RU2795168C2 - Compounds containing a cleavable linker and methods for their use - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: this document presents a compound containing a cleavable linker, a method of its use, and an intermediate product for its preparation.

The invention relates to an antibody-active agent conjugate containing a cleavable linker, an SO₂ functional group that is capable of selectively releasing the active agent, and a functional group that triggers a chemical reaction, a physicochemical reaction and/or a biological reaction by external stimulation. Intermediate products and methods for preparing the conjugate are also provided.

EFFECT: conjugate is intended for the treatment of proliferative diseases.

22 cl, 7 dwg, 5 tbl, 144 ex

Description

RELATED APPLICATIONS

This application claims priority of U.S. Provisional Application No. 62/597226, filed Dec. 11, 2017, and Korean Patent Application No. 10-2017-0084805, filed July 4, 2017. The contents of each of these applications are hereby incorporated herein in their entirety by links.

BACKGROUND OF THE INVENTION

Antibody-drug conjugates (ADCs) are a new, highly effective class of anticancer agents with efficacy in a number of cancers. ADCs typically consist of three different components: a cell-binding agent or targeting moiety; linker and cytotoxic agent. The linker component of the ADC is an important feature in the development of targeted anticancer agents that have the desired target specificity, ie high activity in tumor cells but low activity in normal cells.

Therefore, there is a need for improved linkers suitable for the preparation of ADC.

SUMMARY OF THE INVENTION

Provided herein are conjugates of formula ( I' ):

or a pharmaceutically acceptable salt thereof,

Where:

CB is a targeting fragment;

cb and n are each independently integers having a value from 1 to about 20, preferably from 1 to about 10;

each of the DLs is independently a group having the structure of the formula ( I'' ):

Q is an active agent linked to L' via a heteroatom, preferably O or N;

Z' is a linking group;

L' is a spacer moiety attached to SO ₂ via a heteroatom selected from O, S and N, preferably O or N, and chosen such that cleavage of the bond between L' and SO ₂ promotes cleavage of the bond between L' and Q for release of the active agent;

X is -O-, -C(R ^b ) ₂ - or -N(R ^c )-, preferably -O-;

Ar is a ring, such as aryl, heteroaryl, cycloalkyl or heterocycloalkyl, preferably aryl or heteroaryl;

Y' is -(CR ^b ₂ ) _y N(R ^a )-, -(CR ^b ₂ ) _y O- or -(CR ^b ₂ ) _y S-, positioned such that an N, O or S atom is attached to TG if y is 1;

X and Y' are located on adjacent Ar atoms;

TG is a trigger group that, when activated, generates a N, O, or S atom capable of reacting with SO ₂ to displace (Q) _q -(L') _w and form a 5- or 6-membered ring, including X-SO ₂ and intermediate Ar atoms;

q is an integer having a value from 1 to about 20, preferably from 1 to about 10;

each w, x and y is independently an integer having a value of 0 or 1;

each of R ^a and R ^c independently represents hydrogen or lower alkyl; And

each R ^b is independently hydrogen or lower alkyl; or

two R ^b together with the atom to which they are attached, form a 3-5-membered ring, preferably a 3-4-membered ring;

provided that when w is 0, then q is 1.

The present invention also relates to compositions ( eg, pharmaceutical compositions) containing a compound of formula ( I' ) and a carrier ( eg, a pharmaceutically acceptable carrier).

In one aspect, the invention relates to conjugates of formula ( I' ) and compositions containing such conjugates, for example , for use in therapy, imaging, as sensors, as molecular switches, as molecular machines and/or as nanomachines.

In another aspect, the invention further relates to conjugates of formula ( I' ) and pharmaceutical compositions thereof for use in a method for delivering an active agent to a cell, wherein the targeting moiety is selected to bind to a molecule associated with the target cell. In particular, the present compounds, conjugates, and compositions may be useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal ( e.g., a human), such as where the target cell is a cancer cell and the targeting moiety is selected to bind to a molecule, associated with a cancer cell (and not associated with healthy cells, or at least predominantly associated with tumor cells rather than healthy cells).

The present conjugates of formula ( I' ) and pharmaceutical compositions thereof may be useful in the treatment of conditions such as cancer, rheumatoid arthritis, multiple sclerosis, graft versus host disease (GVHD), transplant rejection, lupus, myositis, infection, immunodeficiency, for example , AIDS and inflammatory diseases in a mammal ( eg human).

BRIEF DESCRIPTION OF GRAPHICS

FIG. 1 illustrates the results of an enzymatic digestion assay of Compound A-1.

FIG. 2 illustrates the results of an enzymatic digestion assay for Compound A-2.

FIG. 3 illustrates the results of an enzymatic digestion assay of Compound A-3.

FIG. 4 illustrates the results of an enzymatic digestion assay of Compound A-4.

FIG. 5 illustrates the results of an enzymatic digestion assay of Compound B-1.

FIG. 6 illustrates the results of an enzymatic digestion assay for Compound B-3.

FIG. 7 illustrates the results of an enzymatic degradation assay (pH 7.4) of Compound B-2.

FIG. 8 illustrates the results of an enzymatic degradation assay (pH 5.0) of Compound B-2.

FIG. 9 illustrates the results of an enzymatic digestion assay for Compound B-4.

FIG. 10 illustrates the results of an enzymatic cleavage assay of Compound C-3.

FIG. 11 illustrates the results of the stability analysis of compound A-1.

FIG. 12 illustrates the results of an in vitro assay of compound D-1-AB.

FIG. 13 illustrates the results of in vivo testing of compound D-1-AB, D-2-AB, D-8-AB and D-16-AB.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compounds and conjugates containing a cleavable linker, and methods for their use. Representative compounds and conjugates described herein include an active agent ( e.g. , chemical factor, biological factor, hormone, oligonucleotide, drug, toxin, ligand, detection probe , etc. ) having the desired function or activity, functional a group that undergoes a chemical reaction ( for example , a physicochemical reaction and/or a biological reaction) under given conditions to release the nucleophilic heteroatom, and an SO ₂ functional group located proximal to the nucleophilic heteroatom in such a way that it can react with the nucleophilic heteroatom during the reaction of intramolecular cyclization with the release of the active agent. In some embodiments, the compounds and conjugates described herein further comprise a targeting moiety ( e.g. , oligopeptide, polypeptide, antibody , etc. ) having binding specificity for the desired target receptor or other molecule associated with the target cell.

Definitions

The meaning of the term "alkyl" is understood in the art. For example, the term "alkyl", used alone or as a larger moiety, such as "alkoxy", "haloalkyl", "cycloalkyl", "heterocycloalkyl", and the like, may refer to a straight or branched chain hydrocarbon that is fully saturated . Generally, a straight or branched alkyl group is an acyclic group having from 1 to about 20 carbon atoms, preferably from 1 to about 10 carbon atoms, unless otherwise indicated. Examples of straight and branched alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n -butyl, sec -butyl, t -butyl, n -pentyl, n -hexyl, n -heptyl, and n -octyl. A C ₁ -C ₆ straight or branched chain alkyl group is also referred to as a "lower alkyl" group. Alkyl groups having two open valences are sometimes denoted by the suffix "ene", as in the term "alkylene". Representative alkylene groups include methylene, ethylene, propylene, and the like.

In addition, the term "alkyl" (or "lower alkyl") can include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl fragments having substituents replacing hydrogen on one or more carbon atoms of the hydrocarbon backbone. . Such substituents, unless otherwise indicated, may include, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (such as thioether, thioacetate or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate , amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. One of skill in the art will appreciate that moieties substituted in the hydrocarbon chain may themselves be substituted if desired. For example, substituted alkyl substituents may include substituted and unsubstituted alkyl, amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl, and sulfonate) and silyl groups, as well as ethers, alkylthio , carbonyls (including ketones, aldehydes, carboxylates and esters), -CF ₃ , -CN and the like. Representative substituted alkyls are described below. Cycloalkyls may be further substituted with alkyls, alkenyls, alkoxy, alkylthio, aminoalkyls, carbonyl-substituted alkyls, -CF ₃ , -CN and the like.

The term "C _x -C _y " when used in conjunction with a chemical moiety such as, for example, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, may include groups that contain x to y carbon atoms in the chain, where "x" and "y" are integers selected from 1 to about 20, and where x is an integer less than y, and x and y are not the same. For example, the term "C _x -C _y -alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight or branched chain alkyl groups that contain from x to y carbon atoms in the chain, including haloalkyl groups such as trifluoromethyl and 2 ,2,2-trifluoroethyl , etc. The terms "C ₂ -C _y -alkenyl" and "C ₂ -C _y -alkynyl" refer to substituted or unsubstituted unsaturated aliphatic groups that are similar in length and possible substitution with alkyls, described above, but which contain at least one double or triple bond, respectively. When applied to heteroalkyls, "C _x -C _y " indicates that the group contains from x to y carbon atoms and heteroatoms in the chain. When applied to carbocyclic structures such as aryl and cycloalkyl groups, "C _x -C _y " indicates that the ring contains from x to y carbon atoms in the ring.

The meaning of the term "alkoxy" is understood in the art and, for example, can refer to an alkyl group, preferably a lower alkyl group, having an oxygen attached to it. Representative alkoxy groups include methoxy, ethoxy, propoxy, t -butoxy, and the like.

The term "halogen" is used throughout this document and refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

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

The meaning of the term "aryl" is understood in the art and, for example, can refer to single ring substituted or unsubstituted aromatic groups in which each ring atom is a carbon. Preferably, the ring is a 5-7 membered ring, more preferably a 6 membered ring. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbon atoms are shared by two adjacent rings, where at least one of the rings is aromatic, e.g. cycloalkyls, cycloalkenyls, cycloalkyls, aryls, heteroaryls and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

The meaning of the terms "heterocyclyl" and "heterocycle" is understood in the art and, for example, may refer to substituted or unsubstituted non-aromatic ring structures, preferably 3 to 10 membered rings, more preferably 3 to 7 membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. Such heterocycles also include polycyclic ring systems having two or more cyclic rings in which two or more carbon atoms are shared by two adjacent rings, wherein at least one of the rings is heterocyclic, e.g. , the other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkyls, aryls, heteroaryls and/or heterocyclyls. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

The meaning of the term "heteroaryl" is understood in the art and, for example, may refer to substituted or unsubstituted aromatic single ring structures, preferably 5-7 membered rings, more preferably 5-6 membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic ring systems having two or more cyclic rings in which two or more carbon atoms are shared by two adjacent rings, at least one of the rings being heteroaromatic, e.g. other cyclic rings may be cycloalkyls, cycloalkenyls, cycloalkyls, aryls, heteroaryls and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine and the like.

The term "substituted" refers to fragments having substituents replacing hydrogen on one or more carbon atoms or heteroatoms of the fragment. Those skilled in the art will appreciate that "substitution" or "substituted" includes the implicit condition that such substitution corresponds to the allowable valency of the substituted atom and substituent, and that the substitution results in a stable compound, e.g. as rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is intended to include all allowable substituents on organic compounds.

In some embodiments, acceptable substituents include acyclic and cyclic, branched and straight, carbocyclic and heterocyclic, aromatic and non-aromatic substituents on organic compounds. Allowable substituents may be one or more and the same or different for the respective organic compounds. For the purposes of this invention, heteroatoms, such as nitrogen, may have hydrogen substituents and/or any valid substituents of the organic compounds described herein that satisfy the valency of the heteroatoms. Substituents may include any of the substituents described herein, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioether, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate , amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, alkyl, aralkyl, or an aromatic or heteroaromatic moiety. One skilled in the art will appreciate that the substituents themselves may be substituted if desired. Unless specifically stated that they are "unsubstituted", it is understood that references to chemical moieties in this description include substituted options. For example, reference to an "aryl" group or moiety is implicitly inclusive of both substituted and unsubstituted variants.

The term "subject" for which administration is intended includes, for example, humans ( i.e. , males or females of any age group, e.g. , pediatric subjects ( e.g. , infants, children, adolescents) or adults ( e.g. , young adults, middle-aged adults, or older adults)) and/or other primates ( e.g. cynomolgus monkeys, rhesus monkeys); mammals, including commercially important mammals such as cattle, pigs, horses, sheep, goats, cats and/or dogs; and/or birds, including commercial birds such as chickens, ducks, geese, quails and/or turkeys. The preferred subjects are humans.

As used herein, a therapeutic agent that "prevents" a disorder or condition may, for example, refer to a compound that, in a statistical sample, reduces the likelihood of occurrence of the disorder or condition in a treated sample compared to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to an untreated control population.

The term "treatment" includes prophylactic and/or therapeutic treatment. The term "prophylactic or therapeutic" treatment is recognized in the art and includes the administration to the host of one or more of the compositions in question. If it is administered before the clinical manifestation of the adverse condition ( e.g. , a disease or other adverse condition in the host animal), then the treatment is prophylactic ( i.e. , it protects the host from the development of the adverse condition), whereas if it is administered after the occurrence of the adverse condition, the treatment is therapeutic ( i.e., it is intended to reduce, improve, or stabilize an existing undesirable condition or its side effects).

In certain embodiments, the compounds and conjugates described herein may be used alone or in conjunction with another type of therapeutic compound or agent. As used herein, "co-administration" refers to any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still active in the body ( e.g. , two compounds are simultaneously effective in a subject, such that may involve a synergistic effect of the two compounds). For example, various therapeutic compounds and conjugates may be administered either in the same composition or in separate compositions, simultaneously or sequentially. In certain embodiments, various therapeutic compounds and conjugates may be administered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or a week after administration of the other. Thus, a subject who receives such treatment may benefit from the combined action of various therapeutic compounds and conjugates.

The terms "abnormal cell growth" and "proliferative disorder" are used interchangeably in this application. The term "abnormal cell growth" in the context of this document, unless otherwise indicated, refers to cell growth that is independent of normal regulatory mechanisms ( eg loss of contact inhibition). This includes, for example, abnormal growth of: (1) tumor cells (tumors) that spread by expressing a mutated tyrosine kinase or overexpressing a receptor tyrosine kinase; (2) benign and malignant cells of other proliferative diseases in which aberrant tyrosine kinase activation occurs; (3) any tumors that spread by receptor tyrosine kinases; (4) any tumors that spread by aberrant serine/threonine kinase activation; and (5) benign and malignant cells of other proliferative diseases in which aberrant serine/threonine kinase activation occurs.

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

Compounds and conjugates of the present invention

The present invention provides conjugates of formula ( I' ):

or a pharmaceutically acceptable salt thereof,

Where:

CB is a targeting fragment;

cb and n are each independently integers having a value from 1 to about 20, preferably from 1 to about 10;

each of the DLs is independently a group having the structure of the formula ( I'' ):

each Q is independently an active agent linked to L' via a heteroatom, preferably O or N;

Z' is a linking group;

L' is a spacer moiety attached to SO ₂ via a heteroatom selected from O, S and N, preferably O or N, and chosen such that cleavage of the bond between L' and SO ₂ promotes cleavage of the bond between L' and Q for release of the active agent;

X is -O-, -C(R ^b ) ₂ - or -N(R ^c )-, preferably -O-;

Ar is a ring, such as aryl, heteroaryl, cycloalkyl or heterocycloalkyl, preferably aryl or heteroaryl;

Y' is -(CR ^b ₂ ) _y N(R ^a )-, -(CR ^b ₂ ) _y O- or -(CR ^b ₂ ) _y S-, positioned such that an N, O or S atom is attached to TG if y is 1;

X and Y' are located on adjacent Ar atoms;

TG is a trigger group that, when activated, generates a N, O, or S atom capable of reacting with SO ₂ to displace (Q) _q -(L') _w and form a 5-6 membered ring, including X-SO ² and intermediate Ar atoms;

q is an integer having a value from 1 to about 20, preferably from 1 to about 10;

each w, x and y is independently an integer having a value of 0 or 1;

each of R ^a and R ^c independently represents hydrogen or lower alkyl; And

each R ^b is independently hydrogen or lower alkyl; or

two R ^b together with the atom to which they are attached form a 3-5 membered ring, preferably a 3-4 membered ring,

provided that when w is 0, then q is 1.

Each active agent may be any suitable active agent, as described in more detail below. Although many conventional conjugation methods require the presence of certain functional groups, such as amines or hydroxyl groups, to form a stable bond, this document discloses strategies for forming compounds using functional groups, such as phenols and tertiary amines, to form stable bonds in the conjugates described herein, while at the same time providing release under predetermined conditions that activate the trigger group.

Many suitable trigger groups are known in the art, and typical trigger groups and the conditions that activate them are described below, such as the moieties described below for Y. Some trigger groups contain N, O, or S atoms, but in a non-nucleophilic form. For example, the NO ₂ group is a trigger group that, under reducing conditions, is reduced to an NH ₂ or NHOH group that can react with SO ₂ , and the acetate group is a trigger group that, under hydrolytic conditions, is hydrolyzed to a hydroxyl group that can react with SO ₂ . Other trigger groups do not include an N, O, or S atom, but are converted to a nucleophilic N, O, or S atom upon activation. in reaction with SO ₂ . Preferably, the trigger group is chosen such that the conditions that activate it do so selectively, without cleavage or degradation of other portions of the conjugate, such as the targeting moiety. After generating a nucleophilic N, O, or S atom, this atom intramolecularly attacks the SO ₂ fragment to form a ring, displacing the (Q) _q -(L') _w -H fragment, where H is bonded to the Q or L' heteroatom that was previously attached to the SO ₂ fragment.

In embodiments where w is 0, q is 1, and Q is directly attached to SO ₂ via a heteroatom. Accordingly, activation of the trigger group generates a nucleophilic heteroatom that intramolecularly attacks the SO ₂ moiety to form a ring, displacing the active agent QH, where H is bonded to the heteroatom previously attached to SO ₂ .

In embodiments where w is 1, L' may be selected to allow multiple Q options to be attached, which may be the same or different. Accordingly, each instance of Q is indirectly attached to SO ₂ via a spacer moiety. In such embodiments, activation of the trigger group generates a nucleophilic heteroatom that intramolecularly attacks the SO ₂ moiety to form a ring, displacing the (Q) _q -L'-H moiety, where H is bonded to the heteroatom in L' that was previously attached to SO ₂ . In such embodiments, the released heteroatom initiates an intramolecular reaction that displaces the active agent(s) Q (eg, if Q has a tertiary amine that has been attached to L' as a quaternary ammonium) or QH. For example, a heteroatom may undergo an intramolecular cyclization reaction with an ester moiety formed with a QH hydroxyl forming a ring and expelling the QH active agent. Alternatively, the heteroatom may undergo intramolecular tautomerization which displaces the active agent Q or QH.

Ar can be any suitable ring, including a bicycle ring or other polycycle, so that fragments that undergo intramolecular cyclization are held in close proximity to facilitate this reaction after activation of the trigger group. The planar nature of aromatic and heteroaromatic rings is preferred because the rigid geometry of the substituents on such rings allows for a favorable arrangement of reactive moieties, although other types of rings, such as cycloalkenyl or heterocycloalkenyl, may provide a similar geometry. The five- or six-membered ring and/or the number or identity of ring heteroatoms and/or substituents (eg, electron-donating or electron-withdrawing substituents) on the other ring can be chosen to modulate the cyclization rate based on the received ring bond angles. Likewise, more flexible cycloalkyl and heterocyclyl ring conformations may be useful when it is desired to slow the rate of intramolecular cyclization.

Z' may be any suitable linking group that links Ar to one or more CB groups. In general, the linking group should be sufficiently hydrophilic to ensure water solubility and prevent aggregation of the conjugate, for example, by including moieties such as polyethylene glycol moieties, peptide sequences, charge bearing moieties (such as carboxylates, amines, nitrogen rings, etc.). . ) etc. to balance the hydrophobic nature of any alkyl chains that may be included. Because it is often advantageous to prepare conjugates in a modular fashion, Z' may contain a linker, a functional group that results from the conjugation of one reactive moiety to another. Typical linkers are described in more detail below ( e.g. in relation to the Z variable), and typical linker groups include amides, triazoles, oximes, carbamates , etc. Representative Z' groups include L ¹ '-Z groups, as described in more detail. below. In some embodiments, all DL groups attached to each CB are identical, while in other embodiments, each CB may be attached to two or more separate DL groups. For example, some DL groups may have a trigger group that is activated on the first condition, while other DL groups may have a trigger group that is activated on the second condition, thus, for example, one active agent may be selectively released on the first condition, and the second active agent may be selectively released under the second condition.

The specification also provides compounds that can serve as intermediates or reagents in the formation of the DL group in formula (I') as described in formula (I''). Thus, in some embodiments, provided herein are compounds of formula ( Ia ):

or a pharmaceutically acceptable salt thereof, wherein:

each Q is independently an active agent linked to L' via a heteroatom, preferably O or N;

Z' is absent or is a linking group;

L' is a spacer moiety attached to SO ₂ via a heteroatom selected from O, S and N, preferably O or N, and chosen such that cleavage of the bond between L' and SO ₂ promotes cleavage of the bond between L' and Q for release of the active agent;

X is -O-, -CR ^a ₂ - or -NR'-, preferably -O-;

Ar is a ring, such as aryl, heteroaryl, cycloalkyl or heterocycloalkyl, preferably aryl or heteroaryl;

Y' is -(CR ^b ₂ ) _y N(R ^a )-, -(CR ^b ₂ ) _y O- or -(CR ^b ₂ ) _y S-, positioned such that an N, O or S atom is attached to TG if y is 1;

X and Y' are located on adjacent Ar atoms;

TG is a trigger group that, when activated, generates a N, O, or S atom capable of reacting with SO ₂ to displace (Q) _q -(L') _w and form a 5-6 membered ring, including X-SO ² and intermediate Ar atoms;

q is an integer having a value from 1 to about 20, preferably from 1 to about 10;

each w, x and y is independently an integer having a value of 0 or 1;

each of R ^a and R ^c independently represents hydrogen or lower alkyl; And

each R ^b is independently hydrogen or lower alkyl; or

two R ^b together with the carbon atom to which they are attached form a 3-5 membered ring, preferably a 3-4 membered ring,

In some embodiments of formulas ( I ') and ( Ia ), -Y' is -(CH ₂ ) _y NR''-, -(CH ₂ ) _y O-, or -(CH ₂ ) _y S- arranged so such that an N, O, or S atom is attached to TG if y is 1; R'' represents hydrogen or C ₁ -C ₆ -alkyl; and y is an integer having a value of 0 or 1. In some such embodiments, TG is β-galactoside, β-glucuronide, or a combination of β-galactoside and β-glucuronide.

In some embodiments of formulas ( I' ) and ( Ia ), (L') _w links each Q with -SO ₂ -; and each Q is an active agent linked to one of the L' groups via a heteroatom, preferably O or N, and forming -O-, -OC(O)-, -OC(O)O- or -OC(O)NH - bond, including the heteroatom Q.

In other embodiments, (Q) _q - (L') _w - is selected from:

;

; и

;

;

; And

;

Where:

Q is the active agent linked to L' via a heteroatom, preferably O or N,

X ⁴ is absent or forms an -O-, -OC(O)-, -OC(O)O- or -OC(O)NH- bond, including a Q heteroatom;

X ¹ represents -O- or -NR ^a -;

X ² is -O-, -OC(O)-, -OC(O)O- or -OC(O)NH-;

X ³ is -OC(=O)-;

w' is an integer having the value 1, 2, 3, 4 or 5;

each of R ⁹ and R ¹⁰ independently represents hydrogen, alkyl, aryl or heteroaryl, where alkyl, aryl and heteroaryl are unsubstituted or substituted with one or more substituents, for example, selected from alkyl, -(CH ₂ ) _u NH ₂ , -( CH ₂ ) _u NR ^u1 R ^u2 and -(CH ₂ ) _u SO ₂ R ^u3 ;

each of R ^u1 , R ^u2 and R ^u3 independently represents hydrogen, alkyl, aryl or heteroaryl; And

u is an integer value between 1 and 10.

In some such embodiments, (Q) _q - (L') _w - is selected from:

;

.

;

.

In addition, the invention relates to intermediates for the preparation of conjugates of formula ( I' ) or compounds according to formula ( Ia ), where (Q) _q -(L') _w in these formulas is replaced by a leaving group such as halogen (preferably fluorine ) to ensure the attachment of (Q) _q -(L') _w .

;

;

;

;

;

; и

In certain such embodiments, Z' includes a reactive group (e.g., a precursor group, as described in more detail below with respect to Z) that can be used to attach the compound to a triggering agent, such as CB ( e.g. , to obtain a compound of formula ( I' ) , as described in more detail above), to a solid surface ( for example to form a matrix on a solid support or sensor particles) or to any other molecule or substrate of interest. In certain preferred embodiments, the compound of formula ( I' ) is selected from:

;

;

;

;

;

; And

Where:

R ¹ represents C ₁ -C ₆ -alkyl; And

each of R ²¹ and R ²² independently represents hydrogen or C ₁ -C ₆ alkyl;

;

;

; и

.In other embodiments, the compound of formula ( I' ) is selected from:

;

;

; And

.

In additional other embodiments, the compound of formula ( I' ) is selected from:

;

;

;

;

;

; и

.

;

;

;

;

;

; And

.

;

;

; и

.In other embodiments, the compound of formula ( I' ) is selected from:

;

;

; And

.

In certain preferred embodiments, Z is a linking group having the structure of formulas ( F ), ( G ), ( H ), ( J ), ( K ), ( L ), ( M ), or ( N ):

Where:

* represents the point of attachment to CB;

** represents the point of attachment to Ar;

R ^e is alkyl;

X'' represents -O-, -S-, -NH- or -CH ₂ -;

X ⁴ is -NHC(O)-(CH ₂ ) _g -NH- or -C(O)NH-(CH ₂ ) _h -NH-;

или

;each of W ^b1 and W ^b2 is independently -C(O)NH-, -NHC(O)-,

or

;

L ² is an optionally present spacer moiety and may be further substituted with one or more substituents such as C ₁ -C ₆ alkyl, C ₅ -C ₁₄ aryl, and C ₃ -C ₈ heteroaryl, where alkyl, aryl, and heteroaryl may be further substituted, for example with one or more substituents selected from the group consisting of C ₁ -C ₁₀ alkyl, -(CH ₂ ) _u NH ₂ , -(CH ₂ ) _u NR ^u1 R ^u2 , -(CH ₂ ) _u CO ₂ H, -(CH ₂ ) _u CO ₂ R ^u1 and -(CH ₂ ) _u SO ₂ R ^u3 , where R ^u1 , R ^u2 and R ^u3 are each independently hydrogen, C ₁ -C ₁₅ alkyl, C ₆ -C ₂₀ aryl or C ₃ -C ₁₀ heteroaryl; and u is an integer having a value from 1 to about 10;

R ¹² is hydrogen, C ₁ -C ₈ alkyl, or an amino acid moiety such as a naturally occurring amino acid moiety;

each of b, c, d, e, g, h, o and qq is independently an integer having a value from 1 to about 10; And

s' is an integer having a value from 1 to about 10.

In other embodiments, Z is a linking group having the structure of formulas ( F' ), ( G' ), ( H' ), ( J' ), ( K' ), ( L' ), ( M' ), or ( N ' ):

In certain preferred embodiments, CB is selected from:

,

, и

,

, And

.

.

In certain preferred embodiments, (Q) _q - (L') _w is selected from:

.

.

This document also provides compounds of formula ( I ):

( I )

or a pharmaceutically acceptable salt thereof, wherein:

X represents -O-, -CH ₂ - or -NR'-;

R' represents hydrogen, C ₁ -C ₆ -alkyl, C ₆ -C ₁₄ -aryl or C ₂ -C ₂₀ -heteroaryl;

Ar is a C ₅ -C ₂₀ aromatic ring, C ₂ -C ₂₀ heteroaromatic ring, C ₂ -C ₃₀ fused ring or C ₅ -C ₂₀ aromatic ring, C ₂ -C ₂₀ heteroaromatic ring;

R is a substituent on Ar or -L ¹ '-Z-(CB) _cb , preferably -L ¹ '-Z-(CB) _cb ;

L ¹ ' is a C ₁ -C ₂₀₀ -alkylene or C ₁ -C ₂₀₀ alkylene, additionally containing at least one of a peptide bond, an amino bond of an ether bond, a triazole bond, a tetrazole bond, a glycosidic bond, a sulfonamide bond, a phosphonate bond, a sulfo -bonds or dendrimeric structure;

Z is a link connecting CB and L ¹ 'or a reactive group ( for example , which provides connection with CB);

CB is a targeting moiety such as a ligand having the property that it binds to a receptor;

cb is an integer having a value of 0, 1, or 2;

n is an integer having a value of 1, 2, 3 or 4;

или -Y'-TG, предпочтительно Y представляет собой -NO₂, -OC(O)(CH₂)_rC(O)R¹, -O(CH₂)_r-Ar¹-NO₂, -NHNH₂, -BR²R³,

или -Y'-TG;Y is -NO ₂ , -OC(O)(CH ₂ ) _r C(O)R ¹ , -O(CH ₂ ) _r -Ar ¹ -NO ₂ , -NHOH, -NHNH ₂ , -BR ² R ³ ,

or -Y'-TG, preferably Y is -NO ₂ , -OC(O)(CH ₂ ) _r C(O)R ¹ , -O(CH ₂ ) _r -Ar ¹ -NO ₂ , -NHNH ₂ , -BR ² R ³ ,

or -Y'-TG;

R ¹ represents C ₁ -C ₆ -alkyl;

r is an integer having a value of 1, 2, 3, 4, or 5;

Ar ¹ is C ₆ -C ₂₀ -arylene;

each of R ² and R ³ independently represents hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ -alkoxy or hydroxy;

each of R ^a , R ^b , R ^c and R ^d independently represents hydrogen or C ₁ -C ₆ alkyl;

Y' represents -(CH ₂ ) _x NR''-, -(CH ₂ ) _x O- or -(CH ₂ ) _x S-;

R'' represents hydrogen or C ₁ -C ₆ -alkyl;

x is an integer having a value of 0 or 1;

TG is a trigger group;

Q is -Q ¹ or -L'-(Q ¹ ) _w ;

L' is a C ₇ -C ₃₀ hydrocarbon spacer having -O- or -NR'''- at one end and -O-,-OC(O)-, -O(CO)O-, -OC( O)NR'''- or -OC(O)NR ⁴ CH ₂ O- at the other end, where -O-, -OC(O)-, -O(CO)O- or -OC(O)NR''''- may be further included in the C ₇ -C ₃₀ hydrocarbon spacer, wherein the C ₇ -C ₃₀ hydrocarbon spacer is further substituted with one or more substituents such as C ₁ -C ₆ alkyl, C ₅ -C ₁₄ - aryl and C ₃ -C ₈ -heteroaryl, and alkyl, aryl and heteroaryl can be additionally substituted, for example , with one or more substituents selected from the group consisting of C ₁ -C ₁₀ -alkyl, -(CH ₂ ) _u NH ₂ , -(CH ₂ ) _u NR ^u1 R ^u2 , -(CH ₂ ) _u CO ₂ H, -(CH ₂ ) _u CO ₂ R ^u1 and -(CH ₂ ) _u SO ₂ R ^u3 , where each of R ^u1 , R ^u2 and R ^u3 are independently hydrogen, C ₁ -C ₁₅ alkyl, C ₆ -C ₂₀ aryl, or C ₃ -C ₁₀ heteroaryl; and u is an integer having a value from 1 to about 10;

Q ¹ is an active agent comprising at least one functional group from -OH, -NH-, -NR ₅ R ₆ , -SH, -SO ₂ NH ₂ or -COOH;

R ⁴ represents hydrogen, C ₁ -C ₆ -alkyl, C ₅ -C ₁₄ -aryl or C ₃ -C ₈ -heteroaryl, and alkyl, aryl and heteroaryl are substituted or unsubstituted;

each of R ⁵ and R ⁶ independently represents hydrogen, C ₁ -C ₆ -alkyl, C ₃ -C ₉ -cycloalkyl or C ₅ -C ₁₀ -heteroaryl, and heteroaryl is substituted or unsubstituted;

each of R''' and R'''' independently represents hydrogen or C ₁ -C ₆ alkyl; And

w is an integer having the value 1, 2, 3, 4, or 5.

In some embodiments, a compound of formula ( I ) contains a functional group ( eg , Y) capable of inducing intramolecular cyclization via external stimulation. In certain embodiments, the implementation of the specified functional group is introduced in the ortho position relative to X.

In some embodiments, R' is C ₁ -C ₆ alkyl, C ₆ -C ₁₄ aryl, or C ₂ -C ₂₀ heteroaryl.

In some embodiments, Ar is a C ₅ -C ₂₀ aromatic ring, C ₂ -C ₂₀ heteroaromatic ring, C ₂ -C ₃₀ fused ring, or C ₅ -C ₂₀ aromatic ring - C ₂ -C ₂₀ heteroaromatic ring. For example, Ar may be a benzene ring, a naphthalene ring, a pyridine ring, or a quinolone ring. Preferably Ar is a benzene ring or a naphthalene ring. In some embodiments, a compound of formula ( I ) is a compound having a structure according to formula ( II ):

or a pharmaceutically acceptable salt thereof.

In other embodiments, a compound of formula ( I ) is a compound having a structure according to formula ( III ):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of formula ( I ), ( II ), or ( III ), wherein R is selected from hydrogen, halogen (halogen), aldehyde, acetal, ketal, -R*, -OR*, -SR*, -NR*R**, -C(halogen) ₃ , -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO ₂ , -N ₃ , -NC, -C( O)R*, -OC(O)R*, -OS(O)R*, -S(O) ₂ R*, -S(O) ₂ OR*, -OS(O)OR*, -OS( O) ₂ OR*, -S(O)NR*R**, -S(O) _2NR *R**, -S(O)R*, -OP(O)(OR*) ₂ , -P (O)(OR*) ₂ , -OP(OR*) ₂ , -OP(OR*)N(R**) ₂ , -OP(O)(OR*)N(R**) ₂ , -PR *, -P(O) ₂ , -P(O)R*, -C(O) halogen, -C(S)R*, -CO ₂ R*, -C(S)OR*, -C(O )SR*, -C(S)SR*, -C(O)NR*R**, -C(S)NR*R**, -C(=NR*)NR*R**, -NR* C(O)R**, -NR*S(O) ₂ OR**, -NR*S(O)R**, -NR*C(O)NR**, -SS-R* or -R *SSR**, where: each of R* and R** independently represents hydrogen, C ₁ -C ₁₈ alkyl, C ₆ -C ₂₀ aryl, C ₃ -C ₁₅ heterocycle or C ₃ -C ₂₀ heteroaryl.

In some embodiments, the compound is a compound of formula ( I ), ( II ), or ( III ), where R is hydrogen or *-(L ^a -A ₁ -L ^b -L ^c -Z) _m -CB; Where:

L ^a represents a single bond or C ₁ -C ₂₀ -alkylene;

A ¹ is -C(O)NR*-, -NR*C(O)-, -NR*-, -O-, -PO ₃ -, -OPO ₃ -, -SO-, -SO ₂ - or -SO ₃ -;

L ^b is -(CH ₂ CH ₂ O) _a - or -(CH ₂ ) _a -;

R* is hydrogen, C ₁ -C ₁₈ alkyl, C ₆ -C ₂₀ aryl, C ₃ -C ₁₅ heterocycle, or C ₃ -C ₂₀ heteroaryl;

a is an integer having a value from 1 to about 20;

L ^c represents a single bond or C ₁ -C ₂₀ -alkylene;

n is an integer having a value of 1 or 2; And

Z is a link connecting CB and L ^c ; or

,

, фосфоновой кислоты (-P(=O)(OH)₂), кетона,C₈-C₁₀ циклоалкинила, -OH, -NHOH, -NHNH₂, -SH, карбоновой кислоты (-COOH), ацетилена (-C≡CH), азида (-N₃), амино (-NH₂), сульфоновой кислоты (-SO₃H), производного алкинона (-C(O)C≡C-R^a, где R^a представляет собой C₁-C₁₀-алкил) и дигидрофосфат (-OP(=O)(OH)₂);Z is a precursor selected from isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH ₂ -halogen), maleimide, diene, alkene, halide, tosylate (TsO ^- ), aldehyde, sulfonate (R-SO ₃ ^- ),

,

, phosphonic acid (-P (= O) (OH) ₂ ), ketone, C ₈ -C ₁₀ cycloalkynyl, -OH, -NHOH, -NHNH ₂ , -SH, carboxylic acid (-COOH), acetylene (-C≡ CH), azide (-N ₃ ), amino (-NH ₂ ), sulfonic acid (-SO ₃ H), alkynone derivative (-C(O)C≡CR ^a , where R ^a is C ₁ -C ₁₀ - alkyl) and dihydrophosphate (-OP(=O)(OH) ₂ );

CB is a targeting moiety, such as a ligand capable of binding to a receptor; And

m is an integer having a value of 0, 1 or 2;

In some embodiments, the implementation of the connection is a compound of formula ( I ), ( II ) or ( III ), where R represents hydrogen or *-L ^a -A ₁ -L ^b -L ^c -Z; Where:

L ^a represents a single bond or C ₁ -C ₂₀ -alkylene;

A ¹ is -C(O)NR*-, -NR*C(O)-, -NR*-, -O-, -PO ₃ -, -PO ₄ -, -SO-, -SO ₂ - or -SO ₃ -;

L ^b is -(CH ₂ CH ₂ O) _a - or -(CH ₂ ) _a -;

R* is hydrogen, C ₁ -C ₁₈ alkyl, C ₆ -C ₂₀ aryl, C ₃ -C ₁₅ heterocycle, or C ₃ -C ₂₀ heteroaryl;

a is an integer having a value from 1 to about 20;

L ^c represents a single bond or C ₁ -C ₂₀ -alkylene;

n is an integer having a value of 1 or 2; And

,

, фосфоновой кислоты (-P(=O)(OH)₂), кетона,C₈-C₁₀ циклоалкинила, -OH, -NHOH, -NHNH₂, -SH, карбоновой кислоты (-COOH), ацетилена (-C≡CH), азида (-N₃), амино (-NH₂), сульфоновой кислоты (-SO₃H), производного алкинона (-C(O)C≡C-R^a, где R^a представляет собой C₁-C₁₀-алкил) и дигидрофосфат (-OP(=O)(OH)₂).Z is a precursor selected from isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH ₂ -halogen), maleimide, diene, alkene, halide, tosylate (TsO ^- ), aldehyde, sulfonate (R-SO ₃ ^- ),

,

, phosphonic acid (-P (= O) (OH) ₂ ), ketone, C ₈ -C ₁₀ cycloalkynyl, -OH, -NHOH, -NHNH ₂ , -SH, carboxylic acid (-COOH), acetylene (-C≡ CH), azide (-N ₃ ), amino (-NH ₂ ), sulfonic acid (-SO ₃ H), alkynone derivative (-C(O)C≡CR ^a , where R ^a is C ₁ -C ₁₀ - alkyl) and dihydrophosphate (-OP(=O)(OH) ₂ ).

In some embodiments, the compound is a compound of formula ( I ), ( II ), or ( III ), where R is *(-L ^a -A ₁ -L ^b -L ^c -Z) _m -CB; Where:

L ^a represents a single bond or C ₁ -C ₂₀ -alkylene;

A ¹ is -C(O)NR*-, -NR*C(O)-, -NR*-, -O-, -PO ₃ -, -PO ₄ -, -SO-, -SO ₂ - or -SO ₃ -;

L ^b is -(CH ₂ CH ₂ O) _a - or -(CH ₂ ) _a -;

R* is hydrogen, C ₁ -C ₁₈ alkyl, C ₆ -C ₂₀ aryl, C ₃ -C ₁₅ heterocycle, or C ₃ -C ₂₀ heteroaryl;

a is an integer having a value from 1 to about 20;

L ^c represents a single bond or C ₁ -C ₂₀ -alkylene;

n is an integer having a value of 1 or 2;

Z is a link connecting CB and L ^c ;

CB is a targeting moiety such as a ligand having the property that it binds to a receptor; And

m is an integer having the value 1 or 2.

In some embodiments, the compound is a compound of formula ( I ), ( II ), or ( III ), where L' is a C ₇ -C ₃₀ hydrocarbon spacer further containing -O-, -OC(O)-, -O( CO)O- or -OC(O)NR''''-.

In some embodiments, the compound is a compound of formula ( I ), ( II ), or ( III ), where Q is -L'-(Q ¹ ) _w selected from:

;

;

;

; и

;

;

;

; And

Where:

Q ¹ is an active agent containing at least one functional group selected from -OH, -NR ⁵ R ⁶ , -SH and -COOH;

Q ² is an active agent containing -NR ⁵ R ⁶ ;

X ¹ is -O- or -NR'''-;

each of X ² and X ⁴ is independently absent or selected from -O-, -OC(O)-, -OC(O)O- and -OC(O)NH-;

X ³ is -OC(=O)-;

R ⁵ and R ⁶ are as defined above;

each of R ⁹ and R ¹⁰ independently represents hydrogen, C ₁ -C ₆ -alkyl, C ₆ -C ₁₄ -aryl or C ₃ -C ₉ heteroaryl, and these alkyl, aryl and heteroaryl R ⁹ and R ¹⁰ may optionally be substituted with one or more substituents selected from the group consisting of C ₁ -C ₁₀ alkyl, -(CH ₂ ) _u NH ₂ , -(CH ₂ ) _u NR ^u1 R ^u2 and -(CH ₂ ) _u SO ₂ R ^u3 , and each of R ^u1 , R ^u2 and R ^u3 independently represents hydrogen, C ₁ -C ₁₅ -alkyl, C ₆ -C ₂₀ -aryl or C ₃ -C ₁₀ -heteroaryl; and u is an integer having a value from 1 to about 10;

R''' represents hydrogen or C ₁ -C ₆ alkyl; And

w is an integer having the value 1, 2, 3, 4, or 5.

In certain embodiments, -L'-(Q ¹ ) _w is selected from:

.

.

At least one functional group from Q, Q ¹ or Q ² is selected from -OH, -NR ⁵ R ⁶ , -SH, and -COOH serves as the connection point of the active agent with L'. The functional group may exist as part of an ester, thioether, carbonate, carbamate, amide, sulfonamide, sulfonate, sulfate, or other suitable bond; that is, the -OH, -NR ⁵ R ⁶ , -SH, and -COOH moiety does not exist as such, while the active agent is part of the conjugate.

In some embodiments, Q ² is an active agent comprising -NR ⁵ R ⁶ , wherein the active agent is capable of binding in a quaternary amine structure, for example, the -NR ⁵ R ⁶ moiety in the active agent is capable of forming a quaternary amine bond with L'.

In some embodiments of formulas ( I ''), ( Ia ), ( I ), ( II ), and ( III ), R ⁴ is substituted alkyl, aryl, or heteroaryl. In some such embodiments, R ⁴ is substituted with one or more substituents selected from C ₁ -C ₁₀ alkyl, -(CH ₂ ) _u NH ₂ , -(CH ₂ ) _u NR ^u1 R ^u2 , -(CH ₂ ) _u CO ₂ H, -(CH ₂ ) _u CO ₂ R ^u1 and -(CH ₂ ) _u SO ₂ R ^u3 , where R ^u1 , R ^u2 and R ^u3 are each independently hydrogen, C ₁ -C ₁₅ alkyl, C ₆ -C ₂₀ -aryl or C ₃ -C ₁₀ -heteroaryl; and u is an integer having a value from 1 to about 10.

In some embodiments of formulas ( I'' ), ( Ia ), ( I ), ( II ) and ( III ) R ⁵ and/or R ⁶ is heteroaryl substituted with -NR ⁷ R ⁸ , where each of R ⁷ and R ⁸ independently, represent hydrogen, C ₁ -C ₆ -alkyl, C ₃ -C ₉ -cycloalkyl or C ₅ -C ₁₄ -aryl.

In some embodiments of formulas ( I'' ), ( Ia ), ( I ), ( II ) and ( III ), Q or -(L') _w -(Q) _q is selected from:

In certain embodiments, a compound of formula ( I ), ( II ), or ( III ) is provided, wherein:

Y is -NO ₂ , -OC(O)(CH ₂ ) _r C(O)R ¹ , -O(CH ₂ ) _r -Ar ¹ -NO ₂ , -NHOH, -BR ² R ³ or -Y' -TG, preferably Y is -NO ₂ , -OC(O)(CH ₂ ) _r C(O)R ¹ , -O(CH ₂ ) _r -Ar ¹ -NO ₂ , -BR ² R ³ or -Y '-TG;

R ¹ represents C ₁ -C ₆ -alkyl;

r is an integer having a value of 1, 2, 3, 4, or 5;

Ar ¹ is phenylene, biphenylene or naphthylene;

each of R ² and R ³ independently represents hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ -alkoxy or hydroxy;

Y' represents -(CH ₂ ) _x NR''-, -(CH ₂ ) _x O- or -(CH ₂ ) _x S-;

R'' represents hydrogen or C ₁ -C ₆ -alkyl;

x is an integer having a value of 0 or 1;

R'' represents hydrogen or C ₁ -C ₆ -alkyl; And

TG is a trigger group such as β-galactoside, β-glucuronide, or a combination of β-galactoside and β-glucuronide.

In certain embodiments, a compound of formula ( I ), ( II ), or ( III ) is selected from:

;

;

;

;

;

; и

;

;

;

;

;

; And

Where:

R ¹ represents C ₁ -C ₆ -alkyl;

each of R ²¹ and R ²² independently represents hydrogen or acetyl;

R represents hydrogen, a linking group, *-L ^a -A ₁ -L ^b -L ^c -Z or a group having the structure of formulas ( F ), ( G ), ( H ), ( J ), ( K ), ( L ), ( M ) or ( N ):

L ^a represents a single bond or C ₁ -C ₂₀ -alkylene;

A ¹ is -C(O)NH-, -NHC(O)-, -NH-, -O-, -PO ₃ -, -PO ₄ -, -SO-, -SO ₂ - or -SO ₃ - ;

L ^b is -(CH ₂ CH ₂ O) _a - or -(CH ₂ ) _a -;

a is an integer having a value from 1 to about 20;

L ^c is C ₁ -C ₂₀ alkylene;

X'' represents -O-, -S-, -NH- or -CH ₂ -;

или

;each of W ^b1 and W ^b2 is independently -C(O)NH-, -NHC(O)-,

or

;

R ¹² is hydrogen, C ₁ -C ₈ -alkyl, amino acid fragment, -(CH ₂ ) _s COR ¹³ or -(CH ₂ ) _p NR ¹⁴ R ¹⁵ ;

R ¹³ is OH or -NH(CH ₂ ) _s' (X''CH ₂ CH ₂ ) _s'' Z;

each of R ¹⁴ and R ¹⁵ independently represents hydrogen or -(C(O)(CH ₂ ) _s' (X''CH ₂ CH ₂ ) _s ''Z) _m -CB;

X'' represents -O-, -S-, -NH- or -CH ₂ -;

R ^e represents C ₁ -C ₈ -alkyl or -(L ¹ '-Z) _m -CB;

X ⁴ is -NHC(O)-(CH ₂ ) _g -NH- or -C(O)NH-(CH ₂ ) _h -NH-;

each of b, c, d, e, g, h, o and q is independently an integer having a value from 1 to about 10;

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

each of s and s'' is independently an integer having a value from 0 to about 10;

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

m is an integer having a value of 0 or 1;

,

, фосфоновую кислоту (-P(=O)(OH)₂), кетон, C₈-C₁₀-циклоалкинил, -OH, -NHOH, -NHNH₂, -SH, карбоновую кислоту (-COOH), ацетилен (-C≡CH), азид (-N₃), амино (-NH₂), сульфоновую кислоту (-SO₃H), производное алкинона (-C(O)C≡C-R^a, где R^a представляет собой C₁-C₁₀-алкил) и дигидрофосфат (-OP(=O)(OH)₂).Z is an isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH ₂ -halogen), maleimide, diene, alkene, halide, tosylate (TsO ^- ), aldehyde, sulfonate (R-SO ₃ ^- ),

,

, phosphonic acid (-P (= O) (OH) ₂ ), ketone, C ₈ -C ₁₀ -cycloalkynyl, -OH, -NHOH, -NHNH ₂ , -SH, carboxylic acid (-COOH), acetylene (-C ≡CH), azide (-N ₃ ), amino (-NH ₂ ), sulfonic acid (-SO ₃ H), alkynone derivative (-C(O)C≡CR ^a , where R ^a is C ₁ -C ₁₀ -alkyl) and dihydrophosphate (-OP(=O)(OH) ₂ ).

CB is a ligand selected from:

Q is selected from:

Release of the active agent

As described above, in certain embodiments, the compounds and conjugates described herein are capable of dissociating into one or more active agents (represented by Q, Q ¹ , Q ² ) via an intramolecular cyclization reaction following a chemical reaction that activates a trigger group. In certain embodiments, the chemical reaction is a physicochemical reaction and/or a biochemical reaction.

In some embodiments, the compounds and conjugates described herein contain a nucleophilic functional group (Y or Y') introduced at an adjacent atom on Ar relative to X ( eg , O). Typically, a trigger group (TG) masks the nucleophilic functional group, as described in more detail below. Upon activation, the trigger group releases a nucleophilic functional group to react with a nearby SO ₂ moiety in intramolecular cyclization, ultimately releasing one or more active agents (Q, Q ¹ or Q ² ). In some such embodiments, one or more active agents are released via an intramolecular cyclization reaction following a chemical reaction, a physicochemical reaction, and/or a biochemical reaction (see, for example, Reaction Scheme 1), or the active agent is released via 1,6-elimination or 1,4-elimination after an intramolecular cyclization reaction (see, for example, Reaction Scheme 2).

As an example, the mechanism when Y is -Y'-TG is illustrated in Reaction Scheme 1:

Reaction scheme 1:

, проиллюстрирован на Схеме реакции 2:Mechanism when Q is

, illustrated in Reaction Scheme 2:

Reaction Scheme 2:

.

.

In some embodiments, upon release, Q ¹ is an active agent containing at least one functional group selected from -OH, -NH-, -SH, and -COOH. According to these embodiments, as further described herein, Q ¹ is conjugated to a compound as described herein, -OH, -NH-, -SH, and -COOH, for example, via a functional group selected from an ester, amide, thioether, carbamate, urea, oxime, hydrazone, etc. In some such embodiments, Q ² is used instead of Q ¹ and Q ² is a drug containing an amino group. In other embodiments, Q ² is an active agent capable of binding to an ammonium unit. In other embodiments, Q ² is capable of dissociating in its original amino form upon release of Q ² , wherein the active agent may be a drug, a toxin, an affinity ligand, a detection probe, or a combination thereof.

In some embodiments, the compounds and conjugates described herein are chemically and physiologically stable. In some such embodiments, the compounds and conjugates described herein reach the desired target cell in a state in which there is little dissociation of the active agent in the blood, thereby selectively releasing the drug.

Trigger groups (TG)

In some embodiments, the implementation of the conjugates of the present invention include a trigger group (TG). TGs are groups that can be cleaved, preferably selectively cleaved, in a chemical reaction such as a biological reaction. Typically, trigger groups serve to mask the nucleophilic nature of the Y or Y' group, thereby providing stability ( e.g. , preventing self-cleavage or intramolecular cyclization before the conjugate reaches the target location or experiences a predetermined trigger condition) for the compounds and conjugates described in this document. Upon activation, the trigger group releases the Y or Y' nucleophilic group and allows the process of self-cleavage or intramolecular cyclization as described above.

In some embodiments, the TG contains a sequence (such as a peptide sequence) or fragment recognized by TEV, trypsin, thrombin, cathepsin B, caspin D, cathepsin K, caspase 1, matrix metalloproteinase (MMP), etc., which can be hydrolyzed enzyme ( for example , oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, etc. ) and / or may include a fragment selected from phosphodiester, phospholipid, ester, β-galactose, β-glucose, fucose, oligosugar, etc. . P.

In some embodiments, the TG contains a reactive chemical moiety or functional group that can be cleaved under conditions using a nucleophilic reagent ( e.g. , silyl ether, 2-N-acylnitrobenzenesulfonamide, unsaturated vinyl sulfide, post-activation sulfonamide, malondialdehyde-indole derivative, levulinoyl ether, hydrazone or acylhydrazone).

In some embodiments, the TG may contain a reactive chemical moiety or functional group that can be cleaved under the conditions of a basic reagent (eg, 2-cyanoethyl ether, ethylene glycol disuccinate, 2-sulfonyl ethyl ether, alkylthioether, or thiophenyl ether).

In some embodiments, the TG may contain a reactive chemical moiety or functional group that can be cleaved by photoirradiation ( e.g. , a 2-nitrobenzyl derivative, a phenacyl ester, 8-quinolinylbenzenesulfonate, coumarin, a phosphotriester, a bis-arylhydrazone, or a biman bi-thiopropionic acid derivative).

In some embodiments, the TG may contain a reactive chemical moiety or functional group that can be cleaved under the conditions of using a reducing agent (eg, hydroxylamine, disulfide, levulinate, nitro, or a 4-nitrobenzyl derivative).

In some embodiments, the TG may contain a reactive chemical moiety or functional group that can be cleaved in an acidic environment ( e.g. , saccharides, tert-butyl carbamate analogue, dialkyl or diaryl dialkoxysilane, orthoester, acetal, aconityl, hydrazone, β-thiopropionate, phosphoramidate derivative , imine, trityl, vinyl ether, polyketal and alkyl 2-(diphenylphosphino)benzoate derivative; alkyl ester, 8-hydroxyquinoline ester and picolinate ester).

In some embodiments, the TG may contain a reactive chemical moiety or functional group that can be cleaved under oxidizing conditions ( eg , a boronate, a vicinal diol, a paramethoxybenzyl derivative, or a selenium compound).

In certain preferred embodiments, the TG contains a saccharide that can be cleaved under acidic or enzymatic conditions. In certain preferred embodiments, the trigger group is -NO ₂ , which can be cleaved under reducing conditions. In certain preferred embodiments, the trigger group is a boronate that can be cleaved under oxidative conditions. In certain preferred embodiments, the trigger group is an ester that can be cleaved under acidic, basic, or enzymatic conditions. In certain preferred embodiments, the trigger group is a hydrazone, which can be cleaved under nucleophilic conditions or in an acidic environment. In certain preferred embodiments, the trigger group is hydroxylamine, which can be cleaved under reducing conditions.

Saccharide trigger groups

In some embodiments, the compounds and conjugates described herein include a saccharide trigger group, for example, a trigger group selected from:

;

; и

;

; And

where each R ²¹ is independently hydrogen or is selected such that OR ²¹ is a hydroxy protecting group ( eg acetyl); and R ²² is hydrogen or lower alkyl ( eg C ₁ -C ₆ alkyl). In certain embodiments, the hydroxy protecting group can be used in organic synthesis, including but not limited to: methyl ether, methoxymethyl ether, methylthiomethyl ether, 2-methoxyethoxymethyl ether, bis(2-chloroethoxy)methyl ether, tetrahydropyranyl ether, tetrahydrothiopyranyl ether, 4- methoxytetrahydropyranyl ether, 4-methoxytetrahydrothiopyranyl ether, tetrahydrofuranyl ether, 1-ethoxyethyl ether, 1-methyl-1-methoxyethyl ether, 2-(phenylselenyl)ethyl ether, tert-butyl ether, allyl ether, benzyl ether, o-nitrobenzyl ether, triphenylmethyl ether, α-naphthyldiphenylmethyl ether, p-methoxyphenyldiphenylmethyl ether, 9-(9-phenyl-10-oxo)anthryl ether, trimethylsilyl ether, isopropyldimethylsilyl ether, tert-butyldimethylsilyl ether, tert-butyldiphenylsilyl ether, tribenzylsilyl ether, triisopropylsilyl ether, formate complex ester, acetate ester, trichloroacetate ester, phenoxyacetate ester, isobutyrate ester, pivaloate ester, adamantoate ester, benzoate ester, 2,4,6-trimethylbenzoate ester, methyl carbonate, 2,2,2-trichloroethyl carbonate, but not limited to allyl carbonate, p-nitrophenyl carbonate, benzyl carbonate, p-nitrobenzyl carbonate, S-benzylthiocarbonate, N-phenyl carbamate, nitrate ester, 2,4-dinitrophenyl sulfenate ester, etc.

Protecting groups as trigger groups

In some embodiments, TG is a group that is capable of being 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 protecting group, an alcohol protecting group, or a thiol protecting group.

Amine protecting groups

In certain embodiments, an amine protecting group is a general protecting group that can be used in organic synthesis, including but not limited to m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methyl carbamate, alkyl carbamate , 9-fluorenylmethylcarbamate, 2,2,2-trichloroethylcarbamate, 2-trimethylsilylethylcarbamate (Teoc), tert-butylcarbamate (Boc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropyllylcarbamate (Ipaoc), 8-quinolylcarbamate, N- hydroxypiperidinylcarbamate, benzylcarbamate, p-methoxybenzylcarbamate, p-nitrobenzylcarbamate, diphenylmethylcarbamate, acetamide, chloroacetamide, trichloroacetamide, phenylacetamide, benzamide, N-phthalimide, N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1- dimethylthiomethyleneamine, N-benzylideneamine, benzenesulfenamide, o-nitrobenzenesulfenamide, triphenylmethylsulfanamide, p-toluenesulfonamide, methanesulfonamide, etc. , but not limited to them.

Alcohol protecting groups

In certain embodiments, an alcohol protecting group is a general protecting group that can be used in organic synthesis, including but not limited to: methyl ether, methoxymethyl ether (MOM ether), benzyloxymethyl ether (BOM ether), 2-(trimethylsilyl)ethoxymethyl ether (SEM ether), phenylthiomethyl ether (PTM ether), 2,2-dichloro-1,1-difluoroethyl ether, p-bromophenacyl ether, chloropropyl methyl ether, isopropyl ether, cyclohexyl ether, 4-methoxybenzyl, 2,6-dichlorobenzyl ether , 4-(dimethylaminocarbonyl)benzyl ether, 9-anthryl methyl 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), tert-butyl ether, allyl ether, benzyl ether , o-nitrobenzyl ether, triphenylmethyl ether, α-naphthyldiphenylmethyl ether, p-methoxyphenyldiphenylmethyl ether, 9-(9-phenyl-10-oxo)anthryl ether, trimethylsilyl ether (TMS ether), isopropyldimethylsilyl ether, tert-butyldimethylsilyl ether (TBDMS ether ), tert-butyldiphenylsilyl ether, tribenzylsilyl ether, triisopropylsilyl ether, formate ester, acetate ester, trichloroacetate ester, phenoxyacetate ester, isobutyrate ester, pivaloate ester, adamantoate ester, benzoate ester, ester 2,4 ,6-trimethylbenzoate (mesitoate), 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, but not limited to dimethylphosphinyl ester (DMP ester), dimethylthiophosphinyl ester (MPT ester), arylmethanesulfonate, aryltoluenesulfonate, etc.

Thiol protecting groups

In certain embodiments, a thiol protecting group can be used in organic synthesis, including but not limited to: S-benzylthioether, S-p-methoxybenzylthioether, So-or p-hydroxyl or acetoxybenzylthioether, S-p-nitrobenzylthioether, S-4- picolylthioether, S-2-picolyl-N-oxide-thioether, S-9-anthrylmethylthioether, S-9-fluorenylmethylthioether, S-methoxymethylmonothioacetal, A-acetyl derivative, S-benzoyl derivative, S-(N-ethylcarbamate), S- (N-methoxymethylcarbamate), etc. , but not limited to.

Link group

In some embodiments, the compounds and conjugates described herein contain a linking group that connects each CB and Ar via a covalent bond. Typical linking groups are stable, non-hydrolysable moieties, such as, for example, linear or branched, saturated or unsaturated C ₁₀ -C ₁₀₀ alkylene. In certain embodiments, the link satisfies at least two, and more preferably at least three of the four following criteria:

(i) at least one -CH ₂ - in the alkylene moiety is substituted (ie replaced) with one or more heteroatoms selected from -NH-, -C(=O), -O-, -S- and -P-;

(ii) at least one heteroarylene is included in the alkylene moiety;

(iii) at least one amino acid moiety, glycosidic bond, peptide bond, or amide bond is included in the alkylene moiety; And

(iv) the alkylene may be further substituted with one or more substituents selected from the group consisting of C ₁ -C ₂₀ alkyl, C ₆ -C ₂₀ aryl, C ₁ -C ₈ alkyl, -(CH ₂ ) _s COOH and (CH ₂ ) _p NH ₂ , where s is an integer having a value of 0 to 10 and p is an integer having a value of 1 to about 10.

In certain embodiments, the implementation of the link contains at least two, and more preferably at least three of the following:

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

(ii) at least one heteroarylene;

(iii) at least one amino acid fragment, glycosidic bond, peptide bond or amide bond; And

(iv) the alkylene may be further substituted with one or more substituents selected from the group consisting of C ₁ -C ₂₀ alkyl, C ₆ -C ₂₀ aryl, C ₁ -C ₈ alkyl, -(CH ₂ ) _s COOH and (CH ₂ ) _p NH ₂ , where s is an integer having a value of 0 to 10 and p is an integer having a value of 1 to about 10.

In other embodiments, the implementation of the linking group connecting each of CB and Ar contains a functional group resulting from a chemical click reaction.

In alternative embodiments, the implementation of the link contains a reactive functional group capable of participating in a chemical click reaction.

The chemical click reaction is a reaction that can be performed under mild conditions and is extremely selective for functional groups not normally found in biological molecules ( eg azide group, acetylene group, etc.). Accordingly, this reaction can be carried out in the presence of complex trigger groups, target fragments, etc. In addition, click chemistry has a high reaction specificity. For example, a chemical click reaction between an azide group and an acetylene group proceeds selectively, without interference from other functional groups present in the molecule. For example, as a result of azide-acetylene click chemistry, a triazole fragment can be obtained in high yield.

Таким образом, в некоторых вариантах осуществления связывающая группа, соединяющая каждый из CB и Ar, содержит

,

или

, V может представлять собой одинарную связь, -O-, -S-, -NR²¹-, -C(O)NR²² -, -NR²³C(O)-, -NR²⁴SO₂- или -SO₂NR²⁵-, каждый из R²¹-R²⁵ могут независимо представлять собой водород, (C₁-C₆)-алкил, (C₁-C₆)-алкил-(C₆-C₂₀)-арил или (C₁-C₆)алкил(C₃-C₂₀)гетероарил, r может представлять собой целое число, имеющее значение от 1 до 10, p может представлять собой целое число, имеющее значение от 0 до 10, q может представлять собой целое число, имеющее значение от 1 до 10, и L'' может представлять собой одинарную связь.

Thus, in some embodiments, the linking group connecting each of CB and Ar contains

,

or

, V may be a single bond, -O-, -S-, -NR ²¹ -, -C(O)NR ²² -, -NR ²³ C(O)-, -NR ²⁴ SO ₂ - or -SO ₂ NR ²⁵ -, each of R ²¹ -R ²⁵ can independently represent hydrogen, (C ₁ -C ₆ )-alkyl, (C ₁ -C ₆ )-alkyl-(C ₆ -C ₂₀ )-aryl or (C ₁ - C ₆ )alkyl(C ₃ -C ₂₀ )heteroaryl, r may be an integer having a value of 1 to 10, p may be an integer having a value of 0 to 10, q may be an integer having a value from 1 to 10, and L'' may be a single bond.

In other embodiments, the linker connecting each of CB and Ar is a linking group represented by formula ( A ):

Where:

* represents the point of attachment to CB;

** represents the point of attachment to Ar;

each of W ^a1 , W ^a2 and W ^a3 independently represents -NH-, -C(=O)- or (-CH ₂ -) _b ;

W ^b1 is an amide bond or triazolylene;

P ¹ is a linker connecting W ^a3 and Y ² and is an amino acid fragment, a peptide bond or an amide bond;

L ^c is alkylene;

Y ² is a single bond, -W ^a4 -(CH ₂ ) _c -W ^b2 -(CH ₂ ) _d -W ^a5 - or -W ^a6 -(CH ₂ ) _e -CR ^e R ^f -X-;

R ^e is C ₁ -C ₈ -alkyl or CB-W _a7 -Y ₃ -W _c1 -(CH ₂ ) _f -;

R ^f is BW ^a7 -Y ³ -W ^c1 -(CH ₂ ) _f -;

X is -NHC(=O)-(CH ₂ ) _g -W ^a8 - or -C(=O)NH-(CH ₂ ) _h -W ^a9 -;

each of W ^a4 , W ^a5 , W ^a6 , W ^a7 , W ^a8 and W ^a9 is independently -NH-, -C(=O)- or -CH ₂ -;

W ^b2 is an amide bond or triazolylene;

W ^c1 is -NHC(=O)- or -C(=O)NH-;

Y ³ is -(CH ₂ ) _i -(X'CH ₂ CH ₂ ) _j -(CH ₂ ) _k -;

X' represents -O-, -S-, -NH- or -CH ₂ -;

CB is as defined above;

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

each of k and y is independently an integer having a value from 0 to about 10;

Y ¹ is -(CH ₂ ) _q -(CH ₂ CH ₂ X'') _o - or -(CH ₂ ) _q -(X''CH ₂ CH ₂ ) _o -;

X'' represents -O-, -S-, -NH- or -CH ₂ -; And

o and q are an integer having a value from 1 to about 10;

In some embodiments, P ¹ contains at least one unit represented by formula ( B ) or ( C ):

Where:

R ¹² is hydrogen, C ₁ -C ₈ alkyl, an amino acid side chain such as a naturally occurring amino acid side chain ( e.g. H, methyl, isopropyl, isobutyl, sec-butyl, S-methylthioether, benzyl, indole, pyrrolidine, pyrroline , hydroxymethyl, tyrosyl, lysyl, imidazole, glycyl, glutamyl, carbamoylbutanoic acid, carboxamide, aspartic acid, 1-hydroxyethyl and 2-hydroxyethyl), -(CH ₂ ) _s COR ¹³ or -(CH ₂ ) _p NR ¹⁴ R ¹⁵ ;

R ¹³ is OH or -NH(CH ₂ ) _s '(X''CH ₂ CH ₂ ) _s'' Z;

each of R ¹⁴ and R ¹⁵ independently represents hydrogen or -(C(O)(CH ₂ ) _s '(X''CH ₂ CH ₂ ) _s'' Z) _m -CB;

X'' represents -O-, -S-, -NH- or -CH ₂ -;

Z and CB are as defined above;

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

s and s'' are an integer having a value from 0 to about 10;

s' is an integer having a value from 1 to about 10; And

m is an integer having the value 0 or 1.

In some embodiments of formula (B) or (C):

R ¹² is hydrogen, alkyl, amino acid side chain, -(CH ₂ ) _s C(O)R ¹³ or -(CH ₂ ) _p NR ¹⁴ R ¹⁵ ;

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

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

R ¹³ is OH or -NH(CH ₂ ) _s '(X'''CH ₂ CH ₂ ) _s'' Z''-(CB) _m ;

each of R ¹⁴ and R ¹⁵ independently represents hydrogen or -C(O)(CH ₂ ) _s '(X'''CH ₂ CH ₂ ) _s'' Z''-(CB) _m ;

s'' is an integer having a value from 0 to about 10;

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

m is an integer having a value of 0 or 1;

X''' represents -O-, -S-, -NH- or -CH ₂ -; And

Z'' represents a linking group connecting CB with the remainder of R ¹⁴ or R ¹⁵ ; or Z'' represents a linking group containing a reactive group.

In some such embodiments of formula (B) or (C):

R ¹³ is OH or -NH(CH ₂ ) _s' (X'''CH ₂ CH ₂ ) _s'' Z'';

each of R ¹⁴ and R ¹⁵ is independently hydrogen or —C(O)(CH ₂ ) _s' (X'''CH ₂ CH ₂ ) _s'' Z''; And

,

, фосфоновой кислоты (-P(=O)(OH)₂), кетона, C₈-C₁₀ циклоалкинила, -OH, -NHOH, -NHNH₂, -SH, карбоновой кислоты (-COOH), ацетилена (-C≡CH), азида (-N₃), амино (-NH₂), сульфоновой кислоты (-SO₃H), производного алкинона (-C(O)C≡C-R^a, где R^a представляет собой C₁-C₁₀-алкил) и дигидрофосфат (-OP(=O)(OH)₂).Z'' is a reactive linker precursor selected from isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH ₂ -halogen), maleimide, diene, alkene, halide, tosylate (TsO ^- ), aldehyde, sulfonate (R-SO ₃ ^- ),

,

, phosphonic acid (-P (= O) (OH) ₂ ), ketone, C ₈ -C ₁₀ cycloalkynyl, -OH, -NHOH, -NHNH ₂ , -SH, carboxylic acid (-COOH), acetylene (-C≡ CH), azide (-N ₃ ), amino (-NH ₂ ), sulfonic acid (-SO ₃ H), alkynone derivative (-C(O)C≡CR ^a , where R ^a is C ₁ -C ₁₀ - alkyl) and dihydrophosphate (-OP(=O)(OH) ₂ ).

In other such embodiments of formula (B) or (C):

R ¹³ is OH-NH(CH ₂ ) _s' (X'''CH ₂ CH ₂ ) _s'' Z''CB;

each of R ¹⁴ and R ¹⁵ independently represents hydrogen or C(O)(CH ₂ ) _s' (X'''CH ₂ CH ₂ ) _s'' Z''CB; And

,

, фосфоновой кислоты (-P(=O)(OH)₂), кетона, C₈-C₁₀ циклоалкинила, -OH, -NHOH, -NHNH₂, -SH, карбоновой кислоты (-COOH), ацетилена (-C≡CH), азида (-N₃), амино (-NH₂), сульфоновой кислоты (-SO₃H), производного алкинона (-C(O)C≡C-R^a, где R^a представляет собой C₁-C₁₀-алкил) и дигидрофосфат (-OP(=O)(OH)₂).Z'' is a link connecting CB to an R ¹⁴ or R ¹⁵ residue formed from a precursor selected from isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH ₂ -halogen), maleimide, diene, alkene , halide, tosylate (TsO ^- ), aldehyde, sulfonate (R-SO ₃ ^- ),

,

, phosphonic acid (-P (= O) (OH) ₂ ), ketone, C ₈ -C ₁₀ cycloalkynyl, -OH, -NHOH, -NHNH ₂ , -SH, carboxylic acid (-COOH), acetylene (-C≡ CH), azide (-N ₃ ), amino (-NH ₂ ), sulfonic acid (-SO ₃ H), alkynone derivative (-C(O)C≡CR ^a , where R ^a is C ₁ -C ₁₀ - alkyl) and dihydrophosphate (-OP(=O)(OH) ₂ ).

In some embodiments, Y ² is a single bond selected from

;

;

;

; и

;

;

;

; And

Where:

или

;W ^b2 is -C(O)NH-, -NHC(O)-,

or

;

R ^e represents C ₁ -C ₈ -alkyl or -(L ¹ '-Z-) _m CB;

R ^f is BW _b2 '-(CH ₂ ) _i -(X''CH ₂ CH ₂ ) _j -NH-C(=O)-(CH ₂ ) _f -;

X ^a is -NHC(=O)-(CH ₂ ) _g -NH- or -C(O)NH-(CH ₂ ) _h -NH-;

W ^b2 is -C(O)NH- or -NHC(=O)-;

c, d, e, f, g, h, i, and j are each independently an integer having a value from 1 to about 10;

X'' represents -O-, -S-, -NH- or -CH ₂ -; And

L ¹ ', Z, m and B are as defined above;

In certain embodiments, the link connecting each of CB and Ar is a link group containing groups (CH ₂ ) _b , L ^c , (P ¹ ) _a , W ^a1 , W ^a2 , W ^a3 , Y ¹ and Y ² , linked to each other by covalent bonds, where:

each of W ^a1 , W ^a2 and W ^a3 independently represents -NH-, -C(O)- or -CH ₂ -;

W ^b1 is an amide bond or triazolylene;

P ¹ is an amide bond, amino acid residue or peptide;

L ^c is alkylene;

Y ¹ is -(CH ₂ ) _q -(CH ₂ CH ₂ X'') _o - or -(CH ₂ ) _q -(X''CH ₂ CH ₂ X'') _o -;

X'' represents -O-, -S-, -NH- or -CH ₂ -;

Y ² is a single bond or a group selected from:

;

;

;

;

; и

; And

W ^b2 is an amide bond or triazolylene;

a is from 0 to 10;

each of b, c and d is independently an integer having a value from 1 to about 10; And

each of o and q is independently an integer having a value from 1 to about 10.

In some embodiments, R ¹² is a naturally occurring amino acid side chain. In other embodiments, R ¹² is a non-natural amino acid side chain.

In some embodiments, the linker connecting each of CB and Ar is a linking group represented by formula ( A ):

Where:

* represents the point of attachment to CB; And

** represents the point of attachment to Ar.

In some such embodiments, P ¹ is

или

or

Where:

R ¹² is hydrogen, alkyl, amino acid side chain, -(CH ₂ ) _s COOH or -(CH ₂ ) _p NH ₂ ;

p is an integer having a value from 1 to about 10; And

each of s and s'' is independently an integer having a value from 0 to about 10.

In some embodiments, P ¹ is

или

or

Where:

R ¹² is hydrogen, alkyl, amino acid side chain, -(CH ₂ ) _s C(O)R ¹³ or -(CH ₂ ) _p NR ¹⁴ R ¹⁵ ;

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

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

R ¹³ is OH or -NH(CH ₂ ) _s' (X'''CH ₂ CH ₂ ) _s'' Z''-(CB) _m ;

each of R ¹⁴ and R ¹⁵ independently represents hydrogen or -C(O)(CH ₂ ) _s' (X'''CH ₂ CH ₂ ) _s'' Z''-(CB) _m ;

s'' is an integer having a value from 0 to about 10;

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

m is an integer having a value of 0 or 1;

X''' represents -O-, -S-, -NH- or -CH ₂ -; And

Z'' represents a linking group connecting CB with the remainder of R ¹⁴ or R ¹⁵ ; or Z'' represents a linking group containing a reactive group.

In some such embodiments, P ¹ :

R ¹³ is OH or -NH(CH ₂ ) _s' (X'''CH ₂ CH ₂ ) _s'' Z'';

each of R ¹⁴ and R ¹⁵ is independently hydrogen or —C(O)(CH ₂ ) _s' (X'''CH ₂ CH ₂ ) _s'' Z''; And

,

, фосфоновой кислоты (-P(=O)(OH)₂), кетона, C₈-C₁₀ циклоалкинила, -OH, -NHOH, -NHNH₂, -SH, карбоновой кислоты (-COOH), ацетилена (-C≡CH), азида (-N₃), амино (-NH₂), сульфоновой кислоты (-SO₃H), производного алкинона (-C(O)C≡C-R^a, где R^a представляет собой C₁-C₁₀-алкил) и дигидрофосфат (-OP(=O)(OH)₂).Z'' is a reactive linker precursor selected from isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH ₂ -halogen), maleimide, diene, alkene, halide, tosylate (TsO ^- ), aldehyde, sulfonate (R-SO ₃ ^- ),

,

, phosphonic acid (-P (= O) (OH) ₂ ), ketone, C ₈ -C ₁₀ cycloalkynyl, -OH, -NHOH, -NHNH ₂ , -SH, carboxylic acid (-COOH), acetylene (-C≡ CH), azide (-N ₃ ), amino (-NH ₂ ), sulfonic acid (-SO ₃ H), alkynone derivative (-C(O)C≡CR ^a , where R ^a is C ₁ -C ₁₀ - alkyl) and dihydrophosphate (-OP(=O)(OH) ₂ ).

In other such embodiments, P ¹ :

R ¹³ is OH-NH(CH ₂ ) _s' (X'''CH ₂ CH ₂ ) _s'' Z''CB;

each of R ¹⁴ and R ¹⁵ independently represents hydrogen or C(O)(CH ₂ ) _s' (X'''CH ₂ CH ₂ ) _s'' Z''CB; And

,

, фосфоновой кислоты (-P(=O)(OH)₂), кетона, C₈-C₁₀ циклоалкинила, -OH, -NHOH, -NHNH₂, -SH, карбоновой кислоты (-COOH), ацетилена (-C≡CH), азида (-N₃), амино (-NH₂), сульфоновой кислоты (-SO₃H), производного алкинона (-C(O)C≡C-R^a, где R^a представляет собой C₁-C₁₀-алкил) и дигидрофосфат (-OP(=O)(OH)₂).Z'' is a link connecting CB to an R ¹⁴ or R ¹⁵ residue formed from a precursor selected from isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH ₂ -halogen), maleimide, diene, alkene , halide, tosylate (TsO ^- ), aldehyde, sulfonate (R-SO ₃ ^- ),

,

, phosphonic acid (-P (= O) (OH) ₂ ), ketone, C ₈ -C ₁₀ cycloalkynyl, -OH, -NHOH, -NHNH ₂ , -SH, carboxylic acid (-COOH), acetylene (-C≡ CH), azide (-N ₃ ), amino (-NH ₂ ), sulfonic acid (-SO ₃ H), alkynone derivative (-C(O)C≡CR ^a , where R ^a is C ₁ -C ₁₀ - alkyl) and dihydrophosphate (-OP(=O)(OH) ₂ ).

In alternative embodiments, the linking link connecting CB and Ar is a linking group represented by formulas ( F ), ( G ), ( H ), ( J ), ( K ), ( L ), ( M ), or ( N ) :

Where:

R ^e is alkyl;

X ⁴ is -NHC(O)-(CH ₂ ) _g -NH- or -C(O)NH-(CH ₂ ) _h -NH-;

each of e, g and h is independently an integer having a value from 1 to about 10; And

s' is an integer having a value from 1 to about 10.

In some embodiments of formula ( F ), ( G ), ( H ), ( I ), ( J ), ( K ), ( L ), or ( M ):

R ¹² is hydrogen, alkyl, amino acid side chain, -(CH ₂ ) _s C(O)R ¹³ or -(CH ₂ ) _p NR ¹⁴ R ¹⁵ ;

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

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

R ¹³ is OH or -NH(CH ₂ ) _s' (X'''CH ₂ CH ₂ ) _s'' Z''-(CB) _m ;

each of R ¹⁴ and R ¹⁵ independently represents hydrogen or -C(O)(CH ₂ ) _s' (X'''CH ₂ CH ₂ ) _s'' Z''-(CB) _m ;

s'' is an integer having a value from 0 to about 10;

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

m is an integer having a value of 0 or 1;

X''' represents -O-, -S-, -NH- or -CH ₂ -; And

Z'' represents a linking group connecting CB with the remainder of R ¹⁴ or R ¹⁵ ; or Z'' represents a linking group containing a reactive group.

In some embodiments of formula ( F ), ( G ), ( H ), ( I ), ( J ), ( K ), ( L ), or ( M ):

R ¹³ is OH or -NH(CH ₂ ) _s' (X'''CH ₂ CH ₂ ) _s'' Z'';

each of R ¹⁴ and R ¹⁵ is independently hydrogen or —C(O)(CH ₂ ) _s' (X'''CH ₂ CH ₂ ) _s'' Z''; And

,

, фосфоновой кислоты (-P(=O)(OH)₂), кетона, C₈-C₁₀ циклоалкинила, -OH, -NHOH, -NHNH₂, -SH, карбоновой кислоты (-COOH), ацетилена (-C≡CH), азида (-N₃), амино (-NH₂), сульфоновой кислоты (-SO₃H), производного алкинона (-C(O)C≡C-R^a, где R^a представляет собой C₁-C₁₀-алкил) и дигидрофосфат (-OP(=O)(OH)₂).Z'' is a reactive linker precursor selected from isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH ₂ -halogen), maleimide, diene, alkene, halide, tosylate (TsO ^- ), aldehyde, sulfonate (R-SO ₃ ^- ),

,

, phosphonic acid (-P (= O) (OH) ₂ ), ketone, C ₈ -C ₁₀ cycloalkynyl, -OH, -NHOH, -NHNH ₂ , -SH, carboxylic acid (-COOH), acetylene (-C≡ CH), azide (-N ₃ ), amino (-NH ₂ ), sulfonic acid (-SO ₃ H), alkynone derivative (-C(O)C≡CR ^a , where R ^a is C ₁ -C ₁₀ - alkyl) and dihydrophosphate (-OP(=O)(OH) ₂ ).

In some embodiments of formula ( F ), ( G ), ( H ), ( I ), ( J ), ( K ), ( L ), or ( M ):

R ¹³ is OH-NH(CH ₂ ) _s' (X'''CH ₂ CH ₂ ) _s'' Z''CB;

each of R ¹⁴ and R ¹⁵ independently represents hydrogen or C(O)(CH ₂ ) _s' (X'''CH ₂ CH ₂ ) _s'' Z''CB; And

,

, фосфоновой кислоты (-P(=O)(OH)₂), кетона, C₈-C₁₀ циклоалкинила, -OH, -NHOH, -NHNH₂, -SH, карбоновой кислоты (-COOH), ацетилена (-C≡CH), азида (-N₃), амино (-NH₂), сульфоновой кислоты (-SO₃H), производного алкинона (-C(O)C≡C-R^a, где R^a представляет собой C₁-C₁₀-алкил) и дигидрофосфат (-OP(=O)(OH)₂).Z'' is a link connecting CB to an R ¹⁴ or R ¹⁵ residue formed from a precursor selected from isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH ₂ -halogen), maleimide, diene, alkene , halide, tosylate (TsO ^- ), aldehyde, sulfonate (R-SO ₃ ^- ),

,

, phosphonic acid (-P (= O) (OH) ₂ ), ketone, C ₈ -C ₁₀ cycloalkynyl, -OH, -NHOH, -NHNH ₂ , -SH, carboxylic acid (-COOH), acetylene (-C≡ CH), azide (-N ₃ ), amino (-NH ₂ ), sulfonic acid (-SO ₃ H), alkynone derivative (-C(O)C≡CR ^a , where R ^a is C ₁ -C ₁₀ - alkyl) and dihydrophosphate (-OP(=O)(OH) ₂ ).

Targeting Fragments

Compounds and conjugates of the present invention may further contain a ligand or targeting moiety, CB. In some embodiments, a ligand or targeting moiety is any molecular recognition element that can undergo a specific interaction with at least one other molecular moiety through, for example, non-covalent bonds such as hydrogen bonds, metal coordination, hydrophobic forces, Van- der Waals, π-π interactions, halogen bonds, electrostatic and/or electromagnetic effects. In certain embodiments, CB is selected from nanoparticles, immunoglobulin, nucleic acid, protein, oligopeptide, polypeptide, antibody, antigenic polypeptide fragment, repebody, and the like.

The compounds and conjugates of the present invention may contain one or more targeting moieties. That is, the variable cb can have an integer value selected from 1, 2, 3, 4, 5, 1-10, or 1-20.

In some embodiments, the CB contains two or more independently selected naturally occurring amino acids or unnatural amino acids conjugated by covalent bonds ( e.g. , peptide bonds) and the peptide may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more natural amino acids or non-natural amino acids that are conjugated by peptide bonds. In some embodiments, the implementation of the ligand contains shorter amino acid sequences ( for example , fragments of natural proteins or fragments of a synthetic polypeptide), as well as full-length proteins ( for example , pre-engineered proteins).

In some embodiments, CB is selected from an antibody, hormone, drug, antibody analog ( eg , non-IgG), protein, oligopeptide, polypeptide , etc. that binds to the receptor. In certain embodiments, CB selectively targets a drug to a specific organ, tissue, or cell. In other embodiments, CB specifically binds to a receptor overexpressed in cancer cells compared to normal cells and can be classified into a monoclonal antibody (mAb) or antibody fragment and a non-antibody small molecule. Preferably, CB is selected from peptides, tumor cell-specific peptides, tumor cell-specific aptamers, tumor cell-specific carbohydrates, tumor cell-specific monoclonal antibodies, polyclonal antibodies, and antibody fragments that have been identified by library screening.

Typical ligands or targeting moieties include, but are not limited to, carnitine, inositol, lipoic acid, pyridoxal, ascorbic acid, niacin, pantothenic acid, folic acid, riboflavin, thiamine, biotin, vitamin B ₁₂ , other water-soluble vitamins (vitamin B), fat-soluble vitamins (vitamin A, D, E, K), RGD (Arg-Gly-Asp), NGR (Asn-Gly-Arg), transferrin, VIP receptor (vasoactive intestinal peptide), APRPG peptide (Ala-Pro-Arg- Pro-Gly), TRX-20 (thioredoxin-20), integrin, nucleolin, aminopeptidase N (CD13), endoglin, vascular growth factor receptor, low density lipoprotein receptor, transferrin receptor, somatostatin receptor, bombesin, neuropeptide Y, hormone receptor , luteinizing hormone releasing hormone, 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), receptor cholecystokinin A, cholecystokinin B receptor, discoidin domain receptor, mucin receptor, opioid receptor, plasminogen receptor, bradykinin receptor, insulin receptor, insulin-like growth factor receptor, angiotensin AT1 receptor, angiotensin AT2 receptor, granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor ), galactosamine receptor, sigma-2 receptor, delta-like ligand-3 (DLL-3), aminopeptidase P, melanotransferrin, leptin, tetanus toxin Tet1, tetanus toxin G23, RVG peptide (rabies virus glycoprotein), HER2 (epidermal receptor 2 human growth factor), GPNMB (non-metastatic glycoprotein b), Ley, CA6, CanAng, SLC44A4 (solute transporter family 44, member 4), CEACAM5 (cell adhesion molecule 5 associated with carcinoembryonic antigen), nectin-4, carboxylic anhydrase 9 , TNNB2, 5T4, CD30, CD37, CD74, CD70, PMEL17, EphA2(ephrin A2 receptor), Trop-2, SC-16, tissue factor, ENPP-3(AGS-16), SLITRK6 (SLIT- and NTRK-like family member 6), CD27, Lewis Y antigen, LIV1, GPR161 (G protein-coupled receptor 161), PBR (peripheral benzodiazeoin receptor), MERTK receptor (Mer receptor tyrosine kinase), CD71, LLT1 (lectin-like transcript 1 or CLED2D), interleukin-22 receptor, sigma-1 receptor, peroxisome proliferator-activated receptor, DLL3, C4.4a, cKIT, EphrinA, CTLA4 (cytotoxic T-lymphocyte-associated protein 4), FGFR2b (fibroblast growth factor receptor 2b), N receptor -acetylcholine, gonadotropin releasing hormone receptor, gastrin-releasing peptide receptor, bone morphogenetic protein receptor, receptor type 1B (BMPR1B), E16 (LAT1, SLC7A5), STEAP1 (prostate transmembrane epithelial antigen sixth), 0772P (CA125, MUC16) , MPF (MSLN, mesothelin), Napi3b (SLC34A2), Sema5b (semaphorin 5b), ETBR (endothelin type B receptor), MSG783(RNF124), STEAP2 (6th prostate transmembrane epithelial antigen 2), TrpM4 (transient receptor potential cation channel 5 , subfamily M, member 4), CRIPTO (growth factor derived from teratocarcinoma), CD21, CD79b, FcRH2 (IFGP4), HER2 (ErbB2), NCA (CEACM6), MDP (DPEP1), IL20R-alpha (IN20Ra), Brevican (BCAN), EphB2R, ASLG659 (B7h), CD276, PSCA (prostate stem cell antigen precursor), GEDA, BAFF-R (BR3), CD22 (BL-CAM), CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY64, FcRH1, IRTA2, TENB2, SSTR2, SSTR5, SSTR1, SSTR3, SSTR4, ITGAV (integrin, alpha 5), ITGB6 (integrin, beta 6), MET, MUC1, EGFRvIII, CD33, CD19, IL2RA (interleukin 2 receptor, alpha), AXL, BCMA, CTA (cancer testicular antigens), CD174, CLEC14A, GPR78, CD25, CD32, LGR5 (GPR49), CD133 (prominine), ASG5, ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase 3), PRR4 (proline -rich protein 4), GCC (guanylate cyclase 2C), Liv-1 (SLC39A6), CD56, CanAg, TIM-1, RG-1, B7-H4, PTK7, CD138, Claudins, Her3 (ErbB3), RON (MST1R ), CD20, TNC (tenascin C), FAP, DKK-1, CD52, CS1 (SLAMF7), annexin A1, V-CAM, gp100, MART-1, MAGE-1 (gene-1 encoding melanoma antigen), MAGE -3 (melanoma-associated antigen 3), BAGE, GAGE-1, MUM-1 (multiple myeloma oncogene 1), CDK4, TRP-1(gp75), TAG-72 (tumor-associated glycoprotein-72), ganglioside GD2, GD3, GM2, GM3, VEP8, VEP9, My1, VIM-D5, D156-22, OX40, RNAK, PD-L1, TNFR1, TNFR2, etc.

targets

In some embodiments, the target or targets of the molecular recognition element are specifically associated with one or more specific cell or tissue types. In some embodiments, targets are specifically associated with one or more specific disease states. In some embodiments, targets are specifically associated with one or more specific developmental stages. For example, a cell type-specific marker is typically expressed at levels at least 2 times greater in that cell type than in a reference cell population. In some embodiments, the cell type-specific marker is present at levels of at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 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 mean expression in the control population. Detection or measurement of a cell type-specific marker may allow the cell type or types of interest to be distinguished from many, most, or all other cell types. In some embodiments, the target may comprise a protein, carbohydrate, lipid, and/or nucleic acid, as described herein.

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

In certain embodiments, the conjugates and compounds described herein contain a targeting moiety that specifically binds to one or more targets ( e.g. , antigens) associated with an organ, tissue, cell, extracellular matrix component, and/or intracellular compartment. In some embodiments, the conjugates and compounds described herein contain a targeting moiety that specifically binds to targets associated with a particular organ or organ system. In some embodiments, the conjugates and compounds described herein contain a targeting moiety that specifically binds to one or more intracellular targets (eg, organelle, intracellular protein). In some embodiments, the conjugates and compounds described herein contain a targeting moiety that specifically binds to targets associated with diseased organs, tissues, cells, extracellular matrix components, and/or intracellular compartments. In some embodiments, the conjugates and compounds described herein contain a targeting moiety that specifically binds to targets associated with particular cell types ( e.g. , endothelial cells, cancer cells, cancer cells, prostate cancer cells , etc. ) .

In some embodiments, the conjugates and compounds described herein contain a targeting moiety that binds to a target that is specific to one or more specific tissue types ( eg , liver tissue or prostate tissue). In some embodiments, the conjugates and compounds described herein contain a targeting moiety that binds to a target that is specific to one or more particular cell types ( eg , T cells versus B cells). In some embodiments, the conjugates and compounds described herein contain a targeting moiety that binds to a target that is specific to one or more particular disease states ( eg , tumor cells versus healthy cells). In some embodiments, the conjugates and compounds described herein contain a targeting moiety that binds to a target that is specific to one or more particular developmental stages ( eg , stem cells versus differentiated cells).

In some embodiments, the target may be a marker that is exclusively or primarily associated with one or more cell types, one or more diseases, and/or one or more developmental stages. A cell type-specific marker is typically expressed at levels at least 2 times greater in that cell type than in a reference cell population, which may consist, for example, of a mixture containing cells from a plurality ( e.g. , 5-10 or more) various tissues or organs in approximately equal amounts. In some embodiments, the cell type-specific marker is present at levels of at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 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 mean expression in the control population. Detection or measurement of a cell type-specific marker may allow the cell type or types of interest to be distinguished from many, most, or all other cell types.

In some embodiments, the target comprises a protein, carbohydrate, lipid, and/or nucleic acid. In some embodiments, the target contains a protein and/or a characteristic portion thereof, such as a tumor marker, an integrin, a cell surface receptor, a transmembrane protein, an intercellular protein, an ion channel, a membrane transport protein, an enzyme, an antibody, a chimeric protein, a glycoprotein, etc. e. In some embodiments, the target contains a carbohydrate and/or a characteristic portion thereof, such as a glycoprotein, a sugar ( e.g. , a monosaccharide, a disaccharide, a polysaccharide), a glycocalyx ( i.e., a carbohydrate-rich peripheral zone on the outer surface of most eukaryotic cells) , etc. e. In some embodiments, the target contains a lipid and/or a characteristic portion thereof, such as an oil, a fatty acid, a glyceride, a hormone, a steroid ( eg , cholesterol, bile acid), a vitamin ( eg , vitamin E), a phospholipid, a sphingolipid, lipoprotein, etc. In some embodiments, the target comprises a nucleic acid and/or a characteristic portion thereof, such as a DNA nucleic acid; nucleic acid RNA; modified nucleic acid DNA; modified nucleic acid RNA; a nucleic acid that includes any combination of DNA, RNA, modified DNA, and modified RNA.

Numerous markers are known in the art. Exemplary markers include cell surface proteins such as receptors. Exemplary receptors include, but are not limited to, the transferrin receptor; LDL receptor; growth factor receptors, such as members of the epidermal growth factor receptor family (eg, 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 that is present exclusively or in large quantities in the malignant cell, for example, in a tumor antigen.

Nanoparticles

In some embodiments, the targeting moiety comprises a particle ( eg , a target particle), preferably a nanoparticle, optionally a target nanoparticle, attached to a targeting molecule that can specifically or preferentially bind to the target. In some embodiments, the targeting particle itself targets the compound of the present invention (eg, by enrichment in tumor cells or tissue) and no additional targeting molecules are attached to it.

The term "nanoparticle" in this document refers to any particle having a diameter of less than 1000 nm. In some embodiments, the implementation of the therapeutic agent and/or targeting molecule can be associated with the particle body, for example, in a polymer matrix. In some embodiments, the targeting molecule may be covalently attached to the surface of the polymer matrix. In some embodiments, the covalent association is mediated by a linker. In some embodiments, the therapeutic agent may be surface bound, internally encapsulated, surrounded, and/or dispersed throughout the polymer matrix. See, for example, US Pat. No. 8,246,968, which is incorporated herein in its entirety.

In general, the nanoparticles of the present invention contain particles of any type. Any particle can be used in accordance with the present invention. In some embodiments, the particles are biodegradable and biocompatible. As a rule, a biocompatible substance is not toxic to cells. In some embodiments, a substance is considered biocompatible if its addition to cells results in less than a certain threshold of cell death. In some embodiments, the implementation of the substance is considered biocompatible if its addition to the cells does not cause side effects. Typically, a biodegradable substance is one that undergoes degradation under physiological conditions over a therapeutically relevant period of time ( eg , weeks, months, or years). In some embodiments, the implementation of the biodegradable substance is a substance that can be destroyed by cellular machinery. In some embodiments, a biodegradable substance is a substance that can be degraded by chemical processes. In some embodiments, the particle is a substance that is both biocompatible and biodegradable. In some embodiments, the particle is a substance that is biocompatible but not biodegradable. In some embodiments, the particle is a substance that is biodegradable but not biocompatible.

It is often desirable to use a population of particles that is relatively uniform in terms of size, shape and/or composition so that each particle has similar properties. For example, at least 80%, at least 90%, or at least 95% of the particles may have a diameter or largest size that is within 5%, 10%, or 20% of the average diameter or largest size. In some embodiments, the implementation of the population of particles may be heterogeneous in size, shape and/or composition. Many different particles can be used in accordance with the present invention. In some embodiments, the particles are spheres or spheroids. In some embodiments, the particles are spheres or spheroids. In some embodiments, the particles are flat or plate-like. In some embodiments, the particles are cubes or cuboids. In some embodiments, the particles are ovals or ellipses. In some embodiments, the particles are cylinders, cones, or pyramids.

In some embodiments, the particles are microparticles (eg, microspheres). In general, "microparticle" refers to any particle having a diameter of less than 1000 microns. In some embodiments, the particles are pico-particles ( e.g. picospheres). In general, "picoparticle" refers to any particle having a diameter less than 1 nm. In some embodiments, the particles are liposomes. In some embodiments, the particles are micelles.

The particles may be solid or hollow and may contain one or more layers ( eg nanoshells, nanorings). In some embodiments, each layer has a unique composition and unique properties with respect to the other layer(s). For example, the particles may have a core/shell structure where the core is one layer and the shell is the second layer. The particles may contain many different layers. In some embodiments, one layer may be substantially crosslinked, the second layer may be substantially uncrosslinked, and so on. In some embodiments, one, more, or all of the different layers may contain one or more therapeutic or diagnostic agents to be delivered. In some embodiments, one layer contains the agent to be delivered, the second layer does not contain the agent to be delivered, and so on. In some embodiments, each individual layer contains a different agent or set of agents to be delivered.

In some embodiments, the implementation of the particle is porous, which means that the particle contains holes or channels, which are usually small compared to the size of the particles. For example, the particle may be a porous silica particle, such as a mesoporous silica nanoparticle, or may be coated with mesoporous silica (Lin et al., 2005, J. Am. Chem. Soc, 17:4570). The particles may have pores ranging from about 1 nm to about 50 nm, for example, from about 1 to 20 nm in diameter. From about 10% to 95% of the volume of the particle may consist of voids within the pores or channels.

The particles may have a coating layer. The use of a biocompatible coating layer can be beneficial, for example, if the particles contain materials that are toxic to cells. Suitable coating materials include, but are not limited to, naturally occurring proteins such as bovine serum albumin (BSA), biocompatible hydrophilic polymers such as polyethylene glycol (PEG) or a PEG derivative, phospholipid-(PEG), silica, lipids, polymers, carbohydrates. such as dextran, other nanoparticles that can be bonded to the nanoparticles of the present invention, etc. Coatings can be applied or assembled in various ways such as dipping, layering, self-assembly, interfacing, etc. Self-assembly refers to the process of spontaneous assembly a higher order structure that depends on the natural attraction of higher order structure components (eg molecules) to each other. This usually occurs as a result of the random movements of molecules and the formation of bonds depending on the size, shape, composition or chemical properties.

Examples of polymers include polyalkylenes ( eg polyethylenes), polycarbonates ( eg poly(1,3-dioxan-2-one)), polyanhydrides ( eg poly(sebacic anhydride)), polyhydroxy acids (eg poly(3-hydroxyalkanoate)) , polyfumarates, polycaprolactones, polyamides ( e.g. polycaprolactam), polyacetals, polyesters, polyesters ( e.g. polylactide, polyglycolide), poly(orthoesters), polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, and polyamines . In some embodiments, the polymers of the present invention include polymers that have been approved for use in humans by the U.S. Food and Drug Administration (FDA) under 21 CFR (Code of Federal Regulations) § 177.2600, including but not limited to them, polyesters ( eg , polylactic acid, polyglycolic acid, poly(lactic-glycolic acid), polycaprolactone, polyvalerolactone, poly(1,3-dioxan-2-one)); polyanhydrides ( e.g. poly(sebacic anhydride)); polyethers ( e.g. polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates; and polycyanoacrylates.

In some embodiments, the particles may be non-polymeric particles ( eg , metal particles, quantum dots, ceramic particles, polymers containing inorganic materials, bone-derived materials, bone substitutes, viral particles, etc. ). In some embodiments, the therapeutic or diagnostic agent to be delivered may be associated with the surface of such a non-polymeric particle. In some embodiments, the implementation of the non-polymeric particle is a collection of non-polymeric components, such as a collection of metal atoms ( for example , gold atoms). In some embodiments, the therapeutic or diagnostic agent to be delivered may be surface bound and/or internally encapsulated, surrounded, and/or dispersed in a plurality of non-polymeric components.

Particles ( eg nanoparticles, microparticles) can be obtained by any method known in the art. For example, particulate formulations can be prepared by methods such as nanoprecipitation, flow focused fluid channels, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, milling, microemulsion procedures, micromachining, nanomachining, sacrificial layers, simple and complex coacervation and other suitable methods. Alternatively or additionally, the synthesis of aqueous and organic solvents for monodisperse semiconductor, conductive, magnetic, organic and other nanoparticles has been described (Pellegrino et al., 2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat Sci., 30:545 and Trindade et al., 2001, Chem. Mat., 13:3843).

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

Targeting nucleic acid fragments

In some embodiments, the targeting fragment comprises a targeting nucleic acid fragment.

Typically, a targeting nucleic acid fragment is any polynucleotide that binds to a component associated with an organ, tissue, cell, extracellular matrix component, and/or intracellular compartment (target).

In some embodiments, targeting nucleic acid fragments are aptamers. An aptamer is typically a polynucleotide that binds to a specific target structure that is associated with a particular organ, tissue, cell, extracellular matrix component, and/or intracellular compartment. Typically, the targeting function of an aptamer is based on the three-dimensional structure of the aptamer. In some embodiments, the binding of the aptamer to the target is typically mediated by the interaction of two- and/or three-dimensional structures of both the aptamer and the target. In some embodiments, the binding of the aptamer to the target is not only based on the primary sequence of the aptamer, but depends on the three-dimensional structure(s) of the aptamer and/or target. In some embodiments, aptamers bind to their targets via complementary Watson-Crick base pairing that is interrupted by structures ( eg , hairpin loops) that disrupt base pairing.

In some embodiments, the targeting nucleic acid fragments are Spiegelmers (PCT Publications WO 98/08856, WO 02/100442 and WO 06/117217). In general, Spiegelmers are synthetic mirror-image nucleic acids that can specifically bind to a target (ie, mirror-image aptamers). Spiegelmers are characterized by structural features that make them resistant to exo- and endonucleases.

One of skill in the art will appreciate that any targeting nucleic acid fragment ( eg , aptamer or spiegelmer) that is capable of specifically binding to a target can be used in accordance with the present invention. In some embodiments, targeting nucleic acid fragments to be used in accordance with the present invention may target a marker associated with a disease, disorder, and/or condition. In some embodiments, targeting nucleic acid fragments to be used in accordance with the present invention may target cancer-related targets. In some embodiments, targeting nucleic acid fragments to be used in accordance with the present invention may target tumor markers. Any type of cancer and/or any tumor marker can be targeted using targeting nucleic acid fragments in accordance with the present invention. To name just a few examples, targeting nucleic acid fragments can target markers associated with prostate cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, endometrial cancer, ovarian cancer, bone cancer, esophageal cancer, liver cancer, stomach cancer, brain tumor, skin melanoma and/or leukemia.

Nucleic acids of the present invention (including targeting nucleic acid fragments and/or functional RNAs to be delivered, e.g. a technique including, but not limited to, chemical synthesis, enzymatic synthesis, enzymatic or chemical cleavage of a longer precursor , etc.

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

The nucleic acid that forms the targeting nucleic acid fragment may include naturally occurring nucleosides, modified nucleosides, naturally occurring nucleosides with hydrocarbon linkers ( e.g. alkylene) or a polyester linker ( e.g. PEG linker) inserted between one or more nucleosides modified nucleosides with hydrocarbon or PEG linkers inserted between one or more nucleosides, or a combination thereof. In some embodiments, the nucleotides or modified nucleotides of the targeting nucleic acid fragment may be replaced with a hydrocarbon linker or a polyester linker, provided that the binding affinity and selectivity of the targeting nucleic acid fragment are not significantly reduced by the substitution ( e.g. , the constant of the targeting nucleic acid fragment on the target should not be exceed about 1 × 10 ^-3 M).

Those skilled in the art will appreciate that the nucleic acids of the present invention may contain nucleotides that are wholly of the types found in naturally occurring nucleic acids, or may instead include one or more nucleotide analogs, or have a structure that is otherwise different. from natural nucleic acid. US Patents No. 6403779; 6399754; 6225460; 6127533; 6031086; 6005087; 5977089; and references cited therein disclose a wide range of specific nucleotide analogs and modifications that may be used. See Crooke, S. (ed.) Antisense Drug Technology: Principles, Strategies, and Applications (1 st ed), Marcel Dekker; ISBN: 0824705661; 1st edition (2001) and references given in it. For example, 2' modifications include halogen, alkoxy, and allyloxy groups. In some embodiments, the 2'-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH ₂ , NHR, NR _{2 ,} or CN, where R is C ₁ -C ₆ alkyl, alkenyl, or alkynyl, and halogen is F, Cl, Br, or I. Examples of modified linkages include phosphorothioate and 5'-N-phosphoramidite linkages.

Nucleic acids containing a variety of different nucleotide analogs, modified backbones, or non-naturally occurring internucleoside linkages can be used in accordance with the present invention. The nucleic acids of the present invention may include naturally occurring nucleosides (ie, adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) or modified nucleosides. Examples of modified nucleotides include a base-modified nucleoside ( e.g. aracitidine, inosine, isoguanosine, nebularine, pseudouridine, 2,6-diaminopurine, 2-aminopurine, 2-thiothymidine, 3-deaza-5-azacytidine, 2'-deoxyuridine, 3- nitorpyrrole, 4-methylindole, 4-thiouridine, 4-thiothymidine, 2-aminoadenosine, 2-thiothymidine, 2-thiouridine, 5-bromocytidine, 5-ioduridine, inosine, 6-azauridine, 6-chloropurine, 7-deazaadenosine, 7- deazaguanosine, 8-azaadenosine, 8-azidoadenosine, benzimidazole, Ml-methyladenosine, pyrrolopyrimidine, 2-amino-6-chloropurine, 3-methyladenosine, 5-propynylcytidine, 5-propynyluridine, 5-bromuridine, 5-fluorouridine, 5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine and 2-thiocytidine), chemically or biologically modified bases ( e.g. methylated bases), modified sugars ( e.g. 2'-fluoribose, 2'-aminoribose, 2'-azidoribose, 2'-O-methylribose, L-enantiomeric nucleosides, arabinose and hexose), modified phosphate groups ( eg phosphorothioates and 5'-N-phosphoramidite linkages) and combinations thereof. Natural and modified nucleotide monomers for the chemical synthesis of nucleic acids are readily available. In some cases, nucleic acids containing such modifications exhibit improved properties compared to nucleic acids consisting only of naturally occurring nucleotides. In some embodiments, the nucleic acid modifications described herein are used to reduce and/or prevent cleavage by nucleases ( eg , exonucleases, endonucleases, etc. ). For example, the structure of a nucleic acid can be stabilized by including nucleotide analogs at the 3' end of one or both strands to reduce cleavage.

Modified nucleic acids need not be uniformly modified throughout the length of the molecule. Various modifications of nucleotides and/or backbone structures may exist at various positions in the nucleic acid. One skilled in the art will appreciate that nucleotide analogs or other modifications may be located at any position(s) of the nucleic acid, such that the function of the nucleic acid is not substantially altered. To give just one example, modifications can be located at any position of the targeting nucleic acid fragment so that it does not significantly affect the ability of the targeting nucleic acid fragment to specifically bind to the target. The modified region may be at the 5' end and/or 3' end of one or both chains. For example, modified targeting nucleic acid fragments have been used in which approximately 1-5 residues at the 5' and/or 3' end of either strand are nucleotide analogs and/or have a backbone modification. The modification may be a 5' or 3' terminal modification. One or both nucleic acid strands may contain at least 50% unmodified nucleotides, at least 80% unmodified nucleotides, at least 90% unmodified nucleotides, or 100% unmodified nucleotides.

Nucleic acids in accordance with the present invention may, for example, contain a modification of the glycosidic, nucleoside or internucleoside linkage, such as described in the publications of applications for US patents 2003/0175950, 2004/0192626, 2004/0092470, 2005/0020525, and 2005/0032733. The present invention encompasses the use of any nucleic acid having any one or more of the modifications described herein. For example, a number of terminal conjugates, such as lipids such as cholesterol, lithocholic acid, aluric acid, or long branched alkyls, are reported to improve cellular uptake. Analogues and modifications can be tested using, for example, any suitable assay known in the art, for example, to select those that result in improved delivery of a therapeutic or diagnostic agent, improved specific binding of a targeting nucleic acid fragment to a target, etc. e. In some embodiments, the implementation of the nucleic acids in accordance with the present invention may contain one or more non-natural nucleoside linkages. In some embodiments, one or more internal nucleotides at the 3' end, 5' end, or both 3' and 5' ends of the targeting nucleic acid fragment are inverted to form a bond, such as a 3'-3' bond or 5' -5' connection.

In some embodiments, the implementation of the nucleic acids in accordance with the present invention are not synthetic, but are natural objects that have been isolated from their natural environments.

Any method can be used to create new targeting nucleic acid fragments ( see, for example , US Pat. Nos. 6,716,583; 6,465,189; 6,482,594; 6,458,543; 958691; 5874218; 5853984; 5843732; 5843653; 5817785; 5789163; 5763177; 5696249; 5660985; 5595877; 5567588; and 5270163; /0043923, 2003/0087301, 2003/0054360 and 2002/0064780).

Targeting nucleic acid fragments that bind to a protein, carbohydrate, lipid and/or nucleic acid can be designed and/or identified. In some embodiments, targeting nucleic acid fragments can be designed and/or identified for use in complexes of the invention that bind to proteins and/or characteristic portions thereof, such as tumor markers, integrins, cell surface receptors, transmembrane proteins, intercellular proteins, ion channels, membrane transport proteins, enzymes, antibodies, chimeric proteins, etc. In some embodiments, targeting nucleic acid fragments can be designed and/or identified for use in complexes of the invention that bind to carbohydrates and/or characteristic portions thereof such as glycoproteins, sugars ( e.g. , monosaccharides, disaccharides, and polysaccharides), the glycocalyx (i.e., the carbohydrate-rich peripheral zone on the outer surface of most eukaryotic cells), etc. In some embodiments, targeting nucleic acid fragments can be designed and or identified to use in complexes according to the invention that bind to lipids and/or their characteristic parts, such as oils, saturated fatty acids, unsaturated fatty acids, glycerides, hormones, steroids ( eg cholesterol, bile acids), vitamins ( eg vitamin E) , phospholipids, sphingolipids, lipoproteins, etc. In some embodiments, targeting nucleic acid fragments can be designed and/or identified for use in complexes of the invention that bind to nucleic acids and/or their characteristic parts, such as DNA nucleic acids ; nucleic acids RNA; modified DNA nucleic acids; modified RNA nucleic acids; and nucleic acids, which include any combination of DNA, RNA, modified DNA, and modified RNA; and so on.

Targeting nucleic acid fragments (eg, aptamers or spiegelmers) can be designed and/or identified using any available method. In some embodiments, targeting nucleic acid fragments are designed and/or identified by identifying targeting nucleic acid fragments from a mixture of candidate nucleic acids.

Methods for preparing compounds of the invention

Compounds and conjugates described in this description, can be obtained by simple methods of obtaining ( see, for example , examples 1-78). Easy purification allows the use of such preparation methods.

Thus, the present document also provides methods for preparing the compounds of the invention. For example, compounds of the invention can be prepared as shown in any of Reaction Schemes 3, 4, 5, 6, or 7:

Reaction Scheme 3:

;

;

Reaction Scheme 4:

;

;

Reaction Scheme 5:

;

;

Reaction Scheme 6:

;

;

Reaction Scheme 7:

;

;

where X, Y, Ar, R and n are as defined above.

Also provided herein are methods for preparing a compound, comprising reacting a compound of formula ( IIc ):

or its pharmaceutically acceptable salt with a sulfonyl halide:

to obtain a compound of formula ( Iaa ):

or its pharmaceutically acceptable salt, where:

X ^a is halogen (preferably fluorine), each R ¹¹ is independently an alkyl, aryl, aralkyl or alkoxy substituent, and the remaining groups may be selected according to any definition provided herein.

For example, in some such embodiments

Q is an active agent linked to L' via a heteroatom, preferably O or N;

Z' is absent or is a linking group containing at least one reactive group such as the precursor described above in connection with Z;

L' is a spacer moiety attached to SO ₂ via a heteroatom selected from O, S and N, preferably O or N, and chosen such that cleavage of the bond between L' and SO ₂ promotes cleavage of the bond between L' and Q for release of the active agent;

Ar is aryl, heteroaryl, cycloalkyl or heterocycloalkyl, preferably aryl or heteroaryl;

Y' is -(CR ^b ₂ ) _y N(R ^a )-, -(CR ^b ₂ ) _y O- or -(CR ^b ₂ ) _y S- such that an N, O or S atom is attached to TG if y is equal to 1;

O and Y' are located on adjacent Ar atoms;

TG is a trigger group that upon activation forms an N, O or S atom capable of reacting with SO ₂ to displace (Q) _q -(L') _w and form a 5-6 membered ring including X-SO ² and intermediate Ar atoms;

each w, x and y is independently an integer having a value of 0 or 1;

each of R ^a and R ^c independently represents hydrogen or lower alkyl; And

each R ^b is independently hydrogen or lower alkyl; or

two R ^b together with the carbon atom to which they are attached form a 3-5 membered ring, preferably a 3-4 membered ring,

The reactive group of the linking group may be a moiety capable of participating in 1,3-dipole cycloaddition reactions, hetero-Diels-Alder reactions, nucleophilic substitution reactions, non-aldol type carbonyl reactions, additions to multiple carbon-carbon bonds, oxidation reactions, click reactions, or any other intermolecular reaction. Preferably, the reactive group is selected to participate in a selective reaction with a reactive partner that is not common in biological molecules, such as a 1,3-dipolar cycloaddition reaction, a hetero-Diels-Alder reaction, an oxime/hydrazone condensation reaction, or a click reaction.

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

In other embodiments described herein, methods for preparing a compound are provided, including:

(a) introducing into the reaction a compound of formula ( IIa ):

or its pharmaceutically acceptable salt with 1,1'-sulfonylbis(1H-imidazole):

to obtain a compound of formula ( IIbb ):

or its pharmaceutically acceptable salt, where the variables can be selected in accordance with any definition given in this document.

For example, in some such embodiments

Z' is absent or represents a linking group containing a reactive group, as described in more detail above;

Ar is aryl, heteroaryl, cycloalkyl or heterocycloalkyl, preferably aryl or heteroaryl;

Y' is -(CR ^b ₂ ) _y N(R ^a )-, -(CR ^b ₂ ) _y O- or -(CR ^b ₂ ) _y S-, positioned such that an N, O or S atom is attached to TG if y is 1;

-SO ₂ - and Y' are located on adjacent Ar atoms;

TG is a trigger group that upon activation forms an N, O or S atom capable of reacting with SO ₂ to displace (Q) _q -(L') _w and form a 5-6 membered ring including X-SO ² and intermediate Ar atoms;

each w, x and y is independently an integer having a value of 0 or 1;

each of R ^a and R ^c independently represents hydrogen or lower alkyl; And

each R ^b is independently hydrogen or lower alkyl; or

two R ^b together with the carbon atom to which they are attached form a 3-5 membered ring, preferably a 3-4 membered ring,

The compound of formula ( IIbb ) can then be further reacted with a compound of formula ( Ia '), Q-(L') _w -H, or a pharmaceutically acceptable salt thereof to give compound ( Ia ):

or its pharmaceutically acceptable salt, where the variables can be selected in accordance with any definition given in this document.

For example, in some embodiments

X is O;

Q is an active agent linked to L' via a heteroatom, preferably O or N;

L' is a linking group attached to SO ₂ via a heteroatom selected from O, S and N, preferably O or N, and chosen such that cleavage of the bond between L' and SO ₂ promotes cleavage of the bond between L' and Q for release of the active agent; And

w is 0 or 1.

In some embodiments, the methods use an intermediate of formula ( IIa ), ( IIb ), or ( IIc ) to produce a compound of formula ( Iaa ), where Ar, TG, Y'Z', and R ^a are as defined above for conjugates of formula ( I ' ) or a compound of formula ( Ia ).

The invention further provides the compounds described above that are useful in these methods.

Intermediates

In some embodiments, the compounds and conjugates described herein can be prepared by a method that uses an intermediate having a structure according to formula ( IV ):

or a pharmaceutically acceptable salt thereof, wherein:

W represents hydrogen, -SiR ¹⁶ R ¹⁷ R ¹⁸ or -SO ₂ -G;

each of R¹⁶, R¹⁷ and R¹⁸ independently represents C₁-C₆-alkyl;

G is halogen (preferably fluorine), imidazole or N-methylimidazolium;

R is a Deputy or -L ¹ '-Z;

L ¹ ' is a C ₁ -C ₂₀₀ alkylene optionally containing at least one of a peptide bond, an amino group, an ether bond, a triazole bond, a tetrazole bond, a glycosidic bond, a sulfonamide bond, a phosphonate bond, a sulfo bond, or a dendrimeric structure;

,

, фосфоновой кислоты (-P(=O)(OH)₂), кетона,C₈-C₁₀ циклоалкинила, -OH, -NHOH, -NHNH₂, -SH, карбоновой кислоты (-COOH), ацетилена (-C≡CH), азида (-N₃), амино (-NH₂), сульфоновой кислоты (-SO₃H), производного алкинона (-C(O)C≡C-R^a, где R^a представляет собой C₁-C₁₀-алкил) и дигидрофосфат (-OP(=O)(OH)₂).Z is a precursor selected from isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH ₂ -halogen), maleimide, diene, alkene, halide, tosylate (TsO ^- ), aldehyde, sulfonate (R-SO ₃ ^- ),

,

, phosphonic acid (-P (= O) (OH) ₂ ), ketone, C ₈ -C ₁₀ cycloalkynyl, -OH, -NHOH, -NHNH ₂ , -SH, carboxylic acid (-COOH), acetylene (-C≡ CH), azide (-N ₃ ), amino (-NH ₂ ), sulfonic acid (-SO ₃ H), alkynone derivative (-C(O)C≡CR ^a , where R ^a is C ₁ -C ₁₀ - alkyl) and dihydrophosphate (-OP(=O)(OH) ₂ ).

n is an integer having a value from 1 to 4;

или -Y'-TG, такой как -NO₂, -OC(O)(CH₂)_rC(O)R₁, -O(CH₂)_r-Ar₁-NO₂, -NHNH₂, -BR₂R₃,

или -Y'-TG;Y is -NO ₂ , -OC(O)(CH ₂ ) _r C(O)R ₁ , -O(CH ₂ ) _r -Ar ₁ -NO ₂ , -NHOH, -NHNH ₂ , -BR ₂ R ₃ ,

or -Y'-TG such as -NO ₂ , -OC(O)(CH ₂ ) _r C(O)R ₁ , -O(CH ₂ ) _r -Ar ₁ -NO ₂ , -NHNH ₂ , -BR ₂ R ₃ ,

or -Y'-TG;

R ¹ represents C ₁ -C ₆ -alkyl;

r is an integer from 1 to 5;

Ar ¹ is C ₆ -C ₂₀ -arylene;

each of R ² and R ³ independently represents hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ -alkoxy or hydroxy;

each of R ^a , R ^b , R ^c and R ^d independently represents hydrogen or C ₁ -C _6- -alkyl;

Y' represents -(CH ₂ ) _x NR''-, -(CH ₂ ) _x O- or -(CH ₂ ) _x S-;

R'' represents hydrogen or C ₁ -C ₆ -alkyl;

x is an integer having a value of 0 or 1; And

TG is a trigger group.

In some embodiments, the compounds and conjugates described herein can be prepared by a method that uses an intermediate having a structure according to formula ( V ):

or a pharmaceutically acceptable salt thereof, wherein:

W, L ¹ ' and Z correspond to the definition for formula ( IV ); And

TG is a trigger group such as β-galactoside, β-glucuronide, or a combination of β-galactoside and β-glucuronide.

In other embodiments, the compounds and conjugates described herein can be prepared by a method that uses an intermediate having a structure according to formula ( VI ):

or a pharmaceutically acceptable salt thereof, wherein:

W corresponds to the definition for formula (IV);

Y is -NO ₂ , -OC(O)(CH ₂ ) _r C(O)R ¹ , -O(CH ₂ ) _r -Ar ¹ -NO ₂ , -NHOH, -NHNH ₂ , -BR ² R ³ or -O-TG;

R ¹ is C ₁ -C ₆ -alkyl, such as -NO ₂ , -OC(O)(CH ₂ ) _r C(O)R ¹ , -O(CH ₂ ) _r -Ar ¹ -NO ₂ , - NHOH, -NHNH ₂ , -BR ² R ³ or -O-TG;

R ¹ represents C ₁ -C ₆ -alkyl;

r is an integer from 1 to 5;

Ar ¹ is phenylene, biphenylene or naphthalene;

each of R ² and R ³ independently represents hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ -alkoxy or hydroxy;

each of R ^a , R ^b , R ^c and R ^d independently represents hydrogen or C ₁ -C _{6 -} alkyl; And

TG is a trigger group such as β-galactoside, β-glucuronide, or a combination of β-galactoside and β-glucuronide.

This document also provides intermediates of formula ( IIa ), ( IIb ) or ( IIc ):

or their pharmaceutically acceptable salts, where:

G is halogen, imidazole or N-methylimidazolium;

each R ¹¹ is independently C ₁ -C ₆ alkyl;

Ar is aryl, heteroaryl, cycloalkyl or heterocycloalkyl;

TG is a trigger group which upon activation forms an N, O or S atom capable of forming a 5-6 membered ring including X-SO ² and intermediate Ar atoms;

Y' is -(CR ^b ₂ ) _y N(R ^a )-, -(CR ^b ₂ ) _y O- or -(CR ^b ₂ ) _y S-, positioned such that an N, O or S atom is attached to TG if y is 1;

O and Y' are located on adjacent Ar atoms;

each of x and y is independently an integer having a value of 0 or 1;

Z' is absent or is a link containing, for example , a reactive or link; And

each R ^a is independently hydrogen or alkyl; or

the two R ^a , together with the carbon atom to which they are attached, form a three-membered ring.

In some embodiments, the intermediate is a compound of formula ( IIa ), ( IIb ), or ( IIc ), where Ar, TG, Y'Z', and R ^a are as defined above for conjugates of formula ( I ') or a compound of formula ( Ia ) .

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

,

, фосфоновой кислоты (-P(=O)(OH)₂), кетона,C₈-C₁₀ циклоалкинила, -OH, -NHOH, -NHNH₂, -SH, карбоновой кислоты (-COOH), ацетилена (-C≡CH), азида (-N₃), амино (-NH₂), сульфоновой кислоты (-SO₃H), производного алкинона (-C(O)C≡C-R^a и дигидрофосфата (-OP(=O)(OH)₂.In some embodiments, provided herein is an intermediate that is a compound of formula ( IIa ), ( IIb ), or ( IIc ), where Z' is a linking group containing one or more groups selected from isocyanide, isothiocyanide, 2 -pyridyl disulfide, haloacetamide (-NHC(O)CH ₂ -halogen), maleimide, diene, alkene, halide, tosylate (TsO ^- ), aldehyde, sulfonate (R-SO ₃ ^- ),

,

, phosphonic acid (-P (= O) (OH) ₂ ), ketone, C ₈ -C ₁₀ cycloalkynyl, -OH, -NHOH, -NHNH ₂ , -SH, carboxylic acid (-COOH), acetylene (-C≡ CH), azide (-N ₃ ), amino (-NH ₂ ), sulfonic acid (-SO ₃ H), alkynone derivative (-C(O)C≡CR ^a and dihydrophosphate (-OP(=O)(OH) ₂ .

, такой как NO₂, -OC(O)(CH₂)_rC(O)R¹, -NHNH₂, -BR²R³,

, где:In other embodiments, the intermediate is a compound of formula ( IIa ), ( IIb ), or ( IIc ), where x is 0. In some such embodiments, TG is -NO ₂ , -OC(O)(CH ₂ ) _r C (O)R ¹ , -NHOH, -NHNH ₂ , -BR ² R ³ ,

, such as NO ₂ , -OC(O)(CH ₂ ) _r C(O)R ¹ , -NHNH ₂ , -BR ² R ³ ,

, Where:

R ¹ represents C ₁ -C ₆ -alkyl;

each of R ² and R ³ independently represents hydrogen, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -alkoxy or hydroxy;

each of R ⁴ , R ⁵ , R ⁶ and R ⁷ independently represents hydrogen or C ₁ -C ₆ -alkyl; And

r is an integer having a value of 1, 2, 3, 4, or 5;

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

In particular embodiments, the intermediate is:

или

or

or a pharmaceutically acceptable salt thereof.

In some other embodiments, the intermediate is:

;

;

;

;

;

; или

;

;

;

;

;

; or

or a pharmaceutically acceptable salt thereof.

Antibody Drug Conjugates (ADC)

In some embodiments, CB is an antibody and Q is a drug. Accordingly, the compounds and conjugates described herein can be used to conjugate an antibody to a drug moiety to form an antibody-drug conjugate (ADC). Antibody-drug conjugates (ADCs) may increase therapeutic efficacy in the treatment of a disease, such as cancer, due to the ability of ADCs to selectively deliver one or more drug components to target tissues, such as a tumor-associated antigen. Thus, in certain embodiments, the invention provides an ADC for therapeutic use, such as the treatment of cancer.

The ADCs of the present invention comprise an antibody linked to one or more drug moieties. The specificity of the ADC is determined by the specificity of the antibody. In one embodiment, the antibody is linked to one or more cytotoxic drugs that are delivered to the cancer cell.

Examples of drugs that can be used in the ADC according to the invention are given below. The terms "drug", "agent", and "drug moiety" are used interchangeably herein. The terms "bound" and "conjugated" are also used interchangeably herein and indicate that the antibody and fragment are covalently linked.

In some embodiments, the ADC has the following formula (Formula VII):

where Ab is an antibody and (DL) is a linker-drug moiety. The linker-drug moiety consists of a linker L and a drug moiety D. The drug moiety may have, for example, cytostatic, cytotoxic, or other therapeutic activity on the target cell. n is an integer having a value from 1 to about 20, preferably from 1 to about 10. Preferably, DL has the structure of formula ( I'' ):

Q is an active agent linked to L' via a heteroatom, preferably O or N;

Z' is a linking group;

L' is a spacer moiety attached to SO ₂ via a heteroatom selected from O, S and N, preferably O or N, and chosen such that cleavage of the bond between L' and SO ₂ promotes cleavage of the bond between L' and Q for release of the active agent;

X is -O-, -C(R ^b ) ₂ - or -N(R ^c )-, preferably -O-;

Ar is aryl, heteroaryl, cycloalkyl or heterocycloalkyl, preferably aryl or heteroaryl;

Y' is -(CR ^b ₂ ) _y N(R ^a )-, -(CR ^b ₂ ) _y O- or -(CR ^b ₂ ) _y S-, positioned such that an N, O or S atom is attached to TG if y is 1;

X and Y' are located on adjacent Ar atoms;

TG is a trigger group that upon activation forms an N, O or S atom capable of reacting with SO ₂ to displace (Q) _q -(L') _w and form a 5-6 membered ring including X-SO ² and intermediate Ar atoms;

each w, x and y is independently an integer having a value of 0 or 1;

each of R ^a and R ^c independently represents hydrogen or lower alkyl; And

each R ^b is independently hydrogen or lower alkyl; or

two R ^b together with the atom to which they are attached form a 3-5 membered ring, preferably a 3-4 membered ring.

In some embodiments, n is 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or is an integer, having a value of 1. When cb is 1 and n is 1, the drug-antibody ratio (DAR) of the ADC is equivalent to the amount of drugs present in (D-L). When cb is not equal to 1, the drug-antibody ratio (DAR) of the ADC is equivalent to the ratio of the amount of drugs present in (D-L) to the amount of antibodies present in the conjugate.

Examples of drugs for conjugation

The ADCs of the invention provide a targeted therapy that can, for example, reduce the side effects often seen in cancer therapy because one or more active agents or drugs are delivered to a particular cell.

For example, the drug may be selected from the group consisting of erlotinib (TARCEVA; Genentech/OSI Pharm.); bortezomib (VELCADE; MilleniumPharm.); fulvestrant (FASLODEX; AstraZeneca); sutenta (SU11248; Pfizer); letrozole (FEMARA; Novartis); imatinib mesylate (GLEEVEC; Novartis); PTK787 ZK 222584 (Novartis); oxaliplatin (Eloxatin; Sanofi); 5-fluorouracil (5-FU); leucovorin; rapamycin (sirolimus, RAPAMUNE; Wyeth); lapatinib (TYKERB, GSK572016; GlaxoSmithKline); lonafarnib (SCH 66336); sorafenib (BAY43-9006; Bayer Labs.); gefitinib (IRESSA; Astrazeneca); AG1478, AG1571 (SU 5271; Sugen); an alkylating agent ( eg thiotepa or CYTOXAN® cyclophosphamide); an alkylsulfonate ( eg busulfan, improsulfan or piposulfan); aziridine ( eg benzodopa, carbokwon, meturedopa or uredopa); ethyleneimine, methylmelamine, altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolmelamine; acetogenins ( eg bulatacin or bulatacinone); camptothecin, including the synthetic analog of topotecan; bryostatin; callistatin; CC-1065 (including synthetic analogues of adoselesin, carzelesin or bizelesin); cryptophycins ( eg cryptophycin 1 or cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogue, KW-2189 and CB1-TM1); eleuterobine; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards ( eg chlorambucil, chlornaphasine, holofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquine, phenesterin, prednimustine, trofosfamide or uracil mustard); nitrosoureas ( eg carmustine, chlorozotocin, fotemustine, lomustine, nimustine, or ranimnustine); antibiotics ( eg calicheamycin selected from calicheamicin gamma II and calicheamicin omega II or dynemycin, including dynemycin A as enediine antibiotics); a bisphosphonate ( e.g. clodronate); esperamycin, neocarzinostatin chromophore or related antibiotic chromophore chromoprotein enediin, aclacinomycin, actinomycin, anthramycin, azazerin, bleomycin, cactinomycin, carabicin, carninomycin, carsinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5 -oxo-L-norleucine, doxorubicin ADRLIMYCIN® ( e.g. morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin or deoxydoxorubicin), epirubicin, esorubicin, marcellomycin, mitomycin ( e.g. mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin , kelamycin, rhodorubicin, streptomigrine, streptozocin, tubercidin, ubenimex, zinostatin, or zorubicin); antimetabolites ( eg 5-fluorouracil (5-FU)); folic acid analogs ( eg denopterin, methotrexate, pteropterin or trimetrexate); purine analogues ( eg fludarabine, 6-mercaptopurine, thiamiprin or tiguanine); pyrimidine analogs ( eg ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine or floxuridine); an androgen ( eg calusterone, dromostanolone propionate, epitiostanol, mepitiostane or testolactone); an antiadrenal drug ( eg aminoglutethimide, mitotane, or trilostane); folic acid supplement ( eg folic acid); aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; betrabucil; bisantrene; edatraxate; defofamine; demecolcin; diazikvon; elfornithine; elliptinium acetate; epothilone; etoglucida; gallium nitrate; hydroxyureas; lentinan; lonidamine; maytansinoid ( eg maytansine or ansamitocin); trichothecene ( e.g. T-2 toxin, verracurin A, roridin A or anhidine); mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; phenameta; pyrarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; polysaccharide PSK®; razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecene (particularly T-2 toxin, verracurin A, roridin A or anhidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; hacytosine; arabinoside ("Ara-C");cyclophosphamide;thiotepa; taxoids ( e.g. paclitaxel TAXOL® (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE ^™ without cremophor, paclitaxel albumin nanocompositions, American Pharmaceutical Partners, Schaumber, I11. or doxetaxel TAXOTERE® ((Rhone-Poulenc Rorer, Antony, France ))); chloranbucil; gemcitabine; 6-thioguanine; mercaptopurine; a platinum analogue ( eg cisplatin or carboplatin); vinblastine; platinum; etoposide, ifosfamide; mitoxantrone; vincristine; vinorelbine NAVELBINE®; novantron; teniposide; edatrexate; daunomycin; aminopterin; xelodes; ibandronate; SRT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); a retinoid ( eg retinoic acid); capecitabine; and a pharmaceutically acceptable salt, solvate, acid or derivative thereof.

Mitotic inhibitors

In some embodiments, the linkers of the present invention can be used to conjugate an antibody to one or more mitotic inhibitors to form an ADC for cancer treatment. As used herein, the term "mitotic inhibitor" refers to a cytotoxic and/or therapeutic agent that blocks mitosis or cell division, a biological process particularly important in cancer cells. The mitotic inhibitor disrupts microtubules so that cell division is prevented, often affecting microtubule polymerization or microtubule depolymerization. Thus, in certain embodiments, the antibody is conjugated to one or more mitotic inhibitors that interfere with microtubule formation by inhibiting tubulin polymerization. In one embodiment, the mitotic inhibitor used in the ADC of the present invention is ^Taxol® (paclitaxel), ^Taxotere® (docetaxel) or ^Ixempra® (ixabepilone). Examples of mitotic inhibitors that can be used in the ADCs described herein are provided below. The auristatins described above belong to the genus of mitotic inhibitors.

Auristatins

The linkers of the present invention can be used to conjugate an antibody to at least one auristatin. Auristatins are a group of dolastatin analogs that have been shown to have antitumor activity by influencing microtubule dynamics and GTP hydrolysis, thereby inhibiting cell division. For example, auristatin E (US Pat. No. 5,635,483) is a synthetic analogue of the marine natural product dolastatin 10, a compound that inhibits tubulin polymerization by binding to the same site on tubulin as the anticancer drug vincristine (G. R. Pettit, Prog. Chem. Org. Nat Prod, 70: 1-79 (1997)). Dolastatin 10, auristatin PE and auristatin E are linear peptides having four amino acids, three of which are unique to the dolastatin class of compounds. Exemplary embodiments of the auristatin subclass of mitotic inhibitors include, but are not limited to, monomethylauristatin D (MMAD or auristatin D derivative), monomethylauristatin E (MMAE or auristatin E derivative), monomethylauristatin F (MMAF or auristatin F derivative), phenylenediamine auristatin F (AFP) , auristatin EB (AEB), auristatin EFP (AEFP), and 5-benzoylvaleric acid ester AE (AEVB). The synthesis and structure of auristatin derivatives are described in US Patent Application Publication Nos. 2003-0083263, 2005-0238649 and 2005-0009751; International Patent Publication No. WO 04/010957, International Patent Publication No. WO 02/088172 and US Patent No. 6323315; 6239104; 6034065; 5780588; 5665860; 5663149; 5635483; 5599902; 5554725; 5530097; 5521284; 5504191; 5410024; 5.138036; 5076973; 4986988; 4978744; 4879278; 4816444; and 4486414, each of which is incorporated into the present description by reference.

Dolastatins

The linkers of the present invention can be used to conjugate an antibody to at least one dolastatin to form an ADC. Dolastatins are short peptide compounds isolated from the Indian Ocean bearded seal Dolabella auricularia ( see Pettit et al. , J. Am. Chem. Soc. , 1976, 98, 4677). Examples of dolastatins include dolastatin 10 and dolatstin 15. Dolastatin 15, a seven subunit depsipeptide derived from Dolabella auricularia, is a potent antimitotic agent structurally related to the antitubulin agent dolastatin 10, a five subunit peptide derived from the same organism. Thus, in one embodiment, the ADC of the present invention contains an antibody, a linker as described herein, and at least one dolastatin. The auristatins described above are synthetic derivatives of dolastatin 10.

Maytansinoids

The linkers of the present invention can be used to conjugate an antibody to at least one maytansinoid to form an ADC. Maytansinoids are potent antitumor agents that were originally isolated from members of the higher plant family Celastraceae, Rhamnaceae, and Euphorbiaceae, as well as some moss species (Kupchan et al, J. Am. Chem. Soc. 94:1354-1356 [1972]; Wani et al, J. Chem. Soc. Chem. Commun 390: [1973]; Powell et al, J. Nat. Prod. 46:660-666 [1983]; Sakai et al, J. Nat. Prod. 51:845- 850 [1988]; and Suwanborirux et al, Experientia 46:117-120 [1990]). Evidence suggests that maytansinoids inhibit mitosis by inhibiting polymerization of the microtubule protein tubulin, thereby preventing microtubule formation ( see, for example, US Pat. No. 6,441,163 and Remillard et al. , Science , 189, 1002-1005 (1975)). Maytansinoids have been shown to inhibit tumor cell growth in vitro using cell culture models and in vivo using laboratory animal systems. In addition, the cytotoxicity of maytansinoids is 1000 times higher than that of conventional chemotherapeutic agents such as, for example, methotrexate, daunorubicin, and vincristine ( see, for example , US Pat. No. 5,208,020).

Maytansinoids include maytansine, maytansinol, maytansinol C-3 esters, and other maytansinol analogs and derivatives ( see, for example , US Pat. Nos. 5,208,020 and 6,441,163, each of which is incorporated herein by reference). The C-3 esters of maytansinol may be natural or synthetically produced. In addition, both natural and synthetic C-3 esters of maytansinol can be classified as C-3 ester with carboxylic acids or C-3 ester with N-methyl-L-alanine derivatives, the latter being more cytotoxic than the previous one. Synthetic analogues of maytansinoids are described, for example, in Kupchan et al. , J. Med. Chem. , 21, 31-37 (1978).

Suitable maytansinoids for use in the ADCs of the present invention may be isolated from natural sources, synthetically produced, or semi-synthetically obtained. In addition, the maytansinoid may be modified in any suitable manner, provided that sufficient cytotoxicity is retained in the final conjugate molecule. The structure of a typical maytansinoid, mertansine (DM1), is shown below.

Mertansine (DM1)

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

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

plant alkaloids

The linkers of the present invention can be used to conjugate an antibody to at least one plant alkaloid, for example a taxane or a vinca alkaloid. Plant alkaloids are chemotherapeutic agents derived from certain types of plants. Vinca alkaloids are derived from the periwinkle (Catharanthus rosea), while taxanes are derived from the bark of the Pacific yew (Taxus). Both vinca alkaloids and taxanes are also known as antimicrotubule agents and are described in more detail below.

Taxanes

The linkers of the present invention can be used to conjugate an antibody to at least one taxane. As used herein, the term "taxane" refers to a class of anticancer agents having a microtubule mechanism of action and having a structure that includes a taxane ring structure and a stereospecific side chain that is required for cytostatic activity. The term "taxane" also includes various known derivatives, including both hydrophilic derivatives and hydrophobic derivatives. Taxane derivatives include, but are not limited to, the galactose and mannose derivatives described in International Patent Application No. WO 99/18113; piperazino and other derivatives described in WO 99/14209; the taxane derivatives described in WO 99/09021, WO 98/22451 and US Pat. No. 5,869,680; 6-thio derivatives described in WO 98/28288; the sulfenamide derivatives described in US Pat. No. 5,821,263; and the taxol derivative described in US Pat. No. 5,415,869, each of which is incorporated herein by reference. Taxane compounds have also been previously described in US Pat. 9, 4814470, 5438072, 5403858, 4960790, 5433364, 4942184, 5362831, 5705503 , and 5278324, all of which are expressly incorporated herein by reference. Additional examples of taxanes include, but are not limited to, docetaxel ( ^Taxotere® ; Sanofi Aventis), paclitaxel ( ^Abraxane® or ^Taxol® ; Abraxis Oncology), and paclitaxel nanoparticles (ABI-007/ ^Abraxene® ; Abraxis Bioscience).

In one embodiment, the linkers of the present invention can be used to conjugate an antibody to at least one docetaxel. In one embodiment, the linkers of the present invention can be used to conjugate an antibody to at least one paclitaxel.

Vinca alkaloids

In one embodiment, the linkers of the present invention can be used to conjugate an antibody to at least one vinca alkaloid. Vinca alkaloids are a class of cell cycle-specific drugs that work by inhibiting the ability of cancer cells to divide by acting on tubulin and preventing microtubule formation. Examples of vinca alkaloids that can be used in the ADC of the present invention include, but are not limited to, vindesine sulfate, vincristine, vinblastine, and vinorelbine.

Antitumor antibiotics

The linkers of the present invention can be used to conjugate an antibody to one or more antitumor antibiotics for the treatment of cancer. As used herein, the term "antineoplastic antibiotic" means an antitumor drug that blocks cell growth by acting on DNA and is made from a microorganism. Anticancer antibiotics often either destroy DNA strands or slow down or stop DNA synthesis. Examples of anticancer antibiotics that may be included in the ADCs described herein include, but are not limited to, actinomycins ( e.g. , pyrrolo[2,1-c][1,4]benzodiazepines), anthracyclines, calicheamicins, and duocarmycins, as described more details below.

Actinomycins

The linkers of the present invention can be used to conjugate an antibody to at least one actinomycin. Actinomycins are a subclass of antitumor antibiotics isolated from bacteria of the genus Streptomyces. Representative examples of actinomycins include, but are not limited to, actinomycin D (Cosmegen [also known as actinomycin, dactinomycin, actinomycin IV, actinomycin C1], Lundbeck, Inc.), anthramycin, chikamycin A, DC-81, mazethramycin, neotramycin A, neotramycin B, porotramycin, protracarcin B, SG2285, sibanomycin, sibiromycin, and tomaimycin. In one embodiment, D is pyrrolobenzodiazepine (PBD). Examples of PBDs include, but are not limited to, Anthramycin, Chicamycin A, DC-81, Mazethramycin, Neothramycin A, Neothramycin B, Porothramycin, Protracarcin B, SG2000 (SJG-136), SG2202 (ZC-207), SG2285 (ZC-423 ), sibanomycin, sibiromycin, and tomaimycin. Thus, in one embodiment, D is actinomycin, eg actinomycin D, or PBD, eg pyrrolobenzodiazepine dimer (PBD).

PBD structures can be found, for example, in US Patent Application Publication Nos. 2013/0028917 and 2013/0028919, as well as WO 2011/130598 A1, each of which is incorporated herein by reference in its entirety. The general structure of the PBD is shown below.

PBDs differ in the number, type, and position of substituents, both in their aromatic A rings and in the pyrrolo C rings, as well as in the degree of saturation of the C ring. The B-ring usually has an imine (N=C), carbinolamine (NH-CH(OH)) or carbinolamine methyl ester (NH-CH(OMe)) at the N10-C11 position, which is the electrophilic center responsible for DNA alkylation. All known natural products have the (S)-configuration at the chiral C11α position, giving them a right turn when viewed from ring C to ring A. Additional examples of PBDs that can be conjugated to antibodies via linkers described herein can be found 2013/0028917 A1 and 2013/0028919 A1, US Pat. No. 7,741,319 B2, and WO 2011/130598 A1 and WO 2006/111759 A1, each of which is incorporated herein in its entirety through a link.

Anthracyclines

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

Calicheamycins

The linkers of the present invention can be used to conjugate an antibody to at least one calicheamicin. The calicheamycins are a family of enediine antibiotics derived from the soil organism Micromonospora echinospora. Calicheamycins bind the DNA minor groove and induce DNA double-strand breaks resulting in cell death with a 100-fold increase compared to other chemotherapeutic agents (Damle et al. (2003) Curr Opin Pharmacol 3:386). Obtaining calicheamicins, which can be used as drug conjugates in the invention has already been described, see US patent No. 5712374; 5714586; 5739116; 5767285; 5770701; 5770710; 5773001; and 5,877,296. Structural analogs of calicheamicin that may be used include, but are not limited to, γ1I, α2I, α3I, N-acetyl-γ1I, PSAG, and θI1 (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998) and US Pat. Nos. 5,712,374; 5,714,586; 5,739,116; 5,767,285; Thus, in one embodiment, D is calicheamicin.

Duocarmycins

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

Other anticancer antibiotics

In addition to the above, additional antitumor antibiotics that can be used in the ADC of the present invention include bleomycin (Blenoxane, Bristol-Myers Squibb), mitomycin, and plicamycin (also known as mithramycin).

Immunomodulators

In some embodiments, linkers of the invention can be used to conjugate an antibody to at least one immunomodulator. As used herein, the term "immunomodulators" refers to an agent that can stimulate or modify an immune response. In one embodiment, the immunomodulator is an immunostimulant that enhances the subject's immune response. In some embodiments, an immunomodulator is an immunosuppressant that prevents or reduces a subject's immune response. The immunomodulator can modulate myeloid cells (monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) or lymphoid cells (T cells, B cells and natural killer (NK) cells) and any further differentiated cells. Representative examples include, but are not limited to, Bacillus Calmette-Guérin (BCG) and Levamisole (Ergamisol). Other examples of immunomodulators that can be used in the ADC of the present invention include, but are not limited to, cancer vaccines, cytokines, and immunomodulatory gene therapy.

Cancer Vaccines

The linkers of the present invention can be used to conjugate an antibody to a cancer vaccine. As used herein, the term "cancer vaccine" refers to a composition (eg, tumor antigen and cytokine) that elicits a tumor-specific immune response. The response is elicited from the subject's own immune system by administration of a cancer vaccine or, in the case of the present invention, by administration of an ADC comprising an antibody and a cancer vaccine. In preferred embodiments, the implementation of the immune response leads to the eradication of tumor cells in the body (eg, primary or metastatic tumor cells). The use of cancer vaccines usually involves the administration of a specific antigen or group of antigens, which, for example, are present on the surface of a particular cancer cell or are present on the surface of a particular infectious agent that has been shown to stimulate the formation of cancer. In some embodiments, cancer vaccines are used for prophylactic purposes, while in other embodiments, they are used for therapeutic purposes. Non-limiting examples of cancer vaccines that can be used in the ADCs described herein include recombinant bivalent human papillomavirus (HPV) type 16 and 18 vaccine (Cervarix, GlaxoSmithKline), recombinant quadrivalent human papillomavirus (HPV) type 6 vaccine , 11, 16 and 18 (Gardasil, Merck & Company) and sipuleucel-T (Provenge, Dendreon). Thus, in one embodiment, D is a cancer vaccine that is either an immunostimulant or an immunosuppressant.

Cytokines

The linkers of the present invention can be used to conjugate an antibody to at least one cytokine. The term "cytokine" generally refers to proteins released by one cell population that act on another cell as intercellular mediators. Cytokines directly stimulate immune effector cells and stromal cells at the tumor site and improve tumor cell recognition by cytotoxic effector cells (Lee and Margolin (2011) Cancers 3:3856). Numerous studies in animal models of tumors have shown that cytokines have broad antitumor activity, and this has been embodied in a number of cytokine-based approaches to cancer therapy (Lee and Margoli, supra). In recent years, a number of cytokines, including GM-CSF, IL-7, IL-12, IL-15, IL-18, and IL-21, have been included in clinical trials for advanced cancer patients (Lee and Margoli, supra) .

Examples of cytokines that can be used in the ADC of the present invention include, but are not limited to, parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor; müllerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF; platelet growth factor; transforming growth factors (TGF); insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon α, β and γ, colony stimulating factors (CSF); granulocyte-macrophage-C-SF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11 , IL-12; tumor necrosis factor; and other polypeptide factors including LIF and complete ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the original sequence cytokines. Thus, in one embodiment, D is a cytokine.

Colony stimulating factors (CSF)

The linkers of the invention can be used to conjugate an antibody to at least one colony stimulating factor (CSF). Colony stimulating factors (CSFs) are growth factors that help the bone marrow produce red blood cells. Because some cancer treatments ( such as chemotherapy) can affect white blood cells (which help fight infection), colony stimulating factors may be administered as an aid to maintaining white blood cell levels and boosting the immune system. Colon stimulating factors can also be used after a bone marrow transplant as an aid in starting white blood cell production. Typical examples of CSFs that can be used in the ADCs described herein include, but are not limited to, erythropoietin (Epoetin), filgrastim (Neopogen (also known as granulocyte colony stimulating factor (G-CSF); Amgen, Inc.), sargramostim (leukin (granulocyte-macrophage colony stimulating factor and GM-CSF); Genzyme Corporation), promegapoietin and oprelvekin (recombinant IL-11; Pfizer, Inc.) Thus, in one embodiment, D is CSF.

Gene therapy

The linkers of the present invention can be used to conjugate an antibody to at least one nucleic acid (directly or indirectly via a carrier) for gene therapy. Gene therapy generally refers to the introduction of genetic material into a cell, the genetic material being intended to treat a disease. As it relates to immunomodulators, gene therapy is used to stimulate a subject's natural ability to inhibit cancer cell proliferation or kill cancer cells. In one embodiment, the ADC of the invention comprises a nucleic acid encoding a functional therapeutic gene that is used to replace a mutated or otherwise dysfunctional ( eg , truncated) cancer-associated gene. In other embodiments, the ADC of the invention comprises a nucleic acid that encodes or otherwise provides for the production of a therapeutic protein for the treatment of cancer. The nucleic acid that encodes the therapeutic gene may be directly conjugated to the antibody or, alternatively, may be conjugated to the antibody via a carrier. Examples of carriers that can be used to deliver the nucleic acid for gene therapy include, but are not limited to, viral vectors or liposomes.

Alkylating agents

The linkers of the present invention can be used to conjugate an antibody to one or more alkylating agents. Alkylating agents are a class of anticancer compounds that attach an alkyl group to DNA. Examples of alkylating agents that can be used in the ADC of the invention include, but are not limited to, alkylsulfonates, ethyleneimines, methylamine derivatives, epoxides, nitrogen mustards, nitrosoureas, triazines, and hydrazines.

Alkylsulfonates

The linkers of the present invention can be used to conjugate an antibody to at least one alkyl sulfonate. Alkylsulfonates are a subclass of alkylating agents with the general formula: R-SO ₂ -OR ¹ where R and R ¹ are usually alkyl or aryl groups. A typical example of an alkylsulfonate is busulfan ( ^Myleran® , GlaxoSmithKline; Busulfex ^IV® , PDL BioPharma, Inc.).

nitrogen mustards

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

Nitrosoureas

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

Triazines and hydrazines

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

Other alkylating agents

The linkers of the present invention can be used to conjugate an antibody with at least one ethyleneimine, a methylamine derivative, or an epoxide. Ethyleneimines are a subclass of alkylating agents that typically contain at least one aziridine ring. Epoxides are a subclass of alkylating agents that are characterized as cyclic ethers with only three ring atoms.

Representative examples of ethyleneimines include, but are not limited to, thiopetu (Thioplex, Amgen), diazichone (also known as aziridinylbenzoquinone (AZQ)) and mitomycin C. Mitomycin C is a natural product that contains an aziridine ring and appears to cause cytotoxicity through DNA crosslinking (Dorr R T, et al. Cancer Res. 1985; 45:3510; Kennedy K A, et al Cancer Res. 1985; 45:3541). Representative examples of methylamine derivatives and analogues thereof include, but are not limited to, altretamine (Hexalen, MGI Pharma, Inc.), which is also known as hexamethylamine and hexastat. Typical examples of epoxides of this class of anticancer compounds include, but are not limited to, dianhydrogalactitol. Dianhydrogalactitol (1,2:5,6-dianhydrodulcitol) is chemically bonded to aziridines and generally facilitates alkyl group transfer through a mechanism similar to that described above. Dibromodulcitol hydrolyses to dianhydrogalactitol and is thus an epoxide prodrug (Sellei C, et al. Cancer Chemother Rep. 1969; 53:377).

Antiangiogenic agents

In some embodiments, linkers of the invention can be used to conjugate an antibody to at least one anti-angiogenic agent. Anti-angiogenic agents inhibit the growth of new blood vessels. Anti-angiogenic agents exert their effect in a variety of ways. In some embodiments, the implementation of these agents affect the ability of the growth factor to achieve its goal. For example, vascular endothelial growth factor (VEGF) is one of the primary proteins involved in the initiation of angiogenesis by binding to specific receptors on the cell surface. Thus, certain anti-angiogenic agents that prevent the interaction of VEGF with its cognate receptor prevent the initiation of VEGF angiogenesis. In other embodiments, these agents affect intracellular signaling cascades. For example, after triggering a certain receptor on the cell surface, a cascade of other chemical signals is initiated to promote the growth of blood vessels. Thus, certain enzymes, eg some tyrosine kinases, which are known to facilitate intracellular signaling cascades promoting, for example, cell proliferation, are targets for cancer therapy. In other embodiments, these agents affect intercellular signaling cascades. However, in other embodiments, these agents block specific targets that activate and stimulate cell growth or directly interfere with the growth of blood vessel cells. Angiogenesis inhibitory properties have been found in over 300 substances with numerous direct and indirect inhibitory effects.

Typical examples of anti-angiogenic agents that can be used in the ADC of the present invention include, but are not limited to, angiostatin, ABX EGF, C1-1033, PKI-166, EGF vaccine, EKB-569, GW2016, ICR-62, EMD 55900, CP358, PD153035, AG1478, IMC-C225 (Erbitux, ZD1839 (Iressa), OSI-774, Erlotinib (Tarceva), Angiostatin, Arrestin, Endostatin, BAY 12-9566 and with Fluorouracil or Doxorubicin, Canstatin, Carboxyamidotriazole, and Paclitaxel ohm, EMD121974, S-24, Vitaxin, Dimethylxanthenone Acetic Acid, IM862, Interleukin-12, Interleukin-2, NM-3, HuMV833, PTK787, RhuMab, Angiozyme (ribozyme), IMC-1C11, Neovastat, Marimstat, Prinomastat, BMS-275291, COL -3, MM1270, SU101, SU6668, SU11248, SU5416, with paclitaxel, with gemcitabine and cisplatin and with irinotecan and cisplatin, and radiotherapy, tehogalan, temozolomide and PEG interferon α2b, tetrathiomolybdate, TNP-470, thalidomide , CC -5013 and with taxotere, tumstatin, 2-methoxyestradiol, VEGF decoy, mTOR inhibitors (deforolimus, everolimus (Afinitor, Novartis Pharmaceutical Corporation) and temsirolimus (Torisel, Pfizer, Inc.)), tyrosine kinase inhibitors (eg, erlotinib (Tarceva, Genentech) , Inc.), imatinib (Gleevec, Novartis Pharmaceutical Corporation), gefitinib (Iressa, AstraZeneca Pharmaceuticals), dasatinib (Sprycel, Brystol-Myers Squibb), sunitinib (Sutent, Pfizer, Inc.), nilotinib (Tasigna, Novartis Pharmaceutical Corporation) , lapatinib (Tykerb, GlaxoSmithKline Pharmaceuticals), sorafenib (Nexavar, Bayer and Onyx), phosphoinositide 3-kinases (PI3K).

Antimetabolites

The linkers of the present invention can be used to conjugate an antibody to at least one antimetabolite. Antimetabolites are chemotherapy options that are very similar to the normal substances found in the cell. When cells incorporate an antimetabolite into cellular metabolism, the result for the cell is negative, eg, the cell is unable to divide. Antimetabolites are classified according to the substances with which they interfere. Examples of antimetabolites that can be used in the ADC of the present invention include, but are not limited to, folic acid antagonist ( e.g. methotrexate), pyrimidine antagonist ( e.g. 5-fluorouracil, foxuridine, cytarabine, capecitabine and gemcitabine), purine antagonist ( eg 6-mercaptopurine and 6-thioguanine) and an adenosine deaminase inhibitor ( eg cladribine, fludarabine, nelarabine and pentostatin), as described in more detail below.

Antifolates

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

Purine antagonists

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

Pyrimidine antagonists

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

Boron agents

The linkers of the present invention can be used to conjugate an antibody to at least one boron agent. Boron agents include a class of anti-cancer therapeutic compounds that inhibit cell proliferation. Typical examples of boron-containing agents include, but are not limited to, borofycin and bortezomib (Velcade, Millenium Pharmaceuticals).

Chemoprotective agents

The linkers of the present invention can be used to conjugate an antibody to at least one chemoprotective agent. Chemoprotective drugs are a class of compounds that help protect the body from the specific toxic effects of chemotherapy. Chemoprotective agents can be administered during various chemotherapy regimens to protect healthy cells from the toxic effects of chemotherapy drugs while allowing cancer cells to be treated with the administered chemotherapy drug. Typical chemoprotective agents include, but are not limited to, amifostine (Ethyol, Medimmune, Inc.), which is used to reduce renal toxicity associated with cumulative doses of cisplatin, dexrazoxane (Totect, Apricus Pharma; Zinecard), to treat anthracycline-induced extravasation (Totect) and for the treatment of cardiovascular complications caused by the anticancer antibiotic doxorubicin (Zinecard) and mesna (Mesnex, Bristol-Myers Squibb), which is used to prevent hemorrhagic cystitis during chemotherapy with ifosfamide.

Hormonal agents

The linkers of the present invention can be used to conjugate an antibody to at least one hormonal agent. A hormonal agent (including synthetic hormones) is a compound that interferes with the production or activity of endogenously produced endocrine hormones. In some embodiments, these compounds interfere with cell growth or have a cytotoxic effect. Non-limiting examples include androgens, estrogens, medroxyprogesterone acetate (Provera, Pfizer, Inc.) and progestins.

Antihormonal agents

The linkers of the present invention can be used to conjugate an antibody to at least one antihormonal agent. An "anti-hormonal" agent is an agent that suppresses the production and/or prevents the functioning of certain endogenous hormones. In one embodiment, the antihormonal agent interferes with the activity of a hormone selected from the group consisting of androgens, estrogens, progesterone, and gonadotropin-releasing hormone, thereby inhibiting the growth of various cancer cells. Representative examples of antihormonal agents include, but are not limited to, aminoglutethimide, anastrozole (Arimidex, AstraZeneca Pharmaceuticals), bicalutamide (Casodex, AstraZeneca Pharmaceuticals), cyproterone acetate (Cyprostat, Bayer PLC), degarelix (Firmagon, Ferring Pharmaceuticals), exemestane (Aromasin, Pfizer Inc.), flutamide (Drogenil, Schering-Plough Ltd), fulvestrant (Faslodex, AstraZeneca Pharmaceuticals), goserelin (Zolodex, AstraZeneca Pharmaceuticals), letrozole (Femara, Novartis Pharmaceuticals Corporation), leuprolide (Prostap), lupron, medroxyprogesterone acetate ( Provera, Pfizer Inc.), megestrol acetate (Megace, Bristol-Myers Squibb Company), tamoxifen (Nolvadex, AstraZeneca Pharmaceuticals), and triptorelin (Decapetyl, Ferring).

Corticosteroids

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

Photoactive Therapeutic Agents

The linkers of the present invention can be used to conjugate an antibody to at least one photoactive therapeutic agent. Photoactive therapeutic agents include compounds that can be used to kill treated cells when exposed to electromagnetic radiation of a certain wavelength. Therapeutically relevant compounds absorb electromagnetic radiation at wavelengths that penetrate tissues. In preferred embodiments, the compound is administered in a non-toxic form that is capable of producing a photochemical effect that is toxic to cells or tissues when sufficiently activated. In other preferred embodiments, these compounds are retained by cancerous tissue and are easily removed from normal tissues. Non-limiting examples include various chromagens and dyes.

Oligonucleotides

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

Antisense oligonucleotides

The linkers of the present invention can be used to conjugate an antibody to at least one antisense oligonucleotide. Antisense oligonucleotides are designed to bind to RNA via Watson-Crick hybridization. In some embodiments, the antisense oligonucleotide is complementary to a nucleotide encoding a region, domain, portion, or segment of a conjugated antibody. In some embodiments, the antisense oligonucleotide contains from about 5 to about 100 nucleotides, from about 10 to about 50 nucleotides, from about 12 to about 35, and from about 18 to about 25 nucleotides.

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

Another RNase-dependent antisense mechanism that has received much attention recently is RNAi (Fire et al. (1998). Nature, 391, 806-811; Zamore PD. (2002). Science, 296, 1265-1269. ). RNA interference (RNAi) is a post-transcriptional process in which double-stranded RNA inhibits the expression of genes in a particular sequence. In some embodiments, the RNAi effect is achieved by introducing a relatively longer double-stranded RNA (dsRNA), while in preferred embodiments, this RNAi effect is achieved by introducing shorter double-stranded RNAs, such as small interfering RNA (siRNA) and/or miRNA ( miRNA). In yet another embodiment, RNAi can also be achieved by introducing a plasmid that generates a dsRNA that is complementary to the target gene. In each of the above embodiments, the double-stranded RNA is designed to interfere with gene expression of a particular target sequence in cells. Typically, this mechanism involves the conversion of dsRNAs to short RNAs that direct ribonucleases to homologous mRNA targets (summarized by RRuvkun, Science 2294:797 (2001)), which then leads to degradation of the corresponding endogenous mRNA, thereby leading to modulation of gene expression. Notably, dsRNA has anti-proliferative properties, which also allows for therapeutic applications (Aubel et al., Proc. Natl. Acad. Sci., USA 88:906 (1991)). For example, synthetic dsRNA has been shown to inhibit tumor growth in mice (Levy et al. Proc. Nat. Acad. Sci. USA, 62:357-361 (1969)), is actively involved in the treatment of leukemia in mice (Zeleznick et al. , Proc. Soc. Exp. Biol. Med. 130:126-128 (1969)), and inhibits chemically induced tumorigenesis in mouse skin (Gelboin et al., Science 167:205-207 (1970)). Thus, in preferred embodiments, the invention provides for the use of antisense oligonucleotides in ADCs for the treatment of breast cancer. In other embodiments, the invention relates to compositions and methods for initiating antisense oligonucleotide treatment, wherein the dsRNA interferes with the expression of EGFR by target cells at the mRNA level. The term "dsRNA" as used above refers to natural RNA, partially purified RNA, recombinantly produced RNA, synthetic RNA, and altered RNA that differs from natural RNA by the inclusion of non-standard nucleotides, non-nucleotide material, nucleotide analogs (e.g. closed nucleic acid (LNA) ), deoxyribonucleotides, and any combination thereof. The RNA of the invention should be sufficiently similar to native RNA to be able to mediate the oligonucleotide-based antisense modulation described herein.

Aptamers

The linkers of the present invention can be used to conjugate an antibody to at least one aptamer. An aptamer is a nucleic acid molecule that has been selected from random pools based on its ability to bind other molecules. Like antibodies, aptamers can bind target molecules with extraordinary affinity and specificity. In many embodiments, the aptamers take on complex, sequence-dependent, three-dimensional shapes that allow them to interact with the target protein, resulting in a tightly bound complex analogous to an antibody-antigen interaction, thus disrupting the function of said protein. The special ability of aptamers to bind strongly and specifically to a target protein highlights their potential as targeted molecular therapies.

CpG oligonucleotides

The linkers of the present invention can be used to conjugate an antibody to at least one CpG antisense oligonucleotide. It is known that bacterial and viral DNA are powerful activators of both innate and specific immunity in humans. These immunological characteristics have been associated with unmethylated CpG dinucleotide motifs found in bacterial DNA. Because these motifs are rare in humans, the human immune system has evolved the ability to recognize these motifs as an early sign of infection and subsequently initiate immune responses. Therefore, oligonucleotides containing this CpG motif can be used to initiate an antitumor immune response.

Ribozymes

The linkers of the present invention can be used to conjugate an antibody to at least one ribozyme. Ribozymes are catalytic RNA molecules approximately 40 to 155 nucleotides in length. The ability of ribozymes to recognize and cut certain RNA molecules makes them potential candidates for therapeutic agents. A typical example includes angiozyme.

Radionuclide agents (radioactive isotopes)

The linkers of the present invention can be used to conjugate an antibody to at least one adionuclide agent. Radionuclide agents include agents that have an unstable core capable of undergoing radioactive decay. The basis of successful radionuclide treatment depends on sufficient concentration and long-term retention of the radionuclide by the cancer cell. Other factors to consider include the half-life of the radionuclides, the energy of the emitted particles, and the maximum distance that the emitted particle can travel. In preferred embodiments, the therapeutic agent is a radionuclide selected from the group consisting of 111In, 177Lu, 212Bi, 213Bi, 211At, 62Cu, 64Cu, 67Cu, 90Y, 125I, 131I, 32P, 33P, 47Sc, 111Ag, 67Ga, 1 42Pr, 153Sm, 161Tb, 166Dy, 166Ho, 186Re, 188Re, 189Re, 212Pb, 223Ra, 225Ac, 59Fe, 75Se, 77As, 89Sr, 99Mo, 105Rh, 109Pd, 143Pr, 149Pm, 16 9Er, 194Ir, 198Au, 199Au and 211Pb. Also preferred are radionuclides that substantially decay with emitting Auger particles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111 1, Sb-119, 1-125, Ho-161, Os-189m and Ir-192 . The decay energies of the beta emitting nuclides used are preferably Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-21 1, Ac-225, Fr-221, At -217, Bi-213 and Fm-255. The decay energies of the alpha emitting radionuclides used are preferably 2000-10000 keV, more preferably 3000-8000 keV and most preferably 4000-7000 keV. Additional potential radioisotopes in use include 11C, 13N, 150, 75Br, 198Au, 95Ru, 97Ru, 103Ru, 105Ru, 107Hg, 203Hg, 121mTe, 122mTe, 125mTe, 165Tm, 167Tm, 168Tm, 197Pt, 109Pd, 105Rh, 142Pr, 143Pr, 161Tb , 166Ho, 199Au, 57Co, 58Co, 51Cr, 59Fe, 75Se, 201Tl, 225Ac, 76Br, 169Yb, etc.

Radiosensitizers

The linkers of the present invention can be used to conjugate an antibody to at least one radiosensitizer. As used herein, the term "radiosensitizer" is defined as a molecule, preferably a small molecule, administered to animals in therapeutically effective amounts to increase the sensitivity of radiosensitized cells to electromagnetic radiation and/or to stimulate the treatment of diseases that are treatable by electromagnetic radiation. Radiosensitizers are agents that make cancer cells more sensitive to radiation therapy, but usually have much less of an effect on normal cells. Thus, a radiosensitizer can be used in combination with a radiolabeled antibody or ADC. The addition of a radiosensitizer may result in increased efficacy compared to treatment with a radiolabeled antibody or antibody fragment alone. Radiosensitizers are described in D. M. Goldberg (ed.), Cancer Therapy with Radiolabeled Antibodies, CRC Press (1995). Examples of radiosensitizers include gemcitabine, 5-fluorouracil, taxane, and cisplatin.

Radiosensitizers can be activated by electromagnetic radiation from x-rays. Representative examples of X-ray activated sensitizers include, but are not limited to, metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, ethanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, E09, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR), 5 -iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisplatin, as well as their therapeutically effective analogues and derivatives. Alternatively, radiosensitizers can be activated using photodynamic therapy (PDT). Typical examples of photodynamic radiosensitizers include, but are not limited to, hematoporphyrin derivatives, photofrin(r), benzoporphyrin derivatives, NPe6, tin etioporphyrin (SnET2), pheoborbide a, bacteriochlorophyll a, naphthalocyanines, phthalocyanines, zinc phthalocyanines, as well as their therapeutically effective analogues and derivatives.

Topoisomerase inhibitors

The linkers of the present invention can be used to conjugate an antibody to at least one topoisomerase inhibitor. Topoisomerase inhibitors are chemotherapeutic agents designed to interfere with the action of topoisomerase enzymes (topoisomerases I and II), which are enzymes that control changes in DNA structure by catalyzing and then breaking down and reconnecting the phosphodiester backbone of DNA strands during the normal cell cycle. Representative examples of DNA topoisomerase I inhibitors include, but are not limited to, the camptothecins and their derivatives, irinotecan (CPT-11, Camptosar, Pfizer, Inc.) and topotecan (Hycamtin, GlaxoSmithKline Pharmaceuticals). Representative examples of DNA topoisomerase II inhibitors include, but are not limited to, amsacrine, daunorubicin, doxotrubicin, epipodophyllotoxins, ellipticins, epirubicin, etoposide, razoxane, and teniposide.

Tyrosine kinase inhibitors

The linkers of the present invention can be used to conjugate an antibody to at least one tyrosine kinase inhibitor. Tyrosine kinases are enzymes in the cell that function to attach phosphate groups to the amino acid tyrosine. Tumor growth can be inhibited by blocking the ability of protein tyrosine kinases to function. Examples of tyrosine kinases that can be used in the ADC of the present invention include, but are not limited to, Axitinib, Bosutinib, Cediranib, Dasatinib, Erlotinib, Gefitinib, Imatinib, Lapatinib, Lestaurtinib, Nilotinib, Semaxanib, Sunitinib, and Vandetanib.

Other agents

Examples of other agents that can be used in the ADC of the present invention include, but are not limited to, abrin (e.g. abrin A chain), alpha toxin, Aleurites fordii proteins, amatoxin proteins, crotin, curcine, diantine, diphtheria toxin ( e.g. diphtheria toxin A chains, non-binding active diphtheria toxin moieties), deoxyribonuclease (DNase), gelonin, mitogellin, modeccin A chains, Memordica charantia inhibitor, neomycin, onconase, fenomycin, Phytolaca americana proteins (PAPI, PAPII and PAPe S), lakos antiviral protein, Pseudomonas endotoxin, Pseudomonas exotoxin (eg, exotoxin A chain (Pseudomonas aeruginosa)), restrictocin, ricin, ricin A chain, ribonuclease (RNase), Saponaria officinalis inhibitor, saporin, alpha-sarcin, staphylococcal enterotoxin A, tetanus toxin, cisplatin, carboplatin, and oxaliplatin (Eloxatin, Sanofi Aventis), proteasome inhibitors (eg, PS-341 [bortezomib or velcade]), HDAC inhibitors (vorinostat (Zolinza, Merck & Company, Inc.)), belinostat, entinostat , mocetinostat, and panobinostat), COX-2 inhibitors, substituted ureas, heat shock protein inhibitors (eg, geldanamycin and its many analogues), adrenocortical suppressants, and trichothecenes. (See, for example, WO 93/21232). Other agents also include asparaginase (Espar, Lundbeck Inc.), hydroxyurea, levamisole, mitotane (Lysodren, Bristol-Myers Squibb) and tretinoin (Renova, Valeant Pharmaceuticals Inc.).

It should be noted that the aforementioned groups of drug moieties that can be used in the ADC of the present invention are not exclusive, since some examples of drugs can be found in more than one category, for example, ansamitocins are both mitotic inhibitors and antitumor antibiotics.

All stereoisomers of the above drug moieties are considered for the compounds of the present invention, i.e. for any combination of R and S configurations on the chiral carbons D.

The terms "detectable fragment" or "marker" refers to a composition that can be detected spectroscopically, photochemically, biochemically, immunochemically, radioactively or chemically. For example, the label used includes ^32P , ^35S , fluorescent dyes, electron-dense reagents, enzymes (such as enzymes commonly used in ELISA), biotin-streptavidin, dioxygenin, hapten, and proteins for which antisera or monoclonal antibodies are available, or a nucleic acid molecule with a sequence complementary to the target. The detectable fragment often generates a measurable signal, such as a radioactive signal, a color signal, or a fluorescent signal, which can be used to quantify the amount of detectable fragment that binds in the sample. Signal quantification can be done, for example, by scintillation counting, density metering, flow cell analysis, ELISA, or direct analysis by mass spectroscopy of circular or subsequently cleaved peptides (one or more peptides can be analyzed). Those skilled in the art are familiar with methods and means for detecting a label compound of interest. These methods and techniques are conventional and well known in the art.

A detection probe refers to (i) a material capable of providing a detectable signal, (ii) a material capable of interacting with the first probe or the second probe to change the detectable signal provided by the first probe or the second probe, such as fluorescence resonance energy transfer (FRET), (iii) a material capable of stabilizing an interaction with an antigen or ligand or increasing binding affinity, (iv) a material capable of influencing electrical mobility or cell invasiveness through physical parameters such as charge, hydrophobicity, etc., or (v ) a material capable of regulating the affinity of a ligand, the binding of an antigen to an antibody, or the formation of an ionic complex.

Antibodies

An ADC antibody can be any antibody that usually, but not necessarily specifically, binds to an antigen expressed on the surface of a target cell of interest. The antigen is not necessarily, but in some embodiments, capable of internalizing its associated ADC into the cell. Target cells of interest may include cells in which induction of apoptosis is desired. Target antigens can be any protein, glycoprotein, polysaccharide, lipoprotein, etc. expressed in the target cell of interest, but they are usually proteins that are either uniquely expressed in the target cell and not in normal or healthy cells, or which are overexpressed on the target cell relative to normal or healthy cells such that the ADCs are selectively targeted to specific cells of interest, such as, for example, tumor cells. As will be understood by those skilled in the art, the specific antigen, and hence the antibody chosen, will depend on the identity of the desired target cell of interest. In specific embodiments, the ADC antibody is an antibody suitable for administration to a human.

Antibodies (Ab) and immunoglobulins (Igs) are glycoproteins having similar structural characteristics. While antibodies exhibit binding specificity for a particular target, immunoglobulins include both antibodies and other antibody-like molecules that lack target specificity. Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end.

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

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

The antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that is able to recognize and bind to a specific antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology , 5th Ed., Garland Publishing, New York). The target antigen typically has multiple binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody comprises a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, that is, a molecule that contains an antigen-binding site that immunospecifically binds an antigen of a target of interest, or a portion thereof, such targets include, but are not limited to, a cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin described herein may be of any type ( eg , IgG, IgE, IgM, IgD, and IgA), class ( eg , IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of the immunoglobulin molecule. Immunoglobulins can be obtained from any species. However, in one aspect, the immunoglobulin is of human, mouse, or rabbit origin.

The term "antibody fragment" refers to a portion of a full length antibody, usually a binding target or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments. The "Fv" fragment is the minimum antibody fragment that contains the complete target recognition and binding site. This region consists of a dimer of one variable heavy chain and one light chain light chain in a tight non-covalent association (VH-VL dimer). It is in this configuration that the 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 on an antibody. However, in some cases even a single variable domain (or half of an Fv containing only three target-specific CDRs) may have the ability to recognize and bind a target. "Single chain Fv" or "scFv" antibody fragments contain the VH and VL domains of the antibody in a single polypeptide chain. In general, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, allowing the sFv to form the desired structure for binding to the target. "Single domain antibodies" consist of individual VH or VL domains that exhibit sufficient affinity for the target. In a specific embodiment, the single domain antibody is a camel antibody (see, for example , Riechmann, 1999, Journal of Immunological Methods 231:25-38).

The Fab fragment contains a light chain constant domain and a heavy chain first constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of several residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. F(ab') fragments are obtained by cleaving the disulfide bond in the hinge cysteines of the pepsin cleavage product F(ab')2. Additional methods for chemically linking antibody fragments are well known to those skilled in the art.

The variable domains of both the light chain and the heavy chain have complementarity determining regions (CDRs), also known as hypervariable regions. More conservative fragments of variable domains are called framework (FR). As is known in the art, the amino acid position/boundary defining the hypervariable region of an antibody may vary depending on the context and various definitions known in the art. Some positions in the variable region can be considered as hybrid hypervariable positions in the sense that these positions can be considered to be within the hypervariable region on one set of criteria, while they are considered to be outside the hypervariable region on another set of criteria. One or more of these positions can also be found in the extended hypervariable regions. The CDRs in each strand are held together in close proximity by FR regions and, together with the CDRs from the other strand, participate in the formation of an antibody-target binding site (see Kabat et al. , Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. 1987) For the purposes of this document, immunoglobulin amino acid residue numbering follows the immunoglobulin amino acid residue numbering system of Kabat et al. , unless otherwise indicated.

In certain embodiments, the ADC antibodies of the present invention are monoclonal antibodies. The term "monoclonal antibody" (mAb) refers to an antibody that is derived from a single copy or clone, including, for example , any eukaryotic, prokaryotic or phage clone, and not to the method by which it is obtained. Preferably, the monoclonal antibody of the present invention exists in a homogeneous or substantially homogeneous population. A monoclonal antibody includes both intact molecules and antibody fragments (such as, for example, Fab and F(ab')2 fragments) that are capable of specifically binding to a protein. The Fab and F(ab')2 fragments lack the Fc fragment of the intact antibody, are more rapidly cleared from the animal's circulation, and may have less non-specific tissue binding than the intact antibody (Wahl et al. , 1983, J. Nucl. Med 24:316) . Monoclonal antibodies suitable for use in the present invention can be obtained using a wide range of methods known in the art, including the use of hybridoma, recombinant and phage display technologies, or combinations thereof. The antibodies of the present invention include chimeric, primatized, humanized or human antibodies.

Although in most cases antibodies consist only of genetically encoded amino acids, in some embodiments, non-encoded amino acids may be specifically included. Examples of non-encoded amino acids that can be included in antibodies for use in controlling stoichiometry and attachment location, as well as methods for making such modified antibodies, are discussed in Tian et al. , 2014, Proc Nat'l Acad Sci USA 111(5):1766-1771 and Axup et al. , 2012, Proc Nat'l Acad Sci USA 109(40):16101-16106, the entire contents of which are incorporated herein by reference.

In certain embodiments, an ADC antibody described herein is a chimeric antibody. As used herein, the term "chimeric" antibody refers to an antibody having variable sequences derived from a non-human immunoglobulin, such as a rat or mouse antibody, and human immunoglobulin constant regions, typically selected from a human immunoglobulin template. Methods for producing chimeric antibodies are known in the art. See , for example , Morrison, 1985, Science 229(4719):1202-7; Oi et al. , 1986, BioTechniques 4:214-221; Gillies et al. , 1985, J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5807715; 4816567; and 4816397, the contents of which are incorporated herein by reference in their entirety.

In certain embodiments, an ADC antibody described herein is a humanized antibody. "Humanized" forms of non-human ( e.g. murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antibody-target-binding subdomains) that contain minimal sequences derived from non-human immunoglobulin. In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDRs correspond to non-human immunoglobulin domains and all or substantially all of the FRs. are human immunoglobulin sequence sequences. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin consensus sequence. Methods for producing antibody humanization are known in the art. See , for example, Riechmann et al. , 1988, Nature 332:323-7; US patents No. 5530101; 5585089; 5693761; 5693762; and US Pat. No. 6,180,370 to Queen et al. ; EP239400; PCT Publication WO 91/09967; US patent No. 5225539; EP592106; EP519596; Padlan, 1991, Mol. Immunol. , 28:489-498; Studnicka et al. , 1994, Prot. Eng. 7:805-814; Roguska et al. , 1994, Proc. Natl. Acad Sci. USA 91:969-973; and US Pat. No. 5,565,332, all of which are incorporated herein by reference in their entirety.

In certain embodiments, the ADC antibody described herein is a human antibody. Fully "human" antibodies may be desirable for therapeutic treatment of human patients. As used herein, the term "human antibodies" includes antibodies having the amino acid sequence of a human immunoglobulin and includes antibodies isolated from human or animal immunoglobulin libraries transgenic for one or more human immunoglobulins and which do not express endogenous immunoglobulins. Human antibodies can be generated by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See US Patent Nos. 4,444,887; 4,716,111; 6,114,598; 6,207,418; 6,235,883; 7,227,002; Human antibodies can also be generated using transgenic mice that are not capable of expressing functional endogenous immunoglobulins but that can express human immunoglobulin genes. See, for example , US Pat. Nos. 5,413,923; 5625126; 5633425; 5569825; 5661016; 5545806; 5814318; 5885793; 5916771; 5939598; 7723270; 8,809,051 and US Published Application No. 2013/117871, the contents of which are incorporated herein by reference in their entirety. In addition, companies such as Medarex (Princeton, NJ), Astellas Pharma (Deerfield, Ill.), and Regeneron (Tarrytown, N.Y.) can be used to provide human antibodies directed against a selected antigen using technology as described above. Fully human antibodies that recognize the selected epitope can be obtained using a technique called "guided selection". In this approach, a selected non-human monoclonal antibody, such as a mouse antibody, is used to guide the selection of a fully human antibody that recognizes the same epitope (Jespers et al. , 1988, Biotechnology 12:899-903).

In certain embodiments, an ADC antibody described herein is a primatized antibody. The term "primatized antibody" refers to an antibody containing simian variable regions and human constant regions. Methods for producing primatized antibodies are known in the art. See for example US Patent Nos. 5,658,570; 5681722; and 5693780, the contents of which are incorporated herein by reference in their entirety.

In certain embodiments, an ADC antibody described herein is a bispecific or dual variable domain (DVD) antibody. Bispecific antibodies and DVD antibodies are monoclonal, often human or humanized, antibodies that have binding specificities for at least two different antigens. DVDs are described, for example, in US Pat. No. 7,612,181, the disclosure of which is incorporated herein by reference.

In certain embodiments, an ADC antibody described herein is a derivatized antibody. For example, and not by way of limitation, derivatized antibodies are typically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, binding to a cellular ligand or other protein, etc. Any of the many methods of chemical modification can be performed according to known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, the derivative may contain one or more unnatural amino acids, for example , using ambrx technology (see. eg Wolfson, 2006, Chem Biol 13(10):1011-2).

In certain embodiments, an ADC antibody described herein has a sequence that has been modified to alter at least one constant region-mediated biological effector function relative to a corresponding wild-type sequence. For example, in some embodiments, an antibody can be modified to reduce at least one biological effector function mediated by a constant region compared to an unmodified antibody, such as reduced Fc receptor (FcR) binding. FcR binding can be reduced by mutating the immunoglobulin constant region segment of the antibody in certain regions required for FcR interactions ( see , e.g. , Canfield and Morrison, 1991, Med 173:1483-1491; and Lund et al. , 1991, J. Immunol 147 :2657-2662).

In certain embodiments, an ADC antibody described herein is modified to acquire or improve at least one constant region-mediated biological effector function relative to an unmodified antibody, e.g. , to enhance FcγR interactions ( see , e.g. , US 2006/0134709). For example, an antibody with a constant region that binds FcγRIIA, FcγRIIB, and/or FcγRIIIA with greater affinity than the corresponding wild-type constant region can be made according to the methods described herein.

In some specific embodiments, an ADC antibody described herein is an antibody that binds tumor cells, such as an anti-cell surface receptor antibody or a tumor associated antigen (TAA). In an attempt to discover effective cellular targets for cancer diagnosis and treatment, researchers have attempted to identify transmembrane or otherwise 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-cancer cells. Often such tumor-associated polypeptides are expressed on the surface of cancer cells to a greater extent than on the surface of non-cancerous cells. Such cell surface receptors and tumor antigens are known in the art and can be prepared for use in generating antibodies using methods and information that are well known in the art.

Exemplary cell surface receptors and TAA

Examples of cell surface receptors and TAAs that can be targeted by the ADC antibody described herein include, but are not limited to, the various receptors and TAAs listed below in Table 1. For convenience, information relating to these antigens, all of which known in the art is listed below and includes names, alternate names, Genbank access numbers, and primary references, following National Center for Biotechnology Information (NCBI) nucleic acid and protein sequence identification conventions. Nucleic acid and protein sequences corresponding to the listed cell surface receptors and TAAs are available from public databases such as GenBank.

Table 1.

4-1BB 5AC 5T4 Alpha fetoprotein angiopoietin 2 ASLG659 TCL1 BMPR1B Brevican (BCAN, BEHAB) Antigen C2-42 C5 CA-125 CA-125 (imitation) CA-IX (carbonic anhydrase 9) CCR4 CD140a CD152 CD19 CD20 CD200 CD21 (C3DR) 1) CD22 (CD22-B isoform of the B-cell receptor) CD221 CD23 (gE receptor) CD28 CD30 (TNFRSF8) CD33 CD37 CD38 (cyclic ADP-ribose hydrolase) CD4 CD40 CD44v6 CD51 CD52 CD56 CD70 CD72 (Lyb-2, B-cell differentiated antigen CD72) CD74 CD79a (CD79A, CD79α, immunoglobulin-associated alpha) Genbank accession number NP_001774.10) CD79b (CD79B, CD79β, B29) CD80 CEA CEA-associated antigen ch4D5 CLDN18.2 CRIPTO (CR, CR1, CRGF, TDGF1 growth factor derived from teratocarcinoma) CTLA-4 CXCR5 DLL4 DR5 E16 (LAT1, SLC7A5) EGFL7 EGFR Epcam EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5) Episialin ERBB3 ETBR (endothelin type B receptor) FCRH1 (Fc receptor-like protein 1) FcRH2 (IFGP4, IRTA4, SPAP1, SPAP1B, SPAP1C containing SH2-domain phosphatase anchor protein Fibronectin extra domain-B Folate receptor 1 Receptor Frizzled GD2 [0260] Ganglioside GD3 GEDA GPNMB HER1 HER2 (ErbB2) HER2/neu HER3 HGF HLA-DOB HLA-DR Human scatter factor receptor kinase IGF-1 receptor IgG4 IL-13 IL20Ra (IL20Ra, ZCYTOR7) IL-6 ILGF2 ILFR1R integrin α α5β1 integrin αvβ3 integrin IRTA2 (immunoglobulin superfamily receptor associated with translocation 2, gene chromosome 1q21) Lewis Y antigen LY64 (RP105) MCP-1 MDP (DPEP1) MPF (MSLN, SMR, mesothelin, megakaryocyte potentiating factor) MS4A1 MSG783 (RNF124, hypothetical protein FLJ20315) MUC1 Mucin-like cancer-associated antigen Napi3 (NAPI-3B, NPTIIb, SLC34A2, type II sodium dependent phosphate transporter 3b) NCA (CEACAM6) P2X5 (ligand ion channel 5 of the P2X purinergic receptor) PD-1 PDCD1 PDGF-Rα prostate-specific membrane antigen PSCA (prostate stem cell antigen precursor) PSCA hlg RANKL RON SDC1 Sema 5b SLAMF7 (CS-1) STEAP1 STEAP2 (HGNC_8639, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1) TAG-72 TEM1 Tenascin C TENB2, (TMEFF2, tomoregulin, TPEF, HPP1, TR) TGF-β TRAIL-E2 TRAIL-R1 TRAIL-R2 TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4) TA CTAA16.88 TWEAK-R TYRP1 (glycoprotein 75) VEGF VEGF-A EGFR-1 VEGFR-2 Vimentin

Generic antibodies

Exemplary antibodies to be used with the ADCs of the present invention include, but are not limited to, 3F8 (GD2), abagovomab (CA-125 (mock)), adecatumumab (EpCAM), afutuzumab (CD20), alazizumab pegol (VEGFR2), ALD518 (IL-6), alemtuzumnab (CD52), altumomab pentetate (CEA), amatuximab (mesothelin), anatumomnab mafenatox (TAG-72), apolizumab (HLA-DR), arcitumomab (CEA), bavituximab (phosphatidylserine), bektumomab ( CD22), belimumab (BAFF), besilesomab (CEA-associated antigen), bevacizumab (VEGF-A), bivatuzumab mertansine (CD44 v6), blinatumomab (CD19), brentuximab vedotin ((CD30 (TNFRSF8)), cantuzumab mertansine (mucin-like cancer -associated antigen), cantuzumab ravtansin (MUC1), capromab pendetide (prostate cancer cells), carlumab (MCP-1), catumaxomab (EpCAM, CD3), CC49 (Tag-72), cBR96-DOX ADC (Lewis Y antigen) , cetuximab (EGFR), citotuzumab richox (EpCAM), cixutumumab (IGF-1 receptor), clivatuzumab tetraxetane (MUC1), conatumumab (TRAIL-E2), dacetuzumab (CD40), dalotuzumab (insulin-like growth factor receptor 1), deratumumab (( CD38 (cyclic ADP-ribose hydrolase)), demcizumab (DLL4), denosumab (RANKL), detumomab (B cell), drozitumab (DR5), dusigitumab (ILGF2), ecromeximab (ganglioside D3), eculizumab (C5), edrecolomab (EpCAM), elotuzumab (SLAMF7), elsilimoab (IL-6), enavatuzumab (TWEAK receptor), enoticumab (DLL4), encituximab (5AC), epitumomab cituxetan (episialin), epratuzumab (CD22), ertumaxomab ((HER2/neu, CD3)), etanzizumab (αvβ3 integrin), farletuzumab (folate receptor 1), FBTA05 (CD20), ficlatuzumab (HGF), figitumumab (IGF-1 receptor), flanvotumab ((TYRP1 (glycoprotein 75)), fresolimumab (TGF-1 ), galiximab (CD80), ganitumab (IGF-I), gemtuzumab ozogamicin (CD33), gyrentuximab ((carboxylic anhydrase 9 (CA-IX)), glembatumumab vedotin (GPNMB), ibritumomab tiuxetan (CD20), icrucumab (VEGFR-1 ), igovomab (CA-125), IMAB362 (CLDN18.2), imgatuzumab (EGFR), indatuximab ravtansine (SDC1), intetumumab (CD51), inotuzumab ozogamicin (CD22), ipilimumab (CD152), iratumumab ((CD30 (TNFRSF8) ), labetuzumab (CEA), lambrolizumab (PDCD1), lexatumumab (TRAIL-R2), lintuzumab (CD33), lorvotuzumab mertansine (CD56), lucatumumab (CD40), lumiliximab ((CD23 (IgE receptor)), mapatumumab (TRAIL-R1 ), margetuximab (ch4DS), matuzumab (EGFR), milatuzumab (CD74), mitumomab (ganglioside GD3), mogamulizumab (CCR4), moxetumomab pasudotox (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), onartuzumab (human scatter factor receptor kinase), ontuxizumab (TEM1), oportuzumab monato (EpCAM), oregovomab (CA-125), otlertuzumab (CD37), panitumumab (EGFR) pancomab (tumor-specific glycosylation of MUC1), parzatuzumab (EGFL7), patritumab (HER3), pemtumomab (MUC1), pertuzumab (HER2/neu), pidilizumab (PD-1), pinatuzumab vedotin (CD22), pritumumab (vimentin), racotumomab (N-glycolylneuraminic acid), radretumab (fibronectin extra domain-B) , ramucirumab (VEGFR2), rilotumumab (HGF), rituximab (CD20), robatumumab (IGF-1 receptor), samalizumab (CD200), satumomab pendetide (TAG-72), seribantumab (ERBB3), sibrotuzumab (FAP), SGN-CD19A (CD19), SGN-CD33A (CD33), siltuximab (IL-6), solitomab (EpCAM), sonepcizumab (sphingosine-1-phosphate), tabalumb (BAFF), takatuzumab tetraxetane (alpha-fetoprotein), taplitumomab paptox (CD19) , tenatumumab (tenascin C), teprotumumab (CD221), TGN1412 (CD28), ticilimumab (CTLA-4), tigatuzumab (TRAIL-R2), TNX-650 (IL-13), tovetumab (CD40a), trastuzumab (HER2/neu ), TRBS07 (GD2), tremelimumab (CTLA-4), tucotuzumab celmoleukin (EpCAM), ublituximab (MS4A), urelumab (4-1BB), vandetanib (VEGF), vantictumab (Frizzled receptor), volociximab (α5β1 integrin), vorzetuzumab mafodotin (CD70), votumumab (Tumor CTAA16.88), zalutumumab (EGFR), zanolimumab (CD4), and zatuximab (HER1).

Methods for obtaining antibodies

The ADC antibody can be produced by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. For example, to express an antibody in a recombinant manner, a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and optionally secreted into the environment, in which host cells are cultured, from which antibodies can be recovered. Standard recombinant DNA techniques are used to generate antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors, and introduce the vectors into host cells such as those described in Molecular Cloning; A Laboratory Manual, Second Edition (Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, NY, 1989), Current Protocols in Molecular Biology (Ausubel, FM et al. , eds., Greene Publishing Associates, 1989) and in patent USA No. 4816397.

In one embodiment, anti-Fc variant antibodies are similar to their wild-type equivalents, but with changes in their Fc domains. To obtain nucleic acids encoding such anti-Fc variant antibodies, a DNA fragment encoding the Fc domain or part of the Fc domain of a wild-type antibody (referred to as "wild-type Fc domain") can be synthesized and used as a template for mutagenesis to generate an antibody, as described herein using routine mutagenesis techniques; alternatively, the DNA fragment encoding the antibody can be synthesized directly.

Once DNA fragments encoding wild-type Fc domains have been obtained, subsequent manipulations of these DNA fragments can be carried out using standard recombinant DNA techniques, for example, to convert constant region genes into full-length antibody chain genes. During these manipulations, a DNA fragment encoding CH is operably linked to another DNA fragment encoding another protein, such as an antibody variable region or a flexible linker. In the context of this document, the term "operably linked" means that two DNA fragments are combined in such a way that the amino acid sequences encoded by these two DNA fragments remain in frame.

For expression of Fc variant antibodies, DNAs encoding partial or full length light and heavy chains, prepared as described above, are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" means that the antibody gene is ligated into the vector such that the transcriptional and translational control sequences in the vector perform their intended function of regulating transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gne variant and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector.

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

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

In addition to antibody chain genes and regulatory sequences, recombinant expression vectors may carry additional sequences, such as sequences that regulate the vector's replication in host cells ( eg origins of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced ( see , for example , US Pat . For example, a selectable marker gene typically confers resistance to drugs such as G418, puromycin, blastidine, hygromycin, or methotrexate to the host cell into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR host cells with methotrexate selection/amplification) and the neo gene (for G418 selection). For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains are transfected into the host cell by standard methods. The various forms of the term "transfection" are intended to cover a wide range of methods commonly used to introduce exogenous DNA into a prokaryotic or eukaryotic host cell, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, and the like.

You can express antibodies in either prokaryotic or eukaryotic host cells. In some embodiments, antibody expression is performed in eukaryotic cells, such as mammalian host cells, for optimal secretion of a properly folded and immunologically active antibody. Exemplary mammalian host cells for expressing recombinant antibodies include Chinese hamster ovary (CHO) cells (including DHFR-CHO cells described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, eg as described in Kaufman and Sharp (1982) Mol Biol 159:601-621), NS0 myeloma cells, COS cells, 293 cells and SP2/0 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, antibodies are obtained by culturing the host cells for a period of time sufficient to allow expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. . Antibodies can be isolated from the culture medium using standard protein purification methods. Host cells can also be used to obtain parts of intact antibodies such as Fab fragments or scFv molecules.

In some embodiments, the ADC antibody may be a bifunctional antibody. Such antibodies in which one heavy and one light chain are specific for one antigen and the other heavy and light chain are specific for a second antigen can be prepared by crosslinking the antibody to the second antibody by standard chemical crosslinking techniques. Bifunctional antibodies can also be obtained by expression of a nucleic acid designed to encode a bifunctional antibody.

In some embodiments, dual specific antibodies, that is, antibodies that bind one antigen and a second, unrelated antigen using the same binding site, can be generated by mutating amino acid residues in the light chain and/or heavy chain CDRs. Exemplary second antigens include a pro-inflammatory cytokine (such as, for example, lymphotoxin, interferon-γ, or interleukin-1). Dual specific antibodies can be generated, for example , by mutating amino acid residues at the periphery of the antigen-binding site ( see , for example , Bostrom et al. , 2009, Science 323:1610-1614). Dual functional antibodies can be generated by expression of a nucleic acid designed to encode a specific dual antibody.

Antibodies can also be made by chemical synthesis ( eg , the methods described in Solid Phase Peptide Synthesis , 2nd ed., 1984 The Pierce Chemical Co., Rockford, Ill.). Antibodies can also be generated using a cell-free platform ( see , for example , Chu et al. , Biochemia No. 2, 2001 (Roche Molecular Biologicals)).

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

Once an antibody has been produced by recombinant expression, it can be purified by any method known in the art to purify the immunoglobulin molecule, e.g., by chromatography ( e.g. , ion exchange, affinity, in particular, affinity for the antigen after selection of protein A or protein G and size distribution column chromatography), centrifugation, differential solubility, or any other standard protein purification technique.

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

Connections and Imaging Sensors

In certain embodiments, provided herein are methods of use for the disclosed compounds in imaging compositions and as sensors.

The sensor may be a biosensor, a chemical sensor, or a molecular switch. Biosensors are able to identify the presence or amount of a particular material by reacting certain materials ( e.g. cancer cells, viruses, various chemicals, etc.) with bioreceptors (parts designed to adsorb and react with biomaterials such as DNA , RNA, antibodies, enzyme proteins, cells, biological membranes, hormone receptors, etc. ) that have selection specificity and are measured using a signal converter (a device that converts the reaction between a specific material and a biological receptor into an electrical signal using various methods), and can be used for medical, environmental, processing, military (chemical weapons), research, food processing , etc. purposes (see e.g. Biosensors and Bioelectronics , 2016, 32-45 ; Pol. J. Environ. Stud. 2015, 19-25; Analytica Chimica Acta 568 (2006) 200-210; Biosensors and Bioelectronics 2017, 217-231; ACS Appl. mater. Interfaces 2015, 7, 20190-20199; Journal of Coastal Life Medicine 2016; 4(3): 200-202; Artificial Cells, Blood Substitutes, and Biotechnology , 39: 281-288; Journal of Controlled Release 159 (2012) 154-163).

Chemical sensors provide fast and accurate monitoring of specific materials in many fields such as clinical diagnostics, medical research, measurement of chemical materials, environmental measurement , etc. , using the method of using electrical properties such as electrification, resistance, potential difference and etc. , and optical properties such as color, fluorescence , etc. , and include gas sensor (hydrogen, oxygen, carbon monoxide), ion sensor (cation, anion, gas sensitive ion), component sensor (steam phase, liquid phase, luminescent component), humidity sensor (relative humidity, absolute humidity, condensation), dust/soot sensor (suspended dust, dirty dust, soot, turbidity) , etc. ( see e.g. Chem . Soc Rev. , 2015, 44, 3358; Journal of the Korean Chemical Society , 2010, 451-459; Chem. Sci. , 2015, 6, 1150-1158; KR 10-1549347; J. Phys. Chem. B 2016, 120, 7053-7061; ACS Appl. Mater. Interfaces 2015, 7, 704-712; J. Am. Chem. soc. 2011, 133, 10960-10965; J. Am. Chem. soc. 2012, 134, 20412-20420; Org. Lett. 2014, 16, 1680-1683; J. Org. Chem. 2013, 78, 702-705; J. Org. Chem. 2015, 80, 12129-12136; ACS Macro Lett. 2014, 3, 1191-1195; New J. Chem. , 2012, 36, 386-393; Chem. commun. , 2010, 46, 6575-6577; 2013).

A molecular switch is a molecule capable of reversibly switching between two or more stable states. Molecules can switch between states in response to environmental stimuli, such as changes in the chemical environment (eg, pH), exposure to light (eg, light of a certain wavelength), temperature, electrical current, microenvironment, or the presence of a ligand. In some cases, switching between states may depend on a combination of stimuli. The oldest forms of synthetic molecular switches are pH indicators, which display different colors depending on pH. Synthetic molecular switches can be used in molecular computers or adaptive drug delivery systems. Molecular switches are also important in biology because many biological functions are based on them, such as allosteric regulation and vision.

Such biosensors, chemical sensors, and molecular switches may additionally contain additional photoreactive moieties such as rhodamine, phenol red, azo dye orange, papa red, unsulfated cyanine, sulfated cyanine, chemiluminescent fluoride sensor (a derivative of 1,2-dioxetane), and D2A dyes ( NIR-fluorescent dyes). Alternatively, the photoreactive moiety may be selected from a compound having functional groups and structures similar to the following:

Where:

R ₁₀₀ is H or C ₁ -C ₆ alkyl;

R ₁₀₁ is H or SO ₃ H; R ₁₀₂ is C ₁ -C ₆ alkyl or -(CH ₂ ) _z COOH;

z is an integer from 3 to 8;

R ₁₀₃ and R ₁₀₄ are each independently H or C ₁ -C ₆ alkyl; And

R ₁₀₅ and R ₁₀₆ are independently hydrogen COOH or SO ₃ H.

Additional photoreactive moieties are known in the art. See , for example, Org. Lett. 2014, 16, 1680-1683; J. Am. Chem. soc. 2011, 133, 10960-10965; Dye Lasers , 3rd Ed. (Springer-Verlag, Berlin, 1990); J. Am. Chem. soc. 2012, 134, 20412-20420).

Methods of treatment

Targeted Treatment Options

The targeting fragment of the conjugate can be recognized by the cell, thereby providing the so-called targeted treatment.

In some embodiments, the implementation of the conjugate contains an active agent for use in targeted treatment for the treatment of an autoimmune disease. In some such embodiments, the active agents are selected from cyclosporine, cyclosporine A, mycophenylate mofetil, sirolimus, tacrolimus, enanercept, prednisone, azathioprine, methotrexate, cyclophosphamide, aminocaproic acid, chloroquine, hydroxychloroquine, hydrocortisone, dexamethasone, chlorambucil, DHEA, danazol, bromocriptine, meloxicam, infliximab , etc.

In some embodiments, the compound contains active agent Q for use in targeted therapy for the treatment of an infectious disease. In some such embodiments, Q is selected from: the beta-lactam series (penicillin G, penicillin V, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin, ampicillin, amoxicillin, becampicillin, azlocillin, carbenicillin, mezlocillin, piperacillin, ticarcillin), aminoglycoside ( amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin), macrolide series (azithromycin, clarithromycin, erythromycin, lincomycin, clindamycin), tetracycline series (demeclocycline, doxycilin, minocycline, tetracycline), quinolone series (cinoxacin, nalidixic acid), a number of fluoroquinolones (ciprofloxacin, enoxacin, grepafloxacin, levofloxacin, lomefloxacin, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin), a number of polypeptides (bacitracin, colistin, polymyxin B), a number of sulfonamides (sulfisoxazole, sulfamethoxazole, sulfadiazine, sulfamethizole, sulfacetamide), others antibiotics ( trimethoprim, sulfamethoxazole, chloramphenicol, vancomycin, metronidazole, quinupristin, dalfopristin, rifampicin, spectinomycin, nitrofurantoin), common antiviral agents (idoxuradine, vidarabine, aciclovir, famciclovir, penciclovir, valaciclovir, ganciclovir, foscarnet, ribavirin, amantadine, rimantadine, cidofovir, antisense oligonucleotides, immunoglobulins, interferons), therapeutic agents for the treatment of HIV infection (tenofovir, emtricitabine, zidovudine, didanosine, zalcitabine, stavudine, lamivudine, nevirapine, delaviridine, saquinavir, ritonavir, indinavir, nelfinavir) , etc.

In some embodiments, the compounds and conjugates described herein comprise an active agent Q for use in a method for delivering the active agent into a cell to treat a tumor, wherein the targeting moiety is selected to bind to a target cell (i.e., a cancer cell). In particular, the present compounds, conjugates, and compositions may be useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal ( e.g., a human), such as where the target cell is a cancer cell and the targeting moiety is selected to bind to a molecule, associated with a cancer cell (and not associated with healthy cells, or at least predominantly associated with tumor cells rather than healthy cells).

In some such embodiments, the active agent is selected from: a cytotoxic or immunomodulatory agent, an anticancer agent, an antitubulin agent, a cytotoxic agent , etc. Preferably, the cytotoxic or immunomodulatory agent includes an antitubulin agent, auristatin, a DNA minor groove binder, a DNA transcription inhibitor, alkylating agent, anthracycline, antibiotic, antifolate, antimetabolite, calmodulin inhibitor, chemotherapy sensitizer, duocarmycin, etoposide, fluorinated pyrimidine, ionophore, lexitropsin, maytansinoid, nitrosourea, platinol, pore-forming compound, purine antimetabolite, puromycin, radiotherapy sensitizer, rapamycin, steroid, taxane, topoisomerase inhibitor, vinca alkaloid , etc.; anti-cancer agent includes methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan, nitrogen mustards, cytoxan, etoposide, 5-fluorouracil BCNU , irinotecan, camptothecins, bleomycin, doxorubicin, idaribicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel , etc.; antitubulin agent includes taxane ( eg paclitaxel, docetaxel), T67, vinca alkyloid ( eg vincristine, vinblastine, vindesine, vinorelbine), baccatin derivative, taxane derivative, epothionone ( eg epothilone A, epothilone B), nocodazole, colchicine, colcimide , estramustine, critophycins, cemadotin, maytansinoids, combrestatins, discodermolide, eleutherobin, auristatin derivative (AFP, MMAF, MMAE), etc.; cytotoxic agent includes androgen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busulfan, butionine sulfoximine, calicheamicin, calicheamicin derivative, camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucin, cisplatin, colchicine , cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (actinomycin), daunorubicin, decarbazine, DM1, DM4, docetaxel, doxorubicin, etoposide, estrogen, 5-fluorodeoxyuridine, 5-fluorouracil, gemcitabine, gramicidin D, hydroxyurea, and daribicin , ifosfamide, irinotecan, lomustine (CCNU), maytansine, mechlorethamine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel, palitoxin, plicamycin, procarbizin, rizoxin, streptozotocin, tenoposide, 6-thiogua nin, thioTEPA , topotecan, vinblastine, vincristine, vinorelbine, VP-16, VM-26; DNA minor groove binder ( e.g. enediin, lexytropsin, CBI compound), duocarmycin, taxane ( e.g. paclitaxel, docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rizoxin, cyanomorpholino- doxorubicin, echinomycin, combretastatin, netropsin, epothilone A, epothilone B, estramustine, cryptophycins, cemadotin, maytansinoids, discodermolide, eleutherobin, mitoxantrone , etc.

Cell proliferation and apoptosis

The compounds and conjugates described herein can be used in methods for inducing apoptosis in cells.

Dysregulation of apoptosis is associated with various diseases, including, for example, autoimmune disorders ( eg , systemic lupus erythematosus, rheumatoid arthritis, graft-versus-host disease, myasthenia gravis, or Sjögren's syndrome), chronic inflammatory conditions ( eg , psoriasis, asthma, or Crohn's disease) , hyperproliferative disorders ( eg breast cancer, lung cancer), viral infections ( eg herpes, papilloma or HIV) and other conditions such as osteoarthritis and atherosclerosis. The compounds, conjugates and compositions described herein can be used to treat or ameliorate any of these diseases. Such treatments typically include administering to a subject suffering from a disease an amount of a compound, conjugate, or composition described herein sufficient to provide a therapeutic effect. The identity of the antibody of the administered compound, conjugate or composition will depend on the disease being treated, so the antibody must bind a cell surface antigen expressed in a cell type where inhibition would be beneficial. The therapeutic effect achieved will also depend on the particular disease being treated. In certain cases, the compounds and compositions described herein can treat or reduce the disease itself or the symptoms of the disease when used as monotherapy. In other instances, the compounds and compositions described herein may be part of a general treatment regimen, including other agents that, together with an inhibitor or compounds and compositions described herein, treat or ameliorate the disease being treated or the symptoms of the disease. Agents used to treat or ameliorate specific diseases that can be administered in addition to or with the compounds and compositions described herein will be apparent to those skilled in the art.

Although a complete cure is always desirable in any therapeutic regimen, a cure is not required to achieve a therapeutic effect. The therapeutic effect may include stopping or slowing the progression of the disease, regressing the disease without a cure, and/or reducing or slowing the progression of the symptoms of the disease. Extended survival compared to statistical averages and or improved quality of life can also be considered as a therapeutic effect.

One particular class of diseases that involve dysregulation of apoptosis and that represent a truly significant health burden worldwide are various types of cancer. In a specific embodiment, the compounds and compositions described herein can be used to treat various types of cancer. The cancer may be, for example, solid tumors or hematological tumors. Cancers that may be treated with the compounds and compositions described 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, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, myeloma, prostate cancer glands, small cell lung cancer and spleen cancer. The compounds and compositions described herein may be particularly useful in the treatment of cancer, since the antibody can be used to specifically target a tumor cell, thereby potentially preventing or ameliorating unwanted side effects and/or toxicity that may be associated with systemic administration. unconjugated inhibitors. One embodiment relates to a method of treating a disease involving dysregulation of intrinsic apoptosis, comprising administering to a subject suffering from a disease associated with dysregulation of apoptosis an amount of a compound and composition described herein effective to provide a therapeutic effect, wherein the ligand of the compounds and compositions described herein binds a cell surface receptor on a cell with impaired intrinsic apoptosis. One embodiment relates to a method of treating cancer comprising administering to a subject having cancer a compound and composition described herein, wherein the ligand is capable of binding to a cell surface receptor or tumor associated antigen expressed on the surface of cancer cells in an amount effective to providing a therapeutic effect.

In the context of oncogenic cancer, a therapeutic effect, in addition to including the effects discussed above, may also specifically include stopping or slowing the progression of tumor growth, regressing tumor growth, eradicating one or more tumors, and/or improving patient survival compared to statistical averages for type and stage. cancer to be treated. In one embodiment, the cancer being treated is an oncogenic cancer.

The compounds and conjugates described herein may be used as monotherapy to provide a therapeutic effect, or may be used in addition to or with other chemotherapeutic agents and/or radiation therapy. Chemotherapeutic agents, in addition to which the compounds and compositions described herein may be used as adjuvant therapy, may be targeted (eg, ADCs, protein kinase inhibitors , etc. ) or non-targeted (eg, non-specific cytotoxic agents such as as radionucleotides, alkylating agents and intercalating agents). Non-targeted chemotherapeutic agents, in addition to which the compounds and compositions described herein may be administered, include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin , carboplatin, mitomycin, dacarbazine, procarbizin, topotecan, nitrogen mustards, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, calicheamicin and docetaxel.

The compounds and conjugates described herein, which may not be effective as monotherapy for the treatment of cancer, may be administered in addition to or with other chemotherapeutic agents or radiation therapy to provide a therapeutic effect. One embodiment relates to a method in which a compound or composition described herein is administered in an amount effective to sensitize tumor cells to standard chemotherapy and/or radiation therapy. Accordingly, in the context of the treatment of various types of cancer, the term "therapeutic effect" includes the administration of the compounds and compositions described herein in addition to or together with chemotherapeutic agents and/or radiation therapy, or to patients who have not yet begun undergoing such therapy, either in those who have but have not yet shown signs of resistance, or in patients who have begun to show signs of resistance, as a means of sensitizing tumors to chemotherapy and/or radiation therapy.

Pharmaceutical compositions and methods of administration

The compounds and conjugates described herein can be used to treat an individual in need thereof. In certain embodiments of the invention, the individual is a mammal, such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or compound is preferably administered as a pharmaceutical composition containing, for example, the described compound and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline, or other solvents or carriers such as glycols, glycerin, oils such as olive oil, or injectable organic esters. In preferred embodiments of the invention, when such pharmaceutical compositions are intended for administration to a human, especially for invasive routes of administration (i.e., routes such as injection or implantation that provide for overcoming transport or diffusion through the epithelial barrier), the aqueous solution contains no pyrogens or practically no contains pyrogens. Excipients may be selected, for example, to effect a delayed release of the agent or to selectively target one or more cells, tissues, or organs. The pharmaceutical composition may be in the form of a unit dose such as a tablet, a capsule (including an openable capsule and a gelatin capsule), a granule, a lyophilic preparation for reconstitution, a powder, a solution, a syrup, a suppository, an injection, or the like. The composition may also be present in a transdermal delivery system, such as a skin patch. The composition may also be present in a solution suitable for topical application such as an ointment or cream.

A pharmaceutically acceptable carrier may contain physiologically acceptable agents which act, for example, to stabilize, increase solubility, or increase absorption of a compound, such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates such as glucose, sucrose or dextrans, antioxidants such as ascorbic acid or glutathione, chelating agents, small molecular weight proteins, or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The formulation of the pharmaceutical composition may be a self-emulsifying drug delivery system or a spontaneous microemulsion drug delivery system. The pharmaceutical composition (preparation) may also be a liposome or other polymeric matrix, which may include, for example, a compound of the invention. For example, liposomes that contain phospholipids or other lipids are non-toxic, physiologically acceptable and metabolizable carriers that are relatively easy to manufacture and use.

The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and/or dosage forms that, within the framework of rational medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

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

The pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration, including, for example, oral administration (for example, drops, as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including openable capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (for example, sublingually); anally, rectally, or vaginally (eg, as a pessary, cream, or foam); parenterally (including intramuscularly, intravenously, subcutaneously or intrathecally, such as a sterile solution or suspension); intranasally; intraperitoneally; subcutaneously; transdermally (eg, as a patch applied to the skin); and topically (eg, 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 of the invention, the compound may simply be dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable therefor can be found, for example, in US Pat.

The formulations may conveniently be provided in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to form a unit dosage form will vary depending on the organism being treated, the particular route of administration. The amount of active ingredient that can be combined with the carrier material to form a unit dosage form will usually be that amount of the compound that produces a therapeutic effect. In general, out of one hundred percent, this amount will vary from about 1 percent to about ninety-nine percent active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

Methods for preparing these formulations or compositions include the step of bringing into association the active compound, such as a compound of the invention, with the carrier and, optionally, one or more additional ingredients. Typically, formulations are prepared by uniformly and thoroughly combining a compound of the present invention with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Compositions of the invention suitable for oral administration may be in the form of capsules (including openable capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and gum arabic or tragacanth), lyophilic preparations, powders, granules or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as lozenges (using an inert base such as gelatin and glycerin, or sucrose and gum arabic) and/or mouthwashes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. The compounds, conjugates, or compositions may also be administered as a bolus, electuary, or paste.

For the preparation of solid oral dosage forms (capsules (including openable capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), 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 additives such as starches, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or gum arabic; (3) humectants such as glycerin; (4) disintegrants such as agar-agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) retarding dissolution agents such as paraffin; (6) absorption accelerators such as quaternary ammonium compounds; (7) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents such as modified and unmodified cyclodextrins; and (11) dyes. In the case of capsules (including openable capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of this type can also be used as fillers in soft and hard gelatin capsules using excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

Tablets can be obtained by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using a binder (eg gelatin or hydroxypropyl methylcellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surfactant or dispersing agent. Molded tablets can be made by molding in a suitable apparatus a mixture of a powdered compound moistened with an inert liquid diluent.

Tablets and other solid dosage forms of pharmaceutical compositions such as dragees, capsules (including openable capsules and gelatin capsules), pills and granules may be optionally scored or prepared with coatings and shells such as enteric coatings and other coatings well known in the art. the field of pharmaceuticals. They can also be formulated to provide a slow or controlled release of the active ingredient therein using, for example, hydroxypropyl methylcellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporation of sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water or some other sterile injectable medium immediately prior to use. These compositions may also optionally contain opacifiers and may be of a composition from which they release the active ingredient(s) only, or preferably, in a certain part of the gastrointestinal tract, optionally with a delay. Examples of potting compositions that can be used are polymeric materials and waxes. The active ingredient may also be in microencapsulated form, if appropriate, with one or more of the excipients described above.

Liquid dosage forms suitable for oral administration include pharmaceutically acceptable emulsions, lyophilic preparations for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and their derivatives, solubilizing agents, and emulsifiers, such as 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), glycerin, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters with sorbitan and their mixtures.

In addition to inert diluents, oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavors, colors, flavors, and preservatives.

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

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

Formulations of pharmaceutical compositions for oral administration may be presented as a mouthwash or oral spray or oral ointment.

Alternatively or additionally, the compositions may be formulated for delivery through a catheter, stent, guidewire, or other intraluminal device. Delivery through such devices may be particularly useful for delivery to the bladder, urethra, ureter, rectum or intestines.

Formulations suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray forms containing carriers known in the art.

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

Ointments, pastes, creams and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or their mixtures.

Powders and aerosols may contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Aerosols may additionally contain conventional propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

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

Ophthalmic formulations, eye ointments, powders, solutions, and the like are also contemplated as being within the scope of this invention. Typical ophthalmic formulations are described in US Publications 2005/0080056, 2005/0059744, 2005/0031697 and 2005/004074; and US Pat. No. 6,583,124, the contents of which are incorporated herein by reference. If desired, liquid ophthalmic formulations have properties similar to those of lacrimal fluids, intraocular fluid, or vitreous fluid, or are compatible with such fluids.

The phrases "parenteral administration" and "parenteral administration" as used herein mean methods of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac , intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

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

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

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like in the compositions. In addition, prolonged absorption of an injectable pharmaceutical form may be caused by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

In some cases, in order to prolong the action of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be done by using a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug depends on its rate of dissolution, which in turn may depend on the crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered dosage form is accomplished by dissolving or suspending the drug in an oil vehicle.

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

For use in the methods of this invention, the active compounds may be given alone or as a pharmaceutical composition containing, for example, from 0.1% to about 99.5% (more preferably from about 0.5% to about 90.0%) active ingredient in combination with a pharmaceutically acceptable carrier.

In some embodiments of the invention, the compound of the present invention is administered together with one or more additional compounds/agents.

In certain such embodiments, the co-administration is simultaneous. In certain such embodiments, a compound of the present invention is administered in conjunction with one or more additional compounds. In certain other such embodiments, a compound of the invention is administered separately, but simultaneously with one or more additional compounds. In certain such embodiments, the co-administration is sequential, with the administration of a compound of the invention preceding or following the administration of one or more additional compounds over minutes or hours.

Methods for administering a compound of the present invention may also be provided by rechargeable or biodegradable devices. In recent years, various slow release polymeric devices for controlled drug delivery, including protein biopharmaceuticals, have been developed and tested in vivo. Various biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for a sustained release of a compound at a specific target site.

Actual dosage levels of active ingredients in pharmaceutical compositions can be varied to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and route of administration, without toxic effect to the patient.

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

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

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

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

The patient receiving such treatment is any animal in need, including primates, in particular humans; and other mammals such as horses, cattle, pigs and sheep; and pets in general.

In certain embodiments, the compounds or conjugates described herein may be used alone or in conjunction with another type of therapeutic agent. As used herein, "co-administration" refers to any form of administration of two or more different therapeutic compounds or conjugates such that the second compound is administered while the previously administered therapeutic compound or conjugate is still active in the body (e.g., the 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 either in the same composition or in separate compositions, simultaneously or sequentially. In certain embodiments, different therapeutic compounds or conjugates may be administered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or a week after administration of the other. Thus, an individual who receives such treatment may benefit from the combined action of various therapeutic compounds or conjugates.

This invention includes the use of pharmaceutically acceptable salts of the compounds or conjugates disclosed herein. In certain embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl, or tetraalkylammonium salts. In certain embodiments, intended salts of the invention include, but are not limited to, L-arginine, benentamine, benzathine, betaine, calcium hydroxide, choline, dinol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 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, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn, or other metal salts.

Pharmaceutically acceptable acid addition salts may also exist as various solvates such as water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates may also be prepared. The source of such a solvate may be a crystallization solvent belonging to the preparation or crystallization solvent or incidental to such a solvent.

Wetting agents, emulsifiers, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coatings, sweeteners, flavoring and flavoring agents, preservatives, and antioxidants may also be present in the compositions.

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

The invention as generally described will now be more easily understood with reference to the following examples, which are included for the purpose of illustrating certain aspects and embodiments of the present invention only and are not intended to limit the invention.

ILLUSTRATIVE EXAMPLES

Synthesis Protocols

Abbreviations

AcO: acetyl

AcOH: acetic acid

EA: ethyl acetate

DXM: dichloromethane

m-CPBA: metachloroperoxybenzoic acid

TBTf: tert-butyldimethylsilyl triflate

TBDMS: tert-butyldimethylsilyl

DMF: dimethylformamide

EDCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

HOBt: 1-hydroxybenzotriazole hydrate

ACN: acetonitrile

TBDMS-Cl: tert-butyldimethylsilyl chloride

DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene

THF: tetrahydrofuran

DCC: N,N'-dicyclohexylcarbodiimide

DMAP: 4-dimethylaminopyridine

NHS: N-hydroxysuccinimide

DIPEA: diisopropylethylamine

TEA: triethylamine

DEAD: diethyl azodicarboxylate

Boc: tert-butyloxycarbonyl

LAH: lithium aluminum hydride

CDI: 1,1'-carbonyldiimidazole

BEMP: 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazophosphorine

TPSCl: triphenylchlorosilane

tfa: trifluoroacetyl

PyBop: benzotriazol-1-yl-hydroxytripyrrolidinophosphonium hexafluorophosphate

HBTU : N, N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate

TFA: Trifluoroacetic acid

DIC: N,N'-diisopropylcarbodiimide

DMPA: 2,2-dimethoxy-2-phenylacetophenone

TBAF: Tetra-n-butylammonium fluoride

AgOTf: Silver trifluoromethanesulfonate

(BimC4A) ₃ : Tripotassium 5,5',5''-[2,2',2''-nitrilotris (methylene)tris(1 H -benzimidazole-2,1-diyl)]tripentanoate

[Example 1] Getting BGal-Br (hereinafter referred to as "Int-TG")

β-D-galactose pentaacetate (Alfa, CAS 4163-60-4, 5.0 g, 12.81 mmol) was dissolved in 33% HBr in AcOH (20 ml) at 0° under N ₂ atmosphere. The mixture was warmed to room temperature. After stirring at room temperature for 4 hours, the mixture was concentrated under reduced pressure, and then EA (1000 ml) and saturated sodium bicarbonate (1000 ml) were added. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give Int-TG compound (5.2 g, 99%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.70 (d, J =4.0 Hz, 1H), 5.52 (d, J =2.4 Hz, 1H), 5.41 (dd, J =7.6, 2.8 Hz, 1H), 5.05 (dd, J =6.4, 4.0 Hz, 1H), 4.49 (t, J =6.4 Hz, 1H), 4 .22-4.09 (m, 2H), 2.16-2.01 (m, 12H).

[Example 2] Obtaining an Int-TG1 Compound

Getting connection Int-TG1-1

Dried molecular sieve (2 .5 g) and Ag ₂ O (845 mg, 3.65 mmol) under N ₂ atmosphere. After stirring at room temperature for 1 hour, distilled water (50 ml) and EA (50 ml×2) were added. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG1-1 (441 mg, 81%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.37 (s, 1H), 7.88 (t, J =7.6 Hz, 1H), 7.21 (t, J =7.4 Hz, 1H ), 7.14 (t, J = 8.4 Hz, 1H), 5.62 (m, 1H), 5.48 (m, 1H), 5.16 (d, J = 7.2 Hz, 2H ), 4.27-4.23 (m, 1H), 4.18-4.09 (m, 2H), m 2.21 (s, 3H), 2.07 (s, 6H), 2.03 (s, 3H).

Getting connection Int-TG1-2

To a solution of compound Int-TG1-1 (260 mg, 0.575 mmol) in DCM (3 ml) was added m-CPBA (283 mg, 1.149 mmol) at 0° under N ₂ atmosphere. After 5 hours the mixture was concentrated under reduced pressure. Then EA (50 ml × 2) and aqueous sodium bicarbonate solution (30 ml) were added. The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give Int-TG1-2 (270 mg, quant.). Connection Int-TG1-2 directly, without purification, was used in the next reaction.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.18 (s, 1H), 7.90 (d, J =8.0 Hz, 1H), 7.64 (d, J =8.0 Hz, 1H ), 7.46 (t, J = 7.6 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 5.51 (m, 2H), 5.11 (d, J = 8.8 Hz, 1H), 5.04 (d, J = 8.0 Hz, 1H), 4.24 (m, 1H), 4.16 (m, 1H), 4.08 (m, 1H ), 2.18 (s, 3H), 2.09 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H). EI-MS m/z: 491(M ⁺ +Na).

Getting connection Int-TG1-3

To a solution of compound Int-TG1-2 in CHCl ₃ (3 ml) was added hydrazine hydrate (21 μl, 0.427 mmol) at 0° under N ₂ atmosphere. After stirring at 0° for 0.5 hour, EA (30 ml X 2) and 1M aqueous HCl solution (10 ml) were added. The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give compound Int-TG1-3 (161 mg, 86%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.03 (t, J =8.0 Hz, 1H), 6.98-6.95 (m, 2H), 6.83 (t, J =7, 6 Hz, 1H), 6.02 (s, 1H), 5.47 (d, J =3.2 Hz, 2H), 5.13 (dd, J =10.8, 2.8 Hz, 1H) , 4.93 (d, J = 7.6 Hz, 1H), 4.26 (m, 1H), 4.19-4.09 (m, 2H), 4.06 (m, 1H), 2, 21 (s, 3H), 2.13 (s, 3H), 2.08 (s, 3H), 2.03 (s, 3H). EI-MS m/z: 463(M ⁺ +Na).

Obtaining an Int-TG1 Connection

To a solution of Int-TG1-3 (161 mg, 0.366 mmol) in DCM (3 ml) was added Et ₃ N (102 μl, 0.732 mmol) and TBDMS-OTf (126 μl, 0.549 mmol) at 0° under N ₂ . The mixture was stirred at room temperature for 2 hours. DCM (30 ml X 2) and 1M HCl aqueous solution (10 ml) were then added. The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG1 (147 mg, 91%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.02 (d, J =7.6 Hz, 1H), 6.95-6.84 (m, 3H), 5.48-5.43 (m, 2H), 5.15 (d, J = 8.0 Hz, 1H) 5.10 (d, J = 10.4 Hz, 1H), 4.21-4.11 (m, 2H), 4.03 -3.99 (m, 1H), 2.19 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 0.99 ( s, 9H), 0.20 (s, 3H), 0.16 (s, 3H). EI-MS m/z: 555(M ⁺ ).

[Example 3] Obtaining an Int-TG2 Compound

Getting connection Int-TG2-1

To a solution of 3-formyl-4-hydroxybenzoic acid (3 g, 18.06 mmol) and 11-azido-3,6,9-trioxaundecane-1-amine (Aldrich, CAS 134179-38-7, 5.98 g, 23.48 mmol) in DMF (20 ml) were added EDCI (5.19 g, 27.09 mmol), HOBt (4.15 g, 27.09 mmol) and Et ₃ N (10.1 ml, 72.24 mmol) at 0°C in an atmosphere of N ₂ . The mixture was stirred at room temperature under N ₂ overnight. The reaction was quenched with EA (60 ml x 2) and citric acid (60 ml). The organic layer was extracted with aqueous sodium bicarbonate (80 ml). The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG2-1 (2.56 g, 39%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 11.26 (s, 1H), 9.96 (s, 1H), 8.16 (s, 1H), 7.98-7.96 (d, J = 8.4 Hz, 1H), 7.04-7.02 (d, J =9.2 Hz, 1H), 6.91 (s, 1H), 3.68-3.61 (m, 14H), 3.37-3.34 (m, 2H), EI-MS m/z: 367(M ⁺ ).

Getting connection Int-TG2-2

To a solution of compound Int-TG2-1 (1.41 g, 3.85 mmol) and compound Int-TG (1.74 g, 4.24 mmol) in anhydrous ADC (20 ml) was added a molecular sieve (8 g) and Ag ₂ O (2.68 g, 11.55 mmol) at room temperature under N ₂ atmosphere The mixture was stirred at room temperature for 3 hours, then filtered through celite. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG2-2 (1.88 g, 70%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.35 (s, 1H), 8.20-8.17 (m, 2H), 7.26 (s, 1H), 7.20-7.18 ( d, J = 9.2 Hz, 1H), 6.96 (s, 1H), 5.63-5.58 (m, 1H), 5.50-5.49 (m, 1H), 5.23 -5.21(m, 1H), 5.18-5.14(m, 1H), 4.24-4.14(m, 3H), 3.69-3.64(m, 14H), 3 .37-3.35(m, 2H), 2.21(s, 3H), 2.08-2.07(m, 6H), 2.03(s, 3H). EI-MS m/z: 697(M ⁺ ).

Getting connection Int-TG2-3

To a solution of compound Int-TG2-2 (1.69 g, 2.42 mmol) in DCM (15 ml) was added m-CPBA (2.4 g, 9.70 mmol) at 0° under N ₂ atmosphere. After stirring for 7 hours at 0° C., the mixture was quenched by adding saturated sodium bicarbonate (40 ml x 2). The mixture was separated and the organic layers were washed with brine, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give Int-TG2-3 (1.25 g, 76%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.36-7.33 (m, 2H), 7.01-6.99 (d, J =8.4 Hz, 1H), 6.71 (m, 1H), 6.06 (s, 1H), 5.49-5.44 (m, 2H), 5.15-5.12 (m, 1H), 4.99-4.97 (d, J = 8.0 Hz, 1H), 4.24-4.09 (m, 3H), 3.69-3.63 (m, 14H), 3.37-3.34 (m, 2H), 2.20 (s, 3H), 2.13 (s, 3H), 2.12 (s, 3H), 2.03 (s, 3H), EI-MCm/z: 685(M ⁺ ).

Obtaining an Int-TG2 Connection

To a solution of compound Int-TG2-3 (750 mg, 1.09 mmol) in DCM (10 mL) was added TBDMS-OTf (504 μL, 2.19 mmol) and Et ₃ N (458 μL, 3.29 mmol) at 0°C in N ₂ atmosphere. The mixture was stirred overnight at room temperature and then quenched by adding citric acid (20 ml). The organic layer was washed with brine (20 ml), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG2 (799 mg, 91%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.35 (d, J =2.4 Hz, 1H), 7.30 (dd, J =8.4, 2.0 Hz, 1H), 7.02 (d, J =8.0 Hz, 1H), 6.65 (t, J =5.2 Hz, 1H), 5.49-5.44 (m, 2H), 5.20 (d, J= 7.6 Hz, 1H), 5.12 (dd, J =10.0, 3.6 Hz, 1H), 4.20-4.11 (m, 2H), 4.06-4.03 (m , 1H), 3.69-3.62 (m, 15H), 3.37 (t, J =5.2 Hz, 2H), 2.19 (s, 3H), 2.05 (s, 3H) , 2.02 (s, 3H), 2.01 (s, 3H), 1.01 (s, 9H), 0.22 (s, 3H), 0.18 (s, 3H). EI-MS m/z: 799(M ⁺ ).

[Example 4] Obtaining an Int-TG3 Compound

Preparation of the Int-TG3-1 connection

Dried molecular sieve (1.0 g) and Ag ₂ O ( 1.42 g, 4.92 mmol). at room temperature. The mixture was stirred overnight and distilled water (50 ml) and EA (50 ml × 2) were added. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give Int-TG-3-1 (218 mg, 30%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.35 (s, 1H), 7.86 (dd, J=6.0, 1.6 Hz, 1H), 7.56 (td, J=7, 6, 1.6 Hz, 1H), 7.20 (t, J=7.6 Hz, 1H), 7.13 (d, J=8.8 Hz, 1H), 5.39-5.31 ( m, 3H), 5.27-5.25 (m, 1H), 5.24-4.19 (m, 1H), 3.75 (s, 3H), 2.07-2.04 (m, 9H). EI-MS m/z: 461(M ⁺ +Na).

Getting connection Int-TG3-2

To a solution of compound Int-TG3-1 (217.6 mg, 0.50 mmol) in DCM (10 ml) was added m-CPBA (367.1 mg, 1.50 mmol) at 0° C. under atmosphere. The mixture was stirred overnight at room temperature and 40 ml DCM was added. The organic layer was washed with saturated sodium bicarbonate (10 ml) and dried over anhydrous Na ₂ SO ₄ , filtered, concentrated under reduced pressure.

The residue was redissolved in CHCl ₃ (5 ml). Hydrazine (36.2 μl, 0.74 mmol) was then added. After 30 minutes, DCM (20 ml x 2) and water (10 ml) were added. The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure, and the residue was purified by column chromatography to give the compound Int-TG1-3-2 (195 mg, 92%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.09 (t, J=8.0 Hz, 1H), 7.00-6.95 (m, 2H), 6.83 (td, J=6, 8, 1.6 Hz, 1H), 5.38-5.24 (m, 4H), 4.19-4.13 (m, 1H), 3.75 (s, 3H), 2.06-2 .02 (m, 9H). EI-MS m/z: 449(M ⁺ +Na).

Obtaining an Int-TG3 connection

To a solution of compound Int-TG3-2 (194 mg, 0.46 mmol) in DCM (5 ml) was added Et ₃ N (190.8 μl, 1.37 mmol) and TBDMS-OTf (209.8 μl, 0. 91 mmol) at 0° C. under N ₂ atmosphere. After stirring at 0° C. for 1 hour, DCM (30 ml x 2) and water (10 ml) were added. The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG3 (188.6 mg, 76%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.00 (dd, J=6.0, 1.6 Hz, 1H), 6.94-6.88 (m, 2H), 6.84 (dd, J=6.0, 2.0Hz, 1H), 5.38-5.21(m, 5H), 3.72(s, 3H), 2.03(d, J=6.8Hz, 9H ), 0.98 (s, 9H), 0.18 (s, 3H), 0.15 (s, 3H). EI-MS m/z: 563(M ⁺ +Na).

[Example 5] Obtaining an Int-TG4 Compound

To a solution of 2-nitrophenol (500 mg, 3.59 mmol) in anhydrous pyridine (20 ml) was added TBDMS-Cl (650 mg, 4.31 mmol) at room temperature under N ₂ atmosphere. The mixture was stirred overnight at room temperature and DCM (30 ml x 2) and water (20 ml) were added. The resulting organic layer was washed with 2N aqueous HCl, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG4 (410 mg, 94%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.80 (dd, J=6.8, 0.8 Hz, 1H), 7.43 (t, J=7.2 Hz, 1H), 7.04 -6.97 (m, 2H), 1.01 (s, 9H) 0.26 (s, 6H).

[Example 6] Obtaining an Int-TG5 Compound

Preparation of compound Int-TG5-1a

To a solution of 4-hydroxybenzaldehyde (1 g, 8.19 mmol) in DCM (3 ml) was added Et ₃ N (2.28 ml, 16.38 mmol) at room temperature under N ₂ atmosphere. SO ₂ F ₂ gas was introduced through the balloon and the mixture was stirred at room temperature for 2 hours. The mixture was then washed with DCM (30 ml×3) and brine (30 ml), the organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG5-1a (790 mg, 63%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.06 (s, 1H), 8.05 (d, J=8.0 Hz, 2H), 7.55 (d, J=8.8 Hz, 2H ).

Getting connection Int-TG5-1

DBU (4 μl, 25 μmol) was added to a solution of compound Int-TG2 (100 mg, 0.13 mmol) and compound Int-TG5-1a (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 (10 ml x 2). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give Int-TG5-1 (103 mg, 94%).

EI-MS m/z: 869(M ⁺ ).

Preparation of the Int-TG5-2 connection

To a solution of compound Int-TG5-1 (103 mg, 0.12 mmol) in THF (8 ml) was added NaBH ₄ (9 mg, 0.24 mmol) at 0° C. under N ₂ atmosphere. After stirring at room temperature for 2 hours, distilled water (10 ml) and EA (10 ml x 2) were added. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give compound Int-TG5-2 (101 mg, 98%).

EI-MS m/z: 871(M ⁺ ).

Obtaining an Int-TG5 Connection

To a solution of Int-TG5-2 (47 mg, 54 µmol) in DMF (2 ml) was added bis(4-nitrophenyl)carbonate (25 mg, 81 µmol) and DIPEA (14 µl, 81 µmol) at room temperature under atmospheric nitrogen. The mixture was stirred overnight at room temperature. Distilled water (10 ml) and EA (10 ml×2) were then added, the organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG5 (53 mg, 94%).

EI-MS m/z: 1036(M ⁺ ).

[Example 7] Obtaining an Int-TG6 Compound

Obtaining an Int-TG6-1 connection

To a solution of 1,2-dihydroxybenzene (1.0 g, 9.08 mmol) in DMF (15 ml) was added TBDMS-Cl (1.64 g, 10.88 mmol) and imidazole (1.24 g, 18.21 mmol) at 0°C in an atmosphere of N ₂ . The mixture was stirred at room temperature for 2 hours. EA (30 ml x 2) and distilled water (20 ml) were added. The resulting organic layer was washed again with brine, dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give Int-TG6-1 (1.27 g, 64%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.94 (d, J=8.0 Hz, 1H), 6.89-6.82 (m, 2H), 6.76 (t, J=7, 6 Hz, 1H), 5.48 (s, 1H), 1.02 (s, 9H), 0.28 (s, 6H).

Obtaining an Int-TG6 connection

To a solution of Int-TG6-1 (300 mg, 1.34 mmol) and levulinic acid (310.5 mg, 2.67 mmol) in 1,4-dioxane (12 ml) was added DCC (551.7 mg, 2. 67 mmol) and DMAP (13.07 mg, 0.11 mmol). The mixture was stirred at room temperature for 3 hours. Distilled water (50 ml) and EA (50 ml×2) were then added and the organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give Int-TG6 (380.1 mg, 88%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.09 (td, J=6.0, 1.6 Hz, 1H), 7.03 (dd, J=6.4, 1.6 Hz, 1H) , 6.95-6.88 (m, 2H), 2.84 (s, 4H). ), 2.22 (s, 3H), 0.98 (s, 9H), 0.20 (s, 6H).

[Example 8] Obtaining an Int-TG7 Compound

Connection Int-TG1 (80 mg, 0.14 mmol) was dissolved in anhydrous methanol (2 ml), was added to it K ₂ CO ₃ (99.7 mg, 0.72 mmol) at 0°C. The mixture was stirred at 0°C for 1 hour. The residue was diluted with EA (10 ml × 2), the organic layer was washed with 1N aqueous HCl (2 ml) and water (10 ml). The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was subjected to preparative TLC to give compound Int-TG7 (17.4 mg, 31%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.12 (d, J=8.0 Hz, 1H), 6.95-6.85 (m, 3H), 4.74 (d, J=7, 2 Hz, 1H), 4.05 (d, J=3.2 Hz), 1H), 3.97 (dd, J=6.0, 5.6 Hz, 1H), 3.90-3.85 (m, 2H), 3.68 (dd, J=6.8, 2.8 Hz, 1H), 3.63 (t, J=5.6 Hz, 1H), 3.48 (s, 1H) , 1.03 (s, 9H), 0.20 (d, J=12.0 Hz, 6H). EI-MS m/z: 409(M ⁺ +Na).

[Example 9] Obtaining an Int-TG8 Compound

Getting connection Int-TG8-1

K ₂ CO ₃ (401.6 mg, 2.91 mmol ). The mixture was refluxed for 15 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure and diluted with EA (50 ml×2) and aqueous 1N NaOH (20 ml). The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give Int-TG8-1 (300.3 mg, 79%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.19 (dd, J=6.8, 1.2 Hz, 1H), 7.75 (d, J=7.6 Hz, 1H), 7.69 (td, J=7.2, 1.2 Hz, 1H), 7.53 (td, J=6.8, 1.6 Hz, 1H), 6.99 (dd, J=7.2, 1 .2 Hz, 1H), 6.93 (td, J=5.2, 2.4 Hz, 1H), 6.85-6.79 (m, 2H), 5.64 (s, 1H), 5 .58 (s, 2H).

Obtaining an Int-TG8 Connection

To a solution of compound Int-TG8-1 (300 mg, 1.22 mmol) in DCM (5 ml) was added Et ₃ N (342 μl, 2.50 mmol) and TBDMS-OTf (421.8 μl, 1.83 mmol ) at 0°C in an atmosphere of N ₂ . The mixture was stirred at room temperature for 3 hours and diluted with DCM (30 ml x 2) and aqueous 2N HCl (10 ml). The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give Int-TG8 (410 mg, 94%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.19 (dd, J=6.8, 1.2 Hz, 1H), 8.01 (dd, J=8.0, 0.8 Hz, 1H) , 7.67 (td, J=6.4, 1.2 Hz, 1H) 7.48 (t, J=7.6 Hz, 1H), 6.92-6.87 (m, 4H), 5 .49 (s, 2H), 1.01 (s, 9H), 0.18 (s, 6H).

[Example 10] Preparation of Int-TG9 Compound

Obtaining the connection Int-TG9-1

To a solution of 2,3-dihydroxynaphthalene (930 mg, 5.83 mmol) and compound Int-TG (1.0 g, 2.43 mmol) in acetone (10 mL) was added NaOH (230 mg, 5.75 mmol) at room temperature under a nitrogen atmosphere. The mixture was stirred overnight at room temperature and concentrated under reduced pressure. The mixture was diluted with distilled water (20 ml) and EA (30 ml × 2). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give Int-TG9-1 (560 mg, 47%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.67 (t, J=9.2 Hz, 2H), 7.39-7.29 (m, 4H), 6.07 (s, 1H), 5 .53-5.50 (m, 2H), 5.18 (dd, J=7.2, 3.6 Hz, 1H), 5.10 (d, J=7.6 Hz, 1H), 4, 31-4.11 (m, 3H), 2.21 (s, 3H), 2.12 (d, J=8.8 Hz, 6H), 2.05 (s, 3H).

Obtaining an Int-TG9 Connection

To a solution of compound Int-TG9-1 (200 mg, 0.41 mmol) in DCM (7 ml) was added TBDMS-OTf (0.12 ml, 0.53 mmol) and Et ₃ N (0.11 ml, 0. 82 mmol) at 0° C. under N ₂ atmosphere. After stirring overnight at room temperature, the mixture was diluted with DCM (30 ml x 2) and extracted with distilled water (10 ml). The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG9 (230 mg, 96%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.67-7.63 (m, 2H), 7.36-7.33 (m, 3H), 7.20 (s, 1H), 5.52 ( dd, J=8.4, 2.0 Hz, 1H), 5.47 (d, J=3.2 Hz, 1H), 5.31 (d, J=8.0 Hz, 1H), 5, 15 (dd, J=6.8, 3.6 Hz, 1H), 4.23-4.13 (m, 3H), 2.20 (s, 3H), 2.05 (s, 3H), 2 .02 (d, J=3.6 Hz, 6H), 1.03 (s, 9H), 0.26 (s, 3H), 0.22 (s, 3H).

[Example 11] Preparation of Int-TG10 Compound

Preparation of the Int-TG10-1 compound

To a solution of 3-(4-hydroxyphenyl)propionic acid (500 mg, 3.01 mmol) in CHCl ₃ (10 ml) was added 4 M NaOH (7.5 ml, 30 mmol) and then refluxed for 6 hours. After completion of the reaction, the mixture was acidified with 4 M HCl and concentrated to remove CHCl ₃ . EA (30 ml×3), H ₂ O (20 ml) and brine (20 ml) were added to carry out extraction, and the resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound Int-TG10-1 (408 mg, product: SM=4:6 by HPLC).

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.90 (s, 1H), 9.87 (s, 1H), 7.41-7.38 (m, 2H), 6.94 (d, J= 9.6 Hz, 1H), 2.96 (t, J=7.4 Hz, 2H), 2.69 (t, J=7.4 Hz, 2H).

Getting connection Int-TG10-2

To a solution of compound Int-TG10-1 (408 mg, 2.1 mmol) in DMF (10 mL) was added NHS (363 mg, 3.15 mmol) and EDCI (604 mg, 3.15 mmol) and stirred overnight at room temperature. Then 11-azido-3,6,9-trioxaundecane-1-amine (636 mg, 2.5 mmol) dissolved in DMF (3 ml) and DIPEA (3.66 ml, 21 mmol) were added and stirred at room temperature. temperature for 1 hour. After completion of the reaction, the mixture was acidified with 4 M HCl, diluted with EA (30 ml × 5) and washed with H ₂ O (30 ml) and brine (30 ml). The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give Int-TG10-2 (288 mg, 50% purity by HPLC).

EI-MS m/z: 395(M ⁺ ).

Preparation of the Int-TG10-3 connection

To a solution of compound Int-TG10-2 (288 mg, 0.73 mmol) and compound Int-TG (303 mg, 0.74 mmol) in ACN (10 ml) was added a molecular sieve (1.5 g) under N ₂ . After stirring for 10 minutes at room temperature, Ag ₂ O (508 mg, 2.19 mmol) was added thereto. The mixture was stirred at room temperature for 3 hours and diluted with water (5 ml) followed by filtration using celite. The filtrate was washed with EA (20 ml×2), H ₂ O (20 ml) and brine (20 ml). The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG10-3 (195 mg).

EI-MS m/z: 725(M ⁺ ).

Preparation of the Int-TG10-4 compound

To a solution of compound Int-TG10-3 (195 mg, 0.27 mmol) in DCM (5 ml) was added 70% m-CPBA (133 mg, 0.54 mmol) at 0°C under N ₂ atmosphere. The mixture was stirred at 0°C for 3 hours. Then, 70% m-CPBA (66 mg, 0.27 mmol) was added thereto, and the mixture was stirred overnight at 0°C. The reaction was quenched with saturated NaHCO ₃ (20 ml x 3) and diluted with DCM (20 ml). The organic layer was washed with brine (20 ml) and dried over anhydrous Na ₂ SO ₄ , filtered, concentrated under reduced pressure. Compound Int-TG10-4 was used directly in the next reaction without purification (199 mg, crude).

EI-MS m/z: 741(M ⁺ ).

Preparation of the Int-TG10-5 compound

To a solution of compound Int-TG10-4 (199 mg, 0.27 mmol) in CHCl ₃ (4 ml) was added CHCl ₃ (133 mg, 0.54 mmol) at 0° C. under N ₂ atmosphere. After stirring at room temperature for 30 minutes, the mixture was quenched with EA (20 ml) and saturated citric acid (20 ml). The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG10-5 (146 mg).

EI-MS m/z: 713(M ⁺ ).

Getting connection Int-TG10

To a solution of compound Int-TG10-5 (146 mg, 0.21 mmol) in DMF (2 ml) was added TEA (133 μl, 0.62 mmol) and TBDMS-OTf (94 μl, 0.41 mmol) at 0° C in an N ₂ atmosphere. The mixture was stirred at 0°C for 20 minutes and continued to stir at room temperature for 3 hours. The mixture was extracted with EA (20 ml), saturated citric acid (20 ml) and brine (30 ml). The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography followed by preparative TLC purification to give compound Int-TG10 (32 mg, 19%).

EI-MS m/z: 827(M ⁺ ).

[Example 12] Preparation of compound IntA-Q1

To a solution of Boc-Tyr-OMe (300 mg, 1.02 mmol) in DCM (3 ml) was added Et ₃ N (212 μl, 1.52 mmol) at room temperature under N ₂ atmosphere. SO ₂ F ₂ gas was introduced through the balloon and the mixture was stirred at room temperature for 2 hours under SO ₂ F ₂ atmosphere. The mixture was extracted with DCM (30 ml x 3) and brine (30 ml). The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntA-Q1 (363 mg, 95%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.26 (s, 4H), 5.03 (d, J=8.0 Hz, 1H), 4.62 (m, 1H), 3.73 (s , 3H), 3.21 (dd, J=13.2, 4.8 Hz, 1H), 3.07 (dd, J=13.2, 6.0 Hz, 1H), 1.41 (s, 9H). EI-MS m/z: 400(M ⁺ +Na).

[Example 13] Preparation of compound IntA-Q2

Obtaining compound IntA-Q2-1

To a solution of guanosine (1 g, 3.53 mmol) in DMF (30 ml) was added imidazole (1.92 g, 28.45 mmol) and TBDMS-Cl (3.193 g, 21.18 mmol) at room temperature. The mixture was stirred overnight at room temperature and extracted with EA (100 ml×3), H ₂ O (100 ml), NH ₄ Cl (100 ml) and brine (100 ml). The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntA-Q2-1 (1.56 g, 70%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 12.05 (s, 1H), 7.89 (s, 1H), 6.40 (br. s, 1H), 5.82-5.79 (m, 1H), 4.44-4.38 (m, 1H), 4.29-4.26 (m, 1H), 4.10-4.09 (m, 1H), 4.02-3.99 ( m, 1H), 3.80-3.77 (m, 1H), 0.96 (s, 9H), 0.92 (s, 9H), 0.87 (s, 9H), 0.14 (s , 3H), 0.13 (s, 3H), 0.09 (s, 3H), 0.08 (s, 3H), 0.02 (s, 3H), -0.02 (s, 3H). EI-MS m/z: 627(M ⁺ ).

Obtaining compound IntA-Q2-2

To a solution of 4-hydroxybenzyl alcohol (1.00 g, 8.06 mmol) in DCM (40 ml) and H ₂ O (40 ml) was added Et ₃ N (1.70 ml, 12.085 mmol) at room temperature under N ₂ . SO ₂ F ₂ gas was introduced through the balloon and the mixture was stirred at room temperature for 5 hours. After completion of the reaction, the mixture was concentrated under reduced pressure, and the residue was purified by column chromatography to give compound IntA-Q2-2 (711 mg, 43%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.49 (d, J=8.4 Hz, 2H), 7.34 (d, J=8.0 Hz, 2H), 4.76 (d, J =6.0 Hz, 2H), 1.80 (t, J=4.2 Hz, 1H).

Obtaining compound IntA-Q2

To a solution of compound IntA-Q2-1 (350 mg, 0.56 mmol) in THF (10 ml) was added compound IntA-Q2-2 (253 mg, 1.23 mmol) and PPh ₃ (235 mg, 0.90 mmol ) at room temperature. in N ₂ atmosphere. After the mixture was cooled to 0° C., DEAD (0.41 ml, 0.9 mmol) was added dropwise and stirred at 0° C. for 3 hours. The mixture was extracted with EA (30 ml x 3), H ₂ O (30 ml) and brine (30 ml). The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was separated and purified once by preparative HPLC and then purified by column chromatography to give compound IntA-Q2 (62 mg, 14%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.00 (s, 1H), 7.62 (d, J=8.4 Hz, 2H), 7.33 (d, J=8.4 Hz, 2H ), 5.91 (d, J=5.2 Hz, 1H), 5.57 (s, 2H), 4.82-4.78 (m, 2H), 4.52-4.50 (m, 1H), 4.30-4.28 (m, 1H), 4.12-4.08 (m, 1H), 3.98 (dd, J=11.2, 3.6 Hz, 1H), 3 .93 (dd, J=11.2, 2.4 Hz, 1H), 0.94 (s, 9H), 0.93 (s, 9H), 0.82 (s, 9H), 0.13 ( s, 3H) 0.12 (s, 3H), 0.10 (s, 3H), 0.09 (s, 3H), -0.02 (s, 3H), -0.15 (s, 3H) . EI-MS m/z: 815(M ⁺ ).

[Example 14] Preparation of compound IntA-Q3

Preparation of compound IntA-Q3

To a solution of 3-pyridylacetic acid hydrochloride (1.0 g, 5.76 mmol) in THF (20 ml) was added LAH (1.1 g, 28.8 mmol) at 0° C. under N ₂ atmosphere. The mixture was warmed to room temperature and refluxed for 24 hours. The mixture was quenched by adding methanol (10 ml) and water (20 ml). The aqueous layer was subjected to extraction with ether (50 ml × 2), and the resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-Q3a (396.3 mg, 56%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.48-8.45 (m, 2H), 7.58-7.56 (m, 1H), 7.25-7.22 (m, 2H), 3.89 (t, J=6.8 Hz, 2H), 2.87 (t J=6.8 Hz, 2H). EI-MS m/z: 124(M ⁺ +1).

Obtaining an Int-Q3b connection

A solution of Int-Q3a (260 mg, 2.11 mmol) and CDI (684.6 mg, 4.22 mmol) in DCM (5 mL) was stirred overnight at 50° C. under N ₂ atmosphere. After completion of the reaction, the mixture was cooled to room temperature, followed by extraction with DCM (20 ml×2) and water (10 ml). The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-Q3b (296.5 mg, 65%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.53 (dd, J=3.6, 1.6 Hz, 2H), 8.08 (s, 1H), 7.59-7.57 (m, 1H), 7.37 (s, 1H), 7.29-7.26 (m, 1H), 7.06 (s, 1H), 4.63 (t, J=6.8 Hz, 2H), 3.12 (t, J=6.8 Hz, 1H). EI-MS m/z: 218(M ⁺ +1).

Method for preparing the compound IntA-Q3

To a solution of compound Int-Q3b (47.6 mg, 0.22 mmol) and compound IntA-Q2-2 (45.2 mg, 0.22 mmol) in THF (400 μl) was added NaH (2.6 mg, 0 .07 mmol) at 0°C in an atmosphere of N ₂ . After stirring at 0° C. for 30 minutes, the mixture was extracted with diethyl ether (10 ml x 2) and water (5 ml). The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-Q3 (296.5 mg, 65%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.50-8.49 (m, 2H), 7.56-7.54 (m, 1H), 7.48 (d, J=8.8 Hz, 2H), 7.35 (d, J=8.8 Hz, 2H) 7.24-7.22 (m, 1H), 5.30 (s, 2H), 4.38 (t, J=6, 4 Hz, 2H), 3.00 (t, J=6.8 Hz, 1H). EI-MS m/z: 356(M ⁺ +1).

[Example 15] Preparation of compound IntA-Q4

Compound IntA-Q4 (178.2 mg, 0.86 mmol) was prepared from compound IntA-Q2-2 (178.2 mg, 0.86 mmol) and 3-pyridylacetic acid hydrochloride (100 mg, 0.58 mmol) by analogous method of obtaining Int-TG5-1a, shown in example 6. (161 mg, 86%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.55-8.54 (m, 2H), 7.65-7.62 (m, 1H), 7.58 (d, J=8.4 Hz, 2H), 7.33 (d, J=8.4 Hz, 2H) 7.29-7.27 (m, 1H), 5.20 (s, 2H), 3.70 (s, 2H). EI-MS m/z: 326(M ⁺ +1).

[Example 16] Obtaining compound IntA-Q5

Obtaining connection IntB-Q5-1

Compound IntA-Q2-2 (230 mg, 1.12 mmol) was dissolved in ether (10 ml) and 1M PBr ₃ (446 μl, 0.45 mmol) dissolved in DCM was added and stirred at 0°C for 2 hours. After completion of the reaction, ether (50 ml) and aqueous sodium bicarbonate solution (50 ml) were added to extract. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give compound IntB-Q5-1 (201 mg, 67%).

¹ H NMR (400 Hz, CDCl ₃ ) δ 7.51 (d, J=8.4 Hz, 2H), 7.33 (d, J=8.4 Hz, 2H), 4.49 (s, 2H ).

Preparation of compound IntA-Q5-1

Thymidine (100 mg, 0.41 mmol) and 60% NaH (25 mg, 0.62 mmol) were dissolved in DMF (3 ml) under nitrogen and the mixture was cooled to 0°C. Compound IntB-Q5-1 (167 mg, 0.62 mmol) was added at 0°C and the mixture was stirred at the same temperature for 1 hour. After completion of the reaction, EA (30 ml×3) and an aqueous citric acid solution (30 ml) were added to carry out extraction, and the resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntA-Q5-1 (79 mg, 45%).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.84 (s, 1H), 7.54 (d, J=8.8 Hz, 2H), 7.47 (d, J=8.8 Hz , 2H), 6.20 (t, J=6.8 Hz), 1H), 5.25 (d, J=4 Hz, 1H), 5.07-5.02 (m, 3H), 4, 28-4.22 (m, 1H), 3.78-3.76 (m, 1H), 3.64-3.54 (m, 2H), 2.16-2.11 (m, 2H), 1.84 (s, 3H). EI-MS m/z: 431(M ⁺ ).

Preparation of compound IntA-Q5-2

Compound IntA-Q5-1 (40 mg, 0.09 mmol) and compound Int-TG1 (62 mg, 0.11 mmol) were dissolved in ACN (3 ml) under nitrogen atmosphere, and then BEMP (14 μl, 0.11 mmol) was added. 05 mmol). The mixture was stirred overnight at room temperature, then DBU (7 μl, 0.05 mmol) was added and the mixture was stirred at the same temperature for 1 hour. After completion of the reaction, EA (20 ml×3), aqueous citric acid solution (20 ml) and brine (20 ml) were added to perform extraction, and the resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntA-Q5-2 (26 mg, 33%).

EI-MS m/z: 851(M ⁺ ).

Obtaining compound IntA-Q5

Compound IntA-Q5-2 (16 mg, 0.02 mmol) was dissolved in MeOH (2 ml) under nitrogen and then cooled to 0°C. K ₂ CO ₃ (13 mg, 0.09 mmol) was added at 0°C and the mixture was stirred at the same temperature for 1 hour. After completion of the reaction, the reaction solution was subjected to preparative HPLC to obtain compound IntA-Q5 (10.3 mg, 80%).

EI-MS m/z: 705(M ⁺ +Na).

[Example 17] Preparation of compound IntA-Q6

60% NaH (25 mg, 0.62 mmol) was added to a stirred solution of thymidine (100 mg, 0.41 mmol) in DMF (3 ml) under N ₂ atmosphere. The mixture was cooled to 0° C. and compound IntB-Q5-1 (167 mg, 0.62 mmol) was added. The mixture was stirred for 1 hour. After completion of the reaction, the reaction was quenched by adding EA (30 ml x 3) and saturated citric acid (30 ml). The resulting organic layer was intended to obtain the compound IntA-Q5-1 (79 mg, 45%).

[Example 18] Getting connection IntB-Q1

IntB-Q1 (19.5 mg, 89%) was prepared from amanitin (20 mg, 0.022 mmol) in a similar manner to the compound Int-TG5-1a given in Example 6.

EI-MS m/z: 1002(M ⁺ ).

[Example 19] Obtaining an IntB-Q3 compound

IntB-Q3 (19.5 mg, 89%) was prepared from SN-38 (300 mg, 1.02 mmol) in a similar manner to Int-TG5-1a in Example 6. The residue was purified by column chromatography to give IntB- Q3 (340 mg, 94%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.60 (d, J=2.4 Hz, 1H), 8.39 (d, J=9.2 Hz, 1H), 8.09 (dd, J =9.2, 2.4 Hz, 1H), 7.37 (s, 1H), 6.56 (s, 1H), 5.45 (s, 2H), 5.38 (s, 2H), 3 .25 (m, 2H), 1.91-1.84 (m, 2H), 1.32 (t, J=7.6 Hz, 3H), 0.89 (t, J=6.8 Hz, 3H). EI-MS m/z: 475(M ⁺ ).

[Example 20] Obtaining a connection IntB-Q4

Obtaining connection IntB-Q4-1

To a solution of compound IntA-Q2-2 (110 mg, 0.53 mmol) in DMF (3 ml) was added bis(4-nitrophenyl) carbonate (179 mg, 0.59 mmol) and DIPEA (139 μl, 0.80 mmol ) at room temperature. After stirring for 15 hours, the mixture was extracted with distilled water (10 ml) and EA (10 ml x 2). The organic layer was washed with brine (10 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give IntB-Q4-1 (184 mg, 93%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.29-8.27 (d, J=8.4 Hz, 2H), 7.58-7.56 (d, J=8.8 Hz, 2H) , 7.41-7.38 (m, 4H), 5.32 (s, 2H).

Obtaining the IntB-Q4 connection

The compound IntB-Q4-1 (36 mg, 0.097 mmol) and MMAF-OMe (80 mg, 0.107 mmol), which was prepared in a manner similar to that described in ChemPharmBull, 1995, 43 (10), 1706-1718, was dissolved in DMF (1 ml) at room temperature under nitrogen atmosphere. Then HOBt (3 mg, 0.019 mmol), DIPEA (19 μl, 0.107 mmol) and pyridine (330 μl) were added thereto and stirred overnight. The pH of the mixture was adjusted to 2-3 with 1N HCl and then diluted with distilled water (8 ml) and EA (8 ml X 2). The organic layer was washed with brine (12 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntB-Q4 (62 mg, 65%).

EI-MS m/z: 979(M ⁺ ).

[Example 21] Getting the connection IntB-Q5

Obtaining compound IntB-Q5-2a

To a solution of the compound methyl 4-hydroxybenzoate (3.00 g, 19.72 mmol) in 12% aqueous NaOH (20 ml) was added 40% aqueous formaldehyde (20 ml) at room temperature under N ₂ atmosphere. The mixture was stirred at 50°C for 3 days. After completion of the reaction, the mixture was extracted with EA (200 ml) and saturated NH ₄ Cl (200 ml). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntB-Q5-2a (2.30 g, 55%).

¹ H NMR (400 Hz, DMSO-d ₆ ) δ 7.84 (s, 2H), 4.56 (s, 4H), 3.80 (s, 3H).

Obtaining connection IntB-Q5-2

To a solution of compound IntB-Q5-1 (300 mg _, 1.12 mmol) and compound IntB-Q5-2a (237 mg, 1.12 mmol) in DMF (10 ml) was added K ₂ CO 23 mmol) at room temperature under N ₂ atmosphere. After stirring for 6 hours, the mixture was diluted with EA (100 ml) and washed with 2N aqueous HCl (30 ml). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntB-Q5-2 (140 mg, 32%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.07 (s, 2H), 7.58 (d, J=8.4 Hz, 2H), 7.39 (d, J=8.4 Hz, 2H ), 5.07 (s, 2H), 4.73 (s, 4H), 3.91 (s, 3H).

Obtaining connection IntB-Q5-3

To a solution of compound IntB-Q5-2 (68.8 mg, 1.12 mmol) in THF (2 ml) was added 4 M LiBH ₄ (3.8 ml, 15.20 mmol) dissolved in THF at room temperature. The mixture was stirred for 19 hours and EA (50 ml) was added. The organic layer was washed with saturated NH ₄ Cl (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give IntB-Q5-3 (23 mg, 36%).

¹ H NMR (400 MHz, CDCl ₃ +CD ₃ OD) δ 7.59 (d, J=8.8 Hz, 2H), 7.38 (d, J=8.0 Hz, 2H), 7.32 (s, 2H), 4.94 (s, 2H), 4.65 (s, 4H), 4.56 (s, 2H).

Obtaining connection IntB-Q5-4

To a solution of compound IntB-Q5-3 (33 mg, 0.09 mmol) in THF (4 ml) was added 4-nitrophenyl chloroformate (536 mg, 2.66 mmol) and pyridine (0.20 ml, 2.66 mmol) at 0°C in N ₂ atmosphere. The mixture was stirred for 3 hours and diluted with EA (50 ml). The organic layer was washed with 2N aqueous HCl (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give IntB-Q5-4 (57 mg, 72%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.27 (d, J=8.8 Hz, 6H), 7.68 (s, 2H), 7.64 (d, J=8.0 Hz, 2H ), 7.43-7.34 (m, 8H), 5.41 (s, 4H), 5.33 (s, 2H), 5.13 (s, 2H).

Getting the connection IntB-Q5

HOBt (24 mg, 0.16 mmol), pyridine ( 1.5 ml) and DIPEA (55 μl, 0.32 mmol) at room temperature under N ₂ atmosphere. The mixture was stirred for 15 hours and diluted with EA (100 ml). The organic layer was washed with 2N aqueous HCl (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure, followed by separation and purification using preparative HPLC to give compound IntB-Q5 (92 mg, 54%).

EI-MS m/z: 2689(M ⁺ ).

[Example 22] Obtaining the connection IntB-Q6

To a solution of β-estradiol (300 mg, 1.10 mmol) in DCM (3 ml), H ₂ O (2 ml) and DMF (2 ml) was added TEA (230 μl, 1.65 mmol) under nitrogen atmosphere. The mixture was stirred over night at room temperature, with the introduction of gas SO ₂ F ₂ . After completion of the reaction, it was confirmed that the product and starting material were present in a 1:1 ratio by HPLC followed by preparative HPLC to give compound IntB-Q6 (120 mg, 31%, 134 mg of starting material was obtained).

¹ H NMR (400 Hz, DMSO-d ₆ ) δ 7.45 (d, J=8.8 Hz, 1H), 7.29-7.24 (m, 2H), 5.01 (br. s, 1H), 4.93-4.89 (m, 1H), 3.52 (t, J=8.4 Hz, 1H), 2.32-2.16 (m, 2H), 1.93-1 .77 (m, 3H), 1.63-1.54 (m, 1H), 1.47-1.07 (m, 8H), 0.66 (s, 3H). EI-MS m/z: 378 (M ⁺ +Na).

[Example 23] Preparation of compound IntB-Q7

Combretastatin A4 (18 mg, 0.057 mmol) was dissolved in DCM (3 ml) under nitrogen atmosphere, TEA (88 μl, 0.57 mmol) was added and the mixture was stirred at room temperature for 3 hours while introducing SO ₂ F ₂ gas. After completion of the reaction, a saturated aqueous citric acid solution (6 ml) and DCM (6 ml) were added to extract the organic layer, and the organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntB-Q7 (22 mg, 97%).

EI-MS m/z: 399(M ⁺ ).

[Example 24] Preparation of FA-Int Compound

The FA-Int compound was prepared by carrying out the reaction in a manner similar to that described in US Patent Application Publication No. 20070276018.

[Example 25] Preparation of compound IntC-L-1

Preparation of compound IntCl-L-1a

To a solution of 2,2-(ethylenedioxy)bis(ethylamine) (50 g, 337.4 mmol) in DCM (300 ml) was added Boc ₂ O (14.7 g, 67.47 mmol) dissolved in DCM (200 ml ) in an N ₂ atmosphere. The mixture was stirred overnight at room temperature and quenched with water (500 ml) and brine (150 ml x 3). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. After concentration, IntCl-L-1a was used directly in the next reaction without purification (13.01 g, 78%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.20 (s, 1H), 3.62-3.62 (m, 4H), 3.55-3.51 (m, 4H), 3.35- 3.25 (m, 2H), 2.90-2.87 (m, 2H). ), 1.45 (s, 9H).

Preparation of compound IntCl-L-1b

To a solution of compound IntCl-L-1a (6 g, 24.16 mmol) and zL-Glu-OMe (5.94 g, 20.13 mmol) in DMF (30 ml) 20 mmol) and DIPEA (10.52). ml, 60.39 mmol) at 0° C. under N ₂ . The mixture was stirred at room temperature for 2 hours. EA (20 ml x 6), H ₂ O (20 ml) and saline (200 ml) were added to the mixture. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give the compound IntCl-L-1b (10.6 g, quant.).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.40-7.28 (m, 5H), 6.32 (s, 1H), 5.80 (s, 1H), 5.11 (s, 2H) , 5.02 (s, 1H), 4.36 (s, 1H), 3.74 (s, 3H), 3.60 (s, 4H), 3.54 (s, 4H), 3.44- 3.43 (m, 2H), 3.38-3.21 (m, 2H), 2.30-2.20 (m, 3H). 2.04-2.00 (m, 1H), 1.76 (s, 1H), 1.44 (s, 9H). EI-MS m/z: 526 (M ⁺ ).

Preparation of compound IntCl-L-1c

To a solution of compound IntCl-L-1b (3 g, 5.71 mmol) in MeOH (25 ml) was added Pd/C (900 mg) at room temperature under H ₂ atmosphere. The mixture was stirred for 3 hours and filtered through celite and then concentrated under reduced pressure. Compound IntCl-L-1c was used directly in the next step without further purification (2.23 g, crude).

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.56 (s, 1H), 5.20 (s, 1H), 3.73 (s, 3H), 3.61 (s, 4H), 3.57 -3.55(m, 4H), 3.53-3.50(m, 1H), 3.48-3.44(m, 4H), 2.40-2.32(m, 2H), 2 .18-2.10 (m, 1H), 1.88-1.81 (m, 1H), 1.44 (s, 9H). EI-MS m/z: 392 (M ⁺ ).

Preparation of compound IntCl-L-1d

HBTU (2.36 g, 6 .23 mmol) and DIPEA (1.36 ml, 7.78 mmol) at 0° C. under N ₂ atmosphere. The mixture was stirred at room temperature for 2.5 hours and EA (100 ml x 7) and H ₂ O (100 ml) were added. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give the compound IntCl-L-1d (4.06 g, quant.).

¹ H NMR (400 Hz, DMSO-d ₆ ) δ 8.89 (d, J=7.6 Hz, 1H), 8.63 (s, 1H), 7.90 (d, J=8 Hz, 2H ), 7.64 (d, J=8.4 Hz, 2H), 6.76-6.75 (m, 1H), 5.12 (s, 1H), 4.41-4.36 (m, 1H), 3.64 (s, 3H), 3.47 (s, 4H), 3.39-3.35 (m, 4H) 3.20-3.12 (m, 2H), 3.07- 3.02(m, 2H), 2.23(t, J=7.4Hz, 2H), 2.09-2.06(m, 1H), 1.96-1.91(m, 1H) , 1.36 (s, 9H). EI-MS m/z: 782 (M ⁺ ).

Preparation of compound IntCl-L-1

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

EI-MS m/z: 682 (M ⁺ ).

[Example 26] Obtaining an IntC-L connection

Preparation of Compound K-1

One portion of PyBop (7.89 g, 15 .16 mmol), then DIPEA (5.26 ml, 30.32 mmol) at 0° C. under N ₂ . The mixture was stirred overnight at room temperature. EA (80 ml × 4) and saturated citric acid solution (60 ml) were added to the mixture, and the organic layer was washed with NaHCO ₃ (120 ml), brine (100 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated at reduced pressure. The residue was purified by column chromatography to give compound K-1 (3.29 g, 95%).

EI-MS m/z: 341 (M ⁺ ).

Preparation of Compound K-2

To a solution of compound K-1 (3.29 g, 9.66 mmol) in MeOH (15 ml) was added LiOH⋅H ₂ O (2.03 g, 48.32 mmol) dissolved in H ₂ O (15 ml) at 0°C in an atmosphere of N ₂ .

The mixture was stirred at 0° C. for 30 minutes and warmed to room temperature over 2 hours. The mixture was acidified with saturated aqueous citric acid, and EA (40 ml×2) was added to the mixture. The organic layer was washed with H ₂ O (30 ml) and brine (30 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Compound K-2 was used directly in the next step without further purification (3.15 g, crude).

EI-MS m/z: 327 (M ⁺ ).

Preparation of compound K

To a solution of compound K-2 (3.69 g, 11.31 mmol) in DMF (20 ml) was added NHS (1.69 mg, 14.7 mmol) and EDCI (2.93 g, 15.26 mmol) in N ₂ atmosphere. The mixture was stirred overnight at room temperature and concentrated. The residue, Compound K, was used directly in the next step without further purification (4.79 g, crude).

EI-MS m/z: 446 (M ⁺ +Na).

Getting the connection IntC-L-2

DIPEA (5.21 ml, 29.93 mmol) in N ₂ atmosphere. The mixture was stirred overnight at room temperature. H ₂ O (70 ml) and brine (60 ml) were added to the mixture and extracted with EA (70 ml × 7). And the organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntC-L-2 (1.12 g, 19%).

EI-MS m/z: 991 (M+).

Preparation of the IntC-L-3 compound

To a solution of the compound IntA-L-2 (1.12 g, 1.13 mmol) in MeOH (27 ml) was added LiOH⋅H ₂ O (356 mg, 8.48 mmol) dissolved in H ₂ O (10 ml) at 0°C in an atmosphere of N ₂ . The mixture was stirred at 0° C. for 30 minutes and warmed to room temperature over 3 hours. The mixture was acidified with 2 M HCl and concentrated under reduced pressure. The residue, compound IntC-L-3, was used directly in the next step without further purification (996 mg, crude).

EI-MS m/z: 880 (M ⁺ ).

Obtaining an IntC-L connection

To a solution of compound IntC-L-3 (996 mg, 1.13 mmol) in DCM (30 ml) was added TFA (8 ml) at 0° C. under N ₂ atmosphere. After stirring at 0° C. for 1 hour, the mixture was concentrated under reduced pressure. The residue was dissolved in DMSO (5 ml) and purified by preparative HPLC to give IntC-L (409 mg, 32%).

EI-MS m/z: 780 (M ⁺ ).

[Example 27] Obtaining an MPS-D1 Connection

Obtaining an MPS-D1a connection

To a solution of 4-acetylbenzoic acid (9 g, 54.82 mmol) in EtOH (50 ml) was added piperidine hydrochloride (6.66 g, 54.82 mmol), paraformaldehyde (4.95 g, 164.5 mmol) and conc. . HCl (0.6 ml) at room temperature under N ₂ atmosphere. The mixture was stirred at 100° C. for 16 hours and cooled to room temperature, acetone (90 ml) was added dropwise thereto. The mixture was stirred at 0°C for 1 hour. The solid was filtered off and washed with diethyl ether (30 ml x 2) to give compound MPS-D1a (6.11 g, 38%).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.08 (s, 4H), 5.73 (s, 1H), 3.65 (t, J=7.2 Hz, 2H), 3.35 (t, J=7.2 Hz, 2H), 3.31 (m, 6H), 1.74 (s, 4H).

Obtaining an MPS-D1b connection

To a solution of MPS-D1a (6.11 g, 20.52 mmol) in EtOH (40 ml) and MeOH (26 ml) was added 4-methoxybenzenethiol (2.55 g, 20.52 mmol) and piperidine (0.3 ml , 3.08 mmol) at room temperature. The mixture was stirred at 100°C for 16 hours, cooled to 0°C and further stirred for 1 hour. The solid was filtered off and washed with ether (30 ml x 2) to give compound MPS-D1b (5.56 g, 90%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.04-7.99 (m, 4H), 7.27 (d, J=8.4 Hz, 2H), 7.15 (d, J=7, 6 Hz, 2H), 3.39-3.36 (m, 2H) 3.25-3.21 (m, 2H), 2.27 (s, 3H).

Obtaining an MPS-D1 Connection

To a solution of MPS-D1b (5.56 g, 18.51 mmol) in MeOH (90 ml) and distilled water (90 ml) was added oxone (25.03 g, 40.72 mmol) at 0°C under N ₂ . After stirring at room temperature for 14 hours, 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 Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give compound MPS-D1 (5.29 g, 86%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.04-7.99 (m, 4H), 7.81 (d, J=8.4 Hz, 2H), 7.46 (d, J=8, 4 Hz, 2H), 3.63 (t, J=7.2 Hz), 2H), 3.41 (t, J=7.2 Hz, 2H), 2.44 (s, 3H). EI-MS m/z: 333 (M ⁺ ).

[Example 28] Obtaining an MPS-D2 Connection

Preparation of Compound L-1a

To a solution of hexaethylene glycol (5.0 g, 17.71 mmol) in anhydrous DCM (178 ml) were added KI (294 mg, 1.77 mmol) and Ag ₂ O (4.92 g, 19.48 mmol) under N ₂ . The mixture was stirred overnight at room temperature. After completion of the reaction, the mixture was filtered through celite and washed with DCM (100 ml). The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-1a (5.98 g, 73%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.80 (d, J=8.4 Hz, 2H), 7.35 (d, J=8.4 Hz, 2H), 4.16 (t, J =4.8 Hz, 2H), 3.71-3.58 (m, 22H), 2.88 (br. s, 1H), 2.45 (s, 3H).

Preparation of Compound L-1b

To a solution of compound L-1a (5.98 g, 13.7 mmol) in DMF (30 ml) was added NaN ₃ (1.34 g, 20.55 mmol) under N ₂ atmosphere. The mixture was stirred at 110°C for 1 hour and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-1b (4.1 g, 97%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 3.72-3.60 (m, 22H), 3.39 (t, J=4.8 Hz, 2H), 2.78 (br. s, 1H) .

Preparation of Compound L-1c

To a solution of compound L-1b (2 g, 6.51 mmol) in acetone (56 ml) was added slowly dropwise a solution of Jones' reagent (5 ml) at -5° C. under N ₂ atmosphere. The mixture was stirred at room temperature for 2 hours and filtered through Celite, and the filtrate was concentrated under reduced pressure. The filtrate was diluted with DCM (20 ml x 2) and water (5 ml). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-1c (1.85 g, 89%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.15 (s, 2H), 3.76-3.67 (m, 18H), 3.40 (t, J=4.8 Hz, 2H).

Preparation of Compound L-1d

To a solution of compound L-1c (500 mg, 1.56 mmol) in DCM (10 mL) was added t-BuOH (305 μL, 3.11 mmol), DIC (292.5 μL, 1.87 mmol) and DMAP ( 19 mg, 0.16 mmol) under N ₂ atmosphere. The mixture was stirred at room temperature for 4 hours and diluted with DCM (30 ml x 2). The organic layer was washed with water (5 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound L-1d (278.5 mg, 47%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.01 (s, 2H), 3.70-3.66 (m, 18H), 3.38 (t, J=4.8 Hz, 2H), 1 .47 (s, 9H).

Preparation of Compound L-1e

To a solution of compound L-1d (278 mg, 0.74 mmol) in EtOH (5 mL) was added Pd/C (236 mg, 0.11 mmol) and 4M-HCl (in 1,4-dioxane) under N ₂ . The mixture was stirred at room temperature for 1 hour. The mixture was filtered through celite to remove Pd/C and concentrated to give compound L-1e (255.3 mg, 89.2%).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.32 (s, 1H), 3.98 (s, 2H), 3.55-3.40 (m, 18H), 3.86 (t, J=5.6Hz, 2H), 2.70-2.64(m, 2H), 1.42(s, 9H)

Obtaining an MPS-D2 Connection

To a solution of compound L-1e (255.3 mg, 0.66 mmol) and compound MPS-D1 (240.6 mg, 0.72 mmol) in DMF (6 ml) was added HBTU (300 mg, 0.79 mmol) and DIPEA (229.3 µl, 1.32 mmol) under nitrogen atmosphere. The mixture was stirred at room temperature for 2 hours and diluted with EA (20 ml x 2) and water (5 ml). The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound MPS-D2 (306 mg, 71%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.95 (s, 4H), 7.82 (d, J=8.0 Hz, 2H), 7.38 (d, J=8.0 Hz, 2H ), 7.33-7.30 (m, 1H), 3.98 (s, 2H), 3.68-3.63 (m, 18H), 3.55-3.53 (m, 2H), 3.49-3.47 (m, 2H), 2.95 (s, 1H), 2.88 (s, 1H), 2.46 (s, 3H) 1.46 (s, 9H). EI-MS m/z: 666(M ⁺ +1).

[Example 29] Obtaining an MPS-D4 connection

Obtaining an MPS-D3 connection

To a solution of compound MPS-D2 (120 mg, 0.18 mmol) in DCM (8 ml) was added TFA (4 ml) at 0°C. The reaction was left to warm to room temperature for 2 hours under N ₂ atmosphere. After completion of the reaction, the mixture was concentrated three times under reduced pressure using toluene as a co-solvent, thereby removing TFA. The mixture was then redissolved in DMF and NHS (31 mg, 0.27 mmol) and EDCI (52 mg, 0.27 mmol) were added. The mixture was stirred overnight at room temperature. After completion of the reaction, MPS-D3 was used directly in the next step without further purification (127 mg, crude).

EI-MS m/z: 707 (M ⁺ ).

Obtaining an MPS-D4 connection

To a solution of compound IntC-L (60 mg, 0.08 mmol) and compound MPS-D3 (82 mg, 0.12 mmol) in DMF (6 ml) was added DIPEA (112 μl, 0.64 mmol) under N ₂ . The mixture was stirred for 30 minutes and dissolved in DMSO (3 mL) and purified by HPLC to give compound MPS-D4 (77 mg, 73%).

EI-MS m/z: 1373 (M ⁺ ).

[Example 30] Obtaining an MPS-D5 Connection

To a solution of compound MPS-D1 (500 mg, 1.50 mmol) in DMF (8 ml) was added propargylamine (106 μl, 1.65 mmol) at room temperature under N ₂ atmosphere. The reaction mixture was cooled to 0°C and PyBop (1.17 g, 2.26 mmol) and DIPEA (524 μl, 3.01 mmol) were added thereto. The mixture was stirred at room temperature for 2 hours and diluted with EA (30 ml x 2) and distilled water (20 ml). The organic layer was extracted and washed with brine (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound MPS-D5 (510 mg, 92%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.11 (t, J=5.2 Hz, 1H), 7.98-7.89 (m, 4H), 7.79 (d, J=8, 0Hz, 2H), 7.43(d, J=8.4Hz, 2H), 4.05-4.03(m, 2H), 3.60(t, J=7.6Hz, 2H) , 3.39 (t, J=7.2 Hz, 2H), 3.12 (s, 1H), 2.38 (s, 3H).

[Example 31] Preparation of compound IntA-Q7

Preparation of compound IntA-Q7-1

To a solution of 3,5-diiodo-L-tyrosine dihydrate (3 g, 6.39 mmol) in MeOH (20 ml) was added thionyl chloride (836 μl, 11.5 mmol) dropwise at 0° C. under N ₂ atmosphere. The mixture was stirred overnight at room temperature. After completion of the reaction, the mixture was concentrated to give compound IntA-Q7-1 (2.86 g, quant.).

EI-MS m/z: 448(M ⁺ ).

Preparation of compound IntA-Q7-2

To a solution of compound IntA-Q7-1 (1 g, 2.24 mmol) in ACN (10 ml) was added Boc ₂ O (730 mg, 3.36 mmol) and Et ₃ N (940 μl, 6.72 mmol) at 0°C in N ₂ atmosphere. The mixture was stirred at room temperature for 15 hours and quenched with EA (30 ml x 2) and citric acid (30 ml). The organic layer was extracted and washed with brine (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give compound IntA-Q7-2 (1.22 g, quant.).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.38 (s, 1H), 7.60 (s, 2H), 7.30 (d, J=8.4 Hz, 1H), 4.15 (m, 1H), 3.63 (s, 3H) 2.76-2.62 (m, 2H), 1.33 (s, 9H).

Preparation of compound IntA-Q7-3

Pd(OAc) ₂ (23 mg, 0.101 mmol ), P(o-tol) ₃ (43 mg, 0.141 mmol) and DIPEA (1.75 ml) at room temperature under N ₂ atmosphere. The mixture was stirred at 100°C for 3 hours. The mixture was cooled to room temperature, followed by filtration through celite and then washed with EA (20 ml). The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to give compound IntA-Q7-3 (790 mg, 78%).

EI-MS m/z: 502(M ⁺ ).

Preparation of compound IntA-Q7

To a solution of compound IntA-Q7-3 (100 mg, 0.2 mmol) in ACN (6 ml) and DMF (3 ml) was added Et ₃ N (280 μl, 2.0 mmol) at room temperature under N ₂ atmosphere. SO ₂ F ₂ gas was introduced through the balloon and the mixture was stirred at room temperature for 3 hours. The mixture was quenched with saturated NaHCO ₃ (10 ml x 2) and diluted with EA (20 ml). The organic layer was extracted with brine (10 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntA-Q7 (75 mg, 65%).

EI-MS m/z: 584(M ⁺ ).

[Example 32] Preparation of Compound A-15-1

Preparation of Compound A-15-1a

To a solution of z-valine (1.01 g, 3.81 mmol) and N-methylaniline (412 μl, 3.81 mmol) in DCM (15 ml) was added DCC (1.18 g, 5.71 mmol) and DMAP (92 mg, 0.76 mmol) at room temperature under N ₂ followed by stirring at room temperature for 3 hours. The mixture was filtered through Celite and concentrated under reduced pressure. The residue was purified by column chromatography to give compound A-15-1a (1.05 g, 78%).

EI-MS m/z: 584(M ⁺ ).

Preparation of Compound A-15-1b

Compound A-15-1a (1.05 g, 2.96 mmol) was dissolved in MeOH (15 ml) under nitrogen atmosphere, Pd/C (378 mg, 0.18 mmol) was added. After stirring at room temperature for 2 hours under H ₂ atmosphere, the mixture was filtered through celite and washed with MeOH (30 ml). The filtrate was concentrated to give compound A-15-1b (560 mg, 86.0%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.45-7.41 (m, 2H), 7.38-7.36 (m, 1H), 7.19 (d, J=7.6 Hz, 1H), 3.32 (s, 3H), 2.88 (d, J=6.0 Hz, 1H), 2.33 (s, 3H), 1.73 (q, J=6.8 Hz, 1H), 0.86 (d, J=6.8 Hz, 3H), 0.80 (d, J=6.8 Hz, 3H).

Preparation of Compound A-15-1

To a solution of compound A-15-1b (220 mg, 0.99 mmol) in DMF (8 mL) was added 37% formaldehyde (223 μL, 2.99 mmol) and AcOH (1.14 mL, 19.8 mmol) in N ₂ atmosphere. After stirring at room temperature for 5 minutes, NaCNBH ₃ (125 mg, 1.98 mmol) was added. The mixture was stirred at room temperature for 2 hours and quenched with saturated NaHCO ₃ (15 mL×2). EA (20 ml x 2) and brine (20 ml) were added to the mixture. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered, concentrated under reduced pressure. The residue was purified by column chromatography to give compound A-15-1 (189 mg, 81%).

EI-MS m/z: 235(M ⁺ ).

[Example 33] Obtaining compound IntA-Q8

Obtaining compound IntA-Q8-1

To a solution of the compound Int-TG1 (example 2, 120 mg, 0.22 mmol) and IntA-Q2-2 (example 13, 50 mg, 0.24 mmol) in ACN (2 ml) under N ₂ was added BEMP dropwise (13 µl, 44 µmol). The mixture was stirred at room temperature for 2 hours and EA (10 ml x 2) and citric acid (15 ml) were added. The organic layer was washed with brine (50 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntA-Q8-1 (72 mg, 54%).

EI-MS m/z: 649(M ⁺ +Na).

Obtaining compound IntA-Q8

To a solution of compound IntA-Q8-1 (72 mg, 0.12 mmol) in THF (4 ml) was added NBS (31 mg, 0.18 mmol) and PPh ₃ (45 mg, 0.18 mmol) under N ₂ . The mixture was stirred at room temperature for 3 hours and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntA-Q8 (53 mg, 67%).

EI-MS m/z: 713(M ⁺ +Na).

[Example 34] Obtaining compound IntA-Q9

Obtaining compound IntA-Q9-1

To a solution of IntA-Q2-2 (Example 13, 300 mg, 1.45 mmol) in DMF (5 ml) was added 4-nitrophenyl chloroformate (664 mg, 2.18 mmol) and DIPEA (0.51 ml, 2.91 mmol) at room temperature under N ₂ atmosphere. After stirring overnight, EA (50 ml) and water (50 ml) were added. The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give IntA-Q9-1 (461.3 mg, 85%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.29 (d, J=9.2 Hz, 2H), 7.58 (d, J=8.8 Hz, 2H), 7.42-7.38 (m, 4H), 5.33 (s, 2H).

Obtaining compound IntA-Q9-2

To a solution of compound IntA-Q9-1 (461.3 mg, 1.24 mmol) in DMF (10 ml) and pyridine (2 ml) was added 4-N,N-dimethylethylenediamine (0.14 ml, 1.24 mmol) , HOBT (38 mg, 0.25 mmol) and DIPEA (0.22 ml, 1.24 mmol) at room temperature under N ₂ atmosphere. After stirring for 3 hours, EA (60 ml) and water (60 ml) were added. The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntA-Q9-2 (337 mg, 89%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.48 (d, J=8.8 Hz, 2H), 7.33 (d, J=8.4 Hz, 2H), 5.35 (br. s , 1H), 5.12 (s, 2H), 3.30 (kv, J=5.6, 5.2 Hz, 2H), 2.43 (t, J=5.6 Hz, 2H), 2 .24 (s, 6H). EI-MS m/z: 321(M ⁺ ).

Preparation of compound IntA-Q9-3

To a solution of compound IntA-Q9-2 (100 mg, 0.31 mmol) in anhydrous DCM (3 ml) were added paraformaldehyde (13.1 mg, 0.437 mmol) and TMS-Cl (0.06 ml, 0.47 mmol) at room temperature under N ₂ atmosphere. After stirring for 3 hours, paraformaldehyde and TMS-Cl were further added thereto, each having an amount of 10 equivalents, and the mixture was stirred at 50° C. for 30 minutes.

The crude compound IntA-Q9-3 was used directly in the next step without further purification.

EI-MS m/z: 365(M ⁺ ).

Obtaining compound IntA-Q9

To a solution of compound IntA-Q9-3 (20 mg, 0.054 mmol) in anhydrous DCM (1.5 ml) was added an excess amount of phenylethyl alcohol and DIPEA at room temperature under N ₂ atmosphere. After stirring for 2 hours, the mixture was concentrated under reduced pressure. The residue was dissolved in DMSO (4 ml) and purified by preparative HPLC to give IntA-Q9 (11.8 mg, 48%).

EI-MS m/z: 455(M ⁺ ).

[Example 35] Preparation of compound IntB-Q2

Getting connection IntB-Q2-1

To a solution of PNA (380 mg, 0.51 mmol) in MeOH (4 ml) was added dropwise SOCl ₂ (112 μl, 1.54 mmol) under N ₂ atmosphere. After stirring overnight at 40°C, SOCl ₂ (112 μl, 1.54 mmol) was further added thereto, followed by stirring at 40°C for 3 hours. The mixture was concentrated under reduced pressure. The residue was dissolved in DMSO and purified by preparative HPLC to give IntB-Q2-1 (233 mg, 69%).

EI-MS m/z: 546(M ⁺ ).

Getting the connection IntB-Q2

To a solution of IntB-Q2-1 (233 mg, 0.35 mmol) in THF (2 mL) was added IntA-Q2-2 (Example 13, 160 mg, 0.78 mmol) and PPh ₃ (148 mg, 0 .57 mmol) in an N ₂ atmosphere. The mixture was cooled to 0°C. DEAD was added dropwise and the mixture was stirred for 3 hours and diluted with EA (50 ml x 1). The organic layer was washed with H ₂ O (50 ml X 2) and brine (30 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give IntB-Q2 (118 mg, 38%).

EI-MS m/z: 734(M ⁺ ).

[Example 36] Obtaining a POS-D1 Connection

Obtaining a POS-D1a connection

To a solution of ethyl 4-hydrobenzoate (20 g, 120.35 mmol) in EtOH (60 ml) was added NH ₂ NH ₂ ⋅H ₂ O (88 ml, 1805.4 mmol) under N ₂ atmosphere. The mixture was stirred over night under reflux. After completion of the reaction, the mixture was cooled to room temperature and concentrated under reduced pressure, followed by trituration with EtOH to give compound POS-D1a (17.539 g, 96%).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.50 (s, 1H), 7.68 (d, J=8.4 Hz, 2H), 6.78 (d, J=8.8 Hz , 2H), 4.37 (s, 2H). EI-MS m/z: 431(M ⁺ ).

Obtaining a POS-D1b connection

To a solution of compound POS-D1a (17.54 g, 115.28 mmol) in EtOH (200 ml) and DMF (100 ml) was added CS ₂ (45 ml, 749.32 mmol) and KOH (6.5 g, 115 .28 mmol) in an N ₂ atmosphere. After stirring at 85°C for 18 hours, EA (500 ml) and H ₂ O (500 ml) were added and acidified with 1 M HCl. The organic layer was washed with H ₂ O (500 ml) and brine (500 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was triturated with ether/HX to give compound POS-D1b (20.7 g, 93%).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.44 (s, 1H), 7.72 (d, J=8.4 Hz, 2H), 6.94 (d, J=8.0 Hz , 2H). EI-MS m/z: 195(M ⁺ ).

Obtaining a POS-D1c Connection

Et ₃ N (4.3 ml, 30.9 mmol) and MeI (1.76 ml, 28.33 mmol ) at 0°С. After stirring at 0° C. for 10 minutes, the mixture was warmed to room temperature. And then the mixture was stirred for 2 hours and diluted with EA (100 ml×2). The organic layer was washed with H ₂ O (100 ml) and brine (100 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was triturated with ether to give compound POS-D1c (5.15 g, 96%).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.80 (d, J=8.4 Hz, 2H), 6.94 (d, J=8.4 Hz, 2H), 2.74 (s , 3H). EI-MS m/z: 209(M ⁺ ).

Obtaining a POS-D1d Connection

To a solution of compound POS-D1c (3.2 g, 15.37 mmol) in EtOH (150 ml) was added 70% m-CPBA (11.4 g, 46.11 mmol) at 0°C under N ₂ atmosphere. After stirring at room temperature for 5 hours, additional 70% m-CPBA (11.4 g, 46.11 mmol) was added. The mixture was then stirred overnight at room temperature and quenched with H ₂ O (500 ml), saturated NaHCO ₃ (300 ml), diluted with EA (500 ml × 2). The organic layer was washed with brine (300 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was triturated with HX/EA=1:1 (100 ml) to give compound POS-D1d (3.2 mg, 89%).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.95 (d, J=8.8 Hz, 2H), 7.01 (d, J=8.8 Hz, 2H), 3.69 (4 s, 3H). EI-MS m/z: 241(M ⁺ ).

Obtaining a POS-D1 Connection

To a solution of tetraethylene glycol (17.3 ml, 0.10 mol) in THF (50 ml) was added dropwise NaH (2.6 g, 0.065 mmol) at 0°C. After stirring at 0° C. for 1 hour, propargyl bromide (5.95 g, 0.05 mol) was added. The mixture was stirred overnight at room temperature and quenched with ice/water diluted with EA (100 ml x 2). The organic layer was washed with H ₂ O (100 ml) and brine (100 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. 660 mg (2.84 mmol) of the compound obtained by trituration in ether from the residue, i.e. 3,6,9,12-tetraoxapentadec-14-yn-1-ol (5.87 g, 51%, ¹ H NMR (400 MHz, CDCl ₃ ) 4.21 (s, 2H), 3.73-3.66 (m, 14H), 3.59-3.61 (m, 2H), 2.60 (s, 1H), 2 .42 (t, J=2.4 Hz, 1H)) and compound D-4-5 (310 mg, 1.29 mmol) were dissolved in THF (8 ml) and DMF (0.8 ml) and PPh ₃ was added (667 mg, 2.58 mmol). The mixture was cooled to 0°C. To this was added 2.2 M DEAD (1.17 ml, 2.58 mmol) and the mixture was stirred at 0° C. for 3 hours. After completion of the reaction, EA (15 ml×2) and distilled water (15 ml) were added and the organic layer was extracted and washed with brine (20 ml). The resulting organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give compound POS-D1 205 mg, 30%).

EI-MS m/z: 455(M ⁺ ).

[Example 37] Preparation of compound Int-TG11

Int-TG11-3 was synthesized in a similar synthetic route as described in Example 6 and Example 33.

Obtaining the connection Int-TG11-1

Yield 99%

EI-MS m/z: 265(M+). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 10.41 (s, 1H), 6.54 (s, 2H), 3.91 (s, 6H).

Preparation of the Int-TG11-2 connection

Yield 99%

EI-MS m/z: 685(M+). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 10.42 (s, 1H), 7.37 (d, J=8 Hz, 1H), 7.31-7.26 (m, 2H), 7, 15-7.11 (m, 1H), 6.58 (s, 2H), 5.54-5.45 (m, 2H), 5.09 (t, J=8.4 Hz, 2H), 4 .27-4.22(m, 1H), 4.16-4.05(m, 2H), 3.89(s, 6H), 2.19(s, 3H), 2.08(s, 3H ), 2.06 (s, 3H), 2.01 (s, 3H)

Preparation of the Int-TG11-3 connection

Yield 97% EI-MS m/z: 669 (M ⁺ ).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.41 (d, J=8 Hz, 1H), 7.27 (t, J=5.2 Hz, 2H), 7.15-7.11 ( m, 1H), 6.53 (s, 2H), 5.53-5.44 (m, 2H), 5.29 (s, 1H), 5.11-5.06 (m, 1H), 4 .99 (d, J=8 Hz, 1H), 4.77 (s, 2H), 4.26-4.22 (m, 1H), 4.15-4.11 (m, 1H), 4, 06-4.02 (m, 1H), 3.87 (s, 6H), 2.19 (s, 3H), 2.06-2.03 (m, 6H), 1.99 (s, 3H)

Getting connection Int-TG11

To a solution of compound Int-TG11-3 (137 mg, 0.199 mmol) in dry THF (5 ml) was added NBS (53 mg, 0.299 mmol) and triphenylphosphine (78 mg, 0.299 mmol) at room temperature. After stirring for 2 hours, the mixture was concentrated under reduced pressure. The residue was purified by column chromatography to give compound Int-TG11 (129 mg, 86%).

EI-MS m/z: 750 (M ⁺¹ ). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.36 (m, 1H), 7.29-7.28 (m, 2H), 6.56 (s, 1H), 6.53 (s, 1H ), 5.58-5.51 (m, 1H), 5.46 (d, J=2.8 Hz, 1H), 5.12-5.06 (m, 2H), 4.60 (s, 2H), 4.27-4.23(m, 1H), 4.17-4.11(m, 1H), 4.08-4.05(m, 1H), 3.88(s, 3H) , 3.86 (s, 3H), 2.20 (s, 3H), 2.09 (s, 3H), 2.08 (s, 3H), 2.01 (s, 3H)

[Example 38] Preparation of compound Int-TG12

To a solution of compound Int-TG1-3 (530 mg, 1.2 mmol) in dry THF (25 ml) was added 1,1'-sulfonyldiimidazole (477 mg, 2.4 mmol) and Cs ₂ CO ₃ (196 mg, 0 .6 mmol). After 18 hours at reflux, the mixture was quenched with 2 N aqueous HCl (100 ml). The organic layer was extracted with EtOAc (2×20 ml), dried over anhydrous MgSO ₄ , filtered and concentrated. The residue was purified by column chromatography to give Int-TG12 (396 mg, 58%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.84 (s, 1H), 7.35 (s, 1H), 7.34-7.31 (m, 1H), 7.20-7.16 (m, 2H), 7.05 (t, 1H), 6.86 (m, 1H), 5.51-5.45 (m, 2H), 5.10 (dd, J=3.6 Hz, 1H), 4.98 (d, J=8 Hz, 1H), 2.24 (s, 3H), 2.13 (s, 3H), 2.07 (s, 3H), 2.04 (s, 3H).

[Example 39] Preparation of compound Int-TG13

TEA (0.92 ml, 6.61 mmol) and acetyl chloride (0.47 ml, 6.61 mmol) at 0° C. under N ₂ atmosphere. After stirring at room temperature for 2 hours, the mixture was quenched with H ₂ O (7 ml). The organic layer was extracted with DCM (2×8 ml, dried over anhydrous MgSO ₄ , filtered and concentrated. The residue was purified by column chromatography to give compound Int-TG13 (987 mg, 78%).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.28 (m, 5H), 5.70 (m, 1H), 4.86 (s, 1H), 4.34-4.30 (m, 1H ), 3.65 (m, 1H), 2.00 (s, 3H), 1.01 (d, J=7.2 Hz, 3H)

[Example 40] Preparation of compound Int-TG14

Compound Int-TG14 was synthesized in a similar synthetic route as described in Example 6 and Example 37.

Getting connection Int-TG14-1

Yield 99%; EI-MS m/z: 929(M ⁺¹ ).

Getting connection Int-TG14-2

Yield 96%; EI-MS m/z: 931 (M ⁺¹ ).

Obtaining an Int-TG14 connection

Yield 75%; EI-MS m/z: 750 (M ⁺¹ ).

[Example 41] Preparation of compound IntB-Q3

Obtaining connection IntB-Q3-1

To a solution of PNU-1529682 (52 mg, 0.081 mmol) in MeOH (5 ml) / distilled water (3 ml) was added NaIO ₄ (18 mg, 0.081 mmol) at room temperature. After stirring for 2 hours, the mixture was concentrated under reduced pressure to give crude IntB-Q3-1 (51 mg, 99%). EI-MS m/z: 628 (M ⁺¹ ).

Obtaining an IntB-Q3 connection

To a solution of compound IntB-Q3-1 (51 mg, 0.081 mmol) in dry DCM (5 mL) was added 2-(dimethylamino)ethylamine (6.1 μL, 0.089 mmol) and TEA (34 μL, 0.243 mmol), TBTU ( 52 mg, 0.162 mmol) at room temperature. After stirring for 1 hour, the mixture was diluted with DCM (2×8 ml). The organic layer was washed with H ₂ O (8 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntB-Q3 (38 mg, 67%).

EI-MS m/z: 698 (M ⁺¹ ).

[Example 42] Obtaining compound IntB-Q4

Obtaining compound IntA-Q-12-1

To a solution of (3,4-methylenedioxy)-cinnamic acid (1.5 g, 7.8 mmol) in MeOH (200 ml), acetic acid (10 ml) was added 10% Pd/C (500 mg) under N ₂ . The mixture was stirred at room temperature for 22 hours under H ₂ atmosphere. The mixture was then filtered through Celite and washed with MeOH (200 ml). The filtrate was concentrated to give IntA-Q-12-1 as an off-white crystalline solid (1.51 g, 99%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.74-6.64 (m, 3H), 5.92 (s, 2H), 2.87 (t, J =7.8 Hz, 2H), 2 .63 (t, J = 7.2 Hz, 2H).

Obtaining compound IntA-Q-12-2

To a solution of compound IntA-Q-12-2 (750 mg, 3.86 mmol) and TEA (540 μl, 3.86 mmol) in anhydrous toluene (15 ml) was added dropwise diphenylphosphoryl azide (832 μl, 3.86 mmol) at room temperature under N ₂ atmosphere. The mixture was heated to 90°C for 90 minutes. After completion of the reaction, most of the solvent was removed under reduced pressure. The residue was cooled to 0°C in an atmosphere of N ₂ . BF ₃ .OEt ₂ (2.4 ml, 5.79 mmol) was added dropwise to the stirred mixture and then stirred at room temperature for 1 hour. The mixture was diluted with EA (50 ml) and washed with saturated aqueous NaHCO ₃ (100 ml). The organic layer was dried over MgSO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntA-Q-12-2 (650 mg, 88%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.51 (s, 1H), 6.65 (s, 1H), 6.06 (br. s, 1H), 6.00 (s, 2H), 3 .51 (m, 2H), 2.90 (t, J=6.8 Hz, 2H). EI-MS m/z: 192 (M ⁺¹ ).

Obtaining compound IntA-Q-12-3

To a solution of compound IntA-Q-12-2 (200 mg, 1.04 mmol) in THF (10 ml) was added dropwise n-BuLi (2.5 M in hexane, 1 ml) at -78° C. under N ₂ . The light purple solution was stirred at -78° C. for 30 minutes and B(OMe) ₃ (200 μl, 1.77 mmol) was added. The reaction mixture was heated to 0°C, then AcOH (154 μl) was added, followed by the slow addition of 35% H ₂ O ₂ (230 μl). After stirring at room temperature for 5 hours, diluted with EA (50 ml). The organic layer was washed with saturated aqueous NaCl (100 ml), dried over MgSO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntA-Q-12-3 (60 mg, 27%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 12.33 (s, 1H), 6.29 (s, 1H), 6.03 (s, 2H), 5.89 (br. s, 1H), 3 .55 (m, 2H), 2.92 (t, J=). 6.8 Hz, 2H). EI-MS m/z: 208 (M ⁺¹ ).

Obtaining compound IntA-Q-12-4

To a solution of compound IntA-Q-12-3 (38 mg, 0.184 mmol) in THF (3 ml) was added 1 M-tBuOK (1 M in THF, 220 μl, 0.220 mmol) at room temperature. The mixture was stirred at room temperature for 1 hour under SO ₂ F ₂ , quenched with 10% aqueous HCl to pH 7 and diluted with EA (50 ml). The organic layer was saturated with aqueous NaCl (100 ml), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntA-Q-12-4 (17 mg, 32%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.71 (s, 1H), 6.15 (s, 2H), 5.77 (m, 1H), 3.50 (m, 2H), 2.93 (t, J =6.8 Hz, 2H). EI-MS m/z: 290(M ⁺¹ )

[Example 43] Preparation of compound Int-TG15

Compound Int-TG15 was synthesized in a similar synthetic route as described in Example 6 and Example 37.

Yield 75%; EI-MS m/z: 934 (M ⁺¹ ).

[Example 44] Preparation of Compound L-2

Compound L-2 was synthesized in a similar synthetic route as described in Journal of Polymer Science, Part A: Polymer Chemistry, 2012, 50(19), 3986-3995, incorporated herein by reference.

Preparation of Compound L-2a

Yield 30%

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.80 (d, J=8.4 Hz, 2H), 7.34 (d, J=8.4 Hz, 2H), 4.16 (t, J =4.8 Hz, 2H), 3.74-3.58 (m 14H) 2.45 (s, 3H).

Preparation of Compound L-2b

Yield 68%

¹ H NMR (400 MHz, CDCl ₃ ) δ 3.74-3.61 (m, 14H), 3.40 (t, J=4.8 Hz, 2H), 2.45 (t, J=6, 0 Hz, 1H).

Preparation of Compound L-2c

Yield 63%

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.21 (d, J=2.4 Hz, 2H), 3.72-3.67 (m, 14H), 3.39 (t, J=5, 2 Hz, 2H), 2.43 (t, J=2.4 Hz, 1H).

Preparation of Compound L-2

Yield 76%

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.20 (d, J=2.4 Hz, 2H), 3.71-3.61 (m, 12H), 3.51 (t, J=4, 8 Hz, 2H), 2.87 (t, J=5.6 Hz), 2H), 2.43 (t, J=2.4 Hz, 1H).

[Example 45] Preparation of Compound L-3

Compound L-3 was synthesized in a similar synthetic route as described in Journal of Organic Chemistry, 2002, 67, 5032-5035, incorporated herein by reference.

Preparation of Compound L-3a

Yield 92%

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.08 (s, 1H), 7.97 (q, J=8.8 Hz, 8.8 Hz, 4H), 7.16 (br. s, 1H ), 4.14 (d, J=2.4 Hz, 2H), 3.70-3.62 (m, 16H), 2.41 (t, J=2.4 Hz, 1H). EI-MS m/z: 384(M ⁺¹ )

Preparation of Compound L-3b

Yield 69%

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.82 (d, J=8.0 Hz, 2H), 7.60 (d, J=7.6 Hz, 2H), 6.88 (br. s , 1H), 5.47 (br. s, 1H), 4.14 (d. J=2.4 Hz, 2H), 3.70-3.63 (m, 16H), 2.42 (br. s, 1H), 2.19 (s, 3H). EI-MS m/z: 404 (M ⁺¹ )

Preparation of Compound L-3

Yield 81%

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.19 (d, J=7.2 Hz, 2H), 7.92 (d, J=7.6 Hz, 2H), 7.07 (br. s ., 1H), 4.16 (s, 2H), 3.70-3.49 (m, 16H), 2.42 (br. s, 1H), 2.19 (s, 3H). EI-MS m/z: 402(M ⁺¹ )

[Example 46] Preparation of Compound L-4

Compound L-4 was synthesized in a similar synthetic route as described in Journal of Medicinal Chemistry, 52 (19), 5816-5825; 2009, incorporated herein by reference.

Preparation of Compound L-4a

Yield 55%

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.21 (d, J=2.0 Hz, 2H), 3.72-3.60 (m, 24H), 2.79 (br s, 1H) , 2.43 (t, J=2.4 Hz, 1H).

Preparation of Compound L-4

EI-MS m/z: 400(M ⁺¹ )

[Example 47] Obtaining an MPS-D6 connection

Compound L-5 was synthesized in a similar synthetic route as described in Example 44 and Example 45.

Compound MPS-D6a Yield 91%

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.80 (d, J=8.4 Hz, 2H), 7.35 (d, J=8.4 Hz, 2H), 4.16 (t, J =4.8 Hz, 2H), 3.70-3.61 (m, 20H), 3.39 (t, J=4.8 Hz, 2H), 2.45 (s, 3H). EI-MS m/z: 462(M ⁺¹ )

Obtaining an MPS-D6b connection

Yield 93%; EI-MS m/z: 610(M ⁺¹ )

Obtaining an MPS-D6c connection

Yield 54% EI-MS m/z: 584 (M ⁺¹ )

Obtaining an MPS-D6 Connection

Yield 72%; EI-MS m/z: 899(M ⁺¹ )

[Example 48] Obtaining an MPS-D7 Connection

Соединение MPS-D7 синтезировали аналогичным путем синтеза, как описано в Примере 28.

Compound MPS-D7 was synthesized in a similar synthetic route as described in Example 28.

Yield 80%; EI-MS m/z: 546(M ⁺¹ )

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.11-7.94 (m, 4H), 7.83 (d, J=7.6 Hz, 2H), 7.44 (br. s, 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).

[Example 49] Preparation of compound Int-TG16

To a solution of compound Int-TG6-1 (300 mg, 1.34 mmol) and TOM-Cl (310 μl, 1.34 mmol) in DCM (2 ml) was added DIPEA (291 μl, 1.67 mmol) under N ₂ . After stirring at room temperature for 2 hours, TOM-Cl (310 μl, 1.34 mmol) and DIPEA (466 μl, 2.67 mmol) were further added thereto. The mixture was stirred overnight at room temperature. After completion of the reaction, the mixture was purified by preparative HPLC to give compound Int-TG16 (165 mg, 30%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.22-7.20 (m, 1H), 6.92-6.87 (m, 3H), 5.43 (s, 2H), 1.21- 1.08 (m, 21H), 1.03 (s, 9H), 0.19 (s, 6H).

[Example 50] Preparation of compound Int-TG17

Compound Int-TG17 was synthesized in a similar synthetic route as described in Example 37.

Getting connection Int-TG17-1

Yield 84%; EI-MS m/z: 888 (M ⁺¹ ).

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.04 (s, 1H), 7.99-7.97 (m, 1H), 7.56-7.51 (m, 2H), 7.38- 7.34 (m, 2H), 7.31-7.27 (m, 2H), 7.15-7.08 (m, 1H), 5.58-5.51 (m, 1H), 5. 48-5.45(m, 1H), 5.12-5.07(m, 2H), 4.27-4.22(m, 1H), 4.18-4.13(m, 1H), 4.08-4.04 (m, 1H), 2.19 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.01 (s, 3H).

Getting connection Int-TG17-2

Yield 33% EI-MCm/z: 649(M ⁺¹ Na).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.45-7.41 (m, 3H), 7.35-7.33 (m, 2H), 7.30-7.25 (m, 2H), 7.15-7.10 (m, 1H), 5.51-5.44 (m, 2H), 5.08-5.02 (m, 2H), 4.72 (d, J=5.2 Hz, 2H), 4.26-4.21(m, 1H), 4.16-4.12(m, 1H), 4.05-4.01(m, 1H), 2.22(s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 2.00 (s, 3H).

Preparation of the Int-TG17 connection

Yield 71% EI-MCm/z: 713(M ⁺ Na).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.46 (d, J=8.4 Hz, 2H), 7.35-7.32 (m, 3H), 7.28-7.26 (m, 2H), 7.14-7.09 (m, 1H), 5.58-5.54 (m, 1H), 5.46-5.45 (m, 1H), 5.12-5.07 ( m, 2H), 4.48 (s, 2H), 4.27-4.23 (m, 1H), 4.17-4.11 (m, 1H), 4.07-4.04 (m, 1H), 2.18 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.01 (s, 3H).

[Example 51] Preparation of compound IntA-Q10

To a solution of N-methylbenzenesulfonamide (208 mg, 1.21 mmol) in THF (5 ml) was added the compound IntA-Q2-2 (100 mg, 0.48 mmol) and PPh3 (382 mg, 1.45 mmol) under N ₂ . And then DEAD (221 μl, 1.21 mmol) was added dropwise and the mixture was stirred overnight at room temperature and diluted with EA (50 ml). The organic layer was washed with H ₂ O (40 ml) and brine (40 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntA-Q10 (143 mg, 82%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.84 (d, J=7.6 Hz, 2H), 7.66-7.63 (m, 1H), 7.60-7.53 (m, 2H), 7.44 (d, J=8.8 Hz, 2H) 7.32 (d, J=8.4 Hz, 2H), 4.19 (s, 2H), 2.65 (s, 3H ).

EI-MS m/z: 360(M ⁺¹ ).

[Example 52] Preparation of compound IntB-Q8

Compound IntB-Q8 was synthesized in a similar synthetic route as described in Example 12.

Yield 16%; EI-MS m/z: 656(M ⁺¹ ).

[Example 53] Preparation of compound IntB-Q9

Compound IntB-Q9 was synthesized in a similar synthetic route as described in Example 37 and Example 2.

Getting connection IntB-Q9-1

Yield 77%

¹ H NMR (400 MHz, CDCl ₃ ) δ 10.54 (s, 1H), 9.97 (s, 1H), 8.38 (s, 1H), 8.14 (d, J=8.4 Hz , 1H), 7.59 (d, J=8 Hz, 2H), 7.43 (d, J=8.8 Hz, 2H), 7.19 (d, J=8.4 Hz, 1H), 5.33 (s, 2H).

Getting connection IntB-Q9-2

Yield 94%; EI-MS m/z: 365(M ⁺ +Na).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.67-7.61 (m, 4H), 7.37 (s, 1H), 7.12 (d, J=8.8 Hz, 1H) , 6.94 (d, J=8.4 Hz, 1H) 5.19 (s, 2H), 5.06-5.02 (m, 2H), 4.56 (d, J=4.8 Hz , 2H), 4.42 (d, J=5.6 Hz, 2H).

Getting connection IntB-Q9-3

Yield 82%

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.62 (d, J=8.4 Hz, 2H), 7.41-7.38 (m, 3H), 7.32 (d, J=8, 4 Hz, 1H), 6.86 (d, J=8.8 Hz, 1H), 5.19 (s, 2H), 4.57 (s, 2H), 4.47 (s, 2H).

Getting connection IntB-Q9

EI-MS m/z: 1092(M ⁺¹ ).

[Example 54] Preparation of compound IntB-Q10

Getting connection IntB-Q10-1

Compound IntB-Q10-1 was synthesized by a similar synthesis route as described in the document [see. Mol. Pharmaceutics 2015, 12, 1813-1835]

Obtaining connection IntB-Q10-2

Compound IntB-Q10-2 was synthesized by a similar synthesis route as described in the document [see. Angew. Chem. Int. Ed. 2010, 49, 7336-7339 and WO2015110935A1]

Obtaining connection IntB-Q10-3

To a solution of compound IntB-Q10-1 (80 mg, 0.239 mmol) and compound IntB-Q10-2 (118 mg, 0.239 mmol) in DCM (10 ml) was added a molecular sieve and BF ₃ ⋅OEt ₂ (14.8 μl, 0.12 mmol) at 0° C. under N ₂ atmosphere. After stirring for 2 hours, the mixture was filtered through Celite, washed with DCM (50 ml) and concentrated under reduced pressure. The residue was purified by column chromatography to give IntB-Q10-3 (105 mg, 66%) as a white foam.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.12 (d, J =8.0 Hz, 1H), 7.89 (br. s, 1H), 7.63 (d, J=8.0 Hz , 1H), 7.50 (m, 1H), 7.35 (m, 1H), 5.70 (m, 1H), 5.51 (s, 1H), 5.33 (m, 1H), 5.20(m, 1H), 4.23(m, 3H), 4.11(m, 2H), 3.93(m, 2H), 3.42(t, J=10.8Hz, 1H ), 2.18 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H), 2.00 (s, 3H), 1.55 (s, 9H). EI-MS m/z: 564.4(M ⁺¹ ).

Obtaining connection IntB-Q10-4

Connection IntB-Q10-3 (100 mg, 0.15 mmol) was dissolved in DCM (2 ml) and then added 4N HCl in 1,4-dioxane (1 ml) at 0°C under N ₂ atmosphere. After stirring for 4 hours, the reaction mixture was concentrated under reduced pressure.

The reaction mixture was stirred at room temperature for 4 hours under N ₂ atmosphere. Compound IntB-Q10-3 was used directly in the next step without further purification (90 mg, 99%).

EI-MS m/z: 564.2(M ⁺¹ ).

Getting connection IntB-Q10

To a solution of compound IntB-Q10-3 (90 mg, 0.149 mmol) in THF (5 ml) was added glutaric anhydride (18.8 μl, 0.164 mmol), Et ₃ N (52 μl, 0.373 mmol) and 4-DMAP (2 mg, 0.015 mmol) at room temperature under N ₂ atmosphere. The reaction mixture was stirred at room temperature for 2 hours and purified by preparative HPLC to give IntB-Q10 (30 mg, 30%) as a white solid.

EI-MS m/z: 678.3(M ⁺¹ ).

[Example 55] Preparation of the compound of IntB-Q11 and IntB-Q12

Compounds IntB-Q11-2 and IntB-Q12-2 were synthesized in a similar synthetic route as described in Example 6 and Example 6.

Getting connection IntB-Q11-1

Yield 98%

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.37 (br. s, 1H) 8.02 (d, J=8.8 Hz, 1H), 7.75 (d, J=8.4 Hz, 1H), 7.61 (t, J=7.2, 1H), 7.51 (t, J=8.0 Hz, 1H), 4.32 (br. s, 1H), 4.18 (t , J=8.8, 1H), 4.05 (m, 1H), 3.93 (dd, J=11.2, 2.8 Hz, 1H) 3.52 (t, J=10.8 Hz , 1H), 1.61 (s, 9H). EI-MS m/z: 438.2(M ⁺¹ +Na).

Getting connection IntB-Q11-2

Yield 79%

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.09 (br. s, 1H) 7.77 (m, 3H), 7.57 (t, J=7.2 Hz, 1H), 7.46 ( t, J=7.6 Hz, 1H), 7.32 (m, 1H), 6.78 (m, 1H), 5.56 (m, 1H), 5.46 (d, J=2.8 Hz, 1H), 5.22 (d, J=7.6 Hz, 1H), 5.12 (dd, J=10.4, 3.2 Hz, 1H), 4.30 (br. s, 1H ), 4.25-4.02 (m, 5H), 3.93 (m, 1H), 3.60 (m, 15H), 3.31 (m, 2H), 2.17 (s, 3H) , 2.04 (s, 3H), 1.95 (s, 6H), 1.56 (s, 9H). EI-MS m/z: 1080.6(M ⁺¹ ).

Getting connection IntB-Q11

Compound IntB-11-2 (50 mg, 0.046 mmol) was dissolved in 4N HCl in 1,4-dioxane (1 ml) at 0°C under N ₂ atmosphere. After stirring at room temperature for 4 hours, the mixture was diluted with DCM (5 ml) and concentrated. Compound IntB-Q11 was used directly in the next step without further purification (47 mg, 99%).

EI-MS m/z: 980.5(M ⁺¹ ).

Getting connection IntB-Q12-1

Yield 46%; EI-MS m/z: 452.2(M ⁺¹ +Na).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.4 (br. s, 1H) 7.91 (dd, J=6.8, 2.4 Hz, 1H), 7.38 (m, 2H), 4.34 (m, 1H), 4.23 (m, 1H), 4.05 (m, 1H), 3.62 (d, J=10.4, 1H), 3.30 (t, J= 11.2, 1H), 2.83 (s, 1H), 1.61 (s, 9H).

Getting connection IntB-Q12-2

Yield 71%; EI-MS m/z: 1094.4(M ⁺¹ ).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.33 (br. s, 1H) 7.99 (d, J=7.2 Hz, 1H), 7.78 (m, 2H), 7.35 ( m, 3H), 6.87 (m, 1H), 5.54 (m, 1H), 5.46 (d, J=2.8 Hz, 1H), 5.22 (d, J=8.0 Hz, 1H), 5.12 (dd, J=10.4, 3.2 Hz, 1H), 4.33 (m, 1H), 4.26-4.01 (m, 5H), 3.61 (m, 14H), 3.32 (m, 3H), 2.83 (s, 1H), 2.21 (s, 3H), 2.07 (s, 3H), 1.99 (s, 6H) 1.57 (s, 9H).

Getting connection IntB-Q12

Yield 99%; EI-MS m/z: 994.5(M ⁺¹ ).

[Example 55a] Preparation of compound IntB-Q13

Compound IntB-Q13 was synthesized in a similar synthetic route as described in Mol. Pharmaceutics 2015, 12, 1813-1835, incorporated herein by reference.

[Example 56] Preparation of compound IntB-Q14

Compound IntB-Q14 was synthesized in a similar synthetic route as described in WO 2015038426A1, incorporated herein by reference.

[Example 56] Preparation of compound IntB-Q15

Obtaining connection IntB-Q15-1

To a solution of compound IntB-Q10-1 (55 mg, 0.016 mmol) in DCM (2 ml) were added acetyl chloride (26.8 μl, 0.032 mmol) and pyridine (30 μl, 0.032 mmol) at 0°C under N ₂ atmosphere. After stirring for 30 minutes, the reaction mixture was warmed to room temperature and further stirred for 1 hour. The mixture was diluted with EA (20 ml) and washed with H ₂ O (10 ml). Compound IntB-Q15-1 (50 mg, 80%) as a pale yellow foam.

EI-MS m/z: 398.2(M ⁺¹ +Na).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.02 (br. s, 1H) 7.79 (d, J=8.0 Hz, 1H), 7.72 (t, J=8.8 Hz, 1H), 7.51(m, 1H), 7.38(m, 1H), 4.16(m, 1H), 4.04(m, 1H), 3.92(m, 1H), 3, 71 (m, 1H), 3.36 (m, 1H), 2.27 (s, 3H), 1.54 (s, 9H).

Obtaining an IntB-Q15 connection

Compound IntB-Q15 was synthesized in a similar synthetic route as described in Example 55.

Yield 99%; EI-MS m/z: 276.2(M ⁺¹ ).

[Example 57] Preparation of compound IntB-Q16

Getting connection IntB-Q16-1

To a solution of dimethyl 5-hydroxyisophthalate (5.0 g, 23.79 mmol) in THF (300 ml) was added LAH (3.6 g, 95.15 mmol) at 0°C under nitrogen atmosphere. After stirring at room temperature for 17 hours, 15% NaOH aqueous solution (10 ml) and distilled water (30 ml) were added. After the mixture was extracted with EA (500 ml), the organic layer was washed with brine (100 ml), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound IntB-Q16-1 (3.02 g, 82%).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.21 (s, 1H), 6.66 (s, 1H), 6.58 (s, 2H), 5.07 (t, J=5, 6 Hz, 2H), 4.38 (d, J=5.6 Hz, 4H).

Obtaining connection IntB-Q16-2

To a solution of the compound IntB-Q16-1 (881.1 mg, 5.72 mmol) in AcOH (15 ml) was added 33% HBr in AcOH (2.6 ml, 14.29 mmol) at 0° C. under N ₂ . Thereafter, the mixture was heated to 65° C. and stirred for 8 hours, and stirred again at room temperature for 2 days. DCM (50 ml) and water (30 ml) were added. The organic layer was washed with aqueous NaHCO ₃ , dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatogram to give compound IntB-Q16-2 (1.1 g, 71%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.99 (s, 1H), 6.81 (s, 2H), 4.85 (s, 1H), 4.41 (s, 4H).

Getting connection IntB-Q16-3

To a solution of compound IntB-Q16-2 (1.0 g, 3.57 mmol) in DCM (35 ml) was added TEA (0.45 ml, 3.21 mmol) at room temperature under N ₂ atmosphere. The mixture was stirred for 1 hour and DCM (50 ml) and water (30 ml) were added. The organic layer was washed with aqueous NaHCO ₃ , dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give IntB-Q16-3 (941.7 mg, 73%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.47 (s, 1H), 7.32 (s, 2H), 4.46 (s, 4H).

Getting the connection IntB-Q16

K ₂ CO ₃ (183.2 mg, 1.32 mmol) at room temperature under N ₂ atmosphere. The mixture was stirred at room temperature for 2 hours and EA (20 ml) and water (5 ml) were added. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give IntB-Q8 (254 mg, 64%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.66 (d, J=4.4 Hz, 2H), 7.55 (s, 3H), 7.42 (s, 2H), 6.81 (s , 2H), 5.27-5.17 (m, 8H) 4.29 (s, 4), 3.97 (s, 6H), 3.89-3.85 (m, 2H), 3.15 -3.09 (m, 2H), 2.93 (d, J=16 Hz, 2H). EI-MS m/z: 717(M ⁺¹ ).

[Example 58] Preparation of Compound A-1

Preparation of Compound A-1a

To a solution of compound IntA-Q1 (46 mg, 0.135 mmol) and compound Int-TG1 (75 mg, 0.135 mmol) in acetonitrile (3 ml) was added DBU (4 μl, 0.027 mmol). The mixture was stirred at room temperature for 12 hours under N ₂ atmosphere. The mixture was extracted with EA (30 ml x 2). The organic layer was washed with water (30 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound A-1a (105 mg, 98%).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.33-7.26 (m, 5H), 7.21-7.17 (m, 2H), 7.14-7.08 (m, 1H), 5.56 (dd, J=2.4 Hz, 8.0 Hz, 1H) 5.46 (d, J=3.2 Hz, 1H), 5.12-5.08 (m, 2H), 5 .06-5.00(m, 1H), 4.63-4.56(m, 1H), 4.28-4.23(m, 1H), 4.18-4.13(m, 1H) , 4.08-4.04 (m, 1H), 3.72 (s, 3H), 3.18-3.03 (m, 2H), 2.19 (s, 3H), 2.07 (s , 3H), 2.06 (s, 3H), 2.01 (s, 3H), 1.42 (s, 9H). EI-MS m/z: 798(M ⁺¹ ).

Preparation of Compound A-1

To a solution of compound A-1a (100 mg, 0.13 mmol) in THF (4 ml) and methanol (1 ml) was added dropwise LiOH⋅H ₂ O (33 mg, 0.79 mmol) dissolved in water (1 ml) at 0°C. After stirring at 0° C. for 2 hours, the reaction was quenched with 1N aqueous hydrochloric acid (3 ml). The mixture was purified by HPLC to give compound A-1 (65 mg, 85%).

EI-MS m/z: 616(M ⁺¹ ).

[Example 59 ] Preparation of Compound A-2

Compound A-2a was synthesized in a similar synthetic route as described in Example 58.

Yield 95%; EI-MS m/z: 1043(M ⁺¹ ).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.80 (s, 1H), 7.75 (dd, J=8.0 Hz, 1H), 7.33-7.20 (m, 5H), 7 .0 (m, 1H), 5.76 (dd, J=8.0, 10.4 Hz, 1H), 7.47 (d, J=3.2 Hz, 1H), 5.17-5, 10 (m, 3H), 4.58 (m, 1H), 4.25-4.08 (m, 3H), 3.72 (s, 3H), 3.66-3.60 (m, 13H ), 3.34 (t, J=4.4 Hz, 2H), 2.17 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.01 (s , 3H) 1.41 (s, 9H).

Preparation of Compound A-2

Yield 53%; EI-MCm/z: 860(M ⁺¹ ).

[Example 60] Preparation of Compound A-3

Compound A-3 was synthesized in a similar synthetic route as described in Example 58.

Preparation of Compound A-3a

EI-MS m/z: 806(M ⁺¹ +Na).

Preparation of Compound A-3

Yield 33%; EI-MS m/z: 652(M ⁺¹ +Na).

¹ H NMR (400 Hz, CD ₃ OD) δ 7.42-7.31 (m, 7H), 7.14-7.10 (m, 1H), 5.13 (d, J=7.2 Hz , 1H), 4.42-4.38 (m, 1H), 4.03 (d, J=10 Hz, 1H), 3.67 (t, J=9.6 Hz, 1H), 3.55 -3.43 (m, 2H), 3.27 dd, J=9.2, 5.2 Hz, 1H), 3.16-2.97 (m, 1H), 1.39 (s, 9H) .

[Example 61] Preparation of Compound A-4

Preparation of Compound A-4a

Compound A-4a was synthesized in a similar synthetic route as described in Example 58.

EI-MS m/z: 1235(M ⁺¹ ).

Preparation of Compound A-4b

To a solution of compound A-4a (41 mg, 0.03 mmol) in MeOH (2.66 ml) was added K ₂ CO ₃ (14 mg, 0.10 mmol) at 0°C and the mixture was stirred at 0°C for 2 hours. K ₂ CO ₃ (9 mg, 0.07 mmol) was added thereto, and the mixture was stirred at 0° C. and diluted with EA (20 ml×2). The organic layer was washed with H ₂ O (20 ml) and brine (20 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give compound A-4a (35 mg) which was used for further purification.

EI-MS m/z: 1067(M ⁺¹ ).

Preparation of Compound A-4

To a solution of compound A-4b (17 mg, 0.02 mmol) in THF (640 μl) was added AcOH (3 μl, 0.05 mmol) and TBAF (48 μl, 0.05 mmol) at 0° C. under N ₂ . After stirring for 1.5 hours, additional TBAF (48 μl, 0.05 mmol) was added and stirred at 0° C. for 2 hours. The mixture was purified by preparative HPLC to give compound A-4 (3 mg, 22%).

EI-MS m/z: 724(M ⁺¹ ).

[Example 62] Preparation of Compound A-5

Preparation of Compound A-5a

Compound A-5a was synthesized in a similar synthetic route as described in Example 58.

Yield 88%; EI-MCm/z: 630(M ⁺¹ ).

Preparation of Compound A-5b

To a solution of compound A-5a (100 mg, 0.16 mmol) and acetobromo-α-D-glucuronic acid methyl ester (64 mg, 0.16 mmol, CAS # 21085-72-3) in Et ₂ O (10 ml ) was added AgOTf (204 mg, 0.79 mmol) and trimethylpyridine (106 mg, 0.87 mmol) at 0°C. The mixture was stirred at room temperature for 6 hours and diluted with EA (50 ml). Washed with aqueous 2N HCl (10 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was separated and purified by preparative HPLC to give compound A-5b (14 mg, 9%).

EI-MS m/z: 946(M ⁺¹ ).

Preparation of Compound A-5

Compound A-5 was synthesized in a similar synthetic route as described in Example 58.

EI-MS m/z: 792(M ⁺¹ ).

[Example 63] Preparation of Compound A-6

Compound A-6 was synthesized in a similar synthetic route as described in Example 60.

Preparation of Compound A-6a

Yield 53%

EI-MS m/z: 1071(M ⁺¹ ).

Preparation of Compound A-6

Yield 16%

EI-MS m/z: 888(M ⁺¹ ).

[Example 64] Preparation of Compound A-7

Preparation of Compound A-7a

To a solution of compound Int-TG4 (73.9 mg, 0.29 mmol) and compound IntA-Q1 (100 mg, 0.27 mmol) in anhydrous acetonitrile (2 ml) was added BEMP (31 μl, 0.11 mmol) at room temperature in an atmosphere of N ₂ . The mixture was stirred at 100°C for 5 hours under microwave conditions. The mixture was separated and purified by preparative HPLC to give compound A-7a (61.2 mg, 46%).

EI-MS m/z: 519(M ⁺¹ +Na).

Preparation of Compound A-7b

Compound A-7b was synthesized in a similar synthetic route as described in Example 58.

Yield 64%; EI-MCm/z: 505(M ⁺¹ +Na).

Preparation of Compound A-7

To a solution of compound A-7b (39 mg, 0.08 mmol) in methanol (2 ml) were added dropwise Zn dust (6.4 mg, 0.10 mmol) and ammonium formate (8.22 mg, 0.13 mmol ) at room temperature under N ₂ atmosphere. The mixture was stirred at room temperature for 2 hours and filtered through celite, washed with MeOH and concentrated. The residue was separated and purified by preparative HPLC to give compound A-7.

EI-MS m/z: 469(M ⁺¹ ).

[Example 65] Preparation of Compound A-8

Compound A-8 was synthesized in a similar synthetic route as described in Example 60.

EI-MS m/z: 588(M ⁺¹ +Na).

[Example 66] Preparation of Compound A-9

Compound A-9 was synthesized in a similar synthetic route as described in Example 60.

EI-MS m/z: 608(M ⁺¹ ).

[Example 67] Preparation of Compound A-10

To a solution of compound IntA-Q4 (23 mg, 0.07 mmol) and compound Int-TG7 (30 mg, 0.08 mmol) in anhydrous acetonitrile (1 ml) was added BEMP (11.2 μl, 0.04 mmol) at room temperature in an atmosphere of N ₂ .

The mixture was stirred overnight at room temperature, separated and purified by preparative HPLC to give compound A-10 (0.3 mg).

EI-MS m/z: 578(M ⁺¹ ).

[Example 68] Preparation of Compound A-11

Compound A-11 was synthesized in a similar synthetic route as described in Example 67.

Yield 65%; EI-MS m/z: 603(M ⁺¹ ).

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.19 (dd, J=6.8, 1.2 Hz, 1H), 8.02 (d, J=8.0 Hz, 1H), 7.68 (t, J=6.4 Hz, 1H), 7.49 (t, J=7.2 Hz, 1H), 7.33-7.23 (m, 3H), 7.22 (d, J= 8.0 Hz, 1H), 7.17 (d, J=8.8 Hz, 2H), 7.08 (dd, J=7.2, 1.2 Hz, 1H), 7.01 (td, J=6.8, 1.2Hz, 1H), 5.56(s, 2H), 4.99(d, J=8.4Hz, 1H), 4.59(q, J=7.2 , 6.4 Hz, 1H), 3.14 (dd, J=8.4, 5.6 Hz, 1H), 3.03 (dd, J=7.6, 6.0 Hz, 1H), 1 .42 (s, 9H).

[Example 69] Preparation of Compound A-12

Preparation of Compound A-12a

Compound A-12a was synthesized in a similar synthetic route as described in Example 67.

Yield 82%; EI-MS m/z: 870(M ⁺¹ +Na).

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.80-7.75 (m, 3H), 7.61 (s, 1H), 7.52-7.47 (m, 2H), 7.33 ( d, J=8.0 Hz, 2H), 7.21 (d, J=8.1 Hz, 2H), 5.63 (t, J=2.2 Hz, 1H), 5.50 (s, 1H), 5.23 (d, J=7.6 Hz, 1H), 5.15 (d, J=10.4 Hz, 1H) 5.08-4.98 (m, 1H), 4.62 -4.54(m, 1H), 4.36-4.26(m, 1H), 4.24-4.14(m, 2H), 3.72(s, 3H), 3.22-3 .02 (m, 2H), 2.20 (s, 3H), 2.08 (s, 6H), 2.02 (s, 3H), 1.42 (s, 9H)

Preparation of Compound A-12

Compound A-12 was synthesized in a similar synthetic route as described in Example 58.

Yield 93%; EI-MCm/z: 688(M ⁺¹ +Na).

[Example 70] Preparation of Compound A-13

Preparation of Compound A-13a

Compound A-13a was synthesized in a similar synthetic route as described in Example 67.

Yield 43%; EI-MS m/z: 935(M ⁺¹ ).

Preparation of Compound A-13

Compound A-13 was synthesized in a similar synthetic route as described in Example 61.

Yield 61%; EI-MCm/z: 683(M ⁺¹ ).

[Example71] Preparation of Compound A-14

Preparation of Compound A-14a

Compound A-14a was synthesized in a similar synthetic route as described in Example 66.

Output quantity; EI-MCm/z: 1004(M ⁺¹ ).

Preparation of Compound A-14

Compound A-14a was synthesized in a similar synthetic route as described in Example 61.

Yield 88%; EI-MCm/z: 836(M ⁺¹ ).

[Example 72] Preparation of Compound A-15

Preparation of Compound A-15-2

To a solution of compound A-15-1 (53 mg, 76.9 μmol) and IntA-Q8 (22 mg, 92.3 μmol) in DMF (2 ml) N ₂ atmosphere. After stirring overnight at room temperature, the mixture was separated and purified by preparative HPLC to give compound A-15-2 (25.9 mg, 40%).

EI-MS m/z: 843(M ⁺¹ ).

Preparation of Compound A-15

Compound A-15 was synthesized in a similar synthetic route as described in Example 61.

Yield 51%; EI-MS m/z: 675(M ⁺¹ ).

[Example 73] Preparation of Compound A-17

Preparation of Compound A-17a

Compound A-17a was synthesized in a similar synthetic route as described in Example 60.

Yield 51%; EI-MS m/z: 875(M ⁺¹ ).

Preparation of Compound A-17

Compound A-17 was synthesized in a similar synthetic route as described in Example 71.

Yield 52.2%; EI-MCm/z: 707(M ⁺¹ ).

[Example 74] Preparation of Compound A-18

Preparation of Compound A-18a

To a solution of IntA-Q10 (118 mg, 0.16 mmol) and Int-TG1 (107 mg, 0.19 mmol) in ACN (2 mL) and DMF (0.2 mL) was added BEMP (23.3 μL , 0.08 mmol) in an N ₂ atmosphere. After stirring at room temperature for 2.5 minutes, DBU (12 μl, 0.08 mmol) was further added thereto, and the mixture was stirred at room temperature for 2 hours and diluted with EA (30 ml×2). The organic layer was washed with H ₂ O (30 ml) and brine (20 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was separated and purified by preparative HPLC to give compound A-18a (22.5 mg, 14%).

EI-MS m/z: 900(M ⁺¹ ).

Preparation of Compound A-18b

To a solution of compound A-18a (22.5 mg, 0.022 mmol) in ACN (1 ml) was added Boc ₂ O (14.5 mg, 0.066 mmol) and DMAP (2.7 mg, 0.022 mmol) under N ₂ atmosphere. After stirring overnight at room temperature, Boc ₂ O (4.8 mg, 0.022 mmol) and DMAP (1.4 mg, 0.011 mmol) were further added thereto, and the mixture was stirred at room temperature for 5 hours. The mixture was separated and purified by preparative HPLC to give compound A-18 (10 mg, 37%).

EI-MS m/z: 1101(M ⁺¹ ).

Preparation of Compound A-18

Compound A-18 was synthesized in a similar synthetic route as described in Example 58.

EI-MS m/z: 850(M ⁺¹ ).

[Example 75] Preparation of Compound A-19

Compound A-19 was synthesized in a similar synthetic route as described in Example 72.

Preparation of Compound A-19-1

Yield 44%; EI-MS m/z: 904 (M ⁺¹ ).

Preparation of Compound A-19

Yield 70%; EI-MS m/z: 736 (M ⁺¹ ).

[Example 76] Preparation of Compound A-20

Compound A-20 was synthesized in a similar synthetic route as described in Example 71.

Yield 68%; EI-MCm/z: 861(M ⁺¹ ).

[Example 77] Preparation of Compound A-21

Preparation of Compound A-21a

N-Boc-3-(4-pyridyl)-D-alanine (300 mg, 0.06 mmol) and DMAP (275 mg, 2.25 mmol) were dissolved in MeOH (5 at 0°C). DCC (465 mg, 2.25 mmol) was added at 0°C, the mixture was stirred at 0°C for 2 hours. The reaction mixture was warmed to room temperature and stirred overnight. The mixture was diluted with EA (30 ml), filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound A-21a.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.46 (d, J=5.6 Hz, 2H), 7.37 (d, J=8 Hz, 1H), 7.26 (d, J =5.6 Hz, 2H), 4.29-4.23(m, 1H), 3.63(s, 3H), 3.06-3.01(m, 1H), 2.89-2, 83 (m, 1H), 1.31 (s, 9H).

EI-MS m/z: 281(M ⁺¹ ).

Compound A-21 was synthesized in a similar synthetic route as described in Example 72.

Preparation of Compound A-21b

Yield 29%; EI-MS m/z: 950(M ⁺¹ )

Preparation of Compound A-21

Yield 47%; EI-MCm/z: 768(M ⁺¹ ).

[Example 78] Preparation of Compound A-22

Compound A-22 was synthesized in a similar synthetic route as described in Example 72.

Preparation of Compound A-22a

Yield 68% EI-MCm/z: 888(M ⁺¹ ).

Preparation of Compound A-22

Yield 56% EI-MCm/z: 720(M ⁺¹ ).

[Example 79] Preparation of Compound A-23

Compound A-22 was synthesized in a similar synthetic route as described in Example 72.

Preparation of Compound A-22a

Yield 57% EI-MCm/z: 828(M ⁺¹ ).

Preparation of Compound A-22

Yield 72% EI-MCm/z: 660(M ⁺¹ ).

[Example 80] Preparation of Compound A-24

Compound A-24 was synthesized in a similar synthetic route as described in Example 69.

Preparation of Compound A-24a

Yield 33%; EI-MS m/z: 851(M ⁺¹ ).

Obtaining compound IntA-Q5

Yield 80%; EI-MCm/z: 705(M ⁺¹ +Na).

[Example 81] Preparation of Compound A-25

Preparation of Compound A-25-2

To a solution of compound Int-TG12 (100 mg, 0.175 mmol) in DCM (6 ml) was added methyl triflate (30 μl, 0.263 mmol) at 0° C. under N ₂ atmosphere. The mixture was stirred at room temperature for 4 hours and concentrated under reduced pressure. The residue was dissolved in dry ACN (5 ml) and compound A-15-2 (46 mg, 0.21 mmol) was added at room temperature. The mixture was heated at 40° C. for 8 hours, quenched with 2 N aqueous HCl (8 ml) and extracted with EtOAc (2×10 ml). The organic layer was dried over anhydrous MgSO ₄ , filtered and concentrated. The residue was dissolved in DMSO (1 ml) and purified by preparative HPLC to give compound A-25-2 (43.5 mg, 35%).

EI-MS m/z: 723 (M ⁺¹ ).

Preparation of Compound A-25

Compound A-25 was synthesized in a similar synthetic route as described in Example 61.

Yield 60%; EI-MS m/z: 555 (M ⁺¹ ).

[Example 82] Preparation of Compound A-26

Compound A-26 was synthesized in a similar synthetic route as described in Example 81.

Preparation of Compound A-26-1

Yield 8%; EI-MS m/z: 696 (M ⁺¹ ).

Preparation of Compound A-26

Yield 32%; EI-MS m/z: 528 (M ⁺¹ ).

[Example 83] Preparation of Compound A-27

Compound A-27 was synthesized in a similar synthetic route as described in Example 81.

Preparation of Compound A-26-1

Yield 72%; EI-MS m/z: 802 (M ⁺¹ +Na).

Preparation of Compound A-26

Yield 79%; EI-MS m/z: 634 (M ⁺¹ +Na).

[Example 84] Preparation of Compound B-1

Compound B-1 was synthesized in a similar synthetic route as described in Example 67 and Example 61.

Preparation of Compound B-1a

EI-MS m/z: 1666(M ⁺¹ ).

Preparation of Compound B-1

Yield 19% over 2 stages; EI-MCm/z: 1498(M ⁺¹ ).

[Example 85] Preparation of Compound B-2

Compound B-2 was synthesized in a similar synthetic route as described in Example 67 and Example 61.

Preparation of Compound B-2a

Yield 58%; EI-MS m/z: 1140(M ⁺¹ ).

Preparation of Compound B-2

Yield 76%; EI-MS m/z: 971(M ⁺¹ ).

[Example 86] Preparation of Compound B-3

Preparation of Compound B-3a

HOBt (2 mg, 0.013 mmol), DIPEA (12 µl, 0.069 mmol) and pyridine (330 µl) at room temperature under N ₂ . After stirring overnight, the mixture was adjusted to pH 2-3 with 1N HCl, extracted with EA (8 ml x 2). The organic layer was washed with distilled water (8 ml) and brine (12 ml), dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was subjected to column chromatography to give compound B-3a (73 mg, 71%).

EI-MS m/z: 1644(M ⁺¹ ).

Preparation of Compound B-3

Compound B-3 was synthesized in a similar synthetic route as described in Example 61.

Yield 69%; EI-MS m/z: 1462(M ⁺¹ ).

[Example 87] Preparation of Compound B-4

Compound B-4 was synthesized in a similar synthetic route as described in Example 67 and Example 61.

Preparation of Compound B-4a

Yield 68%; EI-MS m/z: 1678(M ⁺¹ /2).

Preparation of Compound B-4

Yield 91%; EI-MS m/z: 1572(M ⁺¹ /2).

[Example 87] Preparation of Compound B-6

Compound B-6 was synthesized in a similar synthetic route as described in Example 67 and Example 61.

Preparation of Compound B-6a

Yield 100%; unrefined; EI-MCm/z: 1020 (M ⁺¹ ).

Preparation of Compound B-6

Yield 17%;

[Example 87] Preparation of Compound B-7

Compound B-7 was synthesized in a similar synthetic route as described in Example 67 and Example 61.

Preparation of Compound B-7a

Yield 99%; EI-MS m/z: 1063 (M ⁺¹ ).

Preparation of Compound B-7

EI-MS m/z: 895 (M ⁺¹ ).

[Example 89] Preparation of Compound B-8

Preparation of Compound B-8a

Compound B-8a was synthesized in a similar synthetic route as described in Example 67.

Yield 16%; EI-MS m/z: 954(M ⁺¹ ), 976 (M ⁺¹ +Na)

Preparation of Compound B-8

To a solution of compound B-8a (3.1 mg, 0.0032 mmol) in methanol (1 ml) was added 25%-NaOMe in MeOH (3 μl, 0.012 mmol) at 0°C. After stirring at room temperature for 1.5 hours, the mixture was neutralized with aqueous 2M-HCl and then purified by preparative HPLC to give Compound B-8 as a pink solid (1.2 mg, 47%).

[Example 90] Preparation of Compound B-9

Compound B-9 was synthesized in a similar synthetic route as described in Example 67 and Example 61.

Preparation of Compound B-9a

Yield 50%; EI-MCm/z: 1077 (M ⁺¹ ).

Preparation of Compound B-9

Yield 32%; EI-MS m/z: 908 (M ⁺¹ ).

[Example 91] Preparation of Compound B-10

Obtaining an AF-OMe connection

To a solution of compound MMAF-OMe (120 mg, 0.161 mmol) in DMF (4 ml) was added 37% formaldehyde (36 μl, 0.483 mmol) and AcOH (184 μl, 3.22 mmol)) at room temperature. After stirring for 5 minutes, NaCNBH ₃ (21 mg, 0.322 mmol) was added to the mixture, stirred for 2 hours and quenched with saturated NaHCO ₃ (10 ml). EtOAc (20 ml) was added. The organic layer was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to give compound AF-OMe (98 mg, 80%). EI-MS m/z: 761(M ⁺¹ ).

Compound B-10 was synthesized in a similar synthetic route as described in Example 72.

Preparation of Compound B-10-1

Yield 27%; EI-MCm/z: 1673 (M ⁺¹ ).

Preparation of Compound B-10

Yield 81%; EI-MS m/z: 1491 (M ⁺¹ ).

[Example 92] Preparation of Compound B-11

Compound B-11 was synthesized in a similar synthetic route as described in Example 91.

Preparation of Compound B-11a

Yield 87%; EI-MS m/z: 1285 (M ⁺¹ ).

Preparation of Compound B-11

Yield 53%; EI-MCm/z: 1117 (M ⁺¹ ).

[Example 93] Preparation of Compound B-12

Compound B-12 was synthesized in a similar synthetic route as described in Example 30 and Example 73.

Preparation of Compound B-12a

Yield 53%; EI-MS m/z: 1254.7 (M ⁺¹ ).

Preparation of Compound B-12

Yield 58%; EI-MS m/z: 1086.6(M ⁺¹ ).

[Example 94] Preparation of Compound B-13

Compound B-13 was synthesized in a similar synthetic route as described in Example 74.

Preparation of Compound B-13a

Yield 9%; EI-MS m/z: 878(M ⁺¹ /2).

Preparation of Compound B-13

Yield 33%; EI-MS m/z: 1610(M ⁺¹ ).

[Example 95] Preparation of Compound B-14

Compound B-14 was synthesized in a similar synthetic route as described in Example 61.

Yield 30%; EI-MCm/z: 1474 (M ⁺ ).

[Example 96] Preparation of Compound B-15

Compound B-15 was synthesized in a similar synthetic route as described in Example 72.

Preparation of Compound B-15a

Yield 45%; EI-MCm/z: 1116 (M ⁺ ).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.73 (s, 1H), 8.01 (m, 1H), 7.97 (s, 1H), 7.68 (m, 1H), 7 .51 (t, J=9.2 Hz, 1H) 7.45 (d, J=8 Hz, 1H), 7.36 (m, 2H), 7.26 (s, 1H), 7.10- 7.07 (m, 1H), 6.89 (s, 2H), 4.97 (d, J=7.2 Hz, 1H), 4.62 (s, 2H), 4.27 (m, 2H ), 4.04-3.95 (m, 7H), 3.96 (s, 6H)

Preparation of Compound B-15

Yield 70%; EI-MS m/z: 948 (M ⁺¹ ).

[Example 97] Preparation of Compound B-16

Compound B-16 was synthesized in a similar synthetic route as described in Example 72.

Preparation of Compound B-16a

Yield 99%; EI-MS m/z: 1103 (M ⁺¹ ).

Preparation of Compound B-16

Yield 85%; EI-MCm/z: 935 (M ⁺¹ ).

[Example 98] Preparation of Compound B-17

Compound B-17 was synthesized in a similar synthetic route as described in Example 72.

Preparation of Compound B-17a

Yield 55%; EI-MS m/z: 991 (M ⁺¹ ).

Preparation of Compound B-17

Yield 60%; EI-MS m/z: 823 (M ⁺¹ ).

[Example 99] Preparation of Compound B-18

Compound B-18 was synthesized in a similar synthetic route as described in Example 72.

Preparation of Compound B-18a

Yield 72%; EI-MS m/z: 960 (M ⁺¹ ).

Preparation of Compound B-18

Yield 54%; EI-MCm/z: 791 (M ⁺¹ ).

[Example 100] Preparation of Compound B-19

Compound B-19 was synthesized in a similar synthetic route as described in Example 92 and Example 70.

Preparation of Compound B-19a

Yield 38%; EI-MCm/z: 914(M ⁺¹ /2).

Preparation of Compound B-19b

Yield 60%; EI-MS m/z: 831(M ⁺¹ /3).

Preparation of Compound B-19

Yield 17%; EI-MS m/z: 1148(M ⁺¹ /2).

[Example 101] Preparation of Compound B-20

Compound B-20 was synthesized in a similar synthetic route as described in Example 72.

Preparation of Compound B-20a

Yield 14%; EI-MS m/z: 1614 (M ⁺¹ ).

Preparation of Compound B-20

Yield 37%; EI-MS m/z: 1432 (M ⁺¹ ).

[Example 102] Preparation of Compound B-21

Compound B-21 was synthesized in a similar synthetic route as described in Example 72.

Preparation of Compound B-21a

Yield 38 EI-MCm/z: 1551(M ⁺¹ ).

Preparation of Compound B-21

Yield 54%; EI-MCm/z: 1383 (M ⁺¹ ).

[Example 103] Preparation of Compound B-22

Preparation of Compound B-22a

To a solution of compound IntB-10 (30 mg, 0.043 mmol) and compound IntB-Q11 (51 mg, 0.05 mmol) in DMF (3 ml) was added EDC⋅HCl (27.2 mg, 0.142 mmol) at 0°C in N ₂ atmosphere. After stirring for 11 hours, the mixture was purified by preparative HPLC to give compound B-22a (20 mg, 28%) as a light brown solid.

EI-MS m/z: 821.7(M ⁺¹ /2).

Preparation of Compound B-22

To a solution of compound B-22a (10 mg, 0.006 mmol) in MeOH (1.5 ml) was added 25% NaOMe in MeOH (11 μl, 0.048 mmol) at 0°C under N ₂ atmosphere. The reaction mixture was stirred at room temperature for 1 hour under N ₂ and adjusted to pH 7 with 5% TFA in ACN solution. The mixture was purified by preparative HPLC to give compound B-22 (5 mg, 63%) as a pale yellow solid.

EI-MS m/z: 1305.3(M ⁺¹ ).

[Example 104] Preparation of Compound B-23

Compound B-23 was synthesized in a similar synthetic route as described in Example 103.

Preparation of Compound B-23a

Yield 53%; EI-MS m/z: 1303.5(M ⁺¹ ).

Preparation of Compound B-23b

To a solution of compound B-23a (30 mg, 0.023 mmol) was added 4N HCl in 1,4-dioxane (1 ml) at room temperature under N ₂ atmosphere. After stirring for 1 hour, the mixture was diluted with DCM (5 ml) and concentrated. Compound B-23b was used directly in the next step without further purification (28 mg, 97%).

EI-MS m/z: 1259.5(M ⁺¹ ).

Preparation of Compound B-23

Yield 68%; EI-MS m/z: 1259.5(M ⁺¹ ).

[Example 105] Preparation of Compound B-24

Compound B-24 was synthesized in a similar synthetic route as described in Example 104.

Receipt connections B-24a

Yield 53%; EI-MS m/z: 1254.7(M ⁺¹ ).

Preparation of Compound B-24

Yield 58%; EI-MCm/z: 1086.6(M ⁺¹ ).

[Example 106] Preparation of Compound B-25

Compound B-25 was synthesized in a similar synthetic route as described in Example 104.

Preparation of Compound B-25a

Yield 33%; EI-MS m/z: 1094.5(M ⁺¹ ).

Preparation of Compound B-25

Yield 36%; EI-MCm/z: 926.4(M ⁺¹ ).

[Example 107] Preparation of Compound B-26

Compound B-26 was synthesized in a similar synthetic route as described in Example 74.

Preparation of Compound B-26a

Yield 51%; EI-MS m/z: 1382(M ⁺¹ ).

Preparation of Compound B-8

Yield 56%; EI-MS m/z: 1214(M ⁺¹ ).

[Example 108] Preparation of the Ligand-Drug Conjugate Compound C-1

To a solution of IntC-L (1.8 mg, 0.0016 mmol) and B-1 (1.6 mg, 0.0011 mmol) in EtOH (2 ml) and distilled water (0.5 ml) M sodium ascorbate (11 µl, 0.011 mmol) and 0.1 M CuSO ₄ (21 µl, 0.0021 mmol) at room temperature under N ₂ nitrogen. After stirring for 12 hours, the mixture was separated and purified by preparative HPLC to give Compound C-1 (1.7 mg, 61%).

EI-MS m/z: 2278(M ⁺¹ ).

[Example 109] Preparation of the Ligand-Drug Conjugate Compound C-2

Compound C-2 was synthesized in a similar synthetic route as described in Example 108.

Yield 54%; EI-MS m/z: 1751 (M ⁺¹ ).

[Example 110] Preparation of the Ligand-Drug Conjugate Compound C-3

Compound C-3 was synthesized in a similar synthetic route as described in Example 108.

Yield 24%; EI-MS m/z: 2242 (M ⁺¹ ).

[Example 111] Preparation of Compound C-4

Compound C-4 was synthesized in a similar synthetic route as described in Example 108.

EI-MS m/z: 1043.3 (M ⁺¹ /2).

[Example 112] Preparation of Compound C-5

Compound C-6 was synthesized in a similar synthetic route as described in Example 108.

EI-MS m/z: 1886.3 (M ⁺¹ ).

[Example 113] Preparation of Compound C-6

Compound C-6 was synthesized in a similar synthetic route as described in Example 108.

EI-MS m/z: 936.7 (M ⁺¹ /2).

[Example 114] Preparation of Compound C-7

Compound C-7 was synthesized in a similar synthetic route as described in Example 61 and Example 108.

Preparation of Compound B-3b

Yield 30%; EI-MS m/z: 1497(M ⁺¹ +Na).

Preparation of Compound C-7

Yield 8.7%; EI-MCm/z: 1128(M ⁺¹ /2).

[Example 115] Preparation of Compound C-8

Compound C-8 was synthesized in a similar synthetic route as described in Example 108.

EI-MS m/z: 934.0 (M ⁺¹ /2).

[Example 116] Preparation of Compound C-9

Compound C-9 was synthesized in a similar synthetic route as described in Example 108.

EI-MS m/z: 983.0 (M ⁺¹ ).

[Example 117] Preparation of the ligand-drug conjugate compound D 1

To a solution of compound B-3 (example 72, 10 mg, 6.85 µmol) in DMSO (685 µl + 7671 µl) was added (BimC ₄ A) ₃ (1142 µl) prepared at a concentration of 50 mmol. Then, CuBr prepared at a concentration of 10 mmol was added thereto in an amount of 685 µl. The mixture was stirred for 2 minutes. Compound MPS-D5 (4.6 mg, 12.3 μmol) was dissolved in DMSO (685 μl) and added thereto, followed by stirring for 10 minutes. After completion of the reaction, the mixed solution was separated and purified by preparative HPLC to give compound D-1 (5.4 mg, 43%).

EI-MS m/z: 916 (M ⁺¹ /2).

[Example 118] Preparation of a ligand-drug conjugate compound D- 2

Compound D-2 was synthesized in a similar synthetic route as described in Example 108.

Yield 35%; EI-MS m/z: 934 (M ⁺¹ /2).

[Example 119] Preparation of Compound D-4

To a solution of compound B-3 (10 mg, 6.85 µmol) in DMSO (685 µl) was added (BimC ₄ A) ₃ (1142 µl 50 mmol stock solution) at room temperature under N ₂ atmosphere. After adding DMSO (6179 μl), CuBr (685 μl) prepared at 100 mmol was added. The mixture was then stirred for 2 minutes. Compound POS-D1 (12.4 mg, 27.4 μmol) was dissolved in DMSO (685 μl) and added thereto, followed by stirring for 10 minutes. After completion of the reaction, the mixed solution was separated and purified by preparative HPLC to give compound D-4 (0.5 mg, 3.8%).

EI-MS m/z: 958 (M ⁺ 1/2).

[Example 120] Preparation of Compound D-5

Compound D-5 was synthesized in a similar synthetic route as described in Example 108.

Yield 53%; EI-MS m/z: 1417 (M ⁺¹ /2).

[Example 121] Preparation of Compound D-6

Compound D-6 was synthesized in a similar synthetic route as described in Example 108.

Yield 42%; EI-MCm/z: 4515(M ⁺ ), 2258(M/2 ⁺ ), 1505(M/3 ⁺ ).

[Example 122] Preparation of Compound D-7

Compound D-7 was synthesized in a similar synthetic route as described in Example 108.

EI-MS m/z: 1673.0(M ⁺ ).

[Example 123] Preparation of Compound D-8

Compound D-8 was synthesized in a similar synthetic route as described in Example 108.

EI-MS m/z: 728.8(M ⁺ /2).

[Example 124] Preparation of Compound D-9

Compound D-9 was synthesized in a similar synthetic route as described in Example 108.

Yield 50%; EI-MS m/z: 1264 (M ⁺ ).

[Example 125] Preparation of Compound D-10

Compound D-10 was synthesized in a similar synthetic route as described in Example 108.

Yield 17%; EI-MS m/z: 1861(M+).

[Example 126] Preparation of Compound D-11

Compound D-11 was synthesized in a similar synthetic route as described in Example 108.

Yield 25%; EI-MS m/z: 1863(M ⁺ ).

[Example 127] Preparation of Compound D-12

Compound D-12 was synthesized by a similar synthetic route as described in Example 108.

EI-MS m/z: 1850.8(M ⁺ ).

[Example 128] Preparation of Compound D-13

Compound D-13 was synthesized in a similar synthetic route as described in Example 108.

Yield 18%; EI-MS m/z: 930 (M/2 ⁺ ).

[Example 129] Preparation of Compound D-14

Preparation of Compound (A)

The complete synthesis of Compound (A) was obtained according to (J. Org. Chem. 2004, 69, 112-121, J. Am. Chem. SOC1. 995,117, 10143-10144).

Preparation of Compound D-14a

To a solution of compound (A) (10 mg, 0.018 mmol) and compound Int-TG15 (16.8 mg, 0.018 mmol) in ACN (1 ml) was added a molecular sieve (100 mg) under N ₂ atmosphere. After stirring at room temperature for 10 minutes, Ag2O (12.5 mg, 0.054 mmol) was added. The mixture was stirred at room temperature for 5 hours, filtered through celite and concentrated under reduced pressure. The residue was purified by preparative HPLC to give compound D-14a (18 mg, 71%).

¹ H NMR (400 MHz, CDCl3) δ 7.99 (m, 2H), 7.85 (s, 1H), 7.77 (d, J=8.8 Hz, 2H), 7.65 (d, J=8.4Hz, 2H), 7.58(m, 1H), 7.44-7.40(m, 2H), 7.36(J=8.4Hz, 2H), 7.29( d, J=8.8 Hz, 1H), 6.94 (m, 1H), 6.46 (s, 1H), 6.11 (s, 1H), 5.94 (m, 2H), 5, 80(s, 1H), 5.56(m, 1H), 5.50-5.46(m, 2H), 5.35-5.31(m, 2H), 5.25~5.22( m, 2H), 5.15-5.13(m, 2H), 4.38(m, 1H), 4.25-4.08(m, 4H), 3.65~3.59(m , 15H), 3.54-3.49 (m, 1H), 3.33 (m, 2H), 2.17 (s, 3H), 2.13 (s, 3H), 2.09 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H), 2.00 (s, 3H), 1.81 (s, 3H). EI-MS m/z: 705 (1/2M+), 1408 (M+), 1430 (M+Na).

Preparation of Compound D-14b

To a solution of compound D-14a (16 mg, 0.011 mmol) in DCM (4 ml) was added 1M-NaOMe in methanol (20 μl) at 0°C. After stirring at 0° C. for 20 minutes, the mixture was quenched with aqueous 1M-HCl (30 μl) and then purified by preparative HPLC to give compound D-14b (4 mg, 32%).

EI-MS m/z: 557 (1/2M+), 1114 (M+), 1136 (M++Na).

Preparation of Compound D-14

Compound D-14 was synthesized in a similar synthetic route as described in Example 56.

Yield 12%; EI-MS m/z: 830 (M+1/2).

[Example 130] Preparation of Compound D-15

Compound D-15 was synthesized in a similar synthetic route as described in Example 119.

Yield 19%; EI-MS m/z: 1556 (M+), 778 (1/2M+).

[Example 131] Preparation of Compound D-16

Compound D-16 was synthesized in a similar synthetic route as described in Example 108.

Yield 13%; EI-MS m/z: 1753 (M ⁺ ).

[Example 132] Preparation of Compound D-17

Compound D-17 was synthesized in a similar synthetic route as described in Example 108.

Yield 12%; EI-MS m/z: 1668 (M ⁺ ).

[Example 133] Preparation of Compound D-18

Compound D-18 was synthesized in a similar synthetic route as described in Example 108.

Yield 7.4%; EI-MS m/z: 1759 (M ⁺ ).

[Example 134] Preparation of a CBI dimer compound

The CBI dimer was synthesized in a similar synthetic manner as described in WO2015110935A.

[Example135] Obtaining a seco-DUBA connection

seco-DUBA was synthesized in a similar way of synthesis as described in the document [see. Mol. Pharmaceutics 2015, 12, 1813-1835].

[Example 136] Preparation of seco-CBI-indole Compound

seco -CBI-indole was synthesized by a similar synthetic method as described in the document [see. ChemMedChem 2008, 3, 1946-1955].

[Example 137] Preparation of Compound D-3

Compound B-8 (15 mg, 0.01 mmol) was dissolved in DMSO (7 ml) at room temperature under nitrogen atmosphere, and then (BimC ₄ A) ₃ (33 mg, 0.04 mmol) was dissolved in DMSO (2 ml ), CuBr (17 mg). 0.12 mmol) dissolved in DMSO (3 mL) and POS-D1 (Preparation of Example 36, 7 mg, 0.01 mmol) dissolved in DMSO (1 mL), followed by stirring for 2 hours. The mixture was separated and purified by preparative HPLC to give compound D-3 (2.5 mg, 12%).

EI-MS m/z: 1668(M ⁺ ).

Biological and biochemical research

[Example 138] Enzymatic degradation assay kinetic study

Method 1) β -galactosidase isolated from Escherichia coli

Compounds of the present invention (compounds A-2, A-4, A-5, A-6, A-9, A-10, A-12, A-13, A-14, A-15, A-17, A-18, A-19, A-21, A-22, A-23, B-1, B-2, B-3, B-4, B-6, B-7, B-8, B- 9, B-10, B-11, B-14, B-15, B-16, B-19 and C-3) were dissolved in DMSO and mixed with PBS buffer to obtain a 500 μM stock solution (5% DMSO) . MPS used as a standard material was dissolved in PBS buffer to obtain a 500 μM solution. 406 µl of PBS buffer solution (pH 7.4) and 140 µl of the compound (500 µM) of the present invention and 140 µl of MPS were mixed. After that, the enzyme solution (14 µl 1 mg/ml) was added. When compared with human β-galactosidase, 21 μl of enzyme solution (1 mg/ml) was added to achieve the same molar concentration, and the compound of the present invention and MPS were mixed, each having an amount of buffer of 140 μl and 399 μl of buffer solution (pH 7 ,4). The resulting reaction mixture was incubated at 37°C. Escherichia coli β-galactosidase enzyme (Sigma G4155) was used in the reaction mixture. The reaction solution of the enzyme was divided into aliquots at 0 min before the reaction and at a predetermined time after the reaction, respectively, where each aliquot was 70 μl. Then, the remaining compound of the present invention or MPS and the material isolated from the enzymatic reaction were quantitatively analyzed by HPLC.

Method 2) human β-galactosidase

Compounds A-3, B-2, and B-10 of the present invention were each dissolved in DMSO, and mixed with PBS buffer to obtain a 500 μM stock solution (5% DMSO). MPS used as a standard material was prepared as a solution at a concentration of 500 μM in PBS buffer. 280 μl of PBS buffer (pH 7.4) and Compound B-2 (500 μM) of the present invention and MPS, each having an amount of 140 μl, were mixed with each other. Then, 140 µl of a 0.1 mg/ml enzyme solution was added thereto, thereby obtaining an enzyme reaction solution in a total of 700 µl. The reaction mixture was incubated at 37°C. Human β-galactosidase enzyme (R&D 6464-GH-020) was used in the reaction mixture. The enzyme reaction mixture was aliquoted at 0 min before the reaction and at a predetermined time after the reaction, respectively, where each aliquot was 70 μl. Then, the remaining compound of the present invention or MPS and SN38 isolated from the enzymatic reaction were quantitatively analyzed by HPLC.

Method 3) β -glucuronidase isolated from Escherichia coli

Compounds A-1 and A-5 of the present invention were dissolved in DMSO, and mixed with PBS buffer to obtain a solution having a concentration of 500 μM (5% DMSO). MPS used as a standard material was prepared as a solution at a concentration of 500 μM in PBS buffer. 406 µl of PBS buffer solution (pH 7.4) and 140 µl of the compound (500 µM) of the present invention and 140 µl of MPS were mixed. Then, 14 μl of an enzyme solution (1 mg/ml) was added thereto, thereby obtaining an enzyme reaction solution in a total of 700 μl. The reaction mixture was incubated at 37°C. Escherichia coli β-glucuronidase enzyme (Sigma G7396) was used in the reaction mixture. The enzyme reaction mixture was aliquoted at 0 min before the reaction and at a predetermined time after the reaction, respectively, where each aliquot was 70 μl. Then, the remaining compound of the present invention or MPS and the material isolated from the enzymatic reaction were quantitatively analyzed by HPLC.

Method 4) β -galactosidase + β -glucuronidase Escherichia coli

Compound A-5 of the present invention was dissolved in DMSO and mixed with PBS buffer to obtain a solution having a concentration of 500 μM (5% DMSO). MPS used as a standard material was prepared as a solution at a concentration of 500 μM in PBS buffer. 392 μl of PBS buffer solution (pH 7.4) and 140 μl of Compound A-5 (500 μM) of the present invention and MPS were mixed. Then, 14 µl of 1 mg/ml β-galactosidase (Sigma G4155) and 14 µl of 1 mg/ml β-glucuronidase (Sigma G7396) were added, thus obtaining an enzyme reaction solution in a total of 700 µl. The reaction mixture was incubated at 37°C. The enzyme reaction mixture was aliquoted at 0 min before the reaction and at a predetermined time after the reaction, respectively, where each aliquot was 70 μl. Then, the remaining compound of the present invention or MPS and the material isolated from the enzymatic reaction were quantitatively analyzed by HPLC.

[Table 1]

Compounds of the present invention t _1/2 TG release (min) t _1/2 release part Q (min) experimental method A-1 0.43 ± 0.11 36.38±0.39 Method 3 A-2 3.71 ± 0.22 123.08 ± 25.96 Method 1 A-3 10.71 38.18 Method 2 A-4 < 5 15.3 Method 1 A-5 < 5 70.87 Method 3 < 5 No reaction Method 1 < 5 139.4 Method 4 A-6 4.60 ± 1.72 40.36 ± 8.03 Method 1 A-9 <5 37.39 Method 1 A-10 <5 15.24 Method 1 A-12 <5 14.34 Method 1 A-13 <5 8.01 Method 1 A-14 N/D 195.2 Method 1 A-15 BUT <5 Method 1 A-17 <1 6.7 Method 1 A-18 <5 7.4 Method 1 A-19 fast < 5 Method 1 A-21 fast < 5 Method 1 A-22 fast < 5 Method 1 A-23 fast < 5 Method 1 B-1 5.92 40.87 Method 1 B-2 0.28 1.85 Method 1/pH7.4 5.75 46.85 Method 1/pH5.0 N/D 112.2 Method 2/pH7.4 5.33 39.41 Method 2/pH5.0 B-3 <10 40.62 Method 1 B-4 20.68 312.9 Method 1 B-6 < 5 629.4 Method 1 B-7 < 5 50.23 Method 1 B-8 BUT 4.3 Method 1 B-9 < 5 6.82 Method 1 B-10 2.1 6.6 Method 1/pH7.4 8.9 27.9 Method 2/pH5.0 B-11 < 5 6.7 Method 1 B-14 BUT 433 Method 1 B-15 BUT 2.4 Method 1 B-16 BUT 9.0 Method 1 B-19 fast 14.4 Method 1 C-3 < 5 52.03 Method 1

(N/O=not detected by HPLC)

[Example 139] Drug release test

Method using a reducing agent

Compound A-8 obtained in Example 65 was dissolved in DMSO to a concentration of 10 mm and mixed with a solution of MeOH to obtain a solution with a concentration of 300 μm (40% DMSO). MPS used as a standard material was prepared as a 300 μM solution with PBS buffer. NaBH ₄ , the reducing agent, was dissolved in MeOH to a concentration of 10 mm and 3.0 equivalents of Compound A-8 was added. Then the reaction mixture was incubated at 37°C. The reduction reaction mixture was divided into aliquots 0 min before the reaction, 5, 10 and 30 min after the reaction, respectively, where each aliquot was 80 μl. Then, the remaining compound A-8 of the present invention or MPS and BOC-Tyr-OMe released by the enzymatic reaction were quantitatively analyzed by HPLC.

Method using photoreaction

Compound A-11 obtained in Example 68 was dissolved in DMSO and mixed with PBS buffer pH 7.4 and DMSO solution to obtain a solution with a concentration of 300 μM (50% DMSO). MPS used as a standard material was prepared as a solution at a concentration of 300 μM in PBS buffer.

The reaction was then initiated by stirring in a 300 W photoreactor. The reaction mixture was aliquoted 0 minutes before the reaction, 5 minutes, 10 minutes, 30 minutes, 180 minutes, 300 minutes, 420 minutes, 24 hours and 48 hours after the reaction, respectively, where each aliquot was 80 μl. Then, the remaining compound A-11 of the present invention or MPS and BOC-Tyr-OH released by photoreaction were quantitatively analyzed by HPLC.

Hydrolysis Conditions in pH 9.2 Buffer (Test Example According to pH)

Compounds A-7 and A-8, obtained in examples 63 and 64, were dissolved in DMSO and mixed with a PBS buffer solution having a pH of 9.2 to obtain a solution having a concentration of 300 μm (40% DMSO). MPS used as a standard material was prepared as a solution at a concentration of 300 μM in PBS buffer. Then the reaction mixture was incubated at 37°C. The pH 9.2 compound A-7 reaction mixture was aliquoted 0 minutes before the reaction, 5 minutes, 10 minutes, 30 minutes, 60 minutes, 120 minutes and 180 minutes after the reaction, respectively, where each aliquot was 80 μl. Then, the remaining compound A-7 of the present invention or MPS and BOC-Tyr-OH released in a pH 9.2 buffer solution was quantitatively analyzed by HPLC. The pH 9.2 compound A-8 reaction mixture was aliquoted 0 minutes before the reaction, 5 minutes, 10 minutes, 30 minutes, 60 minutes, 120 minutes, 180 minutes, 240 minutes and 300 minutes after the reaction, respectively, where each aliquot was 80 µl. Then, the remaining compound A-8 of the present invention or MPS and BOC-Tyr-OMe released in a buffer solution of pH 9.2 was quantitatively analyzed by HPLC.

Hydrolysis Conditions in pH 5.0 Buffer (Example Test According to pH)

Compound A-8 obtained in Example 65 was dissolved in DMSO and mixed with PBS buffer pH 5.0 and DMSO solution to obtain a solution with a concentration of 300 μM (40% DMSO). MPS used as a standard material was prepared as a 300 μM solution with PBS buffer. Then the reaction mixture was incubated at 37°C. The pH 5.0 compound A-8 reaction mixture was aliquoted 0 minutes before the reaction, 20 minutes, 180 minutes, 630 minutes and 1 day after the reaction, respectively, where each aliquot was 80 μl. Then, the remaining compound A-8 of the present invention or MPS and BOC-Tyr-OMe released in a pH 5.0 buffer solution was quantitatively analyzed by HPLC.

Deprotection at Fluorine Ion Concentration

Compound A-20 was dissolved in DMSO to prepare a solution having a concentration of 100 μM (90% DMSO). MPS used as a standard material was prepared as a solution at a concentration of 100 μM in PBS buffer. To a solution of TBAF in THF (100 mm) was added 1.0, 10.0 and 100 equivalents of compound A-20. The reaction mixture was incubated at 37°C and divided into aliquots at 0°C before the reaction, 5, 20, 44, 68 and 116 hours after the reaction, respectively, where each aliquot was 80 μl. Then the remaining compound A-20 or MPS released during the reaction was quantitatively analyzed by HPLC.

[Table 2]

Compounds of the present invention t _1/2 release part Q (min) experimental method A-7 19.27 pH 9.2 A-8 41.68 pH 9.2 Not released pH 5.0 4.42 Restoring agent A-11 52.03 Photoreaction A-20 2016 F ^- ion (1 equiv.) 672 F ^- ion (10 equiv.) 264 F ^- ion (100 equiv.)

[Example 140] In Vitro Assay of Ligand-Drug Conjugate

KB cancer cells were harvested and seeded in 24-well plates at a density of 30,000 cells per well in 2 ml of medium and cultured for 24 hours. Then serial dilutions of Compound C-1 prepared in Example 108 were used for treatment at 30 nM to 0.0096 nM, serial dilutions of Compound C-2 prepared in Example 109 were used for treatment at 1000 nM to 0.32 nM, serial dilutions of Compound C-3 prepared in Example 110 and Compound C-7 prepared in Example 114 were used for treatment at 25 nM to 0.0016 nM, and serial dilutions of MMAF-OMe drug were used for treatment at 10 nM to 0 .0097 nM. Serial dilutions of compounds C-5 and C-6 obtained in Example 112 and Example 113 were used for treatments at 100 nM to 0.0001 nM, and serial dilutions of seco-DUBA drug were used for treatments at 100 nM to 0.001 nM. Serial dilutions of Compounds C-4 and C-9 prepared in Example 111 and Example 116 were used for treatments at 1000 nM to 0.001 nM, and serial dilutions of drug CBI dimer were used for treatments at 10 nM to 0.00001 nM. Serial dilutions of compound C-8 obtained in Example 115 and CBI-indole drug were used for treatment at 100 nM to 0.0001 nM. After 72 hours, 96 hours, or 144 hours of incubation, 0.2 ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye dissolved in PBS buffer (5 mg/ml ) was added to each well of the plates. Formazans formed by reduction of the MTT dye by mitochondrial oxidoreductases in living cells were dissolved in DMSO and measured using absorbance at 550 nm. The results are shown in Table 3 below.

[Table 3] Cellular Cytotoxicity of the Ligand-Drug Conjugate

Compounds of the present invention Cell KB IC ₅₀ (nM) C-1 2.31±0.46 C-2 >500 C-3 0.64±0.13 C-4 0.02 C-5 0.45 C-6 0.45 C-7 0.82 C-8 0.12 C-9 0.10 MMAF-OMe 0.35±0.07 Seco-DUBA 0.18 Dimer CBI 0.0007 CBI-indole 0.07

[Example 141] Mouse, Rat, Dog and Human Plasma Stability Tests

Compound A-1 obtained in Example 58 and MPS used as a standard material was dissolved in DMSO to a concentration of 60 mm. Then, each of human plasma (Biochemed 752R-SC-PMG), mouse plasma (Biochemed 029-APSC-MP), rat plasma (Biochemed 031-APSC-MP), and beagle dog plasma (Biochemed 013-APSC-MP) was mixed with compound and MPS to a final concentration of o300 μM (final conc. 0.5% DMSO). The resulting plasma mixtures were incubated at 37°C in a water bath. Aliquots were taken before the reaction and 1, 2, 4 and 7 days after the reaction, with each aliquot being 300 μl. Two times the volume of acetonitrile was added to complete the reaction, followed by brief vortexing and centrifugation to precipitate plasma proteins. Each supernatant obtained after centrifugation was collected and analyzed by HPLC. Compound A-1 was detected and quantified in mouse, rat, beagle and human plasma for 7 days (>95%). This study demonstrated the excellent stability of the β-galactoside linker in plasma.

[Example 142] Chemical stability

Compound A-1 obtained in Example 58 was dissolved in DMSO and mixed with PBS buffer (pH 7.4) to prepare a solution having a concentration of 500 μM (5% DMSO). MPS used as a standard material was prepared as a solution at a concentration of 500 μM in PBS buffer. 420 µl of buffer solution and 140 µl of compound A-1 solution and 140 µl of MPS solution were mixed, thus obtaining a reaction mixture in a total of 700 µl. The reaction mixture was incubated at room temperature blocking light. The reaction mixture was divided into aliquots 0 days before the reaction and 1 day, 2 days, 4 days and 7 days after the reaction, where the amount of each aliquot was 70 μl. Then, the remaining compound A-1, MPS and released tyrosine were quantified by HPLC analysis. HPLC analysis confirmed that Compound A-1 was present (>95%) during the 7 day incubation period, which was found to be very stable.

[ Test example 6 ] Preparation of conjugate (albumin and antibody)

Compound D-1 obtained in Example 117, compound D-2 obtained in Example 118, compound D-5 obtained in Example 110, and compound D-6 obtained in Example 111 were used to carry out the conjugation reaction with the thiol group of the engineered trastuzumab cysteine (or human serum albumin, etc.), thereby producing D-1-AB, D-2-AB, Albu-D-5 and Albu-D-6 as thiomab drug conjugates (TDC) , respectively, with reference to the methods presented in the document [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]. LC/MS analysis confirmed that the molecular weight of thiomab was 145,450 Da and a small amount (~146.895 Da) of the glycation form was also present. LC/MS analysis of the resulting antibody drug conjugate D-1-AB showed masses of about 148,563 Da and about 150,038 Da. These weight shifts corresponded to the conjugation of two D-1 molecules.

[Example 143] In vitro analysis of protein-drug conjugate

Cancer cells KB, SKBR-3, BT-474, NCI-N87 were seeded in 96-well plates at a density of 2000-8000 cells per well in 100 μl of medium and cultured for 24 hours. The four compounds prepared in Test Example 6 were treated with 1:4 serial dilutions from 600 nM to 0.009 nM, the MMAF-OMe drug was treated with 1:4 serial dilutions from 100 nM to 0.0015 nM, and the antibody drug conjugate T- DM1 was treated with 1:4 serial dilutions from 200 nM to 0.003 nM. Experiments were performed in the same manner as Test Example 3 for live cell quantification after 96 hours, and the results are shown in Tables 4 and 5 below. In addition, the results of the in vitro assay of compound D-1-AB are shown in FIG. 12.

[Table 4] Cellular cytotoxicity of protein-drug conjugate

Compounds of the present invention Cell KB IC ₅₀ (nM) Albu-D-5 1.08 Albu-D-6 0.10* MMAF-OMe 0.44(0.16*)

[Table 5] Cellular Cytotoxicity of the Antibody-Drug Conjugate

ADC DAR IC ₅₀ (nM) SKBR-3 BT-474 NCI-N87 D-1-AB 2.0 0.02 0.10 0.21 D-2-AB 1.71 0.01 0.08 0.14 D-4-AB mix 0.38 D-7-AB 0.83 0.074 D-8-AB 2.0 0.056 D-9-AB 0.99 >500 D-12-AB 2.0 0.021 D-13-AB 1.95 0.138 D-14-AB 1.56 6.01 D-15-AB 1.87 ~200 D-16-AB ~0.5 0.066 D-18-AB 1.91 0.013 0.015 T-DM1 3.5 0.02 0.18 0.02

(*T-DM1 : Roche CAS Number: 1018448-65-1)

[Example 144] In vivo efficacy

The in vivo efficacy of the antibody-drug conjugates of the invention was measured by tumor xenograft studies in mice. A male BALB/c nu/nu mouse was injected subcutaneously into the right flank of a suspension of 1 X10 ⁶ NCI-N87 cells, respectively, in PBS. Mice were randomized into study groups when the tumors reached a size of approximately 100 mm ³ . Conjugates T-DM1 (2 mg/kg), D-1-AB, D-2-AB, D-8-AB and D-18-AB (0.5 mg/kg or 2 mg/kg) were administered intravenously. (single injection on treatment day 0). All treatment groups consisted of 6-10 animals per group, and tumor size was monitored twice a week by caliper measurement. Tumor weight was calculated as volume = (width x width x length)/2. Conjugates D-1-AB, D-2-AB and D-8-AB resulted in tumor regression during the observation period, ie 80 days from the start of the experiment. The control T-DM1 conjugate was less active than the conjugates of the invention.

Inclusion by reference

All publications, patents mentioned herein are incorporated herein by reference to the same extent that each individual publication, patent, is specifically and individually indicated for inclusion by reference. In the event of a conflict, this application, including any definitions herein, will govern.

Equivalents

While specific embodiments of the subject matter have been discussed, the above description is illustrative and not restrictive. Many embodiments of the invention will become apparent to those skilled in the art upon review of this description and the claims below. The full scope of the invention is to be determined by reference to the claims, together with their full scope of equivalents, and the description, together with such variations.

Claims (223)

1. Conjugate of formula ( I ')

or a pharmaceutically acceptable salt thereof, where CB is a targeting fragment containing an antibody selected from an intact polyclonal antibody, an intact monoclonal antibody, an antibody fragment, a single-chain Fv mutant (scFv), a multispecific antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, and a fusion protein containing an antigenic the determinant part of the antibody; cb and n are each independently integers having a value from 1 to about 20, preferably from 1 to about 10; each of the DLs is independently a group having the structure of the formula ( I” )

, where (Q) _q -(L') _w - is selected from:

where * denotes the point of attachment (Q) _q -(L') _w to -SO ₂ -; Z' is a linking group containing

formulas ( F '), ( G '), ( H '), ( J '), ( K '), ( L '), ( M ') or ( N '):

where R ^e represents (C ₁ -C ₆ )alkyl; X'' represents -O-, -S-, -NH- or -CH ₂ -; L ² is a spacer moiety unsubstituted or substituted with one or more C ₁ -C ₆ alkyl, C ₅ -C ₁₄ aryl and C ₃ -C ₈ heteroaryl, where alkyl, aryl and heteroaryl are unsubstituted or substituted with one or more substituents selected from C ₁ -C ₁₀ alkyl, -(CH ₂ ) _u NH ₂ , -(CH ₂ ) _u NR ^u1 R ^u2 , -(CH ₂ ) _u CO ₂ H, -(CH ₂ ) _u CO ₂ R ^u1 and - (CH ₂ ) _u SO ₂ R ^u3 , where R ^u1 , R ^u2 and R ^u3 are each independently hydrogen, C ₁ -C ₁₅ alkyl, C ₆ -C ₂₀ aryl, or C ₃ -C ₁₀ heteroaryl; and u is an integer having a value from 0 to 10; X ⁴ is -NHC(O)-(CH ₂ ) _g -NH- or -C(O)NH-(CH ₂ ) _h -NH-; each of W ^b1 and W ^b2 is independently -C(O)NH-, -NHC(O)-,

or

; R ¹² is hydrogen, alkyl, amino acid side chain, -(CH ₂ ) _s C(O)R ¹³ or -(CH ₂ ) _p NR ¹⁴ R ¹⁵ ; R ¹³ is OH or -NH(CH ₂ ) _s '(X'''CH ₂ CH ₂ ) _s” Z''-(CB) _m ; each of R ¹⁴ and R ¹⁵ independently represents hydrogen or -C(O)(CH ₂ ) _s '(X'''CH ₂ CH ₂ ) _s” Z''-(CB) _m ; each of s and s'' is independently an integer having a value from 0 to about 10; m is an integer having a value of 0 or 1; X''' represents -O-, -S-, -NH- or -CH ₂ -; Z'' represents a linking group connecting CB with the remainder of R ¹⁴ or R ¹⁵ ; or Z'' represents a linking group containing a reactive group; each of a, b, c, d, e, g, h, o, p, and qq is independently an integer having a value from 1 to about 10; And s' is an integer having a value from 1 to about 10, where each V is independently a single bond, -O-, -S-, -NR ²¹ -, -C(O)NR ²² -, -NR ²³ C(O)-, -NR ²⁴ SO ₂ - or -SO _2NR ²⁵ -; each of R ²¹ , R ²² , R ²³ , R ²⁴ and R ²⁵ independently represents hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkylC ₆ -C ₂₀ aryl or C ₁ -C ₆ alkylC ₃ -C ₂₀ heteroaryl; r is an integer having a value from 1 to about 10; p is an integer having a value from 0 to about 10; q is an integer having a value from 1 to about 10; And L" is a single bond; X is -O-; Ar is phenyl; Y' represents -O-; X and Y' are located on adjacent Ar atoms; TG is a trigger group selected from: -NO ₂ , -C(O)-(CH ₂ ) ₂ C(O)-(C ₁ -C ₆ )alkyl, nitrobenzyl,

,

And

, where R ²¹ represents hydrogen; And R ²² is hydrogen or (C ₁ -C ₆ )alkyl; And q is an integer having a value from 1 to about 20, preferably from 1 to about 10; each w and x is independently an integer having a value of 0 or 1; provided that when w is 0, then q is 1. 2. The conjugate of claim 1 wherein Z' is a linking group of the formula ( F '), ( G '), ( H '), ( J '), ( K '), ( L '), ( M ') or ( N '):

where R ^e represents (C ₁ -C ₆ )alkyl; X'' represents -O-, -S-, -NH- or -CH ₂ -; L ² is a spacer moiety unsubstituted or substituted with one or more C ₁ -C ₆ alkyl, C ₅ -C ₁₄ aryl and C ₃ -C ₈ heteroaryl, where alkyl, aryl and heteroaryl are unsubstituted or substituted with one or more substituents selected from C ₁ -C ₁₀ alkyl, -(CH ₂ ) _u NH ₂ , -(CH ₂ ) _u NR ^u1 R ^u2 , -(CH ₂ ) _u CO ₂ H, -(CH ₂ ) _u CO ₂ R ^u1 and - (CH ₂ ) _u SO ₂ R ^u3 , where R ^u1 , R ^u2 and R ^u3 are each independently hydrogen, C ₁ -C ₁₅ alkyl, C ₆ -C ₂₀ aryl, or C ₃ -C ₁₀ heteroaryl; and u is an integer having a value from 0 to 10; X ⁴ is -NHC(O)-(CH ₂ ) _g -NH- or -C(O)NH-(CH ₂ ) _h -NH-; each of W ^b1 and W ^b2 is independently -C(O)NH-, -NHC(O)-,

or

; R ¹² is hydrogen, alkyl, amino acid side chain, -(CH ₂ ) _s C(O)R ¹³ or -(CH ₂ ) _p NR ¹⁴ R ¹⁵ ; R ¹³ is OH or -NH(CH ₂ ) _s '(X'''CH ₂ CH ₂ ) _s” Z''-(CB) _m ; each of R ¹⁴ and R ¹⁵ independently represents hydrogen or -C(O)(CH ₂ ) _s '(X'''CH ₂ CH ₂ ) _s” Z''-(CB) _m ; each of s and s'' is independently an integer having a value from 0 to about 10; m is an integer having a value of 0 or 1; X''' represents -O-, -S-, -NH- or -CH ₂ -; Z'' represents a linking group connecting CB with the remainder of R ¹⁴ or R ¹⁵ ; or Z'' represents a linking group containing a reactive group; each of a, b, c, d, e, g, h, o, p, and qq is independently an integer having a value from 1 to about 10; And s' is an integer having a value from 1 to about 10. 3. The conjugate of claim 1 or 2, wherein the TG can be cleaved under nucleophilic reagent conditions, base reagent conditions, photoirradiation, reducing agent conditions, acidic conditions, enzymatic conditions, or oxidizing conditions. 4. Conjugate according to claim 1 or 2, where x is 0. 5. Conjugate according to claim 1, characterized in that the antibody is selected from muromonab-CD3, abciximab, rituximab, daclizumab, palivizumab, infliximab, trastuzumab (Herceptin), etanercept, basiliximab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalim umaba, alefacepta, omalizumab, efalizumab, tositumomab-I ¹³¹ , cetuximab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, riloncept, certolizumab pegola, romiplostim, AMG-531, CNTO-148, CNTO-1275, ABT-874, LEA -29Y, belimumab, TACI-Ig, second generation anti-CD20, ACZ-885, tocilizumab, atlizumab, mepolizumab, pertuzumab, Humax CD20, tremelimumab (CP-675 206), ticilimumab, MDX-010, IDEC-114, inotuzumab ozogamicin, HuMax EGFR, aflibercept , HuMax-CD4, Ala-Ala, ChAglyCD3, TRX4, catumaxomab, IGN101, MT-201, pregovomab, CH-14.18, WX-G250, AMG-162, AAB-001, motavizumab, MEDI-524, efumgumab, aurograb (Aurograb ), raxibacumab, third-generation anti-CD20, LY2469298, and veltuzumab. 6. Compound of formula ( Ia )

or a pharmaceutically acceptable salt thereof, where (Q) _q -(L') _w - is selected from:

where * means the point of attachment (Q) _q -( L') _w - to -SO ₂ -; Z' is absent or is a linking group containing:

where each V is independently a single bond, -O-, -S-, -NR ²¹ -, -C(O)NR ²² -, -NR ²³ C(O)-, -NR ²⁴ SO ₂ - or -SO _2NR ²⁵ -; each of R ²¹ , R ²² , R ²³ , R ²⁴ and R ²⁵ independently represents hydrogen, (C ₁ -C ₆ )alkyl, (C ₁ -C ₆ )alkyl(C ₆ -C ₂₀ )aryl or (C ₁ -C ₆ )alkyl(C ₃ -C ₂₀ )heteroaryl; r is an integer having a value from 1 to about 10; p is an integer having a value from 0 to about 10; q is an integer having a value from 1 to about 10; And L" is a single bond; X is -O-; Ar is phenyl; Y' represents -O-; X and Y' are located on adjacent Ar atoms; TG is a trigger group selected from: -NO ₂ , -C(O)-(CH ₂ ) ₂ C(O)-(C ₁ -C ₆ )alkyl, nitrobenzyl,

,

And

, where R ²¹ represents hydrogen; And R ²² is hydrogen or (C ₁ -C ₆ )alkyl; q is an integer having a value from 1 to about 20, preferably from 1 to about 10; each w and x is independently an integer having a value of 0 or 1; And provided that when w is 0, then q is 1. 7. The compound according to claim 6, where Z' represents a linking group and additionally contains one or more groups selected from isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH ₂ -halogen), maleimide, diene, alkene, halide, tosylate (TsO ^- ), aldehyde, sulfonate (R-SO ₃ ^- ),

,

, phosphonic acid (-P (= O) (OH) ₂ ), ketone, C ₈ -C ₁₀ cycloalkynyl, -OH, -NHOH, -NHNH ₂ , -SH, carboxylic acid (-COOH), acetylene (-C≡ CH), azide (-N ₃ ), amino (-NH ₂ ), sulfonic acid (-SO ₃ H), alkynone derivative (-C(O)C≡CR ^a and dihydrophosphate (-OP(=O)(OH) ₂ ). 8. Connection according to claim 6 or 7, where x is 0. 9. A method for producing a conjugate according to claim 1, comprising reacting a compound according to any one of claims 6 to 8 with a targeting fragment, where the targeting fragment is an antibody selected from an intact polyclonal antibody, an intact monoclonal antibody, an antibody fragment, a single-chain Fv mutant (scFv), multispecific antibody, bispecific antibody, chimeric antibody, humanized antibody, human antibody, fusion protein containing the antigenic determinant portion of the antibody, and other modified immunoglobulin molecules containing antigen recognition sites. 10. The method according to claim 9, characterized in that the antibody is selected from muromonab-CD3, abciximab, rituximab, daclizumab, palivizumab, infliximab, trastuzumab (Herceptin), etanercept, basiliximab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab , alefacepta, omalizumab, efalizumab, tositumomab-I ¹³¹ , cetuximab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, riloncept, certolizumab pegola, romiplostim, AMG-531, CNTO-148, CNTO-1275, ABT-874, LEA -29Y, belimumab, TACI-Ig, second generation anti-CD20, ACZ-885, tocilizumab, atlizumab, mepolizumab, pertuzumab, Humax CD20, tremelimumab (CP-675 206), ticilimumab, MDX-010, IDEC-114, inotuzumab ozogamicin, HuMax EGFR, aflibercept , HuMax-CD4, Ala-Ala, ChAglyCD3, TRX4, catumaxomab, IGN101, MT-201, pregovomab, CH-14.18, WX-G250, AMG-162, AAB-001, motavizumab, MEDI-524, efumgumab, aurograb (Aurograb ), raxibacumab, third-generation anti-CD20, LY2469298, and veltuzumab. 11. A pharmaceutical composition for the treatment of a proliferative disease, comprising an effective amount of a conjugate according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier or adjuvant for the treatment of a proliferative disease or condition. 12. A method of treating a proliferative disease, comprising administering a conjugate according to any one of claims 1 to 5 to a subject in need thereof. 13. The method of claim 12, wherein the proliferative disease is selected from autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis, graft-versus-host disease, myasthenia gravis, or Sjögren's syndrome), chronic inflammatory conditions (e.g., psoriasis, asthma) or Crohn's disease), hyperproliferative disorders (eg, breast cancer, lung cancer), viral infections (eg, herpes, papilloma or HIV), osteoarthritis, and atherosclerosis. 14. The method of claim 13, wherein the proliferative disease is a cancer selected from carcinoma, lymphoma, blastoma, sarcoma, leukemia, or lymphoid malignancy. 15. The method of claim 14, wherein the cancer is squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung, and squamous cell lung cancer) , peritoneal cancer, hepatocellular carcinoma, stomach cancer (eg, gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, cancer rectal, colorectal, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vaginal cancer, thyroid cancer, liver carcinoma, anal cancer, penile cancer, acute leukemia, and head/brain and neck cancer . 16. The method according to claim 15, wherein the cancer is cervical carcinoma. 17. Connection selected from:

And

or their pharmaceutically acceptable salt. 18. A process for preparing a compound according to claim 1, comprising reacting a compound of formula ( IIc )

or a pharmaceutically acceptable salt thereof with a sulfonyl halide

to obtain a connection according to claim 1 or a pharmaceutically acceptable salt thereof, where X ^a represents a halogen; Q, L', Z', Ar, Y', TG, q, w and x as defined in claim 1; each R ¹¹ is independently C ₁ -C ₆ alkyl. 19. A method for preparing a compound of formula ( Iaa ) or a pharmaceutically acceptable salt thereof, wherein the method includes: (a) reacting a compound of formula ( IIa )

or a pharmaceutically acceptable salt thereof with 1,1'-sulfonylbis(1H-imidazole)

to obtain a compound of formula ( IIbb )

or a pharmaceutically acceptable salt thereof; a compound of formula ( IIbb ) is further reacted with a compound of formula ( Ia '), (Q) _q -(L') _w -H or a pharmaceutically acceptable salt thereof to obtain a compound of formula ( Iaa )

or a pharmaceutically acceptable salt thereof, where (Q) _q -(L') _w - is selected from:

where * means the point of attachment of Q-(L') _w - to -SO ₂ -; Z' is absent or is a linking group containing:

where each V is independently a single bond, -O-, -S-, -NR ²¹ -, -C(O)NR ²² -, -NR ²³ C(O)-, -NR ²⁴ SO ₂ - or -SO _2NR ²⁵ -; each of R ²¹ , R ²² , R ²³ , R ²⁴ and R ²⁵ independently represents hydrogen, (C ₁ -C ₆ )alkyl, (C ₁ -C ₆ )alkyl(C ₆ -C ₂₀ )aryl or (C ₁ -C ₆ )alkyl(C ₃ -C ₂₀ )heteroaryl; r is an integer having a value from 1 to about 10; p is an integer having a value from 0 to about 10; q is an integer having a value from 1 to about 10; L'' represents a single bond; Ar is phenyl; Y' represents -O-; O and Y' are located on adjacent Ar atoms; TG is a trigger group selected from: -NO ₂ , -C(O)-(CH ₂ ) ₂ C(O)-(C ₁ -C ₆ )alkyl, nitrobenzyl,

,

And

, where R ²¹ represents hydrogen; And R ²² is hydrogen or (C ₁ -C ₆ )alkyl; And q is an integer having a value from 1 to about 20, preferably from 1 to about 10; And each w and x is independently an integer value 0 or 1. 20. The method according to any one of claims 17-19, where Z' is a linking group and further contains one or more groups selected from isocyanide, isothiocyanide, 2-pyridyl disulfide, haloacetamide (-NHC(O)CH ₂ -halogen), maleimide, diene, alkene, halide, tosylate (TsO ^- ), aldehyde, sulfonate (R-SO ₃ ^- ),

,

, phosphonic acid (-P (= O) (OH) ₂ ), ketone, C ₈ -C ₁₀ cycloalkynyl, -OH, -NHOH, -NHNH ₂ , -SH, carboxylic acid (-COOH), acetylene (-C≡ CH), azide (-N ₃ ), amino (-NH ₂ ), sulfonic acid (-SO ₃ H), alkynone derivative (-C(O)C≡CR ^a and dihydrophosphate (-OP(=O)(OH) ₂ ). 21. The method according to any one of claims 18-20, where x is 0. 22. Process for the preparation of a compound of formula ( Iaa )

or a pharmaceutically acceptable salt thereof, where (Q) _q -(L') _w - is selected from:

where * means the point of attachment (Q) _q -( L') _w - to -SO ₂ -; Z' is absent or is a linking group containing:

where each V is independently a single bond, -O-, -S-, -NR ²¹ -, -C(O)NR ²² -, -NR ²³ C(O)-, -NR ²⁴ SO ₂ - or -SO _2NR ²⁵ -; each of R ²¹ , R ²² , R ²³ , R ²⁴ and R ²⁵ independently represents hydrogen, (C ₁ -C ₆ )alkyl, (C ₁ -C ₆ )alkyl(C ₆ -C ₂₀ )aryl or (C ₁ -C ₆ )alkyl(C ₃ -C ₂₀ )heteroaryl; r is an integer having a value from 1 to about 10; p is an integer having a value from 0 to about 10; q is an integer having a value from 1 to about 10; And L" is a single bond; Ar is phenyl; Y' represents -O-; O and Y' are located on adjacent Ar atoms; TG is a trigger group selected from: -NO ₂ , -C(O)-(CH ₂ ) ₂ C(O)-(C ₁ -C ₆ )alkyl, nitrobenzyl,

,

And

, where R ²¹ represents hydrogen; And R ²² is hydrogen or (C ₁ -C ₆ )alkyl; q is an integer having a value from 1 to about 20, preferably from 1 to about 10; each w and x is independently an integer having the value 0 or 1, or a pharmaceutically acceptable salt thereof, wherein the method comprises: additionally reacting a compound of formula ( Iaa ) with a targeting moiety to provide a conjugate of formula ( I ')

or a pharmaceutically acceptable salt thereof, where CB is a targeting fragment containing an antibody selected from an intact polyclonal antibody, an intact monoclonal antibody, an antibody fragment, a single-chain Fv mutant (scFv), a multispecific antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, and a fusion protein containing antigenic the determinant part of the antibody; cb and n are each independently integers having a value from 1 to about 20, preferably from 1 to about 10; each of the DLs is independently a group having the structure of the formula ( I” )

, where (Q) _q -(L') _w - is selected from:

where * denotes the point of attachment (Q) _q -(L') _w to -SO ₂ -; Z' is a linking group containing

where each V is independently a single bond, -O-, -S-, -NR ²¹ -, -C(O)NR ²² -, -NR ²³ C(O)-, -NR ²⁴ SO ₂ - or -SO _2NR ²⁵ -; each of R ²¹ , R ²² , R ²³ , R ²⁴ and R ²⁵ independently represents hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkylC ₆ -C ₂₀ aryl or C ₁ -C ₆ alkylC ₃ -C ₂₀ heteroaryl; r is an integer having a value from 1 to about 10; p is an integer having a value from 0 to about 10; q is an integer having a value from 1 to about 10; And L" is a single bond; X is -O-; Ar is phenyl; Y' represents -O-; X and Y' are located on adjacent Ar atoms; TG is a trigger group selected from: -NO ₂ , -C(O)-(CH ₂ ) ₂ C(O)-(C ₁ -C ₆ )alkyl, nitrobenzyl,

,

And

, where R ²¹ represents hydrogen; And R ²² is hydrogen or (C ₁ -C ₆ )alkyl; And q is an integer having a value from 1 to about 20, preferably from 1 to about 10; each w and x is independently an integer having a value of 0 or 1; provided that when w is 0, then q is 1, where the targeting fragment contains an antibody selected from an intact polyclonal antibody, an intact monoclonal antibody, an antibody fragment, a single-chain Fv mutant (scFv), a multispecific antibody, a bispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, a fusion protein containing the antigenic determinant part of the antibody, other modified immunoglobulin molecules containing antigen recognition sites and an antibody selected from muromonab-CD3, abciximab, rituximab, daclizumab, palivizumab, infliximab, trastuzumab (Herceptin), etanercept, basiliximab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adali mumaba, alefacepta, omalizumab , efalizumab, tositumomab-I ¹³¹ , cetuximab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab, rilonacept, certolizumab pegola, romiplostim, AMG-531, CNTO-148, CNTO-1275, ABT-874, LEA-29Y, belimumab, TACI -Ig, second generation anti-CD20, ACZ-885, tocilizumab, atlizumab, mepolizumab, pertuzumab, Humax CD20, tremelimumab (CP-675 206), ticilimumab, MDX-010, IDEC-114, inotuzumab ozogamicin, HuMax EGFR, aflibercept, HuMax-CD4, Ala-Ala, ChAglyCD3, TRX4, Catumaxomaba, IGN101, MT-201, Pregovomaba, CH-14.18, WX-G250, AMG-162, AAB-001, Motavizumab, MEDI-524, Efumgumaba, Aurograb , raxibacumab, third-generation anti-CD20, LY2469298, and veltuzumab.

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