 hours. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain compound Mono-4-4 (2.65 g, 95%).

[0712] 1 ¹H NMR (400 MHz, CDCl₃) δ 7.51 (s, 1H), 6.87 (s, 1H), 3.78-3.69 (m, 4H), 3.63 (s, 3H), 3.09-2.84 (m, 2H). EI-MS m / z: 184 (M + +1).

[0713] Preparation of compound Mono-4-5

[0714] Compound Mono-4-4 (2.65 g, 14.46 mmol) was dissolved in distilled water (100 mL) under an N2 environment, and the reaction mixture was then cooled to 0°C. After adding conc-HCl (2.5 mL) dropwise at 0°C, formaldehyde (37%, 2.2 mL, 28.93 mmol) was added. The reaction mixture was refluxed overnight. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was suspended in IPA (20 mL), and 4 M HCl (4.0 mL in 1,4-dioxane) was added. The reaction mixture was stirred for 2 hours. The solid was filtered and washed with IPA (5 mL) and ether (10 mL x 2) to obtain compound Mono-4-5 (3.14 g, 99%).

[0715] EI-MS m / z: 182 (M + +1).

[0716] Preparation of compound Mono-4-6

[0717] SOCl2 (2.5 mL, 35.15 mmol) was added dropwise at 0°C to a solution of compound Mono-4-5 (3.14 g, 14.43 mmol) in MeOH (100 mL). The reaction mixture was refluxed for 5 hours and then concentrated under reduced pressure. The residue was washed with ether (25 mL x 2) to obtain compound Mono-4-6 (2.18 g, 65%).

[0718] EI-MS m / z: 196 (M + +1).

[0719] Preparation of compound Mono-4-7

[0720] DIPEA (4.9 mL, 28.22 mmol) was added at 0°C 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). After stirring at room temperature for 2 hours, 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 obtain compound Mono-4-7 (2.81 g, 62%).

[0721] EI-MS m / z: 481 (M + +1).

[0722] Preparation of compound Mono-4-8

[0723] DIBAL (10.4 mL, 10.41 mmol, 1.0 M in toluene) was added dropwise 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) at -78°C under an N2 environment. After stirring at -78°C for 5 hours, the mixture was quenched with MeOH (1.0 mL) and 2N HCl (100 mL) at the same temperature. The mixture was diluted with water (100 mL) and DCM (200 mL) in succession, and then 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-4-8 (1.21 g, 52%).

[0724] EI-MS m / z: 451 (M + +1).

[0725] Preparation of compound Mono-4-9

[0726] Na2S2O4 (3.7 g, 21.31 mmol) was added at room temperature to a solution of compound Mono-4-8 (1.2 g, 2.66 mmol) in THF (100 mL) and distilled water (70 mL). After stirring for 6 hours, the reaction was quenched with MeOH (20 mL), and water was removed by concentrating the mixture three times under reduced pressure using toluene as a co-solvent. The obtained yellow solid was suspended in anhydrous MeOH (200 mL), and acetyl chloride (1.9 mL, 26.64 mmol) was added thereto. After stirring for 15 minutes, the reaction mixture was adjusted to pH 8 by adding a 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 obtain compound Mono-4-9 (918 mg, 78%).

[0727] 1 ¹H 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).

[0728] Preparation of compound Mono-4

[0729] Methanesulfonic acid (0.1 mL) in DCM (0.2 mL) was added to a solution of compound Mono-4-9 (50 mg, 0.12 mmol) in anhydrous DCM (2 mL) at 0°C. After stirring at room temperature for 1 hour, the mixture was adjusted to pH 8 by adding a saturated NaHCO3 solution. The residue was purified by preparative HPLC to obtain compound Mono-4 (27 mg, 71%).

[0730] 1 H 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).

[0731] Example 8: Preparation of compound Mono-5

[0732]

[0733] Compound Mono-5 was synthesized in a manner similar to that described in Example 4.

[0734] Compound Mono-5-1

[0735] Yield: 92%; 1 ¹H NMR (400 MHz, DMSO-d6) δ 9.97 (s, 2H), 7.08 (s, 1H), 4.47 - 4.31 (m, 1H), 4.22 (d, J = 16.0 Hz, 1H), 4.12 (d, J = 15.6 Hz, 1H), 3.12 - 3.02 (m, 2H); EI-MS m / z: 263 (M + +1).

[0736] Compound Mono-5-2

[0737] Yield: 95%; 1 1H NMR (400 MHz, DMSO- d6 ) δ 10.1 (s, 2H), 7.09 (s, 1H), 4.66 - 4.56 (m, 1H), 3.80 (s, 3H), 4.25 (d, J = 15.6 Hz, 1H), 4.14 (d, J = 15.6 Hz, 1H), 3.32-3.28 (m, 1H), 3.13-3.04 (m, 1H); EI-MS m / z: 277 (M + +1).

[0738] Compound Mono-5-3

[0739] Yield: 68%; EI-MS m / z: 562 (M + +1).

[0740] Compound Mono-5-4

[0741] Yield: 34%; EI-MS m / z: 532(M + +1).

[0742] Compound Mono-5-5

[0743] Yield: 80%; 1¹H NMR (400 MHz, CDCl₃) δ 7.59 (d, J = 6.0 Hz, 1H), 7.52 (s, 1H), 7.46 - 7.27 (m, 5H), 6.92 (s, 1H), 6.85 (s, 1H), 5.20 (q, J = 12.4, 11.6 Hz, 2H), 4.87 (d, J = 16.4, 1H), 4.38 (d, J = 16.8, 1H), 4.06-4.0 (m, 1H), 3.98 (s, 3H), 3.23 (dd, J = 12.2, 6.4 Hz, 1H ), 3.15 (d, J = 16.0 Hz, 1H); EI-MS m / z: 484(M + +1).

[0744] Compound Mono-5

[0745] Yield: 84%; 1 ¹H NMR (400 MHz, CDCl₃) δ 7.61 (d, J = 5.6 Hz, 1H), 7.52 (s, 1H), 6.91 (d, J = 12.4 Hz, 1H), 6.02 (s, 1H), 4.87 (d, J = 16.4 Hz, 1H), 4.39 (d, J = 16.8 Hz, 1H), 4.07-4.02 (m, 1H), 3.99 (s, 3H), 3.28-3.16 (m, 2H); EI-MS m / z: 394 (M + +1).

[0746] Example 9: Preparation of compound Mono-6

[0747]

[0748] Compound Mono-6 was synthesized in a manner similar to that described in Example 4.

[0749] Compound Mono-6-1

[0750] Yield: 95%;1 H 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).

[0751] Compound Mono-6-2

[0752] 수율: 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).

[0753] Compound Mono-6-3

[0754] 수율: 76%; EI-MS m / z: 483 (M + +1).

[0755] Compound Mono-6-4

[0756] 수율: 74%; EI-MS m / z: 453 (M + +1).

[0757] Compound Mono-6-5

[0758] 수율: 74%; 1 H 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).

[0759] Compound Mono-6

[0760] 수율: 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, 1H), 4.57 (d, J = 16.4, 1H), 4.08-3.99 (m, 1H), 3.98 (s, 3H), 3.19-3.14 (m, 2H ); EI-MS m / z: 315 (M + +1).

[0761] 실시예 10: Preparation of compound Mono-8

[0762]

[0763] Preparation of compound Mono-8-1

[0764] SOCl2 (2.30 mL, 31.04 mmol) was added dropwise to a solution of (s)-(-)-1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid (5.0 g, 28.22 mmol) in MeOH (140 mL) at 0°C under an N2 environment. After stirring at 40°C for 21 hours, the mixture was concentrated under reduced pressure. The residue was washed with ether (50 mL) to obtain compound Mono-8-1 (6.42 g, 99%).

[0765] 1 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).

[0766] Preparation of compound Mono-8-2

[0767] TEA (7.9 mL, 56.43 mmol) was added at 0°C 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). After stirring at room temperature for 2 hours, distilled water (500 mL) and EA (800 mL) were added thereto. 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).

[0768] Preparation of compound Mono-8-3

[0769] DIBAL (16.8 mL, 16.79 mmol, 1.0 M in toluene) was added dropwise to a solution of compound Mono-8-2 (4 g, 8.39 mmol) in anhydrous DCM (18 mL) and toluene (52 mL) under an N2 environment at -78°C. After stirring the reaction mixture at -78°C for 4 hours, the reaction was quenched with MeOH (0.4 mL) and 2N HCl (25 mL) at -78°C, 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 obtain compound Mono-8-3 (3.07 g, 82%). EI-MS m / z: 447 (M + +1).

[0770] Preparation of compound Mono-8-4

[0771] Na2S2O4·2H2O (11.3 g, 53.76 mmol) was added to a solution of compound Mono-8-3 (3 g, 6.72 mmol) in THF (130 mL) and distilled water (86 mL) at room temperature under an N2 environment. After stirring for 5 hours, 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 obtained yellow solid was left overnight under high vacuum to further dry completely. The residue was suspended in anhydrous MeOH (220 mL), and then acetyl chloride (4.8 mL, 67.19 mmol) was added. After stirring for 15 minutes, the turbid solution was filtered, and the solid was washed with anhydrous MeOH (50 mL). After stirring for 2 hours, the reaction mixture was adjusted to pH 7 with a NaHCO3 solution, and distilled water (100 mL) and EA (500 mL) were added thereto. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain the compound Mono-8-4 (2.48 g, 93%).

[0772] 1 ¹H 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). EI-MS m / z: 399 (M + +1).

[0773] Preparation of compound Mono-8

[0774] To a solution of compound Mono-8-4 (1 g, 2.51 mmol) in anhydrous DCM (10 mL), methanesulfonic acid (5 mL) in DCM (10 mL) was added at 0°C. After stirring at 0°C for 3 hours, the mixture was adjusted to pH 7 with a NaHCO3 solution, and then distilled water (100 mL) and EA (400 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 obtain compound Mono-8 (703 mg, 91%).

[0775] 1 ¹H 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, 1H), 4.56 (d, J = 15.6 Hz, 1H), 3.98 (s, 3H), 3.95 - 3.90 (m, 1H), 3.30 - 3.13 (m, 2H). EI-MS m / z: 309 (M + +1).

[0776] Example 11: Preparation of compound Mono-9 (IBD-monomer)

[0777]

[0778] Mono-9 was synthesized via a route similar to that described in WO2010091150.

[0779] 1 ¹H 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).

[0780] Example 12: Preparation of compound Mono-10

[0781]

[0782] Preparation of compound Mono-10-1

[0783] Cbz-Cl (37.4 mL, 260 mmol) was added under an N2 environment to a solution of trans-4-hydroxy-L-proline (30 g, 230 mmol, CAS 51-35-4) in toluene (120 mL) and NaHCO3 (43 g, 570 mmol) in H2O (500 mL). The reaction mixture was stirred overnight at room temperature. 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 the compound Mono-10-1 (57.5 g, 95%) as a light brown oil.

[0784] 1 ¹H 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).

[0785] Preparation of compound Mono-10-2

[0786] 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 an N2 environment at 0°C and stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure to obtain 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.

[0787] 1 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).

[0788] Preparation of compound Mono-10-3

[0789] LiBH4 (3.9 g, 180 mmol) was added to a solution of compound Mono-10-2 (60.5 g, 220 mmol) in anhydrous THF (500 mL) under an N2 environment at 0°C. 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 x 3). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain compound Mono-10-3 (54.6 g, 98%) as a light brown oil.

[0790] 1 ¹H 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).

[0791] Preparation of compound Mono-10-4

[0792] 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 an N2 environment 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 obtain the compound Mono-10-4 (48.2 g, 63%) as a light brown oil.

[0793] 1 ¹H 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).

[0794] Preparation of compound Mono-10-5

[0795] Carbon-phase palladium, 5% Pd / C (1.3 g, 1.23 mmol) was added to a stirred solution of compound Mono-10-4 (15 g, 41.0 mmol) in EA (50 mL) under a N2 environment at room temperature. The flask was flushed at room temperature by bubbling hydrogen gas through the solution. The mixture was stirred at the same temperature for 5 hours. 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, quantitative) as a light brown oil was used directly in the next step without further purification.

[0796] 1 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).

[0797] Preparation of compound Mono-10-6

[0798] A brown solution of compounds 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) under an N2 environment at 0°C and stirred for 5 hours. The reaction mixture was diluted with distilled water (50 mL) and EA (150 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 2 : 1) to obtain compound Mono-10-6 (20 g, 83%) as a yellow foam solid.

[0799] 1 ¹H 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).

[0800] Preparation of compound Mono-10-7

[0801] Carbon-phase palladium, 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 an N2 environment. The flask was flushed at room temperature by bubbling hydrogen gas through the solution. After stirring for 8 hours, the mixture was diluted with EA (50 mL), filtered through CELITE®, and the CELITE® plug was washed with EA (50 mL x 2). The filtrate was concentrated under reduced pressure to obtain the compound Mono-10-7 (18.5 g, 99%) as a yellow foamed solid.

[0802] 1 ¹H 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).

[0803] Preparation of compound Mono-10-8

[0804] 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) at 0°C under an N2 environment and stirred for 3 hours. The reaction mixture was washed with CuSO4 solution (50 mL) and brine (100 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 obtain the compound Mono-10-8 (21.2 g, 82%) as a brown foamed solid.

[0805] 1 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).

[0806] Preparation of compound Mono-10-9

[0807] A homogeneous solution of oxalyl chloride (21 mL, 24.4 mmol) in anhydrous DMC (50 mL) was treated with DMSO (3.5 mL, 48.9 mmol) in anhydrous DCM (20 mL) at -78°C under an N2 environment and stirred for 1 hour. 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 hours. The reaction mixture was treated with TEA (22.7 mL, 162.9 mmol) and stirred at room temperature for 1 hour. The reaction mixture was extracted with NH4Cl solution (30 mL) and DCM (100 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 = 4 :1 to 1 :1) to obtain compound Mono-10-9 (8.2 g, 83%) as a brown foamed solid.

[0808] 1 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).

[0809] Preparation of compound Mono-10-10

[0810] A yellow solution of Mono-10-9 (3.0 g, 4.9 mmol) and 2,6-rutidine (6.9 mL, 58.8 mmol) in anhydrous DCM (150 mL) was treated with triflicate anhydride (8.25 mL, 49 mmol) at -10°C under an N2 environment and stirred for 6 hours. The reaction mixture was heated to room temperature and stirred for 1 hour. The reaction mixture was diluted with distilled water (50 mL) and extracted with DCM (100 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 = 4 : 1) to obtain the compound Mono-10-10 (1.3 g, 36%) as a yellow oil.

[0811] 1 ¹H 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).

[0812] Preparation of compound Mono-10-11

[0813] 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 an N2 environment, followed by stirring for 2 hours. 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 obtain the compound Mono-10-11 (420.9 mg, 59%) as a yellow foamed solid.

[0814] 1 ¹H 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).

[0815] Preparation of compound Mono-10-12

[0816] A yellow solution of Mono-10-11 (420 mg, 0.60 mmol) in THF (4.0 mL) and H2O (2.0 mL) was treated with acetic acid (8.0 mL) under an N2 environment at room temperature 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 obtain the compound Mono-10-12 (279.5 mg, 79%) as a yellow foamed solid.

[0817] 1 ¹H 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).

[0818] Preparation of compound Mono-10-13

[0819] 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 DMC (1.0 mL) at -78°C under an N2 environment and stirred for 15 minutes. A solution of Mono-10-2 (240 mg, 0.40 mmol) in anhydrous DMC (3.0 mL) was added dropwise to the reaction mixture and stirred for 3 hours, after which TEA (569 μL, 4.08 mmol) was added. The mixture was stirred at room temperature for 30 minutes. The reaction mixture was diluted with distilled water (5.0 mL) and extracted with EA (15 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 obtain the compound Mono-10-13 (191.4 mg, 80%) as a yellow foamed solid.

[0820] 1 ¹H 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)

[0821] Preparation of compound Mono-10

[0822] A yellow solution of Mono-10-13 (150 mg, 0.26 mmol) in MeOH (6.0 mL) and H2O (3.0 mL) was treated with K2CO3 (88.5 mg, 0.64 mmol) under an N2 environment at room temperature and stirred for 1 hour. 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 obtain the compound Mono-10 (105 mg, 75%) as a yellow foamed solid.

[0823] 1 ¹H 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)

[0824] Example 13: Preparation of Compound D-101

[0825]

[0826] K2CO3 (2.0 mg, 0.012 mmol) was added 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) under an N2 environment at room temperature. After stirring for 3 hours, the reaction mixture was purified by preparative HPLC to obtain compound D-101 (1.2 mg, 34%).

[0827] EI-MS m / z: 743 (M + +1).

[0828] Example 14: Preparation of Compound D-102

[0829]

[0830] Compound D-102 was synthesized in a manner similar to that described in Example 13.

[0831] Yield 34%; EI-MS m / z: 743 (M + +1).

[0832] Example 15: Preparation of Compound D-103

[0833]

[0834] K2CO3 (9.4 mg, 0.07 mmol) was added 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) under an N2 environment. The mixture was stirred at room temperature for 7 hours. After the reaction was complete, the reaction mixture was purified by preparative HPLC to obtain compound D-103 (5.4 mg, 50%).

[0835] EI-MS m / z: 747 (M + +1).

[0836] Example 16: Preparation of Compound D-104

[0837]

[0838] Preparation of compound D-104a

[0839] K2CO3 (66 mg, 0.477 mmol) was added 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) under an N2 environment at room temperature. After stirring for 3 hours, the reaction mixture was purified by preparative HPLC to obtain compound D-104a (62 mg, 48%).

[0840] EI-MS m / z: 824 (M + +1).

[0841] Preparation of Compound D-104

[0842] 1 M dimethylamine in THF (0.5 mL) was added to a solution of compound D-104a (62 mg, 0.075 mmol) in DMF (1 mL) under an N2 environment at room temperature. After stirring for 1 hour, the reaction mixture was purified by preparative HPLC to obtain compound D-104 (39 mg, 60%).

[0843] EI-MS m / z: 788 (M + +1).

[0844] Example 17: Preparation of Compound D-105

[0845]

[0846] Preparation of compound D-105a

[0847] K2CO3 (4.5 mg, 0.03 mmol) was added 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) under an N2 environment at 40°C. After stirring for 2 hours, the reaction mixture was purified by preparative HPLC to obtain compound D-10a (9 mg, 45%).

[0848] EI-MS m / z: 622 (M + +1).

[0849] Preparation of Compound D-105

[0850] K2CO3 (2 mg, 0.01 mmol) was added 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) under an N2 environment at 40°C. After stirring for 15 hours, the reaction mixture was purified by preparative HPLC to obtain compound D-105 (1.1 mg, 23%).

[0851] EI-MS m / z: 739 (M + ).

[0852] Example 18: Preparation of Compound D-106

[0853]

[0854] Compound D-106 was synthesized in a manner similar to that described in Example 16.

[0855] Compound D-106a

[0856] EI-MS m / z: 820 (M + +1).

[0857] Compound D-106

[0858] (Stage 2 yield 4%). EI-MS m / z: 784 (M + +1)

[0859] Example 19: Preparation of Compound D-107

[0860]

[0861] Compound D-107 was synthesized in a manner similar to that described in Example 16.

[0862] Compound D-107a

[0863] EI-MS m / z: 792 (M + +1).

[0864] Compound D-107

[0865] Stage 2 yield 16%; EI-MS m / z: 756 (M+ +1).

[0866] Example 20: Preparation of Compound D-108

[0867]

[0868] Compound D-108 was synthesized in a manner similar to that described in Example 16.

[0869] Compound D-108a

[0870] EI-MS m / z: 824 (M + +1).

[0871] Compound D-108

[0872] Stage 2 yield 16%; EI-MS m / z: 788 (M + +1).

[0873] Example 21: Preparation of Compound D-109

[0874]

[0875] Compound D-109 was synthesized in a manner similar to that described in Example 16.

[0876] Compound D-109a

[0877] 20% yield

[0878] EI-MS m / z: 982 (M + +1).

[0879] Compound D-109

[0880] 56% yield

[0881] EI-MS m / z: 946 (M + +1).

[0882] Example 22: Preparation of Compound D-110

[0883]

[0884] Iodomethane (0.1 mL) was added to a solution of compound D-102 (3.5 mg, 0.005 mmol) in DMF (1 mL) under an N2 environment at room temperature. After stirring at room temperature for 5 hours, the reaction mixture was purified by HPLC to obtain compound D-110 (1.2 mg, 33%).

[0885] EI-MS m / z: 772 (M + +1).

[0886] Example 23: Preparation of Compound D-111

[0887]

[0888] Compound D-111 was synthesized in a manner similar to that described for Compound D-105 of Example 17.

[0889] Compound D-111a

[0890] Yield 54%, white solid. EI-MS m / z: 628 (M + +1).

[0891] Compound D-111

[0892] Yield 11%, white solid. EI-MS m / z: 745 (M + +1)

[0893] Example 24: Preparation of Compound D-112

[0894]

[0895] Preparation of compound D-112a

[0896] 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 eq) in DMF (1 mL) was treated with K2CO3 (4.4 mg, 0.032 mmol, 1.0 eq) under an N2 environment at room temperature and stirred for 5 hours. The reaction mixture was purified by preparative HPLC (Column: Innoval ODS-2 10 μm, 100 Å, 21.2x250 mm; Flow rate: 15 mL / min, 0.1% formic acid in buffer A water / 0.1% formic acid in buffer B ACN, Method gradient, Solvent A: Solvent B 95:5 to 5:95, 1 hr, Wavelength 214 nm) to obtain compound D-112a (7.5 mg, 40%) as a white solid.

[0897] EI-MS m / z: 591(M + +1).

[0898] Preparation of Compound D-112

[0899] A homogeneous solution of compounds D-112a (7.5 mg, 0.013 mmol) and Mono-4 (3.9 mg, 0.013 mmol, 1.0 eq) in DMF (1 mL) was treated with K2CO3 (1.8 mg, 0.013 mmol, 1.0 eq) at room temperature under an N2 environment and stirred for 6 hours. The reaction mixture was treated with 1 M dimethylamine in THF (0.5 mL) and stirred for 30 minutes. The reaction mixture was purified by preparative HPLC (column: Innoval ODS-2 10 μm, 100 Å, 21.2x250 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 hr, wavelength 214 nm) to obtain compound D-112 (0.9 mg, 9%) as a white solid.

[0900] EI-MS m / z: 786(M ++1).

[0901] Example 25: Preparation of Compound D-113

[0902]

[0903] Compound D-113 was synthesized in a manner similar to the synthesis of compound D-105 of Example 17.

[0904] compound D-113a

[0905] Yield 32%, white solid. EI-MS m / z: 494 (M + +1).

[0906] Compound D-113

[0907] Yield 7%, white solid. EI-MS m / z: 725 (M + +1)

[0908] Example 26: Preparation of Compound D-114

[0909]

[0910] Preparation of compound D-114a

[0911] A homogeneous solution of compounds 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) under an N2 environment at room temperature and stirred for 5 hours. The reaction mixture was treated with 1 M dimethylamine in THF (0.5 mL) and stirred for 30 minutes. The reaction mixture was purified by preparative HPLC (column: Innoval ODS-2 10 μm, 100 Å, 21.2x250 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 hr, wavelength 214 nm) to obtain compound D-114a (3.4 mg, 9.8%) as a white solid.

[0912] EI-MS m / z: 1018(M + +1).

[0913] Preparation of Compound D-114

[0914] 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) under an N2 environment at room temperature and stirred for 3 days. The reaction mixture was purified by preparative HPLC (Column: Innoval ODS-2 10 μm, 100 Å, 21.2 x 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 hr, Wavelength 214 nm) to obtain compound D-114 (0.8 mg, 29%) as a white solid. EI-MS m / z: 824 (M + +1).

[0915] Example 27: Preparation of Compound Ref-1

[0916]

[0917] Preparation of compound Ref-1a

[0918] 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) under an N2 environment at room temperature, and then K2CO3 (45 mg, 0.32 mmol) was added thereto. The mixture was stirred at room temperature for 4 hours. After the reaction was complete, extraction was performed by adding EA (100 mL), H2O (50 mL), and 2N HCl aqueous solution (5 mL), and 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 Ref-1a (54 mg, 42%).

[0919] EI-MS m / z: 812 (M + +1).

[0920] Preparation of Compound Ref-1

[0921] Compound Ref-1a (50 mg, 0.01 mmol) was dissolved in dimethylamine (1 mL) under an N2 environment at room temperature. After stirring for 1 hour, the mixture was purified by preparative HPLC to obtain compound Ref-1 (2.2 mg, 17%).

[0922] EI-MS m / z: 776(M + ).

[0923] Example 28: Preparation of Compound Ref-2

[0924]

[0925] K2CO3 (4.7 mg, 0.034 mmol) was added 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) under an N2 environment. The mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was purified by preparative HPLC to obtain compound Ref-2 (8.0 mg, 71%).

[0926] EI-MS m / z: 719 (M + +1).

[0927] Example 29: Preparation of Compound Ref-3

[0928]

[0929] Compound Ref-3 was synthesized in a manner similar to that described in Example 13.

[0930] Yield 35%; EI-MS m / z: 734 (M + +1).

[0931] Example 30: Preparation of Compound L-2

[0932]

[0933] Preparation of Compound L-2

[0934] TEA (0.45 mL, 3.21 mmol) was added to a solution of compound L-1-2 (1.0 g, 3.57 mmol) in DCM (35 mL) under an N2 environment at room temperature. SO2F2 gas was introduced through a balloon, and the mixture was stirred at room temperature for 1 hour. Subsequently, the mixture was washed with DCM (50 mL) and water (30 mL) was added. The organic layer was washed with an aqueous NaHCO3 solution, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-2 (941.7 mg, 73%).

[0935] 1 H NMR (400 Hz, CDCl3) δ 7.47 (s, 1H), 7.32 (s, 2H), 4.46 (s, 4H).

[0936] Example 31: Preparation of Compound L-3

[0937]

[0938] Compound L-3 included herein by reference Journal of Polymer Science, Part A: Polymer Chemistry It was synthesized in a manner similar to the method described in , 2012, 50(19), 3986-3995.

[0939] Preparation of Compound L-3-1

[0940] 30% yield

[0941] 1 ¹H 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).

[0942] Preparation of Compound L-3-2

[0943] 68% yield

[0944] 1 ¹H 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).

[0945] Preparation of Compound L-3-3

[0946] 63% yield

[0947] 1 ¹H 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).

[0948] Preparation of Compound L-3

[0949] 76% yield

[0950] 1 ¹H 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).

[0951] Example 32: Preparation of Compound L-4

[0952]

[0953] Under an N2 environment at 0°C, hexaethylene glycol (2.6 g, 9.2 mmol) in THF (38 mL) and then propargyl boromide (544 μL, 4.9 mmol) were added to a solution of t-BuOK (575 mg, 4.9 mmol) in dry THF (30 mL). The reaction mixture was heated to room temperature and stirred overnight. After the reaction was complete, the mixture was filtered through CELITE®, and the CELITE® plug was washed with DCM (10 mL). The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-4 (1.38 g, 48%).

[0954] 1 ¹H NMR (400 Hz, CDCl₃) δ 4.21 (s, 2H), 3.7-3.6 (m, 24H), 3.05 (brs, 1H), 2.43 (s, 1H).

[0955] Example 33: Preparation of Compound L-5

[0956]

[0957] Preparation of Compound L-5-1

[0958] KI (294 mg, 1.77 mmol), Ag2O (4.92 g, 19.48 mmol), and p-TsCl (3.7 g, 19.48 mmol) were added to a solution of hexaethylene glycol (5.0 g, 17.71 mmol) in anhydrous DCM (178 mL) under an N2 environment. The mixture was stirred overnight at room temperature. After the reaction was complete, 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 obtain compound L-5-1 (5.98 g, 73%).

[0959] 1 ¹H 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).

[0960] Preparation of Compound L-5-2

[0961] NaN3 (1.34 g, 20.55 mmol) was added to a solution of compound L-5-1 (5.98 g, 13.7 mmol) in DMF (30 mL) under an N2 environment. The mixture was stirred at 110°C for 1 hour and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-5-2 (4.1 g, 97%).

[0962] 1 ¹H NMR (400 Hz, CDCl₃) δ 3.72 - 3.60 (m, 22H), 3.39 (t, J = 4.8 Hz, 2H), 2.78 (br, 1H).

[0963] Preparation of Compound L-4-3

[0964] Compound L-5-2 (1.9 g, 6.18 mmol) was dissolved in DCM (20 mL) under an N2 environment, triethiamine (2.0 mL, 14.22 mmol) and p-TsCl (2.4 g, 12.36 mmol) were added thereto, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-5-3 (2.58 g, 91%).

[0965] 1 ¹H 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).

[0966] EI-MS m / z: 462 (M + +1).

[0967] Preparation of Compound L-5-4

[0968] 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) at 0°C under an N2 environment. After stirring the mixture at the same temperature for 20 minutes, compound L-5-3 (1.56 g, 3.4 mmol) was added thereto. The reaction was heated to room temperature and stirred overnight. After the reaction was complete, the reaction mixture was cooled, quenched with MeOH (5 mL), and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-5-4 (1.91 g, 93%).

[0969] EI-MS m / z: 610 (M + +1).

[0970] Preparation of Compound L-5

[0971] Compound L-5-4 (906.7 mg, 1.49 mmol) was dissolved in EA (4 mL) and ether (4 mL) under an N2 environment and then cooled to 0°C. 5% HCl solution (8 mL) was added thereto, triphenylphosphine (390 mg, 1.49 mmol) was slowly added, 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 obtain compound L-5 (495 mg, 54%).

[0972] EI-MS m / z: 584 (M+ +1).

[0973] Example 34: Preparation of Compounds L-6 and L-7

[0974]

[0975] Preparation of Compound L-6-1

[0976] 5% Pd / C (1.04 g, 0.49 mmol) was added to a solution of compound L-5-2 (1.0 g, 3.25 mmol) in EtOH (5 mL) under an H2 environment. The mixture was stirred at room temperature for 4 hours. 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 hours. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography to produce compound L-6-1 (330 mg, 28%).

[0977] 1 ¹H 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).

[0978] EI-MS m / z: 382 (M + +1).

[0979] Preparation of Compound L-6-2

[0980] 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) at 0°C under an N2 environment. After stirring the mixture at 0°C for 20 minutes, L-5-3 (544.5 mg, 1.18 mmol) was added thereto. The reaction was heated to room temperature and stirred overnight. The reaction was cooled, quenched with MeOH (5 mL), and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound L-6-2 (582.9 mg, 74%).

[0981] Preparation of Compound L-6

[0982] 4M-HCl (in 1,4-dioxane, 1 mL) was added to a solution of compound L-6-2 (582.9 mg, 0.87 mmol) in DCM (3 mL) under an N2 environment at 0°C. The mixture was stirred at room temperature for 2 hours. The mixture was concentrated to obtain compound L-6 (527.6 mg, quantitative).

[0983] EI-MS m / z: 571 (M + +1).

[0984] Preparation of Compound L-7

[0985] Compound L-7 (quantitative, colorless oil) was synthesized in a manner similar to the method of preparing Compound L-5 of Example 33.

[0986] EI-MS m / z: 645(M + +1).

[0987] Example 35: Preparation of Compound L-8

[0988]

[0989] Preparation of Compound L-8-1

[0990] A homogeneous solution of 11-azido-3,6,9-trioxaudecane-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 equivalents) and BOC2O (6.0 g, 27.5 mmol, 1.2 equivalents) at room temperature under an N2 environment, followed by stirring for 6 hours. 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 obtain compound L-8-1 (7.2 g, 99%) as a colorless oil.

[0991] 1 ¹H 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).

[0992] EI-MS m / z: 319 (M + +1).

[0993] Preparation of Compound L-8

[0994] Compound L-8 (quantitative, colorless oil) was synthesized in a manner similar to the method of preparing Compound L-5 of Example 33.

[0995] 95% yield, colorless oil.

[0996] EI-MS m / z: 293 (M + +1)

[0997] Example 36: Preparation of Compound L-9

[0998]

[0999] Preparation of Compound L-9-1

[1000] 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) and para-toluenesulfonyl chloride (5.22 g, 27.4 mmol) at room temperature under an N2 environment and stirred overnight. The reaction was quenched by the addition of water (50 mL) and extracted with EA (100 mL x 3). The combined organic layer was 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 obtain compound L-9-1 (7.0 g, 82%) as a white solid.

[1001] 1 ¹H 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).

[1002] Preparation of Compound L-9-2

[1003] A suspension of CsCO3 (1.05 g, 3.21 mmol, 0.6 equivalents) in DMF (12 mL) was treated with thioacetic acid (498 μL, 6.96 mmol) and L-9-1 (2.0 g, 5.36 mmol) in DMF (8 mL) under an N2 environment at room temperature and stirred overnight. The mixture was quenched by the addition of water (50 mL) and extracted with EA (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 (Hex:EA = 5:1) to obtain compound L-9-2 (1.4 g, 95%) as a white solid.

[1004] 1 ¹H 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).

[1005] Preparation of Compound L-9-3

[1006] 35% hydrogen peroxide (4 mL) was added to a solution of compound L-9-2 (1.2 g, 4.33 mmol) in AcOH (10 mL) under an N2 environment at room temperature. The mixture was stirred for 7 hours and then concentrated under reduced pressure. The residue was diluted with water (5 mL) and basicized to a pH of 9 at 0°C using a saturated aqueous solution of NaHCO3. Boc2O (1.4 g, 6.49 mmol, 1.5 equivalents) 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 obtain compound L-9-3 (521.5 mg, 42%) as a white solid.

[1007] 1¹H 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).

[1008] Preparation of Compound L-9-4

[1009] A homogeneous solution of L-9-3 (71 mg, 0.25 mmol) in THF / H2O (2.0 mL / 4.0 mL) was treated with LiOH (17.3 mg, 0.41) under an N2 environment at room temperature and stirred for 3 hours. The mixture was neutralized with 2N HCl at 0°C and concentrated under reduced pressure to obtain compound L-9-4 (67 mg, 99%) as a white solid.

[1010] 1 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).

[1011] Preparation of Compound L-9

[1012] 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 an N2 environment. After stirring the mixture overnight, compound L-9 was used directly in the next step without further purification.

[1013] EI-MS m / z: 367 (M + +1).

[1014] Example 37: Manufacture of BGal-Br (hereinafter, "Int-TG")

[1015]

[1016] β-D-galactose pentaacetate (Alfa, CAS 4163-60-4, 5.0 g, 12.81 mmol) was dissolved in 33% HBr (20 mL) in AcOH under an N2 environment at 0°C. The mixture was heated to room temperature. After stirring for 4 hours at room temperature, the mixture was concentrated under reduced pressure, followed by the addition of EA (1000 mL) and a saturated aqueous sodium bicarbonate solution (1000 mL). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain the compound Int-TG (5.2 g, 99%).

[1017] 1 ¹H 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).

[1018] Example 38: Preparation of compounds Int-TG1 and Int-TG2

[1019]

[1020] Preparation of compound Int-TG1-1

[1021] Benzyl bromide (5.1 mL, 43.06 mmol) and NaHCO3 (2.53 g, 43.06 mmol) were added to a solution of 3-formyl-4-hydroxybenzoic acid (5 g, 43.06 mmol) in DMF (100 mL) under a N2 environment at room temperature. The mixture was stirred overnight under a N2 environment at room temperature. 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 the compound Int-TG1-1 (2.56 g, 39%).

[1022] 1 ¹H 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).

[1023] Preparation of compound Int-TG1-2

[1024] 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 sieve (8 g) and Ag2O (3.62 g, 15.61 mmol) were added at room temperature under an N2 environment. 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 obtain compound Int-TG1-2 (2.1 g, 92%).

[1025] 1 ¹H NMR (400 Hz, CDCl₃) δ10.34 (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).

[1026] Preparation of compound Int-TG1-3

[1027] m-CPBA (2.65 g, 10.74 mmol) was added to a solution of compound Int-TG1-2 (2.1 g, 3.58 mmol) in DCM (30 mL) under a N2 environment at 0°C. After stirring at 0°C for 7 hours, the mixture was quenched by adding saturated sodium bicarbonate (40 mL x 2). The mixture was separated, 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 under a N2 environment at 0°C. After stirring at 0°C for 1 hour, EA (30 mL x 2) and 1 M HCl aqueous solution (10 mL) were added. The obtained organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the compound Int-TG1-3 (1.1 g, 55%).

[1028] EI-MS m / z: 574 (M + +Na)

[1029] Preparation of compound Int-TG1-4

[1030] TBDMS-OTf (224 μL, 0.97 mmol) and Et3N (207 μL, 1.46 mmol) were added to a solution of compound Int-TG1-3 (280 mg, 0.49 mmol) in DCM (5 mL) under an N2 environment at 0°C. The mixture was stirred at room temperature for 1.5 hours and then quenched by the addition of 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 obtain compound Int-TG1-4 (246.3 mg, 68%).

[1031] 1 ¹H 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).

[1032] Preparation of compound Int-TG1-5

[1033] Pd / C (5%, 87.5 mg, 0.04 mmol) was added to a solution of compound Int-TG1-4 (283.2 mg, 0.41 mmol) in EA (5 mL) under H2 at room temperature. The mixture was stirred for 1 hour, 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).

[1034] 1 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).

[1035] Preparation of compound Int-TG1

[1036] PyBOP (275 mg, 0.53 mmol) and DIPEA (176 μL, 1.02 mmol) were added to a solution of compound Int-TG1-5 (243.2 mg, 0.41 mmol) and 11-azido-3,6,9-trioxaudecane-1-amine (Aldrich, CAS 134179-38-7, 89.5 mg, 0.41 mmol) in DMF (5 mL) under a N2 environment at room temperature. The mixture was stirred for 2 hours under a N2 environment at room temperature. 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 obtain compound Int-TG1 (272.8 mg, 84%).

[1037] 1 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).

[1038] EI-MS m / z: 799 (M + +1).

[1039] Preparation of compound Int-TG2

[1040] PyBOP (278 mg, 0.53 mmol) and DIPEA (179 μL, 1.02 mmol) were added 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) under a N2 environment at room temperature. After stirring the mixture for 2 hours, the reaction mixture was transferred to a preparative HPLC to obtain compound Int-TG2 (384.6 mg, 81%). EI-MS m / z: 1152 (M + +1).

[1041] Example 39: Preparation of compounds Int-TG3 and Int-TG4

[1042]

[1043] Preparation of compound Int-TG3-1

[1044] A homogeneous solution of compounds Int-TG1-5 (1.0 g, 0.26 mmol) and L-8 (586 mg, 2.0 mmol, 1.2 equivalents) in DMF (10 mL) was treated with PyBOP (1.13 g, 2.17 mmol, 1.3 equivalents) and DIPEA (873 μL, 5.01 mmol, 3.0 equivalents) at room temperature under an N2 environment and stirred for 4 hours. 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 obtain compounds Int-TG3-1 (1.05 g, 72%) as a white foamed solid.

[1045] EI-MS m / z: 874 (M + +1).

[1046] Preparation of compound Int-TG3a

[1047] Et3N (2.28 mL, 16.38 mmol) was added to a solution of 4-hydroxybenzaldehyde (1 g, 8.19 mmol) in DCM (3 mL) under an N2 environment at room temperature. SO2F2 gas was introduced through a balloon, and the mixture was stirred at room temperature for 2 hours. Subsequently, the mixture was washed with DCM (30 mL x 3) and brine (30 mL), the organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain the compound Int-TG3a (790 mg, 63%).

[1048] 1 ¹H 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).

[1049] Preparation of compound Int-TG3-2

[1050] A homogeneous solution of compound Int-TG3-1 (500 mg, 0.57 mmol) and compound Int-TG3a (140 mg, 0.69 mmol, 1.2 equivalents) in anhydrous ACN (10 mL) was treated with BEMP (66.3 μL, 0.23 mmol, 0.4 equivalents) under an N2 environment at room temperature and stirred for 4 hours. 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 (4% MeOH in DCM) to obtain compound Int-TG3-2 (495 mg, 85%) as a white foamed solid.

[1051] EI-MS m / z: 869 (M + +1).

[1052] Preparation of compound Int-TG3-3

[1053] NaBH4 (39.7 mg, 1.05 mmol, 2.0 equivalents) was added to a solution of compound Int-TG3-2 (495 mg, 0.52 mmol) in anhydrous THF (5.0 mL) under an N2 environment at 0°C, and the resulting mixture was stirred for 2 hours. 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 (2% to 3% MeOH in DCM) to obtain compound Int-TG3-3 (418 mg, 91%) as a white foamed solid.

[1054] EI-MS m / z: 945 (M + +1).

[1055] Preparation of compound Int-TG3-4

[1056] To a solution of compound Int-TG3-3 (214.2 mg, 0.23 mmol) in anhydrous THF (5.0 mL), methanesulfonyl chloride (24.6 μL, 0.32 mmol, 1.4 equivalents) and TEA (79.2 μL, 0.57 mmol, 1.5 equivalents) were added at 0°C under an N2 environment, and the resulting mixture was stirred overnight at room temperature. The reaction was quenched 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 (100% DCM to 5% MeOH in DCM) to obtain compound Int-TG3-4 (164 mg, 70%) as a white foamed solid.

[1057] EI-MS m / z: 1024 (M + +1).

[1058] Preparation of compound Int-TG3

[1059] LiBr (69.6 mg, 0.80 mmol, 5.0 equivalents) was added to a solution of compound Int-TG3-4 (164 mg, 0.16 mmol) in anhydrous THF (10 mL) under an N2 environment at room temperature, and the resulting mixture was stirred for 3 hours. 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% to 5% MeOH in DCM) to obtain compound Int-TG3 (161 mg, 99%) as a white foamed solid.

[1060] EI-MS m / z: 1008 (M + +1).

[1061] Compound Int-TG4 was synthesized in a manner similar to the method used to prepare compound Int-TG3.

[1062] Preparation of compound Int-TG4-1

[1063] Yield 72%, colorless oil

[1064] EI-MS m / z: 1226 (M + +1).

[1065] Preparation of compound Int-TG4-2

[1066] Yield 82%, colorless oil

[1067] EI-MS m / z: 1296 (M + +1).

[1068] Preparation of compound Int-TG4-3

[1069] Yield 75%, colorless oil

[1070] EI-MS m / z: 1298 (M + +1).

[1071] Preparation of compound Int-TG4-4

[1072] Yield 82%, colorless oil

[1073] EI-MS m / z: 1376 (M+ +1).

[1074] Preparation of compound Int-TG4

[1075] Yield 82%, colorless oil

[1076] EI-MS m / z: 1361 (M + +1).

[1077] Example 40: Preparation of compound Int-TG5

[1078]

[1079] Preparation of compound Int-TG5-1

[1080] DBU (4 μL, 25 μmol) was added 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). The mixture was stirred at room temperature for 1 hour and washed with distilled water (10 mL) and EA (15 mL x 2). The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-TG5-1 (103 mg, 94%).

[1081] EI-MS m / z: 869 (M + +1).

[1082] Preparation of compound Int-TG5-2

[1083] NaBH4 (9 mg, 0.24 mmol) was added to a solution of compound Int-TG5-1 (103 mg, 0.12 mmol) in THF (8 mL) under an N2 environment at 0°C. After stirring for 2 hours at room temperature, distilled water (10 mL) and EA (10 mL x 2) were added. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain compound Int-TG5-2 (101 mg, 98%).

[1084] EI-MS m / z: 871 (M + +1).

[1085] Preparation of compound Int-TG5-3

[1086] 1 M PBr3 (165 μL, 0.19 mmol) in DCM was added to a solution of compound Int-TG5-2 (320.5 mg, 0.0.37 mmol) in DCM (3 mL) under an N2 environment at 0°C. After stirring for 2 hours at 0°C, the mixture was quenched by adding saturated sodium bicarbonate (8 mL x 2). The mixture was separated, the organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to produce compound Int-TG5-3 (202.6 mg, 59%).

[1087] EI-MS m / z: 934 (M + +1).

[1088] Preparation of compound Int-TG5

[1089] Dimethylamine (0.1 mL) was added to a solution of compound Int-TG5-3 (10 mg, 0.01 mmol) in DMF (1 mL) under an N2 environment at room temperature. After stirring at room temperature for 10 minutes, the reaction mixture was purified by preparative HPLC to obtain compound Int-TG5 (6 mg, 63%). EI-MS m / z: 898 (M + +1).

[1090] Example 41: Preparation of Compound MPS-D1

[1091]

[1092] Preparation of Compound MPS-D1-1

[1093] Piperidine hydrochloride (6.66 g, 54.82 mmol), paraformaldehyde (4.95 g, 164.5 mmol), and conc. HCl (0.6 mL) were added to a solution of 4-acetylbenzoic acid (9 g, 54.82 mmol) in EtOH (50 mL) under an N2 environment at room temperature. The mixture was stirred at 100°C for 16 hours, then cooled to room temperature, and acetone (90 mL) was added dropwise. The mixture was stirred at 0°C for 1 hour. The solid was filtered and washed with diethyl ether (30 mL x 2) to obtain compound MPS-D1-1 (6.11 g, 38%).

[1094] 1 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).

[1095] Preparation of Compound MPS-D1-2

[1096] 4-methoxybenzenethiol (2.55 g, 20.52 mmol) and piperidine (0.3 mL, 3.08 mmol) were added at room temperature to a solution of MPS-D1-1 (6.11 g, 20.52 mmol) in EtOH (40 mL) and MeOH (26 mL). The mixture was stirred at 100°C for 16 hours, cooled to 0°C, and stirred for an additional 1 hour. The solid was filtered and washed with ether (30 mL x 2) to obtain compound MPS-D1-2 (5.56 g, 90%).

[1097] 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).

[1098] Preparation of Compound MPS-D1

[1099] Oxone (25.03 g, 40.72 mmol) was added to a solution of MPS-D1b (5.56 g, 18.51 mmol) in MeOH (90 mL) and distilled water (90 mL) under an N2 environment at 0°C. After stirring for 14 hours at room temperature, the mixture was quenched with distilled water (100 mL) and chloroform (150 mL x 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%).

[1100] 1 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).

[1101] Example 42: Preparation of compound mMPS-D1

[1102]

[1103] Compound mMPS-D1 was synthesized in a manner similar to that described in Example 41.

[1104] Preparation of compound mMPS-D1-1

[1105] 21% yield white solid.

[1106] EI-MS m / z: 262 (M + +1).

[1107] Preparation of compound mMPS-D1-2

[1108] Yield 72%, white solid.

[1109] 1 ¹H 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).

[1110] EI-MS m / z: 301 (M + +1).

[1111] Preparation of compound mMPS-D1

[1112] Yield 47%, yellowish solid.

[1113] 1 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).

[1114] EI-MS m / z: 333 (M + +1).

[1115] Example 43: Preparation of compound pyrMPS-D1

[1116]

[1117] compound pyr Manufacture of MPS-D4-1

[1118] 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 under reflux at room temperature under an N2 environment for 3 hours. 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) under an N2 environment at room temperature and left to stand for 2 hours. 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 obtain the compound pyr MPS-D1-1 (2.22 g, 89%) was obtained as a white solid.

[1119] 1 ¹H 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).

[1120] compound pyr Manufacture of MPS-D1-2

[1121] in MeOH (11 mL) pyrA homogeneous solution of MPS-D1-1 (636 mg, 3.55 mmol) was treated with 1N NaOH (10.64 mL) under an N2 environment at room temperature and stirred for 1 hour. After concentrating the mixture under reduced pressure, 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 obtain the compound pyr MPS-D1-2 (unpurified) was obtained as a white solid.

[1122] 1 H NMR (400 MHz, DMSO-d6) δ 9.31 (s, 1H), 9.25 (s, 1H), 8.64 (s, 1H), 2.69 (s, 3H).

[1123] compound pyr MPS-D1-3, pyr MPS-D1-4, and pyr MPS-D1 was synthesized in a manner similar to the method of preparing the compound MPS-D1 of Example 41.

[1124] compound pyr Manufacture of MPS-D1-3

[1125] Yield 30%, white solid.

[1126] EI-MS m / z: 264 (M + +1).

[1127] compound pyr Manufacture of MPS-D1-4

[1128] Yield 11%, white solid.

[1129] 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).

[1130] compound pyr Manufacture of MPS-D1

[1131] Yield 43%, white solid.

[1132] EI-MS m / z: 334 (M + +1).

[1133] Example 44: Preparation of compounds MPS-D2, mMPS-D2, and PyrMPS-D2

[1134]

[1135] Preparation of Compound MPS-D2

[1136] 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 an N2 environment. HBTU (893.3 mg, 2.36 mmol) and DIPEA (0.684 mL, 3.93 mmol) were added thereto, and the mixture was stirred at room temperature for 3 hours. After the reaction was complete, 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 obtain Compound MPS-D2 (854.1 mg, 80%).

[1137] 1 ¹H 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).

[1138] Compound mMPS-D2 and pyr MPS-D2 was synthesized in a manner similar to the synthesis of compound MPS-D2.

[1139] Preparation of compound mMPS-D2

[1140] Yield 60%, white solid.

[1141] EI-MS m / z: 546 (M + +1).

[1142] compound pyr Manufacture of MPS-D2

[1143] Yield 22%, yellowish oil.

[1144] EI-MS m / z: 547 (M + +1).

[1145] Example 45: Preparation of Compound MPS-D3

[1146]

[1147] Compounds MPS-D3 and mMPS-D3 were synthesized via a synthesis route similar to that described in Example 44.

[1148] Preparation of Compound MPS-D3

[1149] 72% yield

[1150] EI-MS m / z: 899 (M + +1).

[1151] Preparation of compound mMPS-D3

[1152] Yield 32%, white solid.

[1153] EI-MS m / z: 899 (M + +1).

[1154] Example 46: Preparation of Compound Mal-1

[1155]

[1156] 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 eq) in dry DMC was treated with DIPEA (44.5 μL, 0.26 mmol) under an N2 environment at room temperature and stirred for 45 minutes at room temperature. The reaction mixture was diluted with DCM (32 mL), washed with 1N HCl (30 mL) and brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC to obtain the title compound Mal-1 (70.8 mg, 61%) as a white gum.

[1157] EI-MS m / z: 451 (M + +1).

[1158] Example 47: Preparation of compound BCN-PNP

[1159]

[1160] (1R,8S,9s)-bicyclo[6.1.0]non-4-phosphorus-9-yl methanol (800 mg, 5.3 mmol) was dissolved in DCM (125 mL) at room temperature under an N2 environment. Pyridine (1.22 mL, 15.9 mmol) and 4-nitrophenyl chloroformate (1.75 g, 8.74 mmol) were added thereto. After stirring the mixture at the same temperature for 4 hours, the reaction was quenched by the addition of a saturated NH4Cl solution (100 mL) and extracted with EA (100 mL x 4). The organic layer was dried over Na2SO4, filtered, and concentrated under vacuum. The residue was purified by column chromatography (Hex:EA = 10:1) to obtain the compound BCN-PNP (1.34 g, 84%) as a white solid.

[1161] 1 ¹H 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).

[1162] Example 48: Preparation of compound T-Int-1

[1163]

[1164] Preparation of compound T-Int-1-1

[1165] K2CO3 (9.3 mg, 0.07 mmol) was added 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) under an N2 environment at 30°C. After stirring for 3 hours, the reaction mixture was purified by prep-PLC to obtain compound T-Int-1-1 (5.4 mg, 29%). EI-MS m / z: 825 (M + +1).

[1166] Preparation of compound T-Int-1-2

[1167] BEMP (1 μL, 0.004 mmol) was added 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) under an N2 environment at room temperature. After stirring at room temperature for 5 hours, the reaction mixture was purified by HPLC to obtain compound T-Int-1-2 (67 mg, 83%). EI-MS m / z: 1490 (M + +1).

[1168] Preparation of compound T-Int-1

[1169] K2CO3 (5.6 mg, 0.04 mmol) was added to a solution of compound T-Int-1-2 (8.1 mg, 0.01 mmol) in MeOH (1 mL) and MC (0.1 mL) under an N2 environment. After stirring at 0°C for 1 hour, the reaction mixture was purified by HPLC to obtain compound T-Int-1 (5.5 mg, 76%). EI-MS m / z: 1322 (M + +1).

[1170] Example 49: Preparation of compound T-Int-2

[1171]

[1172] Preparation of compound T-Int-2-1

[1173] DIPEA (12 μL, 0.07 mmol) was added 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) under an N2 environment at 40°C. After stirring at room temperature for 5 hours, the reaction mixture was purified by preparative HPLC to obtain compound T-Int-2-1 (28.9 mg, 61%).

[1174] EI-MS m / z: 1641 (M + +1).

[1175] Preparation of compound T-Int-2

[1176] K2CO3 (12.2 mg, 0.09 mmol) was added to a solution of compound T-Int-2-1 (28.9 mg, 0.02 mmol) in MeOH (2 mL) under an N2 environment. After stirring for 1 hour at 0°C under an N2 environment, the reaction mixture was purified by preparative HPLC to obtain compound T-Int-2 (18.4 mg, 71%).

[1177] EI-MS m / z: 1473 (M + +1).

[1178] Example 50: Preparation of compound T-Int-3

[1179]

[1180] Preparation of compound T-Int-3-1

[1181] DIPEA (3 μL, 0.018 mmol) was added 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) under an N2 environment at room temperature. After stirring at room temperature for 5 hours, the reaction mixture was purified by preparative HPLC to obtain compound T-Int-3-1 (3.1 mg, 52%).

[1182] EI-MS m / z: 1637 (M + +1).

[1183] Preparation of compound T-Int-3

[1184] K2CO3 (1.2 mg, 0.009 mmol) was added to a solution of compound T-Int-3-1 (3.1 mg, 0.002 mmol) in MeOH (1 mL) under an N2 environment. After stirring for 1 hour at 0°C under an N2 environment, the reaction mixture was purified by preparative HPLC to obtain compound T-Int-3 (2.0 mg, 72%).

[1185] EI-MS m / z: 1469 (M + +1).

[1186] Example 51: Preparation of compound T-Int-4

[1187]

[1188] Compound T-Int-4 was synthesized in a manner similar to that described in Example 49.

[1189] Preparation of compound T-Int-4-1

[1190] 50% yield

[1191] EI-MS m / z: 1609 (M + +1).

[1192] Preparation of compound T-Int-4

[1193] 55% yield

[1194] EI-MS m / z: 1441 (M + +1).

[1195] Example 52: Preparation of compound T-Int-5

[1196]

[1197] Compound T-Int-5 was synthesized in a manner similar to that described in Example 48.

[1198] Preparation of compound T-Int-5-1

[1199] 61% yield

[1200] EI-MS m / z: 1842 (M + +1).

[1201] Preparation of compound T-Int-5

[1202] 62% yield

[1203] EI-MS m / z: 1674 (M + +1).

[1204] Example 53: Preparation of Compounds T-Int-6 and T-Int-7

[1205]

[1206] Compound T-Int-6-2 was synthesized in a manner similar to the method of preparing compound T-Int-2 of Example 49.

[1207] Preparation of compound T-Int-6-1

[1208] Yield: 61% Ivory solid.

[1209] EI-MS m / z: 1716(M + +1), 858(M + / 2+1).

[1210] Preparation of compound T-Int-6-2

[1211] Yield 67%: Ivory solid.

[1212] EI-MS m / z: 1548(M+ +1).

[1213] Preparation of compound T-Int-6-3

[1214] 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) under an N2 environment at 0°C and stirred for 2 hours. The reaction mixture was purified by preparative HPLC (0.1% formic acid in water / 0.1% formic acid in ACN) to obtain compound T-Int-6-3 (72.5 mg, 84%) as a yellow solid.

[1215] EI-MS m / z: 1448(M + +1).

[1216] Preparation of compound T-Int-6

[1217] A homogeneous solution of compounds 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 an N2 environment and stirred for 2 hours. The reaction mixture was purified by preparative HPLC (0.1% formic acid in water / 0.1% formic acid in ACN) to obtain compound T-Int-6 (12.9 mg, 62%) as a bright yellow solid.

[1218] EI-MS m / z: 1624(M + +1), 812(M + / 2+1).

[1219] Compound T-Int-7 was synthesized in a manner similar to the method used to prepare compound T-Int-6-3.

[1220] Preparation of compound T-Int-7-1

[1221] Yield 73%, ivory solid

[1222] EI-MS m / z: 2069 (M + +1), 1035(M + / 2+1).

[1223] Preparation of compound T-Int-7-2

[1224] Yield 72%, ivory solid

[1225] EI-MS m / z: 1901 (M + +1), 951(M + / 2+1).

[1226] Preparation of compound T-Int-7

[1227] Yield 65%, yellow solid

[1228] EI-MS m / z: 1801 (M + +1), 901(M + / 2+1)

[1229] Example 54: Preparation of Compound T-Int-8

[1230]

[1231] Preparation of compound T-Int-8-1

[1232] 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 an N2 environment and stirred for 2 hours. The reaction mixture was purified by preparative HPLC (0.1% formic acid in water / 0.1% formic acid in ACN) to obtain compound T-Int-8-1 (19.9 mg, 85%) as a yellow solid.

[1233] EI-MS m / z: 1699(M + +1), 850(M + / 2+1).

[1234] Preparation of compound T-Int-8-2

[1235] 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) under an N2 environment at 0°C and stirred for 4 hours. The reaction mixture was purified by preparative HPLC (0.1% formic acid in water / 0.1% formic acid in ACN) to obtain compound T-Int-8-2 (13.2 mg, 71%) as an ivory solid.

[1236] EI-MS m / z: 1599(M + +1), 800(M + / 2+1).

[1237] Preparation of compound T-Int-8

[1238] A clear solution of compounds 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) under an N2 environment at room temperature and stirred for 28 hours. The reaction mixture was purified by preparative HPLC (0.1% formic acid in water / 0.1% formic acid in ACN) to obtain compound T-Int-8 (3.6 mg, 35%) as a bright yellow solid.

[1239] EI-MS m / z: 1776(M + +1), 888(M + / 2+1).

[1240] Example 55: Preparation of compound T-Int-9

[1241]

[1242] Compound T-Int-9 was synthesized in a manner similar to the method of preparing compound T-Int-8 of Example 54.

[1243] Preparation of compound T-Int-9-1

[1244] Yield 56%, ivory solid.

[1245] EI-MS m / z: 2052 (M + +1), 1026(M + / 2+1)

[1246] Preparation of compound T-Int-9-2

[1247] Yield 71%, ivory solid.

[1248] EI-MS m / z: 1952 (M + +1), 976(M + / 2+1)

[1249] Preparation of compound T-Int-9

[1250] Yield 31%, white solid.

[1251] EI-MS m / z: 2128(M + +1), 1064(M + / 2+1)

[1252] Example 56: Preparation of compound T-Int-10

[1253]

[1254] Preparation of compound T-Int-10-1

[1255] A yellow solution of compounds 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) under an N2 environment at room temperature and stirred for 7 hours. 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% to 3%) to obtain compound T-Int-10-1 (136.7 mg, 71%) as a yellow solid.

[1256] EI-MS m / z: 590(M + +1).

[1257] Preparation of compound T-Int-10-2

[1258] A clear solution of compounds 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) under an N2 environment at room temperature and stirred for 7 hours. The reaction mixture was purified by preparative HPLC (0.1% formic acid in water / 0.1% formic acid in ACN) to obtain compound T-Int-10-2 (11.1 mg, 32%) as a white solid.

[1259] EI-MS m / z: 821(M + +1).

[1260] Preparation of compound T-Int-10-3

[1261] 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) under an N2 environment at room temperature and stirred for 6 hours. The reaction mixture was purified by preparative HPLC (0.1% formic acid in water / 0.1% formic acid in ACN) to obtain compound T-Int-10-3 (15 mg, 46%) as an ivory solid.

[1262] EI-MS m / z: 1486(M + +1).

[1263] Preparation of compound T-Int-10

[1264] A turbid solution of compound T-Int-10-3 (13.2 mg, 0.009 mmol) in MeOH (2 mL) was treated with K2CO3 (8.6 mg, 0.063 mmol) under an N2 environment at 0°C and stirred for 30 minutes. The reaction mixture was purified by preparative HPLC (0.1% formic acid in water / 0.1% formic acid in ACN) to obtain compound T-Int-10 (7.4 mg, 63%) as a white solid.

[1265] EI-MS m / z: 1318(M + +1).

[1266] Example 57: Preparation of compound T-Int-11

[1267]

[1268] Compound T-Int-11 was synthesized in a manner similar to the method of preparing compound T-Int-10 of Example 56.

[1269] Preparation of compound T-Int-11-1

[1270] Yield 52%, white solid.

[1271] EI-MS m / z: 576 (M + +1).

[1272] Preparation of compound T-Int-11-2

[1273] Yield 29%, yellow solid.

[1274] EI-MS m / z: 807 (M + +1).

[1275] Preparation of compound T-Int-11-3

[1276] Yield 66%, yellow solid.

[1277] EI-MS m / z: 1472 (M + +1).

[1278] Preparation of compound T-Int-11

[1279] Yield 57%, yellow solid.

[1280] EI-MS m / z: 1304(M + +1).

[1281] Example 58: Preparation of compound T-Int-12

[1282]

[1283] Compound T-Int-12 was synthesized in a manner similar to the method of preparing compound T-Int-1 of Example 48.

[1284] Preparation of compound T-Int-12-1

[1285] Yield 66%, ivory solid.

[1286] EI-MS m / z: 829 (M + +1).

[1287] Preparation of compound T-Int-12-2

[1288] Yield 64%, yellow solid.

[1289] EI-MS m / z: 1494 (M + +1).

[1290] Preparation of compound T-Int-12

[1291] Yield 71%, yellow solid.

[1292] EI-MS m / z: 1326 (M + +1).

[1293] Example 59: Preparation of Compound T-1

[1294]

[1295] 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 minutes. Then, compound MPS-D2 (3.7 mg, 0.007 mmol) in DMSO (674 μL) was added, and the mixture was stirred for 10 minutes. After the reaction was complete, the mixed solution was separated and purified by preparative HPLC to obtain compound T-1 (1.0 mg, 32%).

[1296] EI-MS m / z: 1868(M + +1).

[1297] Example 60: Preparation of Compound T-2

[1298]

[1299] 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 minutes. Compound MPS-D3 (5.7 mg, 0.006 mmol) in DMSO (673 μL) was added thereto, and the mixture was stirred for 10 minutes. After the reaction was complete, the mixed solution was separated and purified by preparative HPLC to obtain compound T-2 (1.3 mg, 34%).

[1300] EI-MS m / z: 2371(M + +1).

[1301] Example 61: Preparation of Compound T-3

[1302]

[1303] Compound T-3 was synthesized in a manner similar to the method of preparing compound T-2 of Example 60.

[1304] Yield 34%.

[1305] EI-MS m / z: 2371(M + +1).

[1306] Example 62: Preparation of Compound T-4

[1307]

[1308] Compound T-4 was synthesized in a manner similar to that described in Example 60.

[1309] Yield 24%

[1310] EI-MS m / z: 2339 (M + +1).

[1311] Example 63: Preparation of Compound T-5

[1312]

[1313] Compound T-5 was synthesized in a manner similar to that described in Example 60.

[1314] 15% yield white solid.

[1315] EI-MS m / z: 1869(M + +1).

[1316] Example 64: Preparation of Compound T-6

[1317]

[1318] Compound T-6 was synthesized in a manner similar to the method of preparing compound T-2 of Example 60.

[1319] Yield 79%, white solid.

[1320] EI-MS m / z: 2372(M + +1), 1186(M + / 2+1).

[1321] Example 65: Preparation of Compound T-7

[1322]

[1323] Compound T-7 was synthesized in a manner similar to the method of preparing compound T-1 of Example 59.

[1324] Yield 66%, white solid

[1325] EI-MS m / z: 2224(M + +1), 1112(M + / 2+1).

[1326] Example 66: Preparation of Compound T-8

[1327]

[1328] Compound T-8 was synthesized in a manner similar to the method of preparing compound T-1 of Example 59.

[1329] Yield 65%, white solid

[1330] EI-MS m / z: 2224(M + +1), 1112(M + / 2+1).

[1331] Example 67: Preparation of Compound T-9

[1332]

[1333] A clear solution of compound T-2 (1.3 mg, 0.0005 mmol), H2O (25 μL), and sodium bisulfite (65.7%, 0.16 mg, 0.0010 mmol) in IPA (50 μL) was stirred at room temperature under an N2 environment for 6 hours. The mixture was freeze-dried to obtain compound T-9 (1.3 mg, 92%) as a yellow solid.

[1334] EI-MS m / z: 2580(M + +1)

[1335] Example 68: Preparation of Compound T-10

[1336]

[1337] Compound T-10 was synthesized in a manner similar to the method of preparing compound T-1 of Example 59.

[1338] Yield 59%, white solid

[1339] EI-MS m / z: 1864(M + +1), 932(M + / 2+1).

[1340] Example 69: Preparation of Compounds T-11 and T-12

[1341]

[1342] Compounds T-11 and T-12 were synthesized in a manner similar to the method of preparing compound T-1 of Example 59.

[1343] Preparation of Compound T-11

[1344] Yield 32%, white solid

[1345] EI-MS m / z: 1864(M + +1), 932(M + / 2+1).

[1346] Preparation of Compound T-12

[1347] Yield 50%, white solid

[1348] EI-MS m / z: 2216(M + +1), 1108(M + / 2+1).

[1349] Example 70: Preparation of Compound T-13

[1350]

[1351] Compound T-13 was synthesized in a manner similar to the method of preparing compound T-1 of Example 59.

[1352] Yield 75%, white solid

[1353] EI-MS m / z: 1849(M + +1), 925(M + / 2+1).

[1354] Example 71: Preparation of Compound T-14

[1355]

[1356] Compound T-14 was synthesized in a manner similar to the method of preparing compound T-3 of Example 61.

[1357] Yield 65%, white solid

[1358] EI-MS m / z: 1925(M + +1), 963(M + / 2+1).

[1359] Example 72: Preparation of compound T-Int-13

[1360]

[1361] Compound T-Int-13 was synthesized in a manner similar to the method of preparing compound T-Int-6 of Example 53.

[1362] Preparation of compound T-Int-13-1

[1363] Yield 70%, white solid

[1364] EI-MS m / z: 1704 (M + +1).

[1365] Preparation of compound T-Int-13-2

[1366] Yield 81%, white solid

[1367] EI-MS m / z: 1536 (M + +1).

[1368] Preparation of compound T-Int-13-3

[1369] Yield 81%, ivory solid

[1370] EI-MS m / z: 1436 (M + +1).

[1371] Preparation of compound T-Int-13

[1372] Yield 81%, beige solid

[1373] EI-MS m / z: 1612 (M + +1).

[1374] Example 73: Preparation of Compound T-16

[1375]

[1376] Compound T-16-2 was synthesized in a manner similar to the method of preparing compound T-Int-2 of Example 49.

[1377] Preparation of Compound T-16-1

[1378] Yield 71%.

[1379] EI-MS m / z: 1630 (M +1 ).

[1380] Preparation of Compound T-16-2

[1381] Yield 60%.

[1382] EI-MS m / z: 1462 (M +1 ).

[1383] Compound T-Int-16 was synthesized in a manner similar to the method of preparing compound T-1 of Example 59.

[1384] Preparation of Compound T-16

[1385] 69% yield pale yellow solid.

[1386] EI-MS m / z: 2360 (M+ +1), 1180 (M / 2 + +1).

[1387] Example 74: Preparation of Compound L-10

[1388]

[1389] Compound L-10 was synthesized in a manner similar to the method of preparing compound L-5 of Example 33.

[1390] Preparation of Compound L-10-1

[1391] 96% yield colorless oil.

[1392] 1 ¹H 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).

[1393] Preparation of Compound L-10

[1394] 97% yield colorless oil.

[1395] EI-MS m / z: 439 (M + +1).

[1396] Example 75: Preparation of Compound MPS-D4

[1397]

[1398] Compound MPS-D4 was synthesized in a manner similar to that described in Example 44.

[1399] Preparation of Compound MPS-D4

[1400] Yield 53%, yellow oil.

[1401] EI-MS m / z: 753 (M + +1).

[1402] biological test

[1403] Example 76: In vitro analysis of benzodiazepine dimer derivatives

[1404] 25 x 10⁶ NCI-N87 cancer cells in a 24-well plate 3 Cells were seeded at a rate of 1 mL medium per well. Plates were incubated for 24 hours at 37°C in a humidified 5% air-in-CO2 environment. The benzodiazepine dimer derivative was initially dissolved in DMSO at a concentration of 10 mM. A series of dilutions were prepared in DMSO. The series of compound dilutions in DMSO was added to the triple wells of a 24-well plate at a rate of 5 μL per well. Three wells on each individual plate contained 5 μL of DMSO without the compound as a control. The final concentration of DMSO per well was 0.5%. Plates were incubated for 6 days at 37°C in a humidified 5% air-in-CO2 environment. Cell viability was determined by the MTT assay, and IC50 was calculated using a S-shaped dose-response nonlinear regression curve fit (GraphPad Software Inc.). 50 It was generated. Its results are shown in Figs. 1, 2, 3, and Table 2.

[1405] Table 2. In vitro biological analysis of benzodiazepine dimer derivatives

[1406]

[1407] Example 77: Manufacturing of conjugates

[1408] Reduction / Oxidation of Antibodies for Conjugation: Cysteine-engineered monoclonal antibodies were reduced for 1 hour at 37°C with approximately 20–50 times excess TCEP (Tris(2-carboxyethyl)phosphine hydrochloride) or DTT (Dithiothreitol) in 4 mM Tris pH 7.3 containing 1 mM EDTA. 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 checked by determining the reduced antibody concentration from the absorbance of the solution at 280 nm, the thiol concentration determined by reaction with DTNB (Aldrich, CAS No. D8130), and the absorbance at 412 nm.

[1409] Conjugation method After the reduction and reoxidation reactions, the antibody was dissolved in PBS. A solution of compound T-1 (5.62 μL, 3.0 mmol, as a linker-toxin intermediate) obtained in Example 59 in DMSO was treated with the reduced, 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 solution of the reaction mixture and incubated at 37°C for 8 hours to block the reversible unconjugation 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 centrifugation and ultrafiltration, the conjugate was purified using an HIC NPR column (TOSOH #0007656 TSKgel phenyl-5PW, 21.5 x 150 mm, 13 μm), and eluted at 0.8 ml / min with a linear gradient of 40 to 100% B (A buffer: 1.5 M ammonium sulfate in 50 mM sodium phosphate (pH 7.0); B buffer: 20% acetonitrile in 50 mM sodium phosphate (pH 7.0)).

[1410] Using compounds T-1, T-2, T-3, and T-4 obtained in Examples 59, 60, 61, and 62, T-1-AB, T-2-AB, T-3-AB, and T-4-AB were prepared as thiomab drug conjugates (TDCs), respectively, by performing a conjugation reaction for the thiol group (anti-HER2) of engineered cysteine ​​of trastuzumab with reference to methods presented in the literature. [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 antibody was analyzed by HIC, and the results of the analysis are shown in Table 3.

[1411] Table 3. Antibody-drug conjugates (ADCs)

[1412]

[1413] Example 78: In vitro analysis of protein-drug conjugates

[1414] NCI-N87 cancer cells were seeded into 96-well plates in 100 μL of medium at a cell density of 5,000 cells per well and cultured for 24 hours. The four compound ADCs from Example 77 were treated with a series of 1:4 dilutions ranging from 50 nM to 0.0003 nM, and the antibody-drug conjugate T-DM1 was treated with a series of 1:4 dilutions ranging from 50 nM to 0.0007 nM. A series of compound dilutions in DMSO was added to triple wells of a 24-well plate at a rate of 5 μL per well. Three wells on each individual plate contained 5 μL of DMSO without the compound as a control. The final concentration of DMSO per well was 0.5%. The plates were incubated for 6 days at 37°C in a humidified 5% air-in-CO2 environment. Cell viability was determined by the MTT assay, and IC was calculated using S-shaped dose-response nonlinear regression curve fitting (GraphPad Software Inc.). 50 generated.

[1415] The results of 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.

[1416] Table 4. Cytotoxicity of antibody-drug conjugates

[1417]

[1418] ( * T-DM1: Roche CAS No.; 1018448-65-1)

[1419] Example 79: Solubility test

[1420] A solubility study was conducted, and the relative solubility of D-102, D-103, D-104, D-105, D-106, and D-107 at different retention times was checked using analytical HPLC. The retention times of these compounds were analyzed by HPLC, and the results are shown in Table 5.

[1421] Table 5. HPLC retention time under acidic conditions (0.1% TFA) *

[1422]

[1423] ( * Retention times were determined on a Waters Alliance LC / MS system using a Thermo Scientific RP-C18 analytical HPLC column (Hypersil Gold, 50 x 4.6 mm) and the following analytical HPLC method: injection volume 5 μL; flow rate 1 mL / min; 5 → 95% ACN in water with 0.1% TFA over 15 minutes; Waters diode array detector at γ = 214 nm; room temperature.)

[1424] Inclusion of references

[1425] All published documents and patents mentioned herein are incorporated herein by reference in their entirety, as specifically and individually indicated that each individual published document or patent is incorporated by reference. In the event of a conflict, this application shall prevail, including any definitions herein.

[1426] Equivalents

[1427] Although specific embodiments of the invention have been discussed, the specification is illustrative and not limiting. Many variations of the invention will become apparent to those skilled in the art upon review of the specification and the following claims. The full scope of the invention should be determined by reference to the claims, together with the full scope of equivalents of the claims, and by variations of the specification.

Claims

Claim 1 Compound having the structure of the following chemical formula (I) or its pharmaceutically acceptable salt: Here: ring A is a 5-membered heteroaryl ring; ring B is a heterocyclic ring optionally fused to one or more heteroaryl rings; R 1 and R 2 are each independently alkyl groups; Z 1 methylene, , or and, here:Y 1 is CR Y1 or N and;R Y1 is H or alkyl and R Z1 is absent, or hydroxyl, amino, amido, or (CH2) z R Z1a Igo;R Z1a is an amino, aryl, or heteroaryl; and z is an integer having a value from 1 to 10 and Y 2 is CR Y2 or N and;R Y2 is H or alkyl and R Z2 is (CH2) z' R Z2a Igo;R Z2a is an amino, aryl, or heteroaryl; and z' is an integer having a value from 0 to 10; and n a is 1 and;n 1 and n 2 Each is an integer that independently has a value from 1 to 5. Claim 2 In paragraph 1, ring A and, here: A 1 , A 2 , and A 3 NR each independently a1 , CR a2 , O, N, or S and;R a1 are each independently H, alkyl, halogen, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and R a2 Each is independently a compound or its pharmaceutically acceptable salt that is H or an alkyl. Claim 3 In paragraph 1, ring A , , , , , and Selected from, where, R a1 A compound or its pharmaceutically acceptable salt, each of which is independently H, alkyl, halogen, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl. Claim 4 In paragraph 2, R a1 Each is a compound or its pharmaceutically acceptable salt that is independently a C1-C4-alkyl. Claim 5 In any one of claims 1 to 4, ring B is a 5- or 6-membered heterocyclic ring, and one or more R selected from OH, alkyl, alkylidenyl, halogen, amino, cyano, aryl, or heteroaryl. C A compound or its pharmaceutically acceptable salt, selectively substituted with a group. Claim 6 In paragraph 5, ring B or and, the dotted line indicates that the double bond is optionally present; and each R C A compound or its pharmaceutically acceptable salt independently selected from H, OH, alkyl, alkylidenyl, halogen, amino, cyano, or aryl. Claim 7 In paragraph 6, ring B And, ring C' is independently R 1a Optionally substituted with 1 to 4 substituents selected from; and each R 1a A compound or its pharmaceutically acceptable salt, independently selected from H, OH, SH, alkylthiol, -CH2N(CH3)2, alkyloxyalkylidenyl, halogen, amino, cyano, aryl, or alkyl. Claim 8 A compound or a pharmaceutically acceptable salt thereof, wherein, in any one of claims 1 to 4, ring B is a 5- or 6-membered heterocyclic ring and fused to a heteroaryl ring C'. Claim 9 In paragraph 8, ring C' is independently R 1c A 5- or 6-membered heteroaryl optionally substituted with 1 to 4 substituents selected from; and each R 1c A compound or its pharmaceutically acceptable salt, independently selected from OH, SH, alkylthiol, -CH2N(CH3)2, alkyloxy, alkyl, halogen, amino, or cyano. Claim 10 In paragraph 8, ring C' Igo;B 1 , B 2 , and B 3 NR each independently b1 , CR b2 , O, N, or S and;R b1 Each of these is independently H, alkyl, halogen, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; and R b2 A compound or its pharmaceutically acceptable salt, which is independently absent, H, or an alkyl. Claim 11 In Clause 10, ring C' , , and A compound or its pharmaceutically acceptable salt selected from. Claim 12 In Paragraph 10, R b1 A compound or its pharmaceutically acceptable salt, each of which is independently a C1-C4-alkyl. Claim 13 In paragraph 8, ring C' and, here:B 6 , B 7 , B 8 , and B 9 CR each independently b3 or N and; and R b3 Each is independently H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that B 6 , B 7 , B 8 , and B 9 A compound or its pharmaceutically acceptable salt, in which one or two of them are N. Claim 14 In claim 1, a compound having the structure of the chemical formula (Ia), (Ib-1), (Ib-2), or (Ib-3), or a pharmaceutically acceptable salt thereof: At this time, A 1 , A 2 , and A 3 NR each independently a1 , CR a2 , O, N, or S and, R a1 are each independently H, alkyl, halogen, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, and R a2 are each independently H, or alkyl and;B 1 , B 2 , and B 3 NR each independently b1 , CR b2 , O, N, or S and, R b1 Each of these is independently H, alkyl, halogen, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, and R b2 is independently absent, or is H, or alkyl; and B 6 , B 7 , B 8 , and B 9 CR each independently b3 or N and R b3 Each is independently H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl, provided that B 6 , B 7 , B 8 , and B 9 One or two of them are N. Claim 15 In any one of paragraphs 1 through 4, R 1 and R 2 A compound or its pharmaceutically acceptable salt that is identical. Claim 16 In any one of paragraphs 1 to 4, Z 1 This methylene, a compound, or its pharmaceutically acceptable salt. Claim 17 In any one of paragraphs 1 through 4, this ; ; , or Phosphorus compound or its pharmaceutically acceptable salt. Claim 18 In any one of paragraphs 1 through 4, this or Phosphorus, a compound, or its pharmaceutically acceptable salt. Claim 19 In paragraph 1, a compound selected from the following: ; ; ; ; ; ; ; ; ; ; ; ; ; or ;or his pharmaceutically acceptable salt. Claim 20 A conjugate comprising the compound of claim 1, a cleavable linker, and a targeting moiety, wherein the cleavable linker cleavably connects the compound to the targeting moiety. Claim 21 Conjugate having the structure of chemical formula (II), (IIa), or (IIb) or its pharmaceutically acceptable salt: Here: ring A is a 5-membered heteroaryl ring; ring B is a heterocyclic ring optionally fused to one or more heteroaryl rings; R 1 and R 2 are each independently alkyl groups; Z 1 methylene, , or Igo, Z 1 In formula (II), provides an amine or phenol for conjugation, where Y 1 CR Y1 or N and, R Y1 is H or alkyl, and R Z1 is absent, or hydroxyl, amino, amido, or (CH2) z R Z1a and, R Z1a is an amino, aryl, or heteroaryl, z is an integer having a value from 1 to 10, and Y 2 is CR Y2 or N and, R Y2 is H or alkyl, and R Z2 is (CH2) z' R Z2a and, R Z2a is an amino, aryl, or heteroaryl, and z' is an integer having a value from 0 to 10; n a is 1 and;n 1 and n 2 are each an integer independently having a value from 1 to 5; and Z 2 is absent or is a connecting device;L 1 is a linker attached to SO2 through a heteroatom selected from O, S, and N, and L 1 The cleavage of the bond between and SO2 is L 1 Selected to promote the cleavage of the bond between the and the active agent; X 1 -O-, -CR a 2-, or -NR c' - and; Ar is a ring selected from aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; TG is a trigger group, and the trigger group releases the active agent upon activation and X 1 -Generating N, O, or S atoms capable of reacting with SO2 to form a 5-6 member ring including intervening atoms of SO2 and Ar; Y 1 is a functional group capable of generating a nucleophilic heteroatom that can react with SO2 selected from N, O, and S upon activation of the above-mentioned trigger; where X 1 and Y 1 is placed on an adjacent atom of Ar; w and x are integers each having a value of 0 or 1 independently; and each R a and R c' is independently hydrogen or lower alkyl. Claim 22 In Paragraph 21, Y 1 is N, O, or S, or -(CR b 2) y N(R a )-, -(CR b 2) y O-, or -(CR b 2) y S-, where y is 1, N, O, or S atoms are arranged to be attached to TG; and each R b is independently hydrogen or lower alkyl; or two R b A conjugate or its pharmaceutically acceptable salt that forms a 3-5 member ring together with the carbon atom to which they are attached. Claim 23 In Paragraph 21, TG is selected from the following, and and ,at this time, each R 21 is independently hydrogen or acetyl, and R 22 is hydrogen or a lower alkyl, and* is Y 1 Or a conjugate or its pharmaceutically acceptable salt indicating a link to Ar. Claim 24 In paragraph 21, the conjugate is a conjugate selected from one of the following or a pharmaceutically acceptable salt thereof: ; ; ; ; ; ; ; ; ; ; ; ; ; and . Claim 25 Conjugate having the structure of chemical formula (III): (DL) dl -LG-(CB) cb (III) or its pharmaceutically acceptable salt, where: LG is a linker, CB is a cell-binding agent, said cell-binding agent has binding specificity to a desired target receptor or other molecule associated with a target cell; cb and dl are each independently integers having a value from 1 to 20; and each DL is represented by the structure of formula (II), (IIa), or (IIb): , where: ring A is a 5-membered heteroaryl ring; ring B is a heterocyclic ring optionally fused to one or more heteroaryl rings; R 1 and R 2 are each independently alkyl groups; Z 1 methylene, , or Igo, Z 1 In formula (II), provides an amine or phenol for conjugation, where Y 1 CR Y1 or N and, R Y1 is H or alkyl, and R Z1 is absent, or hydroxyl, amino, amido, or (CH2) z R Z1a and, R Z1a is an amino, aryl, or heteroaryl, z is an integer having a value from 1 to 10, and Y 2 is CR Y2 or N and, R Y2 is H or alkyl, and R Z2 is (CH2) z' R Z2a and, R Z2a is an amino, aryl, or heteroaryl, and z' is an integer having a value from 0 to 10; n a is 1 and;n 1 and n 2 are each an integer independently having a value from 1 to 5; and Z 2 is a connector connecting CB and Ar;L 1 is a linker attached to SO2 through a heteroatom selected from O, S, and N, and L 1 The cleavage of the bond between and SO2 is L 1 Selected to promote the cleavage of the bond between the and the active agent; X 1 -O-, -CR a 2-, or -NR c' - and; Ar is a ring selected from aryl, heteroaryl, cycloalkyl, or heterocycloalkyl; TG is a trigger group, said trigger group releases an active agent upon activation and X 1 -Generating N, O, or S atoms capable of reacting with SO2 to form a 5-6 member ring including intervening atoms of SO2 and Ar; Y 1 is a functional group capable of generating a nucleophilic heteroatom that can react with SO2 selected from N, O, and S upon activation of the above-mentioned trigger; where X 1 and Y 1 is placed on an adjacent atom of Ar; w and x are integers each having a value of 0 or 1 independently; and each R a and R c' is independently hydrogen or lower alkyl. Claim 26 In Paragraph 25, Y 1 is N, O, or S, or -(CR b 2) y N(R a )-, -(CR b 2) y O-, or -(CR b 2) y S-, where y is 1, N, O, or S atoms are arranged to be attached to TG; and each R b is independently hydrogen or lower alkyl; or two R b A conjugate or its pharmaceutically acceptable salt that forms a 3-5 member ring together with the carbon atom to which they are attached. Claim 27 In Paragraph 25, TG is selected from the following, and and ,at this time, each R 21 is independently hydrogen or acetyl, and R 22 is hydrogen or a lower alkyl, and* is Y 1 Or a conjugate or its pharmaceutically acceptable salt indicating a link to Ar. Claim 28 In paragraph 25, the cell-binding agent binds to a target cell, wherein the target cell is a conjugate or its pharmaceutically acceptable salt selected from the following: tumor cells, virus-infected cells, microbial cells, activated cells, bone marrow cells, activated T-cells, B cells, or melanocytes; cells expressing CD4, CD6, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD40, CD44, CD56, EpCAm, CanAg, CALLA, Her-2 antigen or Her-3 antigen; and cells expressing insulin growth factor receptor, epidermal growth factor receptor, and folate receptor. Claim 29 In paragraph 28, the cell-binding agent is an antibody, a single-chain antibody, an antibody fragment that specifically binds to a target cell, a monoclonal antibody, a single-chain monoclonal antibody, a monoclonal antibody fragment that specifically binds to a target cell, a chimeric antibody, a chimeric antibody fragment that specifically binds to a target cell, a domain antibody, a domain antibody fragment that specifically binds to a target cell, a lymphokine, a hormone, a vitamin, a growth factor, a colony-stimulating factor, or a conjugate that is a nutrient-transport molecule or a pharmaceutically acceptable salt thereof. Claim 30 A pharmaceutical composition for treating cancer, infection, immunodeficiency, autoimmune disease, or chronic inflammatory disorder, wherein the pharmaceutical composition comprises a compound of any one of claims 1 to 4 or a conjugate of any one of claims 25 to 27, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete Claim 44 delete Claim 45 delete Claim 46 delete Claim 47 delete Claim 48 delete Claim 49 delete Claim 50 delete Claim 51 delete Claim 52 delete Claim 53 delete Claim 54 delete Claim 55 delete Claim 56 delete Claim 57 delete Claim 58 delete Claim 59 delete Claim 60 delete Claim 61 delete Claim 62 delete Claim 63 delete Claim 64 delete Claim 65 delete Claim 66 delete Claim 67 delete Claim 68 delete Claim 69 delete

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