KR20220142393A - Protein degrader conjugate and the use thereof - Google Patents

Abstract

The present invention relates to protein degrader conjugate and uses thereof. Specifically, the present invention relates to an antigen-specific binding moiety-protein degrader conjugate, more specifically, relates to the use of these antigen specific binding moiety-proteolytic agent conjugates for the treatment and/or prevention of hyperproliferative and/or angiogenic diseases such as cancer diseases.

Description

Protein degrader conjugate and the use thereof

The present invention relates to a protein degrader conjugate and its use, and more particularly, to an antigen-specific binding moiety-protein degrader conjugate and a pharmaceutical composition comprising the same.

In general, drugs express their drug effects in a manner that inhibits the function of the protein by binding to a specific site of a specific protein. In other words, whether a new therapeutic agent is a druggable target is determined according to the possibility of attacking a specific active site or binding pocket of a disease-related protein.

According to a report in 2016, there are about 400 proteins used as targets for drugs approved by the US Food and Drug Administration (FDA), and most of them are enzymes, receptors, transmitters, various channels, and membrane proteins. However, according to what is known so far, about 3,000 proteins are known to cause human diseases, and only 13% of them are being developed as target proteins. The reason is that current drug development methods cannot target most disease proteins and inhibit their functions. For example, c-Myc, a well-known target of anticancer drugs, does not have a hydrophobic pocket structure to which drugs can bind as a transcription factor, and protein aggregates that cause degenerative brain diseases such as Tau tangle are currently It is not possible to remove protein tangles with the technology of Therefore, new attempts to target undiscoverable target proteins for new therapeutic agents are continuously being carried out.

For example, recently, a method for selectively removing a target protein using the ubiquitin-proteasome pathway has been proposed. Most (80%) of cellular proteins are labeled with ubiquitin through the ubiquitin-proteasome pathway and then degraded in the cytoplasm and nucleus by the proteasome. It not only regulates protein conversion and homeostasis, but also regulates physiological functions of cells such as cell cycle, signal regulation, transcriptional regulation, and apoptosis. It also serves to rapidly remove proteins having an abnormal structure, damaged proteins, or proteins having an abnormal structure due to mutation. Recently, it has been reported that ubiquitin-mediated protein degradation is related to various diseases, particularly degenerative brain diseases such as Alzheimer's disease and Parkinson's disease and hereditary diseases, and abnormalities in the proteolytic pathway are the cause of various types of diseases including cancer. considered to be the cause. Accordingly, research on new drug development called PROTAC (proteolysis-targeting chimaera) is being actively conducted as a treatment for removing disease-causing proteins using activation of the ubiquitin-proteasome pathway. PROTAC is a bifunctional organic small molecule composed of a ligand that binds to a target protein, a ligand that binds to E3 ubiquitin ligase, and a linker. By binding the target protein to the target protein, it has a structure in which the target protein can be easily decomposed. It is expected that desired therapeutic efficacy can be expected by decomposing the protein in question by locating the desired disease-related protein near the E3 ubiquitin ligase. However, in that the target protein whose degradation is induced by PROTAC is not a substrate of the E3 ubiquitin ligase, there is a disadvantage that the target protein that can be ubiquitinated by the E3 ubiquitin ligase having substrate specificity is limited. Accordingly, even if PROTAC binds to the E3 ubiquitin ligase and the target protein, there is a possibility that the target protein is not degraded. In addition, the problem that the targeted delivery of drugs is not easy still remains.

Accordingly, the present inventors have completed the present invention by developing an antigen-specific binding moiety-proteolytic agent conjugate as a new drug platform capable of targeted delivery of drugs while increasing the selective degradation efficiency of the target protein.

PROTAC, a drug that has been recently developed as a method for selective removal of a target protein, is composed of a ligand-linker-E3 ubiquitin ligase of the target protein. There is a problem that it is not easy. In addition, there is a problem in that the target protein capable of inducing degradation by PROTAC is limited, and the degradation efficiency of the target protein is low. In addition, there is a problem that the drug may cause toxicity by affecting not only problematic cells but also normal cells.

Therefore, there is a need for a new concept of drug development that can target only problematic cells without toxic to normal cells, while increasing the selective degradation efficiency of target proteins.

The present invention provides a protein degrader that can increase the selective degradation efficiency of a target protein, an antigen-specific binding moiety that can accurately deliver the protein degradation agent to a target cell, and a stable and stable drug during circulation in the body. Provided is an antigen-specific binding moiety-proteolytic agent conjugate grafted with linker technology that can be easily released within a target cell to maximize drug efficacy.

Through this, it is possible to improve the selective degradation efficiency of the target protein, and the proteolytic agent stably and accurately reaches the target cell, effectively exerting the drug effect, and significantly lowering the toxicity - an antigen-specific binding moiety-proteolytic agent conjugate By providing, the said subject was solved.

In one aspect of the present invention, the present invention provides an antigen-specific binding moiety-protein degrader conjugate, which is represented by the following general formula (I):

[General Formula I]

Ab - [Linker - (B) _m ] _n

In the above formula,

Ab is an antigen specific binding moiety;

Linker is a linker;

B is a protein degrader moiety;

m and n are integers selected from 1 to 20.

In addition, the present invention provides an antigen-specific binding moiety according to the present invention - a pharmaceutical composition for preventing and treating hyperproliferative, cancer or angiogenic disease, preventing or treating, containing the proteolytic agent conjugate as an active ingredient.

The antigen-specific binding moiety-proteolytic agent conjugate according to the present invention comprises an antigen-specific binding moiety that binds to an antigen of a target cell, in particular a cancer cell, thereby effectively and specifically and selectively introducing a proteolytic agent into the target cell. can be transmitted as

In addition, the antigen-specific binding moiety-proteolytic agent conjugate according to the present invention comprises a proteolytic agent that binds to E3 ubiquitin ligase, and thus a substrate protein of the E3 ubiquitin ligase, particularly cancer cells. It can induce selective degradation of proteins that activate the process, and can increase degradation efficiency.

In addition, the antigen-specific binding moiety-proteolytic agent conjugate according to the present invention includes a linker capable of maximizing the effect of the proteolytic agent being easily released within the target cell, so that the proteolytic agent stably reaches the target cell, resulting in efficacy can be used effectively.

Accordingly, the antigen-specific binding moiety-proteolytic agent conjugate according to the present invention can be easily used for the prevention or treatment of hyperproliferative, cancer, or angiogenic diseases.

1 is a diagram showing the reaction pathway of the protein degrading agent of the present invention.
2 and 3 are diagrams showing the reaction pathway of the conjugate of the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention provides a protein degrader conjugate, more particularly an antigen specific binding moiety-protein degrader conjugate.

In one aspect of the present invention, there is provided an antigen-specific binding moiety-proteolytic agent conjugate, represented by the following general formula (I):

[General Formula I]

Ab - [Linker - (B) _m ] _n

In the above formula,

Ab is an antigen specific binding moiety;

Linker is a linker;

B is a protein degrader moiety;

m and n are each independently an integer selected from 1 to 20.

The present invention also provides the above conjugate, a pharmaceutically acceptable salt or solvate thereof.

As used herein, "conjugate" refers to a cell binding agent that is covalently bound to one or more molecules of a cytotoxic compound. Here, the "cell binding agent" is a molecule having affinity for a biological target, and may be, for example, a ligand, a protein, an antibody, specifically a monoclonal antibody, a protein or antibody fragment, a peptide, an oligonucleotide, an oligosaccharide, , the binding agent functions to direct the biologically active compound to the biological target. In one aspect of the invention, the conjugate may be designed to target cancer cells via a cell surface antigen. The antigen may be a cell surface antigen that is overexpressed or expressed in an abnormal cell type. Specifically, the target antigen may be expressed only on proliferative cells (eg, cancer cells). Target antigens can usually be selected based on different expression between proliferative and normal tissues.

As used herein, "antigen-specific binding moiety" refers to any molecule or portion of a molecule capable of specifically binding to a target antigen or target epitope. Specifically, the antigen-specific binding moiety may be an antibody construct or an antibody-like protein.

As used herein, the term "antibody construct" refers to an antibody construct that binds to an antigen of a target cell, and includes not only a complete antibody form, but also an antigen-binding fragment of the antibody molecule. Also included are polypeptides comprising an antigen binding domain and an Fc domain.

As used herein, the term “antigen binding domain” refers to a binding domain from an antibody or non-antibody capable of specifically binding an antigen. The antigen binding domains may be numbered if there are multiple antigen binding domains (eg, a first antigen binding domain, a second antigen binding domain, a third antigen binding domain, etc.) in a given conjugate or antibody construct. Different antigen binding domains in the same conjugate or construct may be present on the same antigen (e.g., a first antigen binding domain and a second antigen are capable of specifically binding to the same target cell antigen of the conjugate or construct) or different antigens (e.g., For example, the first antigen binding domain may specifically bind a first target cell antigen and the second antigen binding domain may specifically bind a conjugate or a second target cell antigen). have.

The antigen binding domain may be an antigen binding portion of an antibody or antibody fragment. An antigen binding domain may be one or more fragments of an antibody capable of retaining the ability to specifically bind an antigen. The antigen binding domain may be any antigen binding fragment. An antigen binding domain typically recognizes a single antigen. Antibody constructs typically include, for example, one or two antigen binding domains, although more may be included in the antibody construct.

The antigen binding domain of the antibody construct may be selected from any domain that specifically binds to an antigen, including, but not limited to, an antibody or non-antibody molecule. In some embodiments, the antigen binding domain is a monoclonal antibody, polyclonal antibody, recombinant antibody, or functional fragment thereof, e.g., a heavy chain variable domain (VH) and a light chain variable domain (VL), Fab', F( any of an antibody that specifically binds to an antigen, including, but not limited to, ab')2, Fab, Fv, rlgG, scFv, hcAbs (heavy chain antibodies), single domain antibodies, VHH, VNAR, sdAbs, or Nanobodies. domains may be selected. In some embodiments, the antigen binding domain is a non-antibody scaffold, such as DARPin, apimer, avimer, notin, monobody, lipocalin, anticalin, 'T-body', apibody, peptibody, affinity clamp, ecto domains, receptor ectodomains, receptors, ligands or centrins, including but not limited to, any domain of a non-antibody molecule that specifically binds to an antigen.

The antigen binding domain of an antibody construct, eg, from a monoclonal antibody, may comprise a light chain and a heavy chain. In some embodiments, the monoclonal antibody specifically binds to an antigen present on the surface of a target cell and is specific for a target cell antigen, including a light chain of an anti-target cell antigen antibody and a heavy chain of an anti-target cell antigen antibody to form an antigen-binding domain that binds hostilely.

As used herein, "Fc domain" refers to the fragment crystallizable region or tail region of an antibody. An Fc domain is a structure capable of binding to one or more Fc receptors (FcRs) and interacts with Fc receptors (FcRs) on the surface of a cell.

FcRs may bind to the Fc domain of an antibody. The FcR is capable of binding to the Fc domain of an antibody bound to an antigen. FcRs are organized into classes based on the class of antibody that the FcR recognizes (eg, gamma (γ), alpha (α) and epsilon (ε)). The FcαR class binds IgA and includes multiple isoforms, FcαRI (CD89) and FcαμR. The FcγR class binds IgG and includes multiple isotypes, FcγRI (CD64), FcγRIIA (CD32a), FcγRIIB (CD32b), FcγRIIIA (CD16a) and FcγRIIIB (CD16b). FcγRIIIA (CD16a) may be FcγRIIIA (CD16a) F158 variant or V158 variant.

Modifications in the amino acid sequence of the Fc domain can alter the recognition of FcRs for the Fc domain. However, such modifications may still allow for FcR-mediated signal transduction. The modification may be substituting an amino acid at a residue (eg wild-type) of the Fc domain with a different amino acid at that residue. Modifications may allow binding of an FcR to sites on the Fc domain to which the FcR may not otherwise bind. The modification may increase the binding affinity of the FcR to the Fc domain. Modifications may decrease the binding affinity of an FcR to a site on the Fc domain where the FcR may increase its binding affinity. The modification may increase the subsequent FcR-mediated signaling following binding of the Fc domain to the FcR.

The Fc domain may be a naturally occurring or variant of a naturally occurring Fc domain and may comprise at least one amino acid change compared to the sequence of the wild-type Fc domain. Amino acid changes in the Fc domain may cause the antibody construct or conjugate to bind at least one Fc receptor with greater affinity compared to the wild-type Fc domain.

The Fc domain may be derived from an antibody. The Fc domain may be derived from an IgG antibody. The Fc domain may be derived from an IgG1, IgG2 or IgG4 antibody. The Fc domain may be at least 80% identical to the Fc domain from the antibody. The Fc domain may be part of the Fc domain of an antibody.

The antibody construct may comprise an Fc domain in an antibody. The antibody construct may comprise an Fc domain in a scaffold. The antibody construct may comprise an Fc domain in an antibody scaffold. The antibody construct may comprise an Fc domain in a non-antibody scaffold.

The antibody construct may comprise an antigen binding domain and an Fc domain, wherein the Fc domain may be covalently bound to the antigen binding domain. The antibody construct may comprise a first antigen binding domain and an Fc domain, wherein the Fc domain may be covalently bound to the first antigen binding domain. The antibody construct may comprise an antigen binding domain and an Fc domain, wherein the Fc domain may be covalently linked to the antigen binding domain as an Fc domain-antigen binding domain fusion protein. The antibody construct may comprise an antigen binding domain and an Fc domain, wherein the Fc domain may be bound to the antigen binding domain by a linker.

The first antigen binding domain and the second antigen binding domain, if present, may be attached to the Fc domain as a fusion protein. The first antigen binding domain may be attached to the Fc domain at the N-terminus of the Fc domain, wherein the second antigen binding domain may be attached to the Fc domain at the C-terminus. The first antigen binding domain may be attached to the Fc domain at the N-terminus of the Fc domain, wherein the second antigen binding domain may be attached to the Fc domain at the C-terminus via a polypeptide linker.

Alternatively, the first antigen binding domain may be attached to the Fc domain at the C-terminus of the Fc domain, wherein the second antigen binding domain may be attached to the Fc domain at the N-terminus. The second antigen binding domain and the Fc domain may comprise an antibody and the first antigen binding domain may comprise a single chain variable fragment (scFv). A single chain variable fragment may comprise a heavy chain variable domain and a light chain variable domain of an antibody. The first antigen binding domain of the fusion protein may be attached to the second antigen binding domain in the heavy chain variable domain of a single chain variable fragment of the first antigen binding domain (HL orientation). Alternatively, the first antigen binding domain of the fusion protein may be attached to the second antigen binding domain in the light chain variable domain of a single chain variable fragment of the first antigen binding domain (LH orientation). In either orientation, the first antigen binding domain and the second antigen binding domain may be attached via a polypeptide linker.

The antibody construct as described herein may have a dissociation constant (Kd) of less than 10 nM for the antigen of the first antigen binding domain. The antibody construct may have a dissociation constant (Kd) for the antigen of the second antigen binding domain of less than 10 nM. The antibody construct may have a dissociation constant (Kd) for the antigen of the first antigen binding domain that is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM, or less than 0.1 pM. The antibody construct may have a dissociation constant (Kd) for the antigen of the second antigen binding domain that is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM, or less than 0.1 pM.

The antibody constructs disclosed herein may be non-natural, designed and/or engineered. The antibody constructs disclosed herein may be non-native, designed and/or engineered scaffolds comprising antigen binding domains.

The antibody construct may comprise an antibody which may comprise an antigen binding domain and an Fc domain. An antibody molecule can consist of two identical light chains and two identical heavy chains, all covalently linked by precisely positioned disulfide bonds. The N-terminal regions of the light and heavy chains together may form the antigen recognition site of each antibody. Structurally, the various functions of an antibody may be restricted to distinct protein domains (ie, regions). The site capable of recognizing and binding antigens consists of three complementarity determining regions (CDRs) that may exist within the variable heavy and variable light chain regions at the N-terminus of the two heavy and two light chains. The constant domains may provide the general framework of an antibody and may not be involved in direct binding of the antibody to antigen, but may be involved in various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC). .

The domains of a native light chain variable region and a heavy chain variable region may have the same general structure, and each domain may comprise three hypervariable regions or four framework regions in which the sequences linked by CDRs may be somewhat conserved. The four framework regions can mainly adopt the β-sheet structure and the CDRs can form loops that form part of the β-sheet structure and in some aspects form part of the β-sheet structure. The CDRs of each chain may be held in close proximity by framework regions and, together with the CDRs of the other chain, may contribute to the formation of an antigen binding site.

Antibodies of antibody constructs may comprise any type of antibody, which may be assigned to different classes of immunoglobins, such as IgA, IgD, IgE, IgG and IgM. A number of different classes can be further divided into isotypes, for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Antibodies may further comprise light and heavy chains, often multiple chains. The heavy chain constant regions (Fc) corresponding to different classes of immunoglobulins may be α, δ, ε, γ and μ, respectively. The light chain may be either kappa or κ and lambda or λ based on the amino acid sequence of the constant domain. The Fc region may comprise an Fc domain. An Fc receptor is capable of binding to an Fc domain. Conjugates may also include any fragment or recombinant form thereof, including, but not limited to, scFvs, Fabs, variable Fc fragments, domain antibodies and any other fragment capable of specifically binding to an antigen.

An antibody may comprise an antigen binding domain that refers to the portion of the antibody comprising an antigen recognition moiety, i.e., an antigenic determinant variable region of the antibody sufficient to confer recognition and specific binding of the antigen recognition moiety to a target, such as an antigen, i.e., an epitope. can

The antigen binding domain of an antibody may comprise one or more light chain (LC) CDRs (LCDR) and one or more heavy chain (HC) CDRs (HCDR), one or more LCDRs, or one or more HCDRs. For example, the antibody binding domain of an antibody may comprise one or more of the following: light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2) or light chain complementarity determining region 3 (LCDR3). In another example, the antibody binding domain may comprise one or more of the following: heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), or heavy chain complementarity determining region 3 (HCDR3). In some embodiments, the antibody binding domain comprises all of the following: light chain complementarity determining region 1 (LCDR1), light chain complementarity determining region 2 (LCDR2), light chain complementarity determining region 3 (LCDR3), heavy chain complementarity determining region 1 (HCDR1) , heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3). Unless otherwise stated, the CDRs described herein may be defined according to IMGT (International ImMunoGeneTics Information System). The antigen binding domain may comprise only the heavy chain of an antibody (eg, no other portion of the antibody). The antigen binding domain may comprise only the variable domain of the heavy chain of an antibody. Alternatively, the antigen binding domain may comprise only the light chain of an antibody. The antigen binding domain may comprise only the variable light chain of an antibody.

Antibody constructs may comprise antibody fragments, such as Fab, Fab', F(ab')2 or Fv fragments. Antibodies used in the present invention may be "humanized". A humanized form of a non-human (eg, murine) antibody is an intact (full-length) chimeric immunoglobulin, immunoglobulin chain or antigen-binding fragment thereof (eg, Fv, Fab, Fab', F(ab')2 or other target-binding subdomains of the antibody), which may contain minimal sequence derived from non-human immunoglobulin. In general, a humanized antibody may comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or Substantially all framework (FR) regions are of human immunoglobulin sequences. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence.

The antibodies described herein may be human antibodies. A "human antibody" as used herein may include, for example, an antibody having the amino acid sequence of a human immunoglobulin, and a gene for one or more human immunoglobulins that do not express endogenous immunoglobulins or from a human immunoglobulin library. antibodies isolated from the transducing animal. Human antibodies can be generated using transgenic mice that cannot express functional endogenous immunoglobulin but are capable of expressing human immunoglobulin genes. Fully human antibodies that recognize selected epitopes can be generated using directed selection. In this approach, a selected non-human monoclonal antibody, eg, a mouse antibody, can be used to elicit a selection of fully human antibodies that recognize the same epitope.

The antibodies described herein may be dual specific antibodies or dual variable domain antibodies (DVDs). Bispecific and DVD antibodies may be monoclonal, often human or humanized antibodies, which may have binding specificities for at least two different antigens.

The antibodies described herein may be derived or otherwise modified. For example, the induced antibody can be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, and the like.

Antibody constructs may include antibodies having modifications that occur in at least one amino acid residue. Modifications may be substitutions, additions, mutations, deletions, and the like. Antibody modifications may be insertions of unnatural amino acids.

The antibody construct may comprise an Fc domain of the IgG1 isotype. The antibody construct may comprise an Fc domain of the IgG2 isotype. The antibody construct may comprise an Fc domain of an IgG3 isotype. The antibody construct may comprise an Fc domain of the IgG4 isotype. Antibody constructs may have hybrid isotypes comprising constant regions from two or more isotypes.

Antibodies described herein may have sequences modified to alter at least one constant region-mediated biological effector function relative to the corresponding wild-type sequence. For example, in some embodiments, the antibody is configured to increase or decrease at least one constant region-mediated biological effector function, such as, for example, increased binding to an Fc receptor (FcR), as compared to an unmodified antibody. can be deformed. FcR binding can be reduced or increased, for example, by mutating the immunoglobulin constant region segment of the antibody in a specific region required for FcR interaction.

The antibodies described herein may be modified to acquire or improve at least one constant region-mediated biological effector function, for example to enhance FcγR interactions, relative to an unmodified antibody. For example, antibodies with constant regions that bind FcγRIIA, FcγRIIB and/or FcγRIIIA with greater affinity than the corresponding wild-type constant region can be generated according to the methods described herein.

The antibody constructs disclosed herein may be non-natural, designed and/or engineered antibodies.

As used herein, the term “antibody-like protein” refers to a protein that has been engineered (by mutagenesis of a loop) to specifically bind to a target antigen. Typically, such antibody-like proteins comprise one or more variable peptides attached at least at both ends to a protein scaffold. This dual structural constraint greatly increases the binding affinity of the antibody-like protein to a level corresponding to that of the antibody. The length of a variable peptide loop typically consists of 10 to 20 amino acids. The scaffold protein may be any protein with good dissolution properties. Preferably, the scaffold protein is a small globular protein. Antibody-like proteins include, but are not limited to, affibodies, anticalins, and designed ankyrin repeat proteins. Antibody-like proteins can be derived from multiple mutant libraries, for example, extracted from multiple phage display libraries, and isolated similarly to general antibodies. Antibody-like binding proteins can also be obtained by combinatorially mutagenizing surface-exposed residues in globular proteins.

In one aspect of the invention, the antigen specific binding moiety may be an antibody construct or an antibody-like protein.

In one aspect of the invention, the antibody construct comprises an antigen binding domain and an Fc domain that specifically binds an antigen. For example, an antibody construct may comprise a first antigen binding domain and an Fc domain that specifically binds a first antigen. The antibody construct may comprise a first antigen binding domain that specifically binds a first antigen, a second antigen binding domain that specifically binds a second antigen, and an Fc domain.

In one embodiment of the present invention, the antibody construct may be covalently bound to an antigen binding domain and an Fc domain. For example, in the antibody construct, the Fc domain may covalently bind to the first antigen binding domain. Further, when the antibody construct comprises a second antigen binding domain, the second antigen binding domain is capable of covalent binding at the N-terminus of the second antigen binding domain and at the C-terminus of the first antigen binding domain, or an Fc domain. can be covalently linked at the C-terminus of

In one aspect of the present invention, the first antigen binding domain and/or the second antigen binding domain may comprise an immunoglobulin heavy chain variable region or antigen binding fragment thereof and an immunoglobulin light chain variable region or antigen binding fragment thereof. In addition, the first antigen binding domain or the second antigen binding domain may comprise a single chain variable region fragment (scFv).

In one aspect of the present invention, the Fc domain may be an IgG region. The Fc domain may be an IgG1 Fc region. In addition, the Fc domain may be an Fc domain variant comprising one or more amino acid substitutions in the IgG region compared to the amino acid sequence of the wild-type IgG region, wherein the Fc domain variant has an affinity for one or more Fcγ receptors compared to the wild-type IgG region. may be increased.

In one aspect of the present invention, the Fc domain may be a non-antibody scaffold.

In one aspect of the invention, the Fc domain is

a) as an Fc domain-antigen binding domain fusion protein; or

b) by conjugation through a second linker

It may be covalently bound to an antigen binding domain.

In one aspect of the present invention, the antibody construct may further comprise an effector antigen binding domain that binds to an antigen presented by an antigen presenting cell.

In one aspect of the present invention, the antibody construct may have a Kd of the conjugate for binding antigen to less than 100 nM and up to 100 times the Kd of the unconjugated antibody construct. For example, the Kd of the unconjugated antibody construct may be less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM, or less than 0.1 pM. It may also be less than 100-fold, less than 200-fold, less than 400-fold, less than 800-fold, or less than 1000-fold the Kd of the unconjugated antibody construct.

In one aspect of the invention, the antibody construct may be an antibody or antibody fragment. For example, monoclonal antibody, dAb, scAb, Fab fragment, F(ab') ₂ fragment, scFv, scFv-Fc fragment, single domain heavy chain antibody, single domain light chain antibody, antibody variant, multimeric antibody or It may be a bispecific antibody.

In one aspect of the invention, the antibody construct may be a rabbit, mouse, chimeric, humanized or fully human monoclonal antibody.

In one aspect of the invention, the antibody construct may be of the IgG isotype.

The antibody constructs disclosed herein bind to an antigen of a target cell. In one aspect of the invention, the antibody construct binds to an antigen of a cancer cell. The antibody is present on the surface of the target cell in an amount of at least 10, 100, 1,000, 10,000, 1.0 x 10 ⁵ , 1.0 x 10 ⁶ , 2.5 x 10 ⁶ , 5 x 10 ⁶ or 1 x 10 ⁷ copies of the target antigen. can be coupled to

The antibody constructs disclosed herein bind to an antigen of a target cell. In one aspect of the invention, the antigen is 5T4, A33, ABL, ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA2A, AFP, Aggrecan, AGR2, AICDA, AIF1, AIGI, AKAP1, AKAP2, ALK, AMH, AMHR2 , ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOCl, AR, aromatase, ATX, AX1, AZGP1 (zinc-a-glycoprotein), avB3, B7.1, B7.2, B7-H1, BAD, BAFF, BAG1, BAI1, BCR, BCL2, BCL6, BDNF, BLNK, BLR1 (MDR15), BIyS, BMP1, BMP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, BMPR1A, BMPR1B, BMPR2, BPAG1 (Plectin), BMPR1B, BMPR2, BPAG1 (plectin) (IL27w), C3, C4A, C5, C5R1, CA-125, ,CA19-9 CAMPATH-1, CANT1, CAPRIN-1, CASP1, CASP4, CAV1, CCBP2 (D6/JAB61), CCL1 (1-309), CCLI1 (eotaxin), CCL13 (MCP-4), CCL15 (MIP-Id), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MIP-3b), CCL2 (MCP-1), MCAF, CCL20 (MIP-3a), CCL21 (MEP-2), SLC, exodus-2, CCL22 (MDC/STC-I), CCL23 (MPIF-I), CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-3), CCL27 (CTACK/ILC), CCL28, CCL3 (MIP-Ia), CCL4 (MIPIb), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2) , CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CCR1 (CKR1/HM14 5), CCR2 (mcp-IRB/RA), CCR3 (CKR3/CMKBR3), CCR4, CCR5 (CMKBR5/ChemR13), CCR6 (CMKBR6/CKR-L3/STRL22/DRY6), CCR7 (CKR7/EBI1), CCR8 ( CMKBR8/TERI/CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR), CD164, CD19, CDIC, CD2, CD20, CD21, CD200, CD-22, CD24, CD25, CD27, CD28, CD3, CD30, CD33, CD35, CD37, CD38, CD3E, CD3G, CD3Z, CD4, CD38, CD40, CD40L, CD44, CD45, CD45RB, CD47, CD5, CD52, CD69, CD72, CD74, CD79A, CD79B, CD8, CD80, CD81, CD83, CD86, CD137, CD152, CD274, CDH1 (E-cadherin), CDH1O, CDH12, CDH13, CDH18, CDH19, CDH2O, CDH5, CDH7, CDH8, CDH9, CDK2, CDK3 , CDK4, CDK5, CDK6, CDK7, CDK9, CDKN1A (p21Wap1/Cip1), CDKN1B (p27Kip1), CDKN1C, CDKN2A (p16INK4a), CDKN2B, CDKN2C, CDKN3, CEA, CHGAP, CHGB, CERI, CEBPB, CERI CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, CLDN3, CLDN7 (claudin-7), CLN3, CLU (clusterin), CMKLR1, CMKOR1 (RDC1), CNR1, COLIA1, COL4A1, CR1, COLIA1, CNR1, COL6A1 , CRP, CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (GCSF), CTL8, CTNNB1 (β-catenin), CTSB (cat hepsin B), CX3CL1 (SCYD1), CX3CR1 (V28), CXCL1 (GRO1), CXCL1O (IP-IO), CXCLI1 (1-TAC/IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL16, CXCL2 ( GRO2), CXCL3 (GRO3), CXCL5 (ENA-78/LIX), CXCL6 (GCP-2), CXCL9 (MIG), CXCR3 (GPR9/CKR-L2), CXCR4, CXCR6 (TYMSTR/STRL33/Bonzo) ), cyclin B1, CYB5, CYC1, CYSLTR1, DAB2IP, DES, DKFZp451J0118, DNCL1, DPP4, E2F1, Engel, Edge, Fennel, EFNA3, EFNB2, EGF, EGFR, ELAC2, ENG, ENO3, ENOEPHA2, EPHA2, ENOEPHA2 , EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6, EPHRIN-A1, EPHRIN-A2, EPHRIN-A2, EPHRINA4, EPHRIN-A5, EPHRINA3, EPHB2 , EPHRIN-B1, EPHRIN-B2, EPHRIN-B3, EPHB4, EPG, ERG, ERBB2 (Her-2), EREG, ERK8, Estrogen receptor, Endosialin, Earl, ESR2, F3 (TF), FADD, farnesyltransferase, FasL, FASNf, FBP (Folate-binding protein), FCER1A, FCER2, FCGR3A, FGF, FGF1 (aFGF), FGF10, FGF1 1, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2 (bFGF), FGF20 , FGF21, FGF22, FGF23, FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF8, FGF9, FGFR3, FIGF (VEGFD), FIL1 (EPSILON), FBL1 (ZETA), FLJ12584, FLJ25530, FLRT1 (fibronectin), FLT1, FLT-3, FOS, FOSL1 (FRA-1), FY (DARC), G250, GABRP (GABAa), GAGEB1, GAGEC1, GALNAC4S-6ST, GATA3, GD2, GD3, GDF5, GFI1, GGT1, GM2, GM-CSF, GNAS1, GNRH1, gp100, GPR2 (CCR10), GPR31 , GPR44, GPR81 (FKSG80), GRCC1O (C1O), GRP, GSN (Gelsolin), GSTP1, HAVCR2, HDAC, HDAC4, HDAC5, HDAC7A, HDAC9, Hedgehog, HGF, HIF1A, HIP1, histamine, histamine receptor, HLA-A , HLA-DRA, HLA-E, HLD-DR, HM74, HMOXI, HPV E6, HPV E7, HSP90, hTERT, HUMCYT2A, ICEBERG, ICOSL, ID2, IFN-a, IFNA1, IFNA2, IFNA4, IFNA5, EFNA6, BFNA7 , IFNB1, IFNγ, IFNW1, IGBP1, IGF1, IGFIR, IGF2, IGFBP2, IGFBP3, IGFBP6, DL-1, ILIO, ILIORA, ILIORB, IL-1, IL1R1 (CD121a), IL1R2 (CD121b), IL-IRA, IL -2, IL2RA (CD25), IL2RB (CD122), IL2RG (CD132), IL-4, IL-4R (CD123), IL-5, IL5RA (CD125), IL3RB (CD131), IL-6, IL6RA, ( CD126), IR6RB (CD130), IL-7, IL7RA (CD127), IL-8, CXCR1 (IL8RA), CXCR2, (IL8RB/CD128), IL-9, IL9R (CD129), IL-10, IL10RA (CD2) 10), IL10RB (CDW210B), IL-11, IL-11RA, IL-12, IL-12A, IL-12B, IL-12RB1, IL-12RB2, IL-13, IL-13RA1, IL-13RA2, IL- 14, IL-15, IL-15RA, IL-16, IL-17, IL-17A, IL-17B, IL-17C, IL-17R, IL-18, IL-18BP, IL-18R1, IL-18RAP, IL-19, ILIA, ILIB, ILIF10, ILIF5, IL1F6, ILIF7, IL1F8, DL1F9, ILIHYI, ILIR1, IL1R2, ILIRAP, ILIRAPLI, ILIRAPL2, ILIRL1, IL1RL2, ILIRN, IL-2, IL-20, IL-20RA, IL-21R, IL-22, IL-22R, IL-22RA2, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A, IL-28B, IL-29, IL- 2RA, IL-2RB, IL-2RG, IL-3, IL-30, IL-3RA, IL-4, IL-6ST (Glycoprotein 130), ILK, INHA, INHBA, INSL3, INSL4, IRAK1, IRAK2, ITGA1 , ITGA2, ITGA3, ITGA6 (α6 integrin), ITGAV, ITGB3, ITGB4 (β4 integrin), JAG1, JAK1, JAK3, JTB, JUN, K6HF, KAI1, KDR, KITLG, KLF5 (GC Box BP), KLF6, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, KRT1, KRT19 (Keratin 19), KRT2A, KRTHB6 (hair-specific type II keratin), LAMA5, LEP (leptin), Lingo-p75, Lingo-p75 -Troy, LMP2, LPS, LTA (TNF-b), LTB, LTB4R (GPR16), LTB4R2, LTBR, MelanA/MART1, MACMARCKS, MAG, O Mgp, MAGE A3, MAP2K7 (c-Jun), MCP-1, MDK, MIB1, midkine, MIF, MISRII, MJP-2, MK, MKI67 (Ki-67), ML-IAP, MMP2, MMP9, MS4A1, MSMB , MT3 (metallotionectin-UI), MMPs, mTOR, MTSS1, MUC1 (mucin), MYC, MYD88, NCK2, NA17, neurocan, NFKBI, NFKB2, NGFB (NGF), NGFR, NgR-Lingo, NgRNogo66, (Nogo), NgR-p75, NgR-Troy, NMEI (NM23A), NOTCH, NOTCH1, NOX5, NPPB, NROB1, NROB2, NRID1, NR1D2, NR1H2, NR1H3, NR1H4, NR112, NR113, NR2C1, NR2F1, NR2C1, NR2F1 , NR2F6, NR3C1, NR3C2, NR4A1, NR4A2, NR4A3, NR5A1, NR5A2, NR6A1, NRP1, NRP2, NT5E, NTN4, NY-ESO-1, ODZI, OPRDI, P2RX7, p53 and its variants, PAP1 PAWR, PAX3, PCA3, PCDGF, PCNA, PDGFA, PDGFB, PDGFRA, PDGFRB, PECAMI, peg-asparaginase, PF4 (CXCL4), PGF, PGR, phosphacan, PIAS2, PI3 Kinase, PIK3CG, PLAU (uPLX), PLG, PI , PKC, PKC-beta, PPBP (CXCL7), PPID, PR1, PRKCQ, PRKD1, PRL, PROC, PROK2, PSAP, PSCA, PSM, PTAFR, PTEN, PTGS2 (COX-2), PTN, RAC2 (P21Rac2), RANK, RANK ligand, RARB, Ras variants, RGS1, RGS13, RGS3, RNFI1O (ZNF144), Ron, ROBO2, RXR, S10 0A2, SCGB 1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SCYE1 (endothelial Monocyte-activating cytokine), SDF2, SERPENA1, SERPINA3, SERPINB5 (maspin), SERPINEI (PAPINFI,), SHIP-1, SHIP-2, SHB1, SHB2, SHBG, SfcAZ, SLC2A2, SLC33A1, SLC43A1, SLIT2, SPP1, SPRR1B (Spr1), ST6GAL1, STAB1, STATE, STEAP, STEAP2, TAG-72, TB4R2, TBX21, TCP1OBX , TDGF1, TEK, Tenacin, TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, THIL, THBS1 (thrombospondin-1), THBS2, THBS4, THPO, TIE (Tie-1), TIE (Tie-1) tissue factor, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TNF, TNF-a, TNFAIP2 (B94), TNFAIP3, TNFRSFI1A, TNFRSF1A, TNFRSF1B, TNFRSF5 ( Fas), TNFRSF7, TNFRSF8, TNFRSF9, TNFSF1O (TRAIL), TNFSF1 1 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFRSF14 (HVEM), TNF SF15 (VEGI), TNF SF15 , TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TO LLIP, Toll-like receptor, TOP2A (topoisomerase Iia), TP53, TPM1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TRKA, TREM1, TREM2, TRPC6, TSLP, TWEAK, VEGF Tyrosinase , VEGFB, VEGFC, versican, VHL C5, VLA-4, Wnt-1, XCL1 (lymphotactin), XCL2 (SCM-Ib), XCRI (GPR5/CCXCR1), YY1, ZFPM2, CLEC4C (BDCA-2, DLEC, CD303 , CLECSF7), CLEC4D (MCL, CLECSF8), CLEC4E (Mincle), CLEC6A (Dectin-2), CLEC5A (MDL-1, CLECSF5), CLEC1B (CLEC-2), CLEC9A (DNGR-1), CLEC7A (Dectin- 1), PDGFRa, SLAMF7, GP6 (GPVI), LILRA1 (CD85I), LILRA2 (CD85H, ILT1), LILRA4 (CD85G, ILT7), LILRA5 (CD85F, ILT11), LILRA6 (CD85b, ILT8), NCR1 (CD335, LY94) , NKp46), NCR3 (CD335, LY94, NKp46), NCR3 (CD337, NKp30), OSCAR, TARM1, CD300C, CD300E, CD300LB (CD300B), CD300LD (CD300D), KIR2DL4 (CD158D), KIR2DS, KLRC2 (CD300C) ), KLRK1 (CD314, NKG2D), NCR2 (CD336, NKp44), PILRB, SIGLEC1 (CD169, SN), SIGLEC14, SIGLEC15 (CD33L3), SIGLEC16, SIRPB1 (CD172B), TREM1 (CD354), or TREM2, WT1 NKp80), but is not limited thereto.

More specifically, the antigen may be an FcRγ-bound receptor, examples of FcRγ-bound receptors include GP6 (GPVI), LILRA1 (CD85I), LILRA2 (CD85H, ILT1), LILRA4 (CD85G, ILT7), LILRA5 (CD85F, ILT11), LILRA6 (CD85b, ILT8), NCR1 (CD335, LY94, NKp46), NCR3 (CD335, LY94, NKp46), NCR3 (CD337, NKp30), OSCAR or TARM1. Alternatively, the antigen may be a DAP12-bound receptor, examples of which are DAP12-bound receptors include CD300C, CD300E, CD300LB (CD300B), CD300LD (CD300D), KIR2DL4 (CD158D), KIR2DS, KLRC2 (CD159C, NKG2C), KLRK1 ( CD314, NKG2D), NCR2 (CD336, NKp44), PILRB, SIGLEC1 (CD169, SN), SIGLEC14, SIGLEC15 (CD33L3), SIGLEC16, SIRPB1 (CD172B), TREM1 (CD354) or TREM2. not. Alternatively, the antigen may be a hemITAM-bearing receptor, and examples of the hemITAM-bearing receptor include, but are not limited to, KLRF1 (NKp80).

Alternatively, the antigen is CLEC4C (BDCA-2, DLEC, CD303, CLECSF7), CLEC4D (MCL, CLECSF8), CLEC4E (Mincle), CLEC6A (Dectin-2), CLEC5A (MDL-1, CLECSF5), CLEC1B (CLEC-2) ), CLEC9A (DNGR-1) or CLEC7A (Dectin-1), but is not limited thereto. Alternatively, the antigen is ATP5I (Q06185), OAT (P29758), AIFM1 (Q9Z0X1), AOFA (Q64133), MTDC (P18155), CMC1 (Q8BH59), PREP (Q8K411), YMEL1 (O88967), LPPRC (Q6PB66), LONM (Q8CGK3), ACON (Q99KI0), ODO1 (Q60597), IDHP (P54071), ALDH2 (P47738), ATPB (P56480), AATM (P05202), TMM93 (Q9CQW0), ERGI3 (Q9CQE7), RTN4 (Q99P72), CL041P72 (Q8BQR4), ERLN2 (Q8BFZ9), TERA (Q01853), DAD1 (P61804), CALX (P35564), CALU (O35887), VAPA (Q9WV55), MOGS (Q80UM7), GANAB (Q8BHN3), ERO1A (Q8R180), UGGG1 (Q6P5E4), P4HA1 (Q60715), HYEP (Q9D379), CALR (P14211), AT2A2 (O55143), PDIA4 (P08003), PDIA1 (P09103), PDIA3 (P27773), PDIA6 (Q922R8), CLH (Q68FD5), PPIB (P24369), TCPG (P80318), MOT4 (P57787), NICA (P57716), BASI (P18572), VAPA (Q9WV55), ENV2 (P11370), VAT1 (Q62465), 4F2 (P10852), ENOA (P17182), ILK (O55222), GPNMB (Q99P91), ENV1 (P10404), ERO1A (Q8R180), CLH (Q68FD5), DSG1A (Q61495), AT1A1 (Q8VDN2), HYOU1 (Q9JKR6), TRAP1 (Q9CQN1), GRP3847) (P08113), CH60 (P63038) or CH10 (Q64433), but is not limited thereto. Alternatively, the antigen may be, but is not limited to, CDH1, CD19, CD20, CD29, CD30, CD38, CD40, CD47, EpCAM, MUC1, MUC16, EGFR Her2, SLAMF7 or gp75.

Antibody constructs that bind to the antigen include, but are not limited to, the following antibodies or Fc fusion proteins: avagobumab, abatacept (also known as ORENCIA ^™ ), absiximab (REOPRO ^™ , also known as c7E3 Fab) , adalimumab (also known as HUMIRA ^™ ), adekatumumab, alemtuzumab (CAMPATH ^™ , also known as MabCampath or Campath-1H), altumomab, apelimomab, anatumomab mafenatox, anetumumab, anrukizumab, apolizumab, arsitumomab, acelizumab, atlizumab, atrolimumab, bafinuzumab, basiliximab (also known as SIMULECT ^™ ), babituximab, VEC tumomab (also known as LYMPHOSCAN ^™ ), belimumab (also known as LYMPHO-STAT-B ^™ ), vertilimumab, besilesomab, bevacizumab (AVASTIN ^™ ), bicirumab bralobarbital, vibatuzumab Mertansine, Campas, Canakinumab (also known as ACZ885), Cantuzumab Meratansine, Capromap (also known as PROSTASCINT ^™ ), Katumaxomab (also known as REMOVAB ^™ ), Sedelizumab (also known as CIMZIA ^™ ), Ser Tolizumab pegol, cetuximab (also known as ERBITUX ^™ ), clenoliximab, dacetuzumab, dacliximab, daclizumab (also known as ZENAPAX ^™ ), denosumab (also known as AMG 162), detumo Mab, dorlimomab aritox, dorlixizumab, duntumumab, durimulumab, durmulumab, ecromeximab, eculizumab (also known as SOLIRIS ^™ ), edovacomab, edrecolomab ( Mab17-1A, also known as PANOREX ^TM ), efalizumab (also known as RAPTIVA ^TM ), epungumab (also known as MYCOGRAB ^TM ), elcilimomab, enrimomab pegol, epitumomab cituxetane, efalizumab, epitumo Mab, epratuzumab, erlizumab, ertumaxomab (also known as REXOMUN ^™ ), etatercept (also known as ENBREL ^™ ), etaracizumab (etaratuzumab) Mab, VITAXIN ^™ , also known as ABEGRIN ^™ ), exbivirumab, panolesomab (also known as NEUTROSPEC ^™ ), paralimomab, felbizumab, pontolizumab (also known as HUZAF ^™ ), galliximab, ganteneru Mab, Gabilimomab (also known as ABXCBL ^™ ), Gemtuzumab ozogamicin (also known as MYLOTARG ^™ ), Golimumab (also known as CNTO 148), Gomiliximab, Ivalizumab (also known as TNX-355), Eve Ritumomab tiuxetan (also known as ZEVALIN ^™ ), igobumab, imimirumab, infliximab (also known as REMICADE ^™ ), inolimomab, inotuzumab ozogamicin, ipilimumab (MDX-010, Also known as MDX-101), iratumumab, keliximab, rabetuzumab, remalesomab, lebrilizumab, lerdelimumab, lexatumumab (HGS-ETR2, also known as ETR2-ST01), lexitumumab, Ribivirumab, Lintuzumab, Lukatumumab, Lumiliximab, Mapatumumab (also known as HGSETR1, TRM-1), Maslimomab, Matuzumab (also known as EMD72000), Mepolizumab (also known as BOSATRIA ^™ ) ), metelimumab, milatuzumab, minretumomab, mitumomab, morolimumab, motavizumab (also known as NUMAX ^TM ), muromonap (also known as OKT3), nacolomab tafenatox, naptumo Mab estafenatox, natalizumab (also known as TYSABRI ^™ , ANTEGREN ^™ ), nebacumab, nererimomab, nimotuzumab (THERACIM hR3 ^™ , THERA-CIM-hR3 ^™ , also known as THERALOC ^™ ), nofetumomab Merpentan (also known as VERLUMA ^™ ), okrelizumab, odulimomab, ofatumumab, omalizumab (also known as XOLAIR ^™ ), oregobomab (also known as OVAREX ^™ ), otelixizumab, pagivacimab, parli Bizumab (also known as SYNAGIS ^™ ), panitumumab (ABX-EGF, also known as VECTIBIX ^™ ), pascolizumab, femtumomab (THERAGYN ^™ ) ), pertuzumab (2C4, also known as OMNITARG ^™ ), pexelizumab, pintumomab, priliximab, pretumumab, ranibizumab (also known as LUCENTIS ^™ ), laxibacumab, regavirumab, reslizumab, rituximab (also known as RITUXAN ^™ , MabTHERA ^™ ), lobelizumab, luflizumab, satumomab, cevirumab, cibrotuzumab, ciflizumab (also known as MEDI-507), sontuzumab, Stamulumab (also known as MYO-029), sulesomab (also known as LEUKOSCAN ^™ ), tacatuzumab tetraxetane, tadozumab, talizumab, taplitumomab POPTOX, tefivazumab (also known as AUREXIS ^™ ) ), telimomab aritox, teneliximab, teflizumab, ticilimumab, tocilizumab (also known as ACTEMRA ^™ ), toralizumab, tositumomab, trastuzumab (also known as HERCEPTIN ^™ ), tre melimumab (also known as CP-675,206), tucotuzumab celmorleukin, tuvirumab, urtoxazumab, ustekinumab (also known as CNTO 1275), bafaliximab, beltuzumab, befalimomab, non cilizumab (also known as NUVION ^™ ), voloxicimab (also known as M200), botumumab (also known as HUMASPECT ^™ ), zalutumumab, zanolimumab (also known as HuMAX-CD4), ziralimumab, Zolimomab aritox, daratumumab, erlotuxumab, obintunzumab, olalatumab, brentuximab vedotin, aflibercept, avatacept, belatacept, apibercept, etanercept, lomiflostim, SBT- 040 (sequence listed in US Patent Publication No. 2017/0158772).

As used herein, "linker" refers to a compound that covalently binds a cytotoxic compound to a ligand.

The linkers described herein may be cleavable, non-cleavable, hydrophilic or hydrophobic.

The cleavable linker is cleavable under intracellular conditions such that cleavage of the linker releases the proteolytic agent from the antibody construct-proteolytic agent conjugate in the intracellular environment.

A cleavable linker is cleavable by a cleaving agent present in the intracellular environment (eg, in a lysosome or endosome or caveolea). The cleavable linker may be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme including, but not limited to, a lysosomal or endosomal protease. Generally, the peptidyl linker is at least 2 amino acids in length or at least 3 amino acids in length. Cleavage agents may include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release the active drug in target cells (eg, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Peptidyl linkers cleavable by enzymes present in antigen-expressing cells are most common. For example, a peptidyl linker cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancer tissues, can be used (eg, a Phe-Leu or Gly-Phe-Leu-Gly linker). Other such linkers are described, for example, in US Pat. No. 6,214,345. In addition, the peptidyl linker cleavable by an intracellular protease is described in, for example, a Val-Cit linker, a Phe-Lys linker (eg, U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin using a Val-Cit linker). see) or a Val-Ala linker. The Val-Cit linker or Val-Ala linker may contain a pentafluorophenyl group and may contain a succinimide group or a maleimide group. Alternatively, it may contain a PABA group and a pentafluorophenyl group, and may contain a PABA (4-aminobenzoic acid) group and a maleimide group, and may contain a PABA group and a succinimide group.

As used herein, the terms "intracellularly cleaved" and "intracellular cleavage" refer to a metabolic process or response inside a cell to an antibody construct-proteolytic agent conjugate, thereby The covalent attachment between the proteolytic agent (B) and the antibody construct (Ab), eg, the linker, is broken, resulting in free drug or other metabolites of the conjugate separated from the intracellular antibody.

In addition, the cleavable linker is pH-sensitive, i.e., can be readily hydrolyzed at certain pH values. In general, the pH-sensitive linker can be hydrolyzed under acidic conditions. For example, acid-labile linkers capable of being hydrolyzed in lysosomes (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconic amide (cis) -aconitic amide), orthoesters, acetals, ketals, etc.) can be used (e.g., U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83). :67-123; see Neville et al., 1989, Biol. Chem. 264:14653-14661). These linkers are relatively stable at neutral pH conditions, such as blood, but are unstable below pH 5.5 or 5.0, the approximate pH of lysosomes. Examples of hydrolysable linkers include thioether linkers (eg, thioethers attached to a therapeutic agent via an acylhydrazone bond (see, eg, US Pat. No. 5,622,929)).

In addition, the linker is cleavable under reducing conditions (eg, a disulfide linker). For example, SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl- Formed using 3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-thio)toluene)-, SPDB and SMPT A variety of disulfide linkers are known, including those capable of being (see, eg, Thorpe et al., 1987, Cancer Res. 47:5924-5931; US Pat. No. 4,880,935).

In addition, linkers include malonate linkers (Johnson et al., 1995, Anticancer Res. 15:1387-93), maleimidobenzoyl linkers (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299- 1304), 3'-N-amide analogs (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12), beta-glucuronide linker (Jeffery et al) ., 2006, Bioconjug Chem. 17(3):832-40), or a beta-galactoside linker (Kolodych et al., 2017, Eur J Med Chem. Dec 15;142:376-382) ) can be

The non-cleavable linker may be a maleimidocaproyl linker. The maleimidocaproyl linker may comprise N-maleimidomethylcyclohexane-1-carboxylate. The maleimidocaproyl linker may contain a succinimide group. The maleimidocaproyl linker may contain a pentafluorophenyl group. The linker may be a combination of a maleimide group and one or more polyethylene glycol molecules. The linker may be a combination of a maleimidocaproyl group and one or more polyethylene glycol molecules. The linker may be a maleimide-PEG4 linker. The linker may be a combination of a maleimidocaproyl linker containing a succinimide group and one or more polyethylene glycol molecules. The linker may be a combination of a maleimidocaproyl linker containing a pentafluorophenyl group and one or more polyethylene glycol molecules. The linker may contain a maleimide linked to a polyethylene glycol molecule, wherein the polyethylene glycol allows for more linker flexibility or may use a longer linker. The linker may be a (maleimidocaproyl)-(valine-citrulline)-(para-aminobenzyloxycarbonyl) linker.

In one aspect of the invention, the linker may be a cleavable linker.

In one aspect of the invention, the linker is a protease cleavable linker, an acid-cleavable linker, a disulfide linker, a self-immolative linker or a self-stabilizing linker, a malonate linker, maleimidobenzoyl linker, 3'-N-amide analog, beta-glucuronide linker, or beta-galactoside linker.

In one embodiment of the present invention, the protease cleavable linker may include a thiolreactive spacer or a dipeptide, and more specifically, the protease cleavable linker is a thiol-reactive maleimidocaproyl spacer, valine-citrulline dipeptide or p-amino-benzyloxycarbonyl spacer.

In one embodiment of the present invention, the acid-cleavable linker may be a hydrazine linker or a quaternary ammonium linker.

In one embodiment of the present invention, the linker may have a structure of Formula II.

[General Formula Ⅱ]

In the above formula,

The tilde indicates a site linked to Ab, * indicates a site linked to a proteolytic agent;

이고, 상기 R³은 수소 또는 카복실 보호기이며, 상기 각각의 R⁴는 독립적으로 수소 또는 하이드록실 보호기이고;G is a glucuronic acid moiety or

, wherein R ³ is hydrogen or a carboxyl protecting group, and each R ⁴ is independently hydrogen or a hydroxyl protecting group;

R ¹ and R ² are each independently hydrogen, C _1-8 alkyl or C _3-8 cycloalkyl;

W is -C(O)-, -C(O)NR'-, -C(O)O-, -SO ₂ NR'-, -P(O)R''NR'-, -SONR'- or -PO ₂ NR'-, wherein C, S or P is directly bonded to a phenyl ring, and R' and R'' are each independently hydrogen, C _1-8 alkyl, C _3-8 cycloalkyl, C _1-8 alkoxy, C _1-8 alkylthio, mono- or di- C _1-8 alkylamino, C _3-20 heteroaryl or C _6-20 aryl;

Z is independently C _1-8 alkyl, halogen, cyano or nitro;

n is an integer from 0 to 3;

L is a covalent linker.

In one aspect of the present invention, the linker may have a structure of the following formula (IIa):

[Formula IIa]

In the above formula,

G is a sugar, sugar acid, or sugar derivatives,

W is -C(O)-, -C(O)NR'-, -C(O)O-, -S(O) ₂ NR'-, -P(O)R''NR'-, -S (O)NR'-, or -PO ₂ NR'-,

When C(O), S, or P is directly connected to a phenyl ring, R' and R'' are each independently hydrogen, C _1-8 alkyl, C _3-8 cycloalkyl, C _{1 - 8} alkoxy, C _1-8 alkylthio, mono- or di-C _1-8 alkylamino, C _3-20 heteroaryl, or C _6-20 aryl;

each Z is independently hydrogen, C _1-8 alkyl, halogen, cyano or nitro;

n is an integer from 0 to 3,

m is 0 or 1,

L is the same as defined above,

R ₁ and R ₂ are each independently hydrogen, C _1-8 alkyl or C _3-8 cycloalkyl, or

R ₁ and R ₂ together with the carbon atom to which they are attached form a C _3-8 cycloalkyl ring,

In the above formula, the tilde indicates a site connected to Ab,

* indicates a site connected to a proteolytic agent.

In one aspect of the present invention, the sugar or sugar acid is a monosaccharide.

In one embodiment of the present invention, G is a glucuronic acid moiety or a compound of formula (IIIa):

[Formula (IIIa)]

From here,

R ³ is hydrogen or a carboxyl protecting group,

each R ⁴ is each independently hydrogen or a hydroxyl protecting group.

In one aspect of the present invention, R ³ is hydrogen, and each R ⁴ is hydrogen.

Further, in one embodiment of the present invention, R ¹ and R ² are each hydrogen.

Further, in one embodiment of the present invention, each Z is independently C _1-8 alkyl, halogen, cyano or nitro.

In one embodiment of the present invention, W is -C(O)-, -C(O)NR'-, -C(O)O-, -SO ₂ NR'-, -P(O)R''NR '-, -SONR'- or -PO ₂ NR'-, wherein C, S or P is directly bonded to a phenyl ring, and R' and R'' are each independently hydrogen, C _{1 -8} alkyl, C _3-8 cycloalkyl, C _1-8 alkoxy, C _1-8 alkylthio, mono- or di- C _1-8 alkylamino, C _3-20 heteroaryl or C _6-20 aryl.

Further, in one embodiment of the present invention, W is -C(O)-, -C(O)NR'- or -C(O)O-, and more specifically, W is -C(O)NR' -, where C(O) is connected to a phenyl ring and NR' is bonded to L.

Further, in one embodiment of the present invention, W is -C(O)NR'-, and the nitrogen of W is a nitrogen atom of a hydrophilic amino acid.

Further, in one embodiment of the present invention, W is -C(O)NR'-, and the nitrogen of W is the nitrogen atom of the N-terminal amino acid of the peptide. In addition, the peptide may include 2 to 20 amino acids.

Further, in one embodiment of the present invention, n is 0, 1, 2 or 3, more specifically 0, 1 or 2, and even more specifically 0.

More specifically, G is a compound of formula (IIIa):

[Formula (IIIa)]

From here,

R ³ is hydrogen or a carboxyl protecting group,

each R ⁴ is each independently hydrogen or a hydroxyl protecting group,

W is -C(O)NR'-, wherein C(O) is connected to a phenyl ring and NR' is bonded to L, each Z is C _1-8 alkyl, halogen, cyano or nitro, n is 0, m is 1, and R ₁ and R ₂ are each hydrogen.

In one embodiment of the present invention, L is an unbranched linker or a branched linker.

In one aspect of the invention, at least one L is alkylene having 1 to 100 carbon atoms, wherein the carbon atoms of the alkylene are one or more heteroatoms selected from the group consisting of N, O and S may be substituted, and the alkylene may be further substituted with one or more alkyl having 1 to 20 carbon atoms.

In one aspect of the invention, L is any one of:

A) C _1-50 alkylene or 1-50 membered heteroalkylene, satisfying one or more of the following:

(i) L contains one or more unsaturated bonds;

(ii) two atoms in L are substituted with a divalent substituent, such as a substituent, completing heteroarylene;

(iii) L is 1 to 50 membered heteroalkylene;

(iv) said alkylene is substituted with one or more C _1-20 alkyl;

B) at least one isoprenyl derivative unit of the general formula III, which can be recognized by an isoprenoid transferase:

[General Formula Ⅲ]

Specifically, at least one L is C _1-50 alkylene or 1-50 membered heteroalkylene, and may satisfy one or more of the following:

(i) contains one or more unsaturated bonds;

(ii) two atoms in L are substituted with a divalent substituent, such as a substituent; This completes heteroarylene,

(iii) L is 1 to 50 membered heteroalkylene;

(iv) said alkylene is substituted with one or more C _1-20 alkyl.

Also, the at least one L is a nitrogen-containing 1-50 membered heteroalkylene, the linker comprises two or more atoms of a hydrophilic amino acid, and the nitrogen forms a peptide bond with the carbonyl of the hydrophilic amino acid.

In one embodiment of the present invention, L is covalently bonded to the antibody through a thioether bond, and the thioether bond includes a sulfur atom of cysteine of the antibody.

In one aspect of the invention, L is C _2-50 alkylene, C _2-50 heteroalkylene, hydrophilic amino acid, -C(O)-, -C(O)NR''-, -C(O)O -, -(CH ₂ ) _s -NHC(O)-(CH ₂ ) _t -, -(CH ₂ ) _u -C(O)NH-(CH ₂ ) _v -, -(CH ₂ ) _s -NHC( O)-(CH ₂ ) _t -C(O)-, -(CH ₂ ) _u -C(O)NH-(CH ₂ ) _v -C(O)-, -S(O) ₂ NR''- , -P(O)R'''NR''-, -S(O)NR''-, or -PO ₂ NR''-,

wherein R'' and R''' are each independently hydrogen, C _1-8 alkyl, C _3-8 cycloalkyl, C _1-8 alkoxy, C _1-8 alkylthio, mono- or di-C _{1 -8} alkylamino, C _3-20 heteroaryl or C _5-20 aryl, and s, t, u and v are each independently an integer from 0 to 10.

In one aspect of the invention, at least one L is a hydrophilic amino acid.

In one aspect of the present invention, the hydrophilic amino acid may be arginine, aspartate, asparagine, glutamate, glutamine, histidine, lysine, ornithine, proline, serine, or threonine.

In one aspect of the present invention, the hydrophilic amino acid may be an amino acid comprising a side chain having a residue having a charge at neutral pH in aqueous solution.

In one aspect of the invention, the hydrophilic amino acid is aspartate or glutamate.

In one aspect of the invention, the hydrophilic amino acid is ornithine or lysine.

In one aspect of the invention, the hydrophilic amino acid is arginine.

In one aspect of the invention, at least one L is -C(O)-, -C(O)NR''-, -C(O)O-, -S(O) ₂ NR''-, -P (O)R'''NR''-, -S(O)NR''-, or -PO ₂ NR''-, wherein R'' and R''' are each independently hydrogen, C _1-8 alkyl, C _3-8 cycloalkyl, C _1-8 alkoxy, C _1-8 alkylthio, mono- or di-C _1-8 alkylamino, C _3-20 heteroaryl, or C _6-20 aryl.

In one aspect of the invention, at least one L is -C(O)NR''-, -(CH ₂ ) _s -NHC(O)-(CH ₂ ) _t -, -(CH ₂ ) _u -C( O)NH-(CH ₂ ) _v -, -(CH ₂ ) _s -NHC(O)-(CH ₂ ) _t -C(O)-, or -(CH ₂ ) _u -C(O)NH-( CH ₂ ) _v —C(O)—, wherein R′′ is hydrogen, C _1-5 alkyl, C _3-8 cycloalkyl, C _1-5 alkoxy, C _3-20 heteroaryl, or C _{6 -20} aryl, and s, tu and v are each independently an integer from 0 to 10.

In one aspect of the invention, at least one L is -C(O)NR'- and R' is hydrogen.

In one aspect of the present invention, L may further include a connection unit.

In one aspect of the invention, the at least one linking unit is represented by the general formula VIII or IX:

[General Formula VIII]

-(CH ₂ ) _r (V(CH ₂ ) _p ) _q -

[General Formula IX]

-(CH ₂ CH ₂ X) _w -

V is a single bond, -O-, -S-, -NR ²¹ -, -C(O)NR ²² -, -NR ²³ C(O)-, -NR ²⁴ SO ₂ - or -SO ₂ NR ²⁵ - ego;

X is —O—, C ₁ -C ₈ alkylene or —NR ²¹ —;

R ²¹ to R ²⁵ are each independently hydrogen, C _1-6 alkyl, C _1-6 alkyl - C _6-20 aryl or C _1-6 alkyl - C _3-20 heteroaryl;

r is an integer from 0 to 10;

p is an integer from 0 to 10;

q is an integer from 1 to 20; and

w is an integer from 1 to 20;

In one aspect of the present invention, q may be 4 to 20, more specifically 6 to 20. Also, q may be 1 to 10 or 2 to 12, and more specifically 2, 5, or 11. Also, r may be 1 or 2. Also, p may be 1 or 2. Also, V may be -O-.

More specifically, r may be 2, p may be 2, q may be 2, 5 or 11, and V may be -O-.

Further, in one aspect of the present invention, X may be -O-. Also, w may be an integer of 1 to 10 or 6 to 20.

More specifically, X is -O-, and w may be 1-10 or 6-20.

또는

의 구조를 가지며, 여기서 n은 1 내지 12이다.In one aspect of the invention, the at least one linking unit is at least one polyethylene glycol unit,

or

has the structure of , where n is 1 to 12.

In one aspect of the invention, the at least one linking unit is 1 to 12 -OCH ₂ CH ₂ -units, or 3 to 12 -OCH ₂ CH ₂ -units, or 5 to 12 -OCH ₂ CH ₂ -units, or 6 to 12 -OCH ₂ CH ₂ -unit, or 3 -OCH ₂ CH ₂ -unit.

In one aspect of the invention, at least one linking unit is -(CH ₂ CH ₂ X) _w -,

wherein X is a single bond, —O—, C _1-8 alkylene, or —NR ₂₁ —;

R ₂₁ is hydrogen, C _1-6 alkyl, C _1-6 alkyl-C _6-20 aryl, or C _1-6 alkyl-C _3-20 heteroaryl, w is an integer from 1 to 20, specifically 1, 3, 6, or 12.

In one embodiment of the present invention, X is -O-, and w is an integer from 6 to 20.

In one aspect of the invention, the linker is 1,3-dipolar cycloaddition reactions, hetero-Diels-Alder reactions, nucleophilic substitution reactions, Bonding units formed by non-aldol type carbonyl reactions, addition to carbon-carbon multiple bond, oxidation reactions or click reactions further includes.

In one aspect of the invention, the binding unit is formed by a reaction between an alkynyl group and an azide, or between an aldehyde or ketone group and a hydrazine or alkoxyamine.

In one aspect of the present invention, the binding unit is represented by the following general formula IV _a , IV _b , IV _c , IV _d or IV _e .

[General formula Ⅳ _a ]

[General formula IV _b ]

,

,

[General formula Ⅳ _c ]

,

,

[General formula Ⅳ _d ]

[General formula IVe]

From here,

L ₁ and L ₂ are a single bond or alkylene having 1 to 30 carbon atoms,

R ₁₁ is hydrogen or alkyl having 1 to 10 carbon atoms, specifically methyl.

In one aspect of the invention, L ₁ is a single bond, or alkylene having 11 carbon atoms, or alkylene having 12 carbon atoms.

Also, in one aspect of the present invention, the binding unit is

또는

을 포함하고,

or

including,

V is a single bond , -O-, -S-, -NR ²¹ -, -C(O)NR ²² -, -NR ²³ C(O)-, -NR ²⁴ SO ₂ -, or -SO ₂ NR ²⁵ - is;

R ²¹ to R ²⁵ are independently hydrogen, C _1-6 alkyl, C _1-6 alkyl - C _6-20 aryl, or C _1-6 alkyl - C _3-20 heteroaryl;

r is an integer from 1 to 10;

p is an integer from 0 to 10;

q is an integer from 1 to 20; and

L ¹ is a single bond.

In one aspect of the present invention, r may be 2 or 3. Also, p may be 1 or 2. Also, q may be 1 to 6.

More specifically, in the binding unit, r is 2 or 3; p is 1 or 2; q may be 1 to 6.

In addition, in one aspect of the present invention, the linker comprising the binding unit is

,

,

, 또는

일 수 있고, 여기에서 물결표시는 Ab와 연결되는 부위, * 표시는 단백질 분해제와 연결되는 부위를 나타내며, n은 0 내지 20의 정수이다.

,

,

, or

, wherein the tilde indicates a site connected to Ab, * indicates a site connected to a proteolytic agent, and n is an integer from 0 to 20.

또는

을 포함한다.In one aspect of the present invention, L is

or

includes

를 추가로 포함한다. 또한, 상기 구조에서 알키닐 및 아자이드 사이의 반응, 상세하게는 클릭 반응에 의해 결합 유닛을 형성할 수 있다.In addition, in one aspect of the present invention, L is

further includes. In addition, in the above structure, a bonding unit may be formed by a reaction between an alkynyl and an azide, specifically, a click reaction.

를 포함하고, 상기 구조를 통해 결합 유닛을 형성하며, 결합 유닛은

를 포함한다.In a specific aspect of the present invention, L is

comprising, forming a coupling unit through the structure, the coupling unit comprising:

includes

의 구조를 갖는 최소 하나 이상의 이소프레닐 유닛을 추가로 함유할 수 있으며, 여기에서 n은 최소 2 이상이다.In addition, in one aspect of the present invention, the linker is

It may further contain at least one isoprenyl unit having the structure of , wherein n is at least 2 or more.

In one aspect of the invention, the at least one isoprenyl unit is a substrate of an isoprenoid transferase or a product of an isoprenoid transferase.

In one embodiment of the present invention, the isoprenyl unit of the linker is covalently bonded to Ab by a thioether bond, wherein the thioether bond comprises the sulfur atom of the cysteine of Ab.

In one embodiment of the present invention, L is covalently bonded to Ab through a thioether bond, and the thioether bond includes a sulfur atom of cysteine of Ab.

Further, in one embodiment of the present invention, L includes at least one isoprenyl derivative unit of the following general formula III, which can be recognized by isoprenoid transferase.

[General Formula Ⅲ]

.

.

In addition, the isoprenyl unit can covalently bond the oxime included in the linker to the Ab.

In one aspect of the present invention, L is 3 to 50 heteroalkylene comprising an oxime,

the oxygen atom of the oxime is on the side of L linked to W and the carbon atom of the oxime is on the side of L linked with Ab; Alternatively, the carbon atom of the oxime may be on the side of L connected to W, and the oxygen atom of the oxime may be on the side of L connected to Ab.

In one embodiment of the present invention, L comprises an oxime, and at least one isoprenyl unit covalently bonds the oxime to Ab.

In one embodiment of the present invention, Ab comprises an amino acid motif recognized by isoprenoid transferase, and the thioether bond comprises a sulfur atom of a cysteine of the amino acid motif. In addition, an amino acid motif is included at the C-terminus of the Ab.

In one aspect of the invention, the amino acid motif is a sequence selected from the group consisting of CXX, CXC, XCXC, XXCC, and CYYX, wherein C represents cysteine; Y at each occurrence independently represents an aliphatic amino acid; X independently for each occurrence represents glutamine, glutamate, serine, cysteine, methionine, alanine, or leucine; The thioether linkage contains the sulfur atom of the cysteine of the amino acid motif.

In one aspect of the invention, the amino acid motif is the sequence CYYX, and Y for each occurrence is independently alanine, isoleucine, leucine, methionine or valine.

In one aspect of the invention, the amino acid motif is a CVIM or CVLL sequence.

In one aspect of the present invention, one or more of the 1 to 20 amino acids preceding the amino acid motif may each be independently selected from glycine, arginine, aspartic acid and serine. For example, in one aspect of the invention, at least one of the seven amino acids preceding the amino acid motif is glycine. Alternatively, at least three of the seven amino acids preceding the amino acid motif are each independently selected from glycine, arginine, aspartic acid and serine. Alternatively, 1 to 10 amino acids preceding the amino acid motif are glycine, specifically, at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids preceding the amino acid motif are glycine.

In one aspect of the present invention, Ab may comprise the amino acid sequence of GGGGGGGCVIM.

In one aspect of the invention, the Linker comprises one or more branched linkers covalently bonded to the Ab,

i) each branched linker comprises a branched unit covalently linked to the Ab by a first linker (PL);

ii) each branched linker comprises a first branch (B1) wherein the first proteolytic agent is covalently linked to the branched unit by a second linker (SL) and a cleavage group (CG); and

iii) each branched linker comprises: a) a second branch (B2) in which a second proteolytic agent is covalently linked to the branched unit by a second linker (SL) and a cleaving group (CG); or b) a second branch in which the polyethylene glycol moiety is covalently bonded to the branched unit,

Each of the above cleaving groups may be hydrolyzed to release the proteolytic agent from the antigen-specific binding moiety-proteolytic agent conjugate.

In one aspect of the present invention, said at least one linker is a nitrogen-containing 1-100 membered heteroalkylene, and the linker comprises at least two atoms of a hydrophilic amino acid, wherein said nitrogen forms a peptide bond with a carbonyl of a hydrophilic amino acid. .

,

,

또는

이고,In one aspect of the present invention, the branched unit comprises

,

,

or

ego,

The L ² , L ³ , L ⁴ are each independently a direct bond or -C _n H _2n -, wherein n is an integer from 1 to 30,

,

,

또는

이며,The G ¹ , G ² , G ³ are independently a direct bond,

,

,

or

is,

wherein R ³⁰ is hydrogen or C _1-30 alkyl,

R ⁴⁰ is a direct bond, C _1-10 alkyl or L ⁵ -COOR ₆ ;

L ⁵ is a direct bond or -C _n' H _2n' -, where n' is an integer from 1 to 10, and R ₆ is hydrogen or C _1-30 alkyl.

In one aspect of the invention, the cleavage group is cleavable in a target cell.

In one aspect of the invention, the cleavage group is capable of releasing one or more active agents.

In one aspect of the present invention, the proteolytic agent is bound to the first branch or the second branch by a cleaving group having the formula:

(In the above formula, each part is as defined in formula II.)

In one aspect of the present invention, said first linker comprises an alkylene having 1 to 100, preferably 1 to 50 carbon atoms;

alkylene contains at least one unsaturated bond; alkylene includes at least one heteroarylene; a carbon atom of the alkylene is replaced by one or more heteroatoms selected from nitrogen (N), oxygen (O), and sulfur (S); or

Alkylene is further substituted with one or more alkyl having 1 to 20 carbon atoms.

In one embodiment of the present invention, the first linker or the second linker has the structure -(CH ₂ ) _r (V(CH ₂ ) _p ) _q -, -((CH ₂ ) _p V) _q -,-(CH ₂ ) ) _r (V(CH ₂ ) _p ) _q Y-, -((CH ₂ ) _p V) _q (CH ₂ ) _r -, -Y((CH ₂ ) _p V) _q - or -(CH ₂ ) _r (V(CH ₂ ) _p ) _q YCH ₂ -, wherein r is an integer from 0 to 10; p is an integer from 1 to 10; q is an integer from 1 to 20;

V and Y are each independently a single bond, -O-, -S-, -NR ²¹ -, -C(O)NR ²² -, -NR ²³ C(O)-, -NR ²⁴ SO ₂ -, or - SO ₂ NR ²⁵ -; R ²¹ to R ²⁵ are each independently hydrogen, C _1-6 alkyl, C _1-6 alkyl-C _6-20 aryl or C _1-6 alkyl-C _3-20 heteroaryl. In one embodiment, each The branched linker comprises a branching unit, each active agent being linked to the branching unit via a second linker, and the branching unit being linked to the antibody by a first linker. In embodiments where the Branching Unit is an amide, the first linker may comprise the carbonyl of the amide, or the second linker may comprise the carbonyl of the amide.

In one aspect of the present invention, [Linker - (B) _m ] comprising the branched linker is

이고,

ego,

wherein B and B' are proteolytic agents, which may be the same or different;

n1 and n2 each independently represent an integer of 1 to 20;

L' represents a portion of L capable of binding to Ab.

이다.In a specific aspect of the present invention, L' is

to be.

In one aspect of the invention, the conjugate comprises Ab;

1, 2, 3 or 4 or more branched linkers covalently attached to the Ab;

The branched linker comprises one or two or more proteolytic agents.

In one aspect of the invention, L comprises an oxime and at least one polyethylene glycol unit covalently bonds the oxime to the proteolytic agent.

In one aspect of the present invention, the cleaving group is capable of cleaving in a target cell and releasing one or more proteolytic agents.

In one aspect of the invention, it comprises at least one branched linker covalently attached to the Ab; and two or more proteolytic agents covalently linked to a branched linker.

Specifically, one branched linker may be bound to Ab.

In addition, two or more branched linkers may be bound to Ab, and each branched linker may be bound to two or more proteolytic agents. More specifically, three branched linkers may be bound to Ab. Alternatively, four branched linkers may be bound to Ab.

In one aspect of the invention, each branched linker may be associated with two or more identical or different proteolytic agents. In one aspect of the invention, each proteolytic agent may be bound to a branched linker by a cleavable bond.

In one aspect of the present invention, each branched linker comprises a branched unit, each proteolytic agent binds to the branched unit via a secondary linker and binds to the Ab by the branched unit primary linker can be

In one aspect of the present invention, the branched unit may be a nitrogen atom. Alternatively, the branched unit may be an amide, and the primary linker or secondary linker may include a carbonyl of the amide. Alternatively, the branched unit may be a lysine unit.

In one embodiment of the present invention, the Binding Unit is covalently bound to Ab by means of a thioether bond, wherein the thioether bond comprises the sulfur atom of the cysteine of Ab.

In one embodiment of the present invention, Ab comprises an amino acid motif recognized by isoprenoid transferase, and the thioether bond comprises a sulfur atom of a cysteine of the amino acid motif.

In one embodiment of the present invention, the isoprenoid transferase is farnesyl protein transferase (FTase) or geranylgeranyl transferase (GGTase).

In one aspect of the invention, the amino acid motif is a sequence selected from the group consisting of CXX, CXC, XCXC, XXCC, and CYYX, wherein C represents cysteine; Y at each occurrence independently represents an aliphatic amino acid; X independently for each occurrence represents glutamine, glutamate, serine, cysteine, methionine, alanine, or leucine; The thioether linkage contains the sulfur atom of the cysteine of the amino acid motif.

In one aspect of the invention, the amino acid motif is the sequence CYYX, and Y for each occurrence is independently alanine, isoleucine, leucine, methionine or valine.

In one aspect of the invention, the amino acid motif is a CVIM or CVLL sequence.

In one aspect of the invention, at least one of the seven amino acids preceding the amino acid motif is glycine.

In one aspect of the invention, at least three of the seven amino acids preceding the amino acid motif may each be independently selected from glycine, arginine, aspartic acid and serine.

In one aspect of the invention, 1 to 10 amino acids preceding the amino acid motif are glycine, specifically at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids preceding the amino acid motif is glycine.

In one aspect of the present invention, Ab may comprise the amino acid sequence of GGGGGGGCVIM.

As used herein, the term “proteolytic agent” refers to a low-molecular compound that induces target protein degradation, and may also be referred to as “protein eraser”. Alternatively, as used herein, the term “proteolytic agent” refers to a low-molecular compound that induces modulation of a target protein, and may be referred to as a “protein modulator”. Here, the target protein is, for example, a disease-causing protein.

As used herein, "proteolytic agent moiety" refers to a compound covalently bound to all or part of a linker, including all or part of a proteolytic agent. The proteolytic agent moiety does not necessarily include a linker, and may mean a case in which all or part of the linker is not included.

In the present invention, the proteolytic agent moiety may be directly bound to the linker, and one or more, specifically, two or more, three or more, four or more proteolytic agent moieties to the linker may be directly bound to the linker. .

In addition, in the present invention, the proteolytic agent moiety may include a compound unit for directly binding the proteolytic agent to the linker. In one example, the proteolytic agent moiety may refer to the whole including the following structure and proteolytic agent, through which the proteolytic agent and the linker may be directly bonded. However, this is illustrative and not limited thereto.

(Here, each part has the same definition as above)

When a proteolytic agent acts as a small molecule compound that induces the regulation of a target protein, it can be divided into two types according to the mechanism of action (MoA). Specifically, a monovalent modulator that binds only to a target protein and regulates the activity of the target protein, and a PPI modulator that directly binds to both the protein involved in the regulation of the target protein and the target protein to regulate the activity of the target protein (protein- protein interaction modulator). Here, the target protein is a native substrate or a non-native substrate of a protein involved in the regulation of the target protein. The intrinsic substrate is an endogenous substrate specific to a protein, and the PPI modulator directly binds to both the protein involved in the regulation of the target protein and its intrinsic substrate, and the protein involved in the regulation of the target protein and its intrinsic substrate can regulate the activity of the substrate by stabilizing the interaction of A non-native substrate is a substrate that is recruited by an exogenous ligand, and the non-native substrate may also be referred to as a “neosubstrate”. The PPI modulator may act as an exogenous ligand of a protein, and the non-native substrate may bind to the binding site of the protein involved in the regulation of the PPI modulator and the target protein, thereby modulating the activity of the non-native substrate.

When a proteolytic agent acts as a low-molecular compound that induces target protein degradation, it can be divided into three types according to the mechanism of action. Specifically, it can be divided into a bivalent degrader, a monovalent degrader, and a molecular glue degrader.

Intracellular protein degradation occurs through two pathways by lysosomes and proteasomes. Most (80%) of cellular proteins are labeled with ubiquitin and then degraded in the cytoplasm and nucleus by proteasome, a process called ubiquitin-proteasome system.

A series of enzyme systems (E1 ubiquitin-activating enzyme), E2 ubiquitin-conjugating enzyme (E2 ubiquitin-conjugating enzyme), E3 ubiquitin linking Enzyme (E3 ubiquitin ligase) is involved, and the labeled protein is degraded by the ATP-dependent protease complex, 26S proteasome.The binding of ubiquitin to the substrate is between Lys of the substrate molecule and Gly at the ubiquitin-C-terminus. Another ubiquitin can be linked to the ubiquitin-C-terminal Lys bound to the substrate protein, and by repeating this process, several ubiquitin molecules are When they are linked together to form a polyubiquitin chain, the protein is recognized and selectively degraded by the 26S proteasome.

More than 600 types of E3 ubiquitin ligase are known to humans and provide substrate specificity for recognizing substrate proteins to be labeled with ubiquitin by binding to both E2 ubiquitin ligases and substrate proteins. That is, the selection of the protein to be degraded is determined by the E3 ubiquitin ligase in the ubiquitination process, and the substrate protein has a recognition site by the E3 ubiquitin ligase and a ubiquitin linking site.

A bivalent degrader is a heterobifunctional small molecule in which a ligand specific for a target protein and a ligand specific for the E3 ubiquitin ligase involved in the above process are linked with a linker. It is termed "proteolysis targeting chimera" or "PROTAC". Accordingly, the bivalent cleavage agent includes a ligand specific for the target protein and induces degradation of the target protein by locating the target protein, not the substrate protein, near the E3 ubiquitin ligase.

A monovalent degrader is a small molecule that binds only to a target protein and induces degradation of the bound target protein. The monovalent lysing agent binds to the target protein and induces degradation thereof by changing the target protein to be easily recognized by the ubiquitin-proteasome system. For example, it binds to a target protein and causes a conformation change or folding change of the target protein, or post-translational modification (PTM) of the target protein, or the target protein -Inducing degradation of target proteins by altering protein-protein interactions such as chaperone interactions (Cornella-Taracido et al., Bioorganic & Medicinal Chemistry Letters 30 (2020) 127202).

The target protein of the monovalent degradation agent may be, for example, an estrogen receptor (ER) or an androgen receptor (AR), but is not limited thereto.

More specifically, the monovalent degradation agent that binds to the ER and induces its degradation may include a selective estrogen receptor degrader (SERD), and examples of the SERD include fulvestrant, brilanestrant (GDC-0810), GDC-0927, elacestrant, giredestrant, amcenestrant (SAR439859), AZD9833, AZD9496, rintodestrant, LSZ102, LY3484356, ZN-c5, D-0502 or SHR9549. In addition, monovalent degradation agents that bind to AR and induce its degradation include selective androgen receptor degrader (SARD), and examples of SARD include bicalutamide, dimethylcurcumin (ASC-J9), AZD-3514 or ARN-509. , but is not limited thereto.

The molecular adhesive degrading agent may exhibit activity through binding to E3 ubiquitin ligase and/or a target protein. For example, the molecular adhesive degrading agent may modify the structure of the ligase or the like through binding to the ligase and the like, and induce protein degradation by inducing binding of a target protein to the modified structure. Molecular adhesive degrading agent is a small molecule that binds to the substrate protein of E3 ubiquitin ligase and induces substrate proteolysis of E3 ubiquitin ligase, and may also be referred to as "molecular glue". Here, the substrate of the E3 ubiquitin ligase as the target protein may specifically be a native substrate or a non-native substrate of the E3 ubiquitin ligase.

The native substrate of E3 ubiquitin ligase is an endogenous substrate specific for E3 ubiquitin ligase, and the molecular adhesive degrading agent binds to E3 ubiquitin ligase and its native substrate to form E3 ubiquitin ligase and E3 ubiquitin ligase and its native substrate. It can induce substrate degradation by stabilizing its intrinsic substrate interaction.

A non-native substrate of E3 ubiquitin ligase is a substrate that is recruited by an exogenous ligand, and the non-native substrate may also be referred to as a “neosubstrate”. Molecular adhesive disintegrants act as exogenous ligands of E3 ubiquitin ligase. Accordingly, the non-native substrate may be bound to the binding site of the molecular adhesive degrading agent and the E3 ubiquitin ligase to enhance the bond between the E3 ubiquitin ligase and the non-native substrate and induce degradation of the non-native substrate. More specifically, as shown in the schematic diagram below, when the molecular adhesive degrading agent binds to the binding site of the E3 ubiquitin ligase, the structure of the E3 ubiquitin ligase is modified so that a non-native substrate can bind, and the modified structure is non- Intrinsic substrate binding can enhance binding between E3 ubiquitin ligase and non-native substrate and induce degradation of non-native substrate (Essays Biochem. 2017 Nov 8;61(5):505-516) .

Examples of E3 ubiquitin ligases to which molecular adhesive breakers bind are von Hippel-Lindau (VHL), cereblon, XIAP, XIAP/cIAP, E3A, MDM2. cullin ring ubiquitin ligase (CRL), including APC (Anaphase-promoting complex), APC/cdc20, APC/cdh1, Cul1-based, Cul2/5-based, Cul3-based, Cul4 based, Cul7-based and Cul7, UBR5 (EDD1), beta-TrCP, SOCS/BCbox/eloBC/CUL5/RING, LNXp80, LNX1, CBX4, CBL, CBLL1, C6orf157, CHFR, DTL, E6-AP, HACE1, HECTD1, HECTD2, HECTD3, HECW1, HECW2, HERC1, HERC2, HERC3, HERC4, HERC5, HUWE1, HYD, ITCH, mahogunin, MARCH-I, MARCH-II, MARCH-III, MARCH-IV, MARCH-VI, MARCH-VII, MARCH-VIII, MARCH-X, MEKK1, MIB1, MIB2, MycBP2, NEDD4, NEDD4L, PELI1, Pirh2, PJA1, PJA2, PPIL2, PRPF19, PIAS1, PIAS2, PIAS3, PIAS4, RANBP2, RFFL, RFWD2, Rictor, RNF4, RNF190 RNF20, RNF34, RNF40, RNF125, RNF128, RNF138, RNF168, RBX1, SMURF1, SMURF2, STUB1, TOPORS, TRIP12, UBE3A, UBE3B, UBE3C, UBE4A, UBE4B, UBOX5, UBR5, WWP1, WWP20/TNFA AMFR/gp78, ARA54, beta-TrCP1/BTRC, SCF/beta-TrCP, BRCA1, CHIP, CHIP/STUB1, E6;E6AP/UBE3A, F-box protein 15/FBXO15, FBXW7/Cdc4, SCF/FBW7, GRAIL/ RNF128, HOIP/RNF31, cIAP-1/HIAP-2, cIAP -2/HIAP-1, cIAP(pan), ITCH/AIP4, KAP1, MARCH8, Mind Bomb 1/MIB1, Mind Bomb 2/MIB2, MuRF1/TRIM63, NDFIP1, NleL, Parkin, RNF2, RNF4, RNF8, RNF168, RNF43, SART1, Skp2, SCF/Skp2, SHPRH, SIAH1, SIAH2, SMURF1, SMURF2, TOPORS, TRAF-1, TRAF-2, TRAF-3, TRAF-4, TRAF-5, TRAF-6, TRAF-7, TRIM5, TRIM21, TRIM32, TRIM63, UBE3B, UBE3C, UBR1, UBR2, UHRF2, WWP1, WWP2, ZNRF1 or ZNRF3.

In addition, the E3 ubiquitin ligase to which the molecular adhesive degrading agent binds may be divided by type, which may be described in PCT Publication No. WO2017/201449, but is not limited thereto.

In addition, examples of native substrates or non-native substrates of E3 ubiquitin ligase to which molecular adhesive degrading agents bind are NKX3.1, β-Catenin, aiolos, Akt1, Aurora A, B7-2, Bim, Caspase-3, Caspase -8, Caspase-10, CD147, Cdc20, Cdc25A, CDH1, CHK1, CHK2, c-Jun, CK1-α, Claspin, C-Myc, CRY2, Cyclin A, Cyclin B, Cyclin E, CDK12/Cyclin K, Delta , Jagged, Dlg, DR4, DVL1, ELF, ErbB4, Erk, Fbxo45, Foxo1, Foxp3, gp190, GSPT1, GSPT2, H2A,H2AX, HIF-1a, HIF-1α, HIF-2a, HIPK2, Histone H2A, Histone H2B , HSF1, HSP70/90, ikaros, IKZF1/3, IL-1β, IL-6, iNOS, IRAK, IRS1 IκBα, JAMP, KAI1, KLFs LRRK2, Mad2, Mcl-1, MDM2, MED13, MEDL, MEIS2, MHC class I, MHC class II, MKK4, MTA1, MyBP-C, MyLC1/2, NEMO, NF-κB, N-Myc, Nrf2, NOTCH, NUMB, p21, p27, p53, p73, P-gp paxillin, PCNA, PCNP, PHD1/3, PLK1, Rbm39, Rbm23, RIP2, RhoA, RNF8, Runx1, SALL4, Securin, Skp2, Skp2-CKS1, SGK1, SMAC, Smad2, Smads, SOD1 and variants thereof, TCF/LEF, TNF-α , TopBP1, TP53, TRAF2, TRB3, TRIP, Troponin I, TSC2, UCK1, Wee1, WNK1 or WNK4, but are not limited thereto.

Specifically, the native substrate or non-native substrate of AMFR/gp78 as E3 ubiquitin ligase may be KAI1, and the native substrate or non-native substrate of APC is Aurora A, Cyclin A, Cyclin B, Cdc20, Claspin, Securin or Skp2, the native substrate or non-native substrate of C6orf157 may be Cyclin B, the native substrate or non-native substrate of CBLL1 may be CDH1, the native substrate of CHFR or The non-native substrate may be Aurora A or PLK1, the native substrate or non-native substrate of CHIP may be HSP70/90, iNOS, Runx1 or LRRK2, the native substrate or non-native substrate of DTL may be p21, the native substrate or non-native substrate of E6-AP may be p53 or Dlg, the native substrate or non-native substrate of HECW1 may be a DVL1, p53 or SOD1 mutation, and the native substrate or non-native substrate of HECW2 The native or non-native substrate may be p73, the native or non-native substrate of HERC2 may be RNF8, and the native or non-native substrate of HUWE1 may be N-Myc, C- It may be Myc, p53, Mcl-1 or TopBP1, the native substrate or non-native substrate of HYD may be CHK2, the native substrate or non-native substrate of ITCH may be MKK4, RIP2 or Foxp3 , the native substrate or non-native substrate of LNX1 may be NUMB, the native substrate or non-native substrate of MARCH-IV may be MHC class I, and the native substrate or non-native substrate of MARCH-VII The antagonistic substrate may be gp190, the native substrate or non-native substrate of MARCH-VIII may be B7-2 or MHC class II, the native substrate or non-native substrate of MDM2 may be p53, The native or non-native substrate of MEKK1 is c-Jun or may be Erk, the native substrate or non-native substrate of MIB1 or MIB2 may be Delta and Jagged, the native substrate or non-native substrate of MycBP2 may be Fbxo45 or TSC2, and the native substrate or non-native substrate of NEDD4L The substrate or non-native substrate may be Smad2, the native substrate or non-native substrate of PELI1 may be TRIP or IRAK, the native substrate or non-native substrate of Pirh2 may be TP53, PJA1 The native substrate or non-native substrate of may be ELF, the native substrate or non-native substrate of PJA1 may be ELF, the native substrate or non-native substrate of RFFL may be p53, The native substrate or non-native substrate of RFWD2 may be MTA1, p53 or FoxO1, the native substrate or non-native substrate of Rictor may be SGK1, and the native substrate or non-native substrate of RNF5 is may be JAMP or paxillin, the native substrate or non-native substrate of RNF8 or RNF168 may be H2A or H2AX, the native substrate or non-native substrate protein of RNF19 may be SOD1, RNF20 or RNF40 The native substrate or non-native substrate may be Histone H2B, the native substrate or non-native substrate may be Caspase-8 or Caspase-10, and the native substrate or non-native substrate of RNF138 may be TCF/LEF, the native substrate or non-native substrate of RNF168 may be Histone H2A or H2A.X, the native substrate or non-native substrate of SCF/beta-TrCP may be IκBα, Weel, Cdc25A or β-Catenin, the native substrate or non-native substrate of SCF/FBW7 may be Cyclin E, c-Myc or c-Jun, the native substrate or non-native substrate of SCF/Skp2 is p27, p21 or Fo x01, the native substrate or non-native substrate of SHPRH may be PCNA, the native substrate or non-native substrate of SIAH1 may be β-catenin, Bim or TRB3, the native substrate of SIAH2 or the non-native substrate may be HIPK2 or PHD1/3, the native substrate or non-native substrate of SMURF1 or SMURF2 may be Smads, and the native substrate or non-native substrate protein of SMURF2 may be Mads and the native substrate or non-native substrate of TOPORS may be p53 or NKX3.1, the native substrate or non-native substrate of TRAF-6 may be NEMO or Akt1, and the native substrate of TRIM63 or the non-native substrate may be Troponin I, MyBP-C or MyLC1/2, the native substrate or non-native substrate of UBR2 may be Histone H2B, the native substrate or non-native substrate of UHRF2 may be PCNP, the native substrate or non-native substrate of VHL may be HIF-1α or HIF-2α, the native substrate or non-native substrate of WWP1 may be Erb4, and the native substrate or non-native substrate of WWP2 The substrate or non-native substrate may be Oct-4, the native substrate or non-native substrate of beta-TrCP may be β-Catenin or IκBα, the native substrate or non-native substrate of XIAP or cIAP The substrate may be Caspase-3 or SMAC, the native substrate or non-native substrate of CRL4-DCAF15 may be Rbm39 or Rbm23, the native substrate or non-native substrate of cereblon may be aiolos, ikaros, MEIS2, ILZF1/3, CK1α, GSPT1 or GSPT2, but is not limited thereto.

More specifically, the native substrate or non-native substrate of beta-TrCP with E3 ubiquitin ligase may be β-Catenin or IκBα, and the molecular adhesive degrading agent binding to beta-TrCP is NRX-1532, NRX-1933 , NRX-2663, NRX-103094, NRX-103095, NRX-252114 or NRX-252262 (Nature Communications volume 10, Article number: 1402 (2019)), but is not limited thereto.

In addition, the native substrate or non-native substrate of CRL4-DCAF15 as an E3 ubiquitin ligase may be Rbm39 or Rbm23, and the molecular adhesive degrading agent binding to CRL4-DCAF15 is indisulam, E7820 or tasisulam (tasisulam) or a derivative thereof (Nature Chemical Biology volume 16, pages7-14 (2020)), but is not limited thereto.

In addition, the native substrate or non-native substrate of cereblon with E3 ubiquitin ligase may be aiolos, ikaros, MEIS2, ILZF1/3, CK1α, GSPT1 or GSPT2, and the molecular adhesive degrading agent binding to cereblon is thalidomide (thalidomide). ), lenalidomide, pomalidomide, or an analog thereof, or a compound described in U.S. Patent Publication No. 2016/0058872 and U.S. Patent Publication No. 2015/0291562, but is not limited thereto.

In one aspect of the present invention, the proteolytic agent does not have heterobifunctionality, and may be a monovalent degradation agent or a molecular adhesive degradation agent.

In one aspect of the present invention, the E3 ubiquitin binding enzyme to which the molecular adhesive disintegrant binds is von Hippel-Lindau (VHL), cereblon, XIAP, XIAP/cIAP, E3A, MDM2. APC (Anaphase-promoting complex), APC/cdc20, APC/cdh1, CRL (cullin ring ubiquitin ligase), UBR5 (EDD1), beta-TrCP, SOCS/BCbox/eloBC/CUL5/RING, LNXp80, LNX1, CBX4, CBL , CBLL1, C6orf157, CHFR, DTL, E6-AP, HACE1, HECTD1, HECTD2, HECTD3, HECW1, HECW2, HERC1, HERC2, HERC3, HERC4, HERC5, HUWE1, HYD, ITCH, mahogunin, MARCH-II, MARCH-II , MARCH-III, MARCH-IV, MARCH-VI, MARCH-VII, MARCH-VIII, MARCH-X, MEKK1, MIB1, MIB2, MycBP2, NEDD4, NEDD4L, PELI1, Pirh2, PJA1, PJA2, PPIL2, PRPF19, PIAS1 , PIAS2, PIAS3, PIAS4, RANBP2, RFFL, RFWD2, Rictor, RNF4, RNF5, RNF8, RNF19, RNF190, RNF20, RNF34, RNF40, RNF125, RNF128, RNF138, RNF12, RBX1, SMURF1, SMURF2, STUB1, TOPORS, STUB , UBE3A, UBE3B, UBE3C, UBE4A, UBE4B, UBOX5, UBR5, WWP1, WWP2, Parkin, A20/TNFAIP3, AMFR/gp78, ARA54, beta-TrCP1/BTRC, SCF/beta-TrCP, BRCA1, CHIP, CHIP/STUB1 , E6;E6AP/UBE3A, F-box protein 15/FBXO15, FBXW7/Cdc4, SCF/FBW7, GRAIL/RNF128, HOIP/RNF31, cIAP-1/HIAP-2, cIAP-2/HIAP-1, cIAP (pan ), ITCH/AIP4, KAP1, MARCH8, Mind Bomb 1/MIB1, Mind Bomb 2/MIB2, MuRF1/TRIM63, NDFIP1, NleL, Parkin, RNF2, RNF4, RNF8, RNF168, RNF43, SART1, Skp2, SCF/Skp2, SPRH, SIAH1, SIAH2, SMURF1, SMURF2, TOPORS, TRAF-1, TRAF 2, TRAF-3, TRAF-4, TRAF-5, TRAF-6, TRAF-7, TRIM5, TRIM21, TRIM32, TRIM63, UBE3B, UBE3C, UBR1, UBR2, UHRF2, WWP1, WWP2, ZNRF1 or ZNRF3; , more specifically cereblon.

In one aspect of the present invention, the target protein to which the molecular adhesive degrading agent binds may be a native substrate or a non-native substrate of E3 ubiquitin binding enzyme, and the native substrate or non-native substrate is NKX3.1, β- Catenin, aiolos, Akt1, Aurora A, B7-2, Bim, Caspase-3, Caspase-8, Caspase-10, CD147, Cdc20, Cdc25A, CDH1, CHK1, CHK2, c-Jun, CK1-α, Claspin, C -Myc, CRY2, Cyclin A, Cyclin B, Cyclin E, CDK12/Cyclin K, Delta, Jagged, Dlg, DR4, DVL1, ELF, ErbB4, Erk, Fbxo45, Foxo1, Foxp3, gp190, GSPT1, GSPT2, H2A,H2AX , HIF-1a, HIF-1α, HIF-2a, HIPK2, Histone H2A, Histone H2B, HSF1, HSP70/90, ikaros, IKZF1/3, IL-1β, IL-6, iNOS, IRAK, IRS1 IκBα, JAMP, KAI1, KLFs LRRK2, Mad2, Mcl-1, MDM2, MED13, MEDL, MEIS2, MHC class I, MHC class II, MKK4, MTA1, MyBP-C, MyLC1/2, NEMO, NF-κB, N-Myc, Nrf2 , NOTCH, NUMB, p21, p27, p53, p73, P-gp paxillin, PCNA, PCNP, PHD1/3, PLK1, Rbm39, Rbm23, RIP2, RhoA, RNF8, Runx1, SALL4, Securin, Skp2, Skp2-CKS1, SGK1, SMAC, Smad2, Smads, SOD1 and variants thereof, TCF/LEF, TNF-α, TopBP1, TP53, TRAF2, TRB3, TRIP, Troponin I, TSC2, UCK1, Wee1, WNK1 or WNK4, more specifically It may be GSPT1 or GSPT2.

In an embodiment of the present invention, a molecular adhesive degrading agent that binds to cerelon as an E3 ubiquitin ligase and to GSPT1, a non-native substrate of E3 ubiquitin ligase as a target protein, was used.

In one aspect of the present invention, the molecular adhesive decomposing agent may be a compound represented by the following general formula X1.

[General formula X1]

In the above formula,

D ^x1 is C(=O) or CH ₂ ;

D ^x2 is O, NC≡N or NH;

a1 is an integer of 0, 1, 2 or 3;

a2 is an integer of 0 or 1;

a3 is an integer of 0, 1, or 2;

A is C, N, or O;

R ^xa is hydrogen, halo or C _1-6 alkyl;

R ^xb and R ^xc are each independently hydrogen, halo, C _1-10 alkyl, CF ₃ , or R ^x10 -(R ^x11 ) _a -R ^x12 ;

or together with the moiety to which R ^xb and R ^xc are attached form C _5-8 cycloalkyl, C _4-8 heterocycloalkyl, C _6-10 aryl, or C _6-10 heteroaryl;

From here,

R ^x10 is a direct bond, -NH-, or -O-,

R ^x11 is each independently a direct bond, -C(O)-, C _1-5 alkylene, C _2-5 alkenylene, C _6-10 arylene, C _6-10 heteroarylene, C _3-8 cyclo selected from the group consisting of alkylene, or C _3-8 heterocycloalkylene,

R ^x12 is OH, NH ₂ , NH-R ^x12' , C _6-10 aryl, C _6-10 heteroaryl, C _3-8 cycloalkyl, or C _3-8 heterocycloalkyl;

wherein R ^x12′ is C _1-5 alkyl, C _2-5 alkenyl, C _6-10 aryl, C _6-10 heteroaryl, C _3-8 cycloalkyl, or C _3-8 heterocycloalkyl;

a is an integer from 1 to 5;

Any one of R ^xb or R ^xc forms a monovalent moiety and is capable of binding to Linker,

However, when a2 is 0, NH binds directly to the Linker.

In one embodiment of the present invention, D ^x1 is CH ₂ . In one embodiment of the present invention, D ^x1 is C(=O).

In one embodiment of the present invention, D ^x2 is O. In one embodiment of the present invention, D ^x2 is NC≡N. In one embodiment of the present invention, D ^x2 is NH.

In one embodiment of the present invention, a1 is 0. In one embodiment of the present invention, a1 is 1. In one embodiment of the present invention, a1 is 2. In one embodiment of the present invention, a1 is 3.

In one embodiment of the present invention, a2 is 0. In one embodiment of the present invention, a2 is 1.

In one embodiment of the present invention, a3 is 0. In one embodiment of the present invention, a3 is 1. wherein a3 is 2.

In one aspect of the present invention, A is C or N.

In one embodiment of the present invention, R ^xa is hydrogen. In one aspect of the invention, R ^xa is F, Br, Cl or I. In one embodiment of the present invention, R ^xa is C _1-6 alkyl. In one embodiment of the present invention, R ^xa is methyl.

In one aspect of the present invention, at least one of R ^xb and R ^xc is C _1-10 alkyl.

In one embodiment of the present invention, at least one of R ^xb and R ^xc is R ^x10 -(R ^x11 ) _a -R ^x12 . Here, a is an integer of 1 to 3.

Further, in one embodiment of the present invention, R ^x10 is a direct bond. In one embodiment of the present invention, R ^x10 is -NH-. In one embodiment of the present invention, R ^x10 is -O-.

Further, in one embodiment of the present invention, R ^x11 is each independently selected from the group consisting of a direct bond, -C(O)-, C _1-5 alkylene, or C _2-5 alkenylene. In one embodiment of the present invention, R ^x11 is a direct bond. In one embodiment of the present invention, R ^x11 is -C(O)-. Wherein R ^x11 is C _1-3 alkylene.

Further, in one embodiment of the present invention, R ^x11 is each independently C _6-10 arylene, C _6-10 heteroarylene, C _3-8 cycloalkylene, or C _3-8 heterocycloalkylene consisting of selected from the group. In one embodiment of the present invention, R ^x11 is C _5-6 cycloalkylene.

Also, in one embodiment of the present invention, R ^x12 is NH ₂ . In one embodiment of the present invention, R ^x12 is -NH-CH ₃ It is.

Further, in one embodiment of the present invention, R ^x12 is C _6-10 heteroaryl containing one or more N atoms or C _3-8 heterocycloalkyl containing one or more N atoms.

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In one embodiment of the present invention, at least one of R ^xb or R ^xc comprises -NH ₂ or -NH-R ^x12' , and -NH ₂ or -NH-R ^x12' forms a monovalent moiety to form a monovalent moiety with Linker combine

In one embodiment of the present invention, R ^xb and R ^xc together with the moiety to which R ^xb and R ^xc are bonded form C _4-8 heterocycloalkyl or C _6-10 heteroaryl.

In one aspect of the present invention, a is 1. In one aspect of the present invention, a is 2. In one aspect of the present invention, a is 3.

In one aspect of the present invention, the molecular adhesive degrading agent

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In one embodiment of the present invention, the molecular adhesive disintegrant binds to the Linker at the terminal NH or NH ₂ .

,In one aspect of the invention, the linker-proteolytic agent that binds to Ab

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The present invention also provides a pharmaceutical composition for preventing or treating a hyperproliferative disease or angiogenic disease, comprising the above-described conjugate, a pharmaceutically acceptable salt or solvate thereof.

The present invention also provides the use of the above-described conjugate, a pharmaceutically acceptable salt or solvate thereof for use as a pharmaceutical composition for the prevention or treatment of a hyperproliferative disease or angiogenic disease.

Also, one or more therapeutic co-agents; and pharmaceutically acceptable excipients.

In one embodiment of the present invention, the therapeutic co-agent is an agent that has a preventive, ameliorated or therapeutic effect on a hyperproliferative disease, or an agent that can reduce the expression of side effects that occur when a therapeutic agent for a proliferative disease is administered, or an agent that enhances immunity It may be an agent showing an effect, but is not limited thereto, and exhibits a therapeutically useful effect when applied in the form of a formulation together with a proteolytic agent, and/or further improves the stability of the proteolytic agent, and/or protein This means that any formulation that can reduce the side effects that may appear during release administration and/or maximize the therapeutic effect by enhancing immunity can be combined and applied.

In one aspect of the present invention, the hyperproliferative disease refers to a cell proliferation-related disease in which undesirably excessive or abnormal cells such as neoplasia or hyperplastic growth, whether in vitro or in vivo, are not undesirably controlled. The hyperproliferative disease may be selected from the group consisting of neoplasia, tumor, cancer, leukemia, psoriasis, bone disease, fibroproliferative disorder, and atherosclerosis. Examples of neoplasms and tumors include histiocytoma, glioma, astrocytoma, osteoma, and the like.

In one aspect of the present invention, the cancer is lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, bowel cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma And it may be selected from the group consisting of melanoma, but all of the cancer types in which the proteolytic agent can exhibit a therapeutic effect are applicable.

The present invention also relates to hyperproliferative disease in a subject having a hyperproliferative disease comprising administering to the subject an effective amount of an antibody construct-proteolytic agent conjugate, a pharmaceutically acceptable salt or solvate thereof for treating the hyperproliferative disease. A method for preventing or treating sexually transmitted diseases is provided.

In one aspect of the present invention, there is provided a method for preventing or treating cancer, comprising administering the pharmaceutical composition to a patient.

In one aspect of the present invention, there is provided a method for preventing or treating angiogenesis, comprising administering the pharmaceutical composition to a patient.

The present invention is suitable for use in providing a proteolytic agent to a target site in a subject. The conjugates according to the present invention release an active proteolytic agent that does not have any linker moieties, and nothing that can affect the reactivity of the proteolytic agent is present.

[Justice]

The following definitions also apply here:

A "therapeutic agent" as used herein is an agent that exerts a cytotoxic, cytostatic and/or immunomodulatory effect on a proliferative disease, for example, cancer cells or activated immune cells. Examples of therapeutic agents include cytotoxic agents, chemotherapeutic agents, cytostatic agents, and immunomodulatory agents.

A "chemotherapeutic agent" as used herein is a chemical compound useful for the treatment of cancer.

As used herein, "subject" is intended to include human and non-human animals, particularly mammals. An example of a subject is a human subject, such as a concept including a human patient or normal subject having a disorder described herein, more specifically cancer. "Non-human animal" refers to all vertebrates, eg, non-mammals (eg, chickens, amphibians, reptiles) and mammals, eg, non-human primates, livestock and/or useful in agriculture. animals (eg, sheep, dogs, cats, cattle, pigs, etc.) and rodents (eg, mice, rats, hamsters, guinea pigs, etc.). In certain embodiments, the subject is a human patient.

As used herein, “treatment” or “treat” refers to both therapeutic treatment and prophylactic or prophylactic measures. Subjects in need of treatment include those already with the disease, and those prone to have the disease or those in which the disease is to be prevented. Thus, when used in reference to a subject in need of a disease or treatment, the term includes arresting or slowing disease progression, preventing symptoms, reducing disease and/or symptom severity, or reducing disease duration, as compared to an untreated subject However, it is not limited thereto.

As used herein, "administration" or "administering" refers to providing, contacting and/or delivering a compound or compounds by any suitable route to achieve a desired effect. Administration may be oral, sublingual, parenteral (eg, intravenous, subcutaneous, intradermal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional or intracranial injection), transdermal, topical, administration may include, but are not limited to, buccal, rectal, vaginal, nasal, ophthalmic, inhalation and via implantation.

As used herein, "unsubstituted or substituted" refers to a parent group that may be unsubstituted or substituted, "substituted" refers to a parent group having one or more substituents, and a substituent refers to a parent group. group) or a chemical moiety fused to a parent group.

As used herein, "halo" refers to fluorine, chlorine, bromine, iodine, and the like.

As used herein, "alkyl" is a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of an aliphatic or alicyclic, saturated or unsaturated (unsaturated, fully unsaturated) hydrocarbon compound, and examples of saturated alkyl include methyl, ethyl, propyl, butyl. , pentyl, hexyl, heptyl, etc., examples of saturated straight chain alkyl include methyl, ethyl, n-propyl, n-butyl, n-pentyl (amyl), n-hexyl, n-heptyl, etc., examples of saturated branched chain alkyl isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl and the like.

As used herein, "alkoxy" means -OR [wherein R is an alkyl group], examples of which include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy and the like.

As used herein, "aryl" means a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound having a ring atom.

As used herein, "alkenyl" is an alkyl having one or more carbon-carbon double bonds, and examples of unsaturated alkenyl groups include ethenyl (vinyl, -CH=CH ₂ ), 1-propenyl (-CH=CH-CH ₃ ). ), 2-propenyl, isopropenyl, butenyl, pentenyl, hexenyl, and the like.

As used herein, "alkynyl" is an alkyl group having at least one carbon-carbon triple bond, and examples of the unsaturated alkynyl group include ethynyl and 2-propynyl.

As used herein, "carboxy" refers to -C(=O)OH.

As used herein, "formyl" refers to -C(=O)H.

As used herein, "aryl" relates to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound. For example, "C _5-7 aryl" means a monovalent moiety in which the moiety has 5 to 7 ring atoms and is obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, and "C ₅ - " ₁₀ aryl" means a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, wherein the moiety has 5 to 10 ring atoms. A prefix (C _5-7 , C _5-10 , etc.) herein refers to a number of ring atoms or a range of number of ring atoms, whether carbon atoms or heteroatoms. For example, “C _5-6 aryl” relates to an aryl group having 5 or 6 ring atoms. Here, the ring atoms may all be carbon atoms as in "carboaryl group". Examples of carboaryl groups include, but are not limited to, those derived from benzene, naphthalene, azulene, anthracene, phenanthrene, naphthacene, and pyrene. Examples of aryl groups comprising a fused ring in which at least one is an aromatic ring include groups derived from indane, indene, isoindene, tetralin, acenaphthene, fluorene, phenalene, acephenanthrene and aceanthrene, but with not limited Alternatively, the ring atom may contain one or more heteroatoms as in "heteroaryl group".

As used herein, "heteroaryl" is an aryl containing one or more heteroatoms, for example, pyridine, pyrimidine, benzothiophene, furyl, dioxalanyl, pyrrolyl, oxazolyl, pyridyl, pyridazinyl, pyrimidi Neil et al., more specifically benzofuran, isobenzofuran, indole, isoindole, indolizine, indoline, isoindoline, purine (adenine or guanine), benzimidazole, indazole, benzoxazole, benzisoxazole, C ₉ having two fused rings derived from benzodioxol, benzofuran, benzotriazole, benzothiofuran, benzothiazole, benzothiadiazole, chromene, isochromen, chromen, isochroman, benzo Two fused rings derived from dioxane, quinoline, isoquinoline, quinolizine, benzoxazine, benzodiazine, pyridopyridine, quinoxaline, quinazoline, cinnoline, phthalazine, naphthyridine, and pteridine C ₁₀ having two fused rings derived from benzodiazepine, C ₁₁ having two fused rings derived from carbazole, dibenzofuran, dibenzothiophene, carboline, perimidine, and C ₁₃ having three fused rings derived from pyridoindole, three fused rings derived from acridine, xanthene, thioxanthene, oxantrene, phenoxatiin, phenazine, phenoxazine, phenothiazine, thiantrene, phenanthridine, phenanthroline, and phenazine; and C ₁₄ having

As used herein, "cycloalkyl" is an alkyl group that is a cyclyl group, and relates to a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon compound. Examples of cycloalkyl groups include, but are not limited to, those derived from:

saturated monocyclic hydrocarbon compounds: cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, methylcyclopropane, dimethylcyclopropane, methylcyclobutane, dimethylcyclobutane, methylcyclopentane, dimethylcyclopentane and methylcyclohexane;

unsaturated monocyclic hydrocarbon compounds: cyclopropene, cyclobutene, cyclopentene, cyclohexene, methylcyclopropene, dimethylcyclopropene, methylcyclobutene, dimethylcyclobutene, methylcyclopentene, dimethylcyclopentene and methylcyclohexene; and

Saturated heterocyclic hydrocarbon compounds: norcharan, norphinane, norbornane.

As used herein, "heterocyclyl" relates to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound.

As used herein, a prefix (eg C _1-12 , C _3-8 , etc.) refers to a number of ring atoms or a range of number of ring atoms, whether carbon atoms or heteroatoms. For example, "C _3-6 heterocyclyl" herein relates to a heterocyclyl group having 3 to 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from:

N ₁ : aziridine, azetidine, pyrrolidine, pyrroline, 2H- or 3H-pyrrole, piperidine, dihydropyridine, tetrahydropyridine, azepine;

N ₂ : imidazolidine, pyrazolidine, imidazoline, pyrazoline, piperazine;

O ₁ : oxirane, oxetane, oxolane, oxol, oxane, dihydropyran, pyran, oxepin;

O ₂ : dioxolane, dioxane and dioxepane;

O ₃ : trioxane;

N ₁ O ₁ : tetrahydrooxazole, dihydrooxazole, tetrahydroisoxazole, dihydroisoxazole, morpholine, tetrahydrooxazine, dihydrooxazine, oxazine

S ₁ : thiirane, thietane, thiolane, thiane, thiepane;

N ₁ S ₁ : thiazoline, thiazolidine, thiomorpholine;

N ₂ O ₁ : oxadiazine;

O ₁ S ₁ : oxathiol, oxathian; and

N ₁ O ₁ S ₁ : Oxatiazine.

As used herein, "prodrug" refers to pyrrolobenzodia by the action of an enzyme, gastric acid, under physiological conditions in vivo (eg, enzymatic oxidation, reduction and/or hydrolysis, etc.) A compound that can be converted directly or indirectly into a zepine drug.

As used herein, an acid addition salt formed by a pharmaceutically acceptable free acid may be used as the "pharmaceutically acceptable salt", and an organic acid or an inorganic acid may be used as the free acid.

The organic acid is not limited thereto, but citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, glutamic acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid.

For example, if a compound is an anion or has a functional group that can be anionic (eg -COOH can be -COO-), it can form a salt with the appropriate cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na ⁺ and K ⁺ , alkaline earth metal cations such as Ca ²⁺ and Mg ²⁺ and other cations such as Al ³⁺ . Examples of suitable organic cations include, but are not limited to, ammonium ions (ie NH ₄ ⁺ ) and substituted ammonium ions (eg NH ₃ R ⁺ , NH ₂ R ₂ ⁺ , NHR ₃ ⁺ , NR ₄ ⁺ ).

Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine , phenylbenzylamine, choline, meglumine and tromethamine, as well as amino acids such as lysine and arginine. An example of a typical quaternary ammonium ion is N(CH ₃ ) ₄ ⁺ .

If the compound is a cation or has a functional group that can be a cation (eg -NH ₂ can be -NH ₃ ⁺ ), it can form a salt with an appropriate anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid and phosphorous acid.

Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyloxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethane Disulfonic acid, ethanesulfonic acid, fumaric acid, glutamic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalene carboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, Malic acid, methanesulfonic acid, mucoic acid, oleic acid, oxalic acid, palmitic acid, palmic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid and valeric acid and the like can be given. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose, and the like.

As used herein, "solvate" refers to a molecular complex between the compound according to the present invention and solvent molecules, and examples of the solvate include water, isopropanol, ethanol, methanol, dimethyl sulfoxide. (dimethylsulfoxide), ethyl acetate, acetic acid, ethanolamine, or a compound according to the present invention combined with a mixed solvent thereof, but is not limited thereto.

It may be convenient or desirable to prepare, purify and/or handle equivalent solvates of the active compounds. The term "solvate" is used herein in its conventional sense to refer to a complex of a solute (eg, an active compound, a salt of an active compound) and a solvent. When the solvent is water, the solvate may conveniently be referred to as a hydrate, such as a monohydrate, dihydrate, trihydrate, or the like.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include macromolecules that are typically slowly metabolized, for example, proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates, and the like, and these pharmaceutically Acceptable carriers can be appropriately selected and used by those skilled in the art.

The composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include one or more compounds and at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. prepared by mixing In addition to simple excipients, lubricants such as magnesium stearate, talc and the like may also be used.

Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The pharmaceutical composition is selected from the group consisting of injections, tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories It can have any one of the formulations.

For intravenous, dermal or subcutaneous injection and the like, the active ingredient may be in the form of an acceptable aqueous solution for parenteral administration, which is pyrogen-free and has appropriate pH, isotonicity and stability. Those skilled in the art can prepare suitable solutions using isotonic vehicles such as, for example, aqueous sodium chloride solution, Ringer's solution, lactate Ringer's solution, and the like, and may include preservatives, stabilizers, buffers, antioxidants or other additives as required. Solid forms suitable for injection may also be prepared as emulsions or in the form of polypeptides encapsulated in liposomes.

As used herein, the phrase “effective amount” or “therapeutically effective amount” refers to the amount necessary (as for the dosage and duration and means of administration) to achieve the desired therapeutic result. An effective amount is at least the minimum amount of an active agent necessary to confer a therapeutic benefit to a subject and is less than a toxic amount. For example, dosages may range from about 100 ng to about 100 mg/kg per patient, more typically from about 1 μg/kg to about 10 mg/kg. When the active compound is a salt, ester, amide, prodrug, etc., the dosage is calculated on the basis of the parent compound, so the actual weight used increases proportionally. The pyrrolobenzodiazepine compound according to the present invention may be formulated to contain 0.1 mg to 3000 mg, 1 mg to 2000 mg, or 10 mg to 1000 mg of the active ingredient per dosage form, but is not limited thereto. The active ingredient may be administered to obtain a peak plasma concentration of the active compound of about 0.05 μM to 100 μM, 1 μM to 50 μM, 5 μM to 30 μM. For example, it may be administered by intravenous injection of a 0.1 w/v% to 5 w/v% solution of the active ingredient, optionally in saline.

The concentration of active compound in a pharmaceutical composition can be determined by the rate of absorption, inactivation and excretion of the drug and other factors known to those skilled in the art. The dosage may vary depending on the severity of the condition/disease. In addition, the dosage and administration regimen for a specific patient may be adjusted according to the professional judgment of the administration supervisor in consideration of the patient's symptoms/disease severity, necessity, age, responsiveness to drugs, etc., and the concentration suggested in the present invention The ranges are exemplary only and are not intended to limit the embodiments of the claimed compositions thereto. In addition, the active ingredient may be administered once or may be administered in smaller doses divided into several doses.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples.

The following Examples and Experimental Examples are provided to aid the understanding of the present invention, and are not intended to limit the scope of the present invention.

<Example 1> Preparation of compound 2

Preparation of compound 1

2-methyl-5-nitroaniline (10 g, 65.7 mmol) in dichloromethane (80 mL)

After dissolution, a diluted solution of di- t -butyl dicarbonate (28.7 g, 131.9 mmol) in dichloromethane (10 mL) was added. Pyridine (10.7 mL, 131.9 mmol) was added under nitrogen atmosphere and the reaction solution was stirred at room temperature for 19 hours. The reaction solution was diluted with dichloromethane (100 mL) and washed with 0.5 N aqueous hydrochloric acid solution (50 mL). The collected organic layers were dried over anhydrous sodium sulfate, dissolved in a tetrahydrofuran/methanol (3:1) solution, and then 4 M aqueous sodium hydroxide solution (24.7 mL, 98.6 mmol) was added at 0° C. under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 1.5 hours, concentrated, diluted with dichloromethane (200 mL), washed with distilled water (100 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain Compound 1 (14.8 g, 89%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.83 (s, 1H), 7.84 (d, 1H), 7.28 (d, 1H), 2.34 (s, 3H), 1.55 (s, 9H).

Preparation of compound 2

Compound 1 (9.5 g, 25.9 mmol) was dissolved in methanol (20 mL) and ethyl acetate (200 mL), and palladium/charcoal (10 % w/w, 1.0 g) was added thereto. The reaction solution was stirred at room temperature under a hydrogen atmosphere for 18 hours. The reaction solution was filtered through celite and concentrated to obtain compound 2 (8.2 g, 95%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.34 (s, 1H), 6.89 (d, 1H), 6.33 (d, 1H), 2.12 (s, 3H), 1.52 (s, 9H).

<Example 2> Preparation of compound 10

Preparation of compound 3

After dissolving 4-bromo-2-methylbenzoic acid (3 g, 13.95 mmol) in dimethylformamide (20 mL), sodium bicarbonate (2.3 g, 27.90 mmol) and iodomethane at 0° C. under a nitrogen atmosphere (1.7 mL, 27.90 mmol) was added sequentially and stirred at 60 °C for 3 h. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous ammonium chloride solution (50 mL) and distilled water (50 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 3 (3.1 g, 99%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.78 (d, 1H), 7.41 (s, 1H), 7.38 (d, 1H), 3.88 (s, 3H), 2.57 (s, 3H).

Preparation of compound 4

Compound 3 (3.1 g, 13.88 mmol) was dissolved in dimethylformamide (10 mL) and then zinc cyanide (Zn(CN) ₂ , 8.2 g, 69.44 mmol) and tetrakis(triphenylphosphine)palladium ( 0) (3.2 g, 2.78 mmol) was added sequentially and stirred at 120 °C for 17 h. After cooling the reaction solution to room temperature, it was filtered using Celite. The filtered solution was diluted with ethyl acetate (100 mL), washed with distilled water (50 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 4 (2.0 g, 85%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.97 (d, 1H), 7.55-753 (m, 2H), 3.93 (s, 3H), 2.62 (s, 3H).

Preparation of compound 5

After dissolving compound 4 (2.0 g, 11.75 mmol) in benzene (100 mL) under a nitrogen atmosphere, N -bromosuccinimide (NBS, 3.1 g, 17.62 mmol) and azobisisobutyronitrile (AIBN, 1.9 g) , 11.75 mmol) were added sequentially and stirred under reflux for 17 h. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous sodium hydrogen carbonate solution (50 mL), distilled water (50 mL) in that order, and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 5 (2.5 g, 84%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.04 (d, 1H), 7.78 (s, 1H), 7.66 (d, 1H), 4.91 (s, 2H), 3.98 (s, 3H).

Preparation of compound 6

Compound 5 (2.5 g, 9.83 mmol) and 3-aminopiperidine-2,6-dione hydrochloride (1.6 g, 9.83 mmol) were dissolved in dimethylformamide (30 mL) at 0° C. under a nitrogen atmosphere. N,N' -diisopropylethylamine (2.2 mL, 12.92 mmol) was added and stirred at room temperature for 16 hours. After concentration under reduced pressure and dilution with dichloromethane (100 mL), the resulting solid was filtered and compound 6 (1.9 g, 71%) was obtained. ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 11.02 (s, 1H), 8.16 (s, 2H), 7.99 (d, 1H), 7.91 (d, 1H), 5.17-5.12 (m, 1H) , 4.57-4.40 (m, 2H), 2.94-2.86 (m, 1H), 2.62-2.58 (m, 1H), 2.44-2.39 (m, 1H), 2.19-2.16 (m, 1H).

Preparation of compound 7

Compound 6 (1 g, 3.37 mmol) was dissolved in tetrahydrofuran (30 mL), and then Raney Ni (Raney Ni, 2 g) and di- t -butyl dicarbonate (2.1 mL, 9.06 mmol) were sequentially added and the reaction solution was stirred at room temperature under a hydrogen atmosphere for 48 hours. The reaction solution was filtered through Celite, concentrated under reduced pressure, and purified by column chromatography to obtain compound 7 (576 mg, 45%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.96 (s, 1H), 7.65 (d, 1H), 7.49 (t, 1H), 7.42 (s, 1H), 7.35 (d, 1H) 5.10-5.06 (m, 1H), 4.44-4.26 (m, 2H), 4.22-4.20 (d, 2H), 2.89-2.84 (m, 1H), 2.60-2.55 (m, 1H), 2.38-2.34 (m, 1H) , 1.99-1.97 (m, 1H), 1.37 (s, 9H).

Preparation of compound 8

Compound 7 (200 mg, 0.53 mmol) was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (1 mL) was added at 0° C. under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 1 hour and concentrated under reduced pressure to obtain compound 8 (181 mg). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 11.00 (s, 1H), 8.24 (br, 2H), 7.80 (d, 1H), 7.68 (s, 1H), 7.59 (d, 1H) 5.15-5.10 (m, 1H), 4.51-4.33 (m, 2H), 4.19-4.15 (m, 2H), 2.99-2.87 (m, 1H), 2.63-2.59 (m, 1H), 2.43-2.40 (m, 1H) , 2.03-2.00 (m, 1H).

Preparation of compound 9

Compound 2 (159 mg, 0.69 mmol) was dissolved in dry toluene (15 mL), and triphosgene (82 mg, 0.28 mmol) and triethylamine (0.15 mL, 1.04 mmol) were added thereto, and then 80° C. under a nitrogen atmosphere for 1 hour. After heating and stirring for a while, it was concentrated under reduced pressure. This product was dissolved in dry tetrahydrofuran (10 mL), and a solution of compound 8 (181 mg) and triethylamine (0.22 mL, 1.60 mmol) in dry tetrahydrofuran (10 mL) at 0 ° C under a nitrogen atmosphere was added. was added to the reaction solution. After stirring for 1.5 hours, the reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 9 (222 mg, 79%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.99 (s, 1H), 8.64 (s, 1H), 8.41 (s, 1H), 7.68 (d, 1H), 7.51 (s, 1H), 7.43 ( d, 1H), 7.40 (s, 1H), 7.13 (d, 1H), 6.98 (d, 1H), 6.69 (t, 1H), 5.12-5.08 (m, 1H), 4.47-4.28 (m, 4H) , 2.92-2.86 (m, 1H), 2.61-2.57 (m, 1H), 2.40-2.35 (m, 1H), 2.09 (s, 3H), 2.00-1.97 (m, 1H), 1.48 (s, 9H) .

Preparation of compound 10

Compound 9 (50 mg, 0.09 mmol) was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (1 mL) was added at 0° C. under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 1.5 hours and concentrated under reduced pressure to obtain Compound 10 (44 mg). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.99 (s, 1H), 8.74 (s, 1H), 7.69 (d, 1H), 7.51 (s, 1H), 7.48 (s, 1H), 7.44 ( d, 1H), 7.06 (d, 1H), 6.96 (d, 1H), 6.82 (t, 1H), 5.13-5.08 (m, 1H), 4.46-4.28 (m, 4H), 2.95-2.87 (m, 1H), 2.62-2.57 (m, 1H), 2.40-2.36 (m, 1H), 2.14 (s, 3H), 2.01-1.98 (m, 1H). EI-MS m/z: [M+H] ⁺ 422.4.

<Example 3> Preparation of compound 14

Preparation of compound 12

Compound 10 (60 mg, 0.11 mmol) and compound 11 (91 mg, 0.10 mmol, compound 11 was prepared by the method described in WO2017/089890) were mixed with dimethylformamide (4 mL) and pyridine (1 mL) After dissolving in 0 ℃, 1-hydroxy-7-azabenzotriazole (6.9 mg, 0.05 mmol) and N,N' -diisopropylethylamine (0.04 mL, 0.25 mmol) were sequentially added under nitrogen atmosphere. Stirred at 50 °C for 16 hours. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous ammonium chloride solution (50 mL), saturated aqueous sodium hydrogen carbonate solution (50 mL), and distilled water (50 mL) in that order, and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 12 (80 mg, 67%). EI-MS m/z: [M+H] ⁺ 1078.59.

Preparation of compound 13

After dissolving compound 12 (80 mg, 0.07 mmol) in 2-propanol (1 mL) and tetrahydrofuran (1 mL), lithium hydroxide (8.6 mg, 0.20 mmol) aqueous solution and tetramethylammonium hydroxide (1.1 M aqueous solution, 0.06 mL, 0.07 mmol) were sequentially added and stirred at -10 °C for 3 hours. After adjusting the pH to about 4-5 with acetic acid, it was freeze-dried to obtain compound 13 (83 mg, crude). EI-MS m/z: [M+H] ⁺ 1038.63.

Preparation of compound 14

Compound 13 (83 mg, crude) was dissolved in dichloromethane (4 mL), and then trifluoroacetic acid (1 mL) was added at 0 ℃ under nitrogen atmosphere. The reaction solution was stirred at room temperature for 1.5 hours, concentrated and purified by HPLC to obtain compound 14 (41 mg). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.98 (s, 1H), 8.88 (s, 1H), 8.59 (s, 1H), 8.31 (t, 1H), 7.86 (d, 1H), 7.69 ( d, 1H), 7.55-7.51 (m, 2H), 7.44-7.42 (m, 2H), 7.28 (d, 1H), 7.14 (d, 1H), 7.00 (d, 1H), 6.64 (t, 1H) , 5.16-5.08 (m, 4H), 4.47-4.28 (m, 4H), 4.05 (t, 2H), 3.98 (d, 1H), 3.65-3.52 (m, 10H) 2.96-2.87 (m, 1H), 2.65-2.58 (m, 1H), 2.40-2.32 (m, 1H), 2.09 (s, 3H), 2.03-1.98 (m, 1H). EI-MS m/z: [M+H] ⁺ 938.54.

<Example 4> Preparation of compound 21

Preparation of compound 15

After dissolving 3-bromo-2-methylbenzoic acid (5 g, 23.25 mmol) in dimethylformamide (30 mL), sodium bicarbonate (3.9 g, 46.50 mmol) and iodomethane at 0 °C under a nitrogen atmosphere (2.9 mL, 46.50 mmol) was added sequentially and stirred at 60 °C for 3 h. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous ammonium chloride solution (50 mL) and distilled water (50 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 15 (5.2 g, 97%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.73-7.68 (m, 2H), 7.09 (t, 1H), 3.90 (s, 3H), 2.63 (s, 3H).

Preparation of compound 16

After dissolving compound 15 (2.6 g, 11.35 mmol) in benzene (100 mL), under a nitrogen atmosphere, N -bromosuccinimide (NBS, 4.4 g, 24.96 mmol) and azobisisobutyronitrile (AIBN, 1.8) g, 11.35 mmol) were added sequentially and stirred under reflux for 17 h. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous sodium hydrogen carbonate solution (50 mL) and distilled water (50 mL) in that order, and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 16 (3.2 g, 91%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.88 (d, 1H), 7.76 (d, 1H), 7.23 (t, 1H), 5.13 (s, 2H), 3.95 (s, 3H).

Preparation of compound 17

Compound 16 (3.2 g, 10.39 mmol) and 3-aminopiperidine-2,6-dione hydrochloride (1.9 g, 11.43 mmol) were dissolved in dimethylformamide (20 mL) at 0 °C under a nitrogen atmosphere. N,N' -diisopropylethylamine (2.7 mL, 15.58 mmol) was added and stirred at room temperature for 16 hours. The reaction solution was concentrated under reduced pressure, diluted with dichloromethane (100 mL), and the resulting solid was filtered and compound 17 (2.2 g, 67%) was obtained. ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 11.01 (s, 1H), 7.87 (d, 1H), 7.77 (d, 1H), 7.51 (t, 1H), 5.17-5.12 (m, 1H) , 4.44-4.24 (m, 1H), 2.98-2.87 (m, 1H), 2.61-2.57 (m, 1H), 2.46-2.44 (m, 1H), 2.03-1.99 (m, 1H).

Preparation of compound 18

Compound 17 (2.2 g, 6.99 mmol) was dissolved in dimethylformamide (10 mL), and zinc cyanide (Zn(CN) ₂ , 4.1 g, 34.96 mmol) and tetrakis(triphenylphosphine)palladium under a nitrogen atmosphere. (0) (1.4 g, 1.39 mmol) was added sequentially and stirred at 120 °C for 17 hours. After cooling the reaction solution to room temperature, it was filtered using Celite. After concentration under reduced pressure and dilution with dichloromethane (100 mL), the resulting solid was filtered and compound 18 (1.2 g, 63%) was obtained. EI-MS m/z: [M+H] ⁺ 270.32.

Preparation of compound 19

Compound 18 (1.2 g, 4.45 mmol) was dissolved in methanol (20 mL) and hydrochloric acid (4 M 1,4-dioxane solution, 5 mL) at 0 ° C., under a nitrogen atmosphere, palladium/charcoal (10% w/w, 1.5 g) was added. The reaction solution was stirred at 50 psi under a hydrogen atmosphere for 18 hours. The reaction solution was filtered through celite and concentrated under reduced pressure to obtain compound 19 (732 mg, crude). EI-MS m/z: [M+H] ⁺ 273.97.

Preparation of compound 20

Compound 2 (798 mg, 3.48 mmol) was dissolved in dry toluene (50 mL), and triphosgene (413 mg, 1.39 mmol) and triethylamine (0.7 mL, 5.22 mmol) were added thereto, and 80 °C under nitrogen atmosphere for 1 hour. After stirring for a while, it was concentrated under reduced pressure. This product was dissolved in dry tetrahydrofuran (25 mL), and a solution of compound 19 (732 mg) and triethylamine (1.1 mL, 8.03 mmol) in dry tetrahydrofuran (25 mL) at 0 ° C under a nitrogen atmosphere was added. was added to the reaction solution. The reaction solution was stirred for 1.5 hours, concentrated under reduced pressure, and purified by column chromatography to obtain compound 20 (160 mg, 11%). EI-MS m/z: [M+Na] ⁺ 544.31, [M-Boc+H] ⁺ 422.24.

Preparation of compound 21

Compound 20 (160 mg, 0.30 mmol) was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (1 mL) was added at 0 °C under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 1.5 hours and then concentrated under reduced pressure to obtain compound 21 (157 mg). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 11.01 (s, 1H), 8.68 (s, 1H), 7.63 (d, 1H), 7.56-7.47 (m, 3H), 7.04 (d, 1H) , 6.94 (d, 1H) 6.76 (t, 1H), 5.17-5.12 (m, 1H), 4.55-4.35 (m, 4H), 2.97-2.88 (m, 1H), 2.67-2.60 (m, 1H), 2.40-2.33 (m, 1H), 2.14 (s, 3H), 2.04-2.01 (m, 1H). EI-MS m/z: [M+H] ⁺ 422.51.

<Example 5> Preparation of compound 24

Preparation of compound 22

After dissolving compound 21 (100 mg, crude) and compound 11 (149 mg, 0.16 mmol) in dimethylformamide (4 mL) and pyridine (1 mL), 1-hydroxy-7- at 0 ℃ under nitrogen atmosphere Azabenzotriazole (11 mg, 0.08 mmol) and N,N' -diisopropylethylamine (0.1 mL, 0.55 mmol) were sequentially added and stirred at 50° C. for 16 hours. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous ammonium chloride solution (50 mL), saturated aqueous sodium hydrogen carbonate solution (50 mL), and distilled water (50 mL) in that order, and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 22 (83 mg, 42%). EI-MS m/z: [M+H] ⁺ 1179.06.

Preparation of compound 23

Compound 22 (83 mg, 0.07 mmol) was dissolved in 2-propanol (1 mL) and tetrahydrofuran (1 mL), and lithium hydroxide (8.8 mg, 0.21 mmol) and tetramethylammonium hydroxide (1.1 M aqueous solution, 0.06 mL, 0.07 mmol) were sequentially added and stirred at -10 °C for 3 hours. After adjusting the pH to about 4-5 with acetic acid, it was purified by HPLC to obtain compound 23 (32 mg). EI-MS m/z: [M+H] ⁺ 1038.95.

Preparation of compound 24

Compound 23 (32 mg) was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (1 mL) was added at 0 °C under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 1.5 hours, concentrated, and purified by HPLC to obtain Compound 24 (24 mg). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 11.00 (s, 1H), 8.87 (s, 1H), 8.57 (s, 1H), 8.31 (t, 1H), 7.86 (s, 1H), 7.62 (d, 1H), 7.54-7.50 (m, 2H), 7.43 (s, 1H), 7.27 (d, 1H), 7.13 (d, 1H), 7.00 (d, 1H) 6.63 (t, 1H), 5.16 -5.10 (m, 3H), 4.55-4.35 (m, 4H), 4.05 (t, 2H), 3.98 (d, 1H), 3.65-3.52 (m, 10H) 2.98-2.88 (m, 1H), 2.63 2.59 (m, 1H), 2.42-2.32 (m, 1H), 2.08 (s, 3H), 2.04-2.00 (m, 1H). EI-MS m/z: [M+H] ⁺ 938.82.

<Example 6> Preparation of compound 26

Compound 26 was synthesized in a similar manner to the synthesis of compound 14 from compound 25 (lenalidomide) and compound 11. ¹ H NMR (400 MHz, MeOH-d ₄ ): δ 7.95 (t, 1H), 7.74 (t, 1H), 7.57 (t, 2H), 7.49 (t, 1H), 7.34 (d, 1H), 5.23 (s, 2H), 5.20-5.11 (m, 2H), 4.42-4.35 (m, 2H), 4.18-4.16 (m, 2H), 4.06 (d, 1H), 3.81 (t, 2H), 3.72-3.52 (m, 12H), 2.94-2.76 (m, 2H), 2.46-2.39 (m, 1H), 2.18-2.46 (m, 1H). EI-MS m/z: [M+H] ⁺ 776.62.

<Example 7> Preparation of compound 30

Preparation of compound 28

Compound 27 (42 mg, 0.09 mmol, compound 27 was prepared by the method described in J. Med. Chem. 2019, 62, 699-726) and compound 11 (92 mg, 0.10 mmol) were mixed with dimethylformamide (4 mL) and pyridine (1 mL), and then 1-hydroxybenzotriazole (2.5 mg, 0.02 mmol) and N,N' -diisopropylethylamine (0.03 mL, 0.19 mmol) at 0 °C under a nitrogen atmosphere. were sequentially added and stirred at 50 °C for 16 hours. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous ammonium chloride solution (50 mL), saturated aqueous sodium hydrogen carbonate solution (50 mL), and distilled water (50 mL) in that order, and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 28 (73 mg, 71%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.99 (s, 1H), 9.98 (s, 2H), 7.94 (t, 1H), 7.84 (t, 1H), 7.69 (d, 2H), 7.61 (s, 1H), 7.44 (d, 1H), 7.33 (d, 2H), 7.15 (d, 1H), 6.95-6.93 (m, 1H), 6.78 (d, 1H), 5.75 (d, 1H), 5.49 (t, 1H), 5.22 (t, 1H), 5.06 (t, 1H), 5.00 (s, 2H) 4.73 (d, 1H), 4.21 (d, 2H), 3.80 (t, 2H), 3.63 ( s, 3H), 3.56- 3.50 (m, 8H), 3.39-3.36 (m, 2H), 2.10-1.99 (m, 9H), 1.38 (s, 9H). EI-MS m/z: [M+H] ⁺ 1092.33.

Preparation of compound 29

Compound 28 (73 mg, 0.07 mmol) was dissolved in 2-propanol (1 mL) and tetrahydrofuran (1 mL), and lithium hydroxide (8.4 mg, 0.20) dissolved in distilled water (1 mL) under a nitrogen atmosphere at -50 ° C. mmol) and stirred at -5 °C for 4 h. After adjusting the pH to about 4-5 with acetic acid, it was freeze-dried to obtain compound 29 (70 mg). EI-MS m/z: [M+H] ⁺ 952.31.

Preparation of compound 30

Compound 29 (70 mg) was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (1 mL) was added at 0 °C under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 2 hours, concentrated, and purified by HPLC to obtain compound 30 (43.6 mg). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 11.03 (s, 1H), 10.00 (s, 1H), 8.31 (t, 1H), 7.88 (t, 1H), 7.80 (s, 1H), 7.70 (d, 2H), 7.48-7.46 (m, 2H), 7.37 (d, 2H), 7.25-7.22 (m, 2H), 6.64 (t, 1H), 6.79 (d, 1H), 5.14 (d, 1H) ), 5.01 (s, 3H), 4.22 (d, 2H), 4.06 (t, 2H), 3.98 (d, 1H) 3.65-3.37 (m, 12H). EI-MS m/z: [M+H] ⁺ 852.21.

<Example 8> Preparation of compound 34

Preparation of compound 31

Compound 2-iodo-4-nitrotoluene (800 mg, 2.59 mmol) was dissolved in toluene (5 mL), followed by palladium acetate (27.3 mg, 0.12 mmol), Xpos (XPhos, 174 mg, 0.36 mmol), cesium carbonate ( 2.97 g, 9.12 mmol), and cyclohexylamine (0.7 ml, 6.08 mmol) were added and reacted at 130 ^o C for 2 hours in a microwave reactor. The reaction solution was purified by column chromatography to obtain compound 31 (160 mg, 22%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.45 (d, 1H), 7.38 (s, 1H), 7.13 (d, 1H), 3.61 (br, 1H), 3.42-3.40 (m, 1H), 2.17 ( s, 3H), 1.77-1.71 (m, 2H), 1.68-.49 (m, 2H), 1.43-1.00 (m, 2H), 1.27-1.19 (m, 4H).

Preparation of compound 32

Compound 31 (800 mg, 3.41 mmol) was dissolved in methanol (10 mL) and ethyl acetate (20 mL), and palladium/charcoal (10 % w/w, 100 mg) was added thereto. The reaction solution was stirred at room temperature under a hydrogen atmosphere for 18 hours. The reaction solution was filtered through celite and concentrated to obtain compound 32 (670 mg). EI-MS m/z: [M+H] ⁺ 205.4.

Preparation of compound 33

After dissolving compound 32 (55 mg, 0.27 mmol) in dichloromethane (3 mL), bis(pentafluorophenyl)carbonate (117 mg, 0.30 mmol) and pyridine (0.03 ml, 0.35 mmol) were added thereto, and 5 at room temperature time was stirred. The reaction solution was diluted with dichloromethane (10 mL), washed with distilled water (10 mL * 2), and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, compound 33 (crude) was obtained.

Preparation of compound 34

Compound 33 (117 mg, 0.28 mmol) was dissolved in dichloromethane (2 mL) at 0 ° C. with N,N' -diisopropylethylamine (0.12 mL, 0.85 mmol) and compound 8 (110 mg, 0.28 mmol) was added. The reaction solution was stirred at room temperature for 2 hours and then concentrated, followed by Prep. Purification by HPLC gave compound 34 (98.7 mg). EI-MS m/z: [M+H] ⁺ 504.2.

<Example 9> Preparation of compound 44

Preparation of compound 35

Compound 32 (802 mg, 3.42 mmol) was dissolved in dichloromethane (10 mL), and pyridine (0.42 mL, 5.13 mmol) and allyl chloroformate (0.4 mL, 3.77 mmol) were added at -78 ^o C, Stirred under nitrogen atmosphere for 2 hours. The reaction solution was concentrated and purified by column chromatography to obtain compound 35 (320 mg, 32%). ¹ H-NMR (400 MHz, CDCl ₃ )δ 6.93 (d, 1H), 6.75 (br, 1H), 6.54-6.49 (m, 2H), 6.01-5.92 (m, 1H), 5.37-5.33 (m, 1H) ), 5.26-5.23 (m, 1H), 4.65 (d, 1H), 3.47 (br, 1H), 3.30-3.28 (m, 1H), 2.11-2.08 (m, 1H), 2.06 (s, 3H), 1.78-1.76(m, 2H), 1.67-1.64(m, 1H), 1.45-1.35 (m, 2H), 1.29-1.15 (m, 4H). EI-MS m/z: [M+H] ⁺ 289.3.

Preparation of compound 36

t -Butyl- N - t -Butoxycarbonyl- N -hydroxycarbamate (10.02 g, 42.9 mmol) was dissolved in dimethylformamide (50 mL) at 0 ° C. Sodium hydride (60% dispersion in mineral) oil, 1.57 g, 39.4 mmol) was added. 1-azido-2-[2-(2-bromoethoxy)ethoxy]ethane at 0 °C under nitrogen atmosphere (8.52 g, 35.8 mmol) was added. The reaction temperature was raised to room temperature, stirred for 4 hours under a nitrogen atmosphere, diluted with ethyl acetate (150 mL), washed with distilled water (100 mL) and saturated aqueous sodium chloride solution (100 mL), and dried over anhydrous sodium sulfate. Filtration, concentration, and purification by column chromatography gave compound 36 (10.9 g, 78%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.55 (s, 1H), 4.05-4.03 (m, 2H), 3.76-3.74 (m, 2H), 3.74-3.69 (m, 6H), 3.42 (t, J = 4.8 Hz, 2H), 1.49 (s, 18H).

Preparation of compound 37

Compound 36 (5.52 g, 14.1 mmol) and triphenylphosphine (4.08 g, 15.6 mmol) were dissolved in dry tetrahydrofuran (50 mL) and distilled water (2.5 mL, 141 mmol) was added at 0 ° C. under a nitrogen atmosphere. After stirring for 10 minutes, the temperature was raised to room temperature and stirred for 16 hours. Concentrated under reduced pressure and purified by column chromatography to obtain compound 37 (4.4 g, 85%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 4.04-4.01 (m, 2H), 3.74-3.62 (m, 7H), 3.55 (t, J = 5.2 Hz, 1H), 2.88 (t, J = 5.2 Hz) , 1H), 2.81 (t, J = 5.2 Hz, 1H), 1.64 (s, 2H), 1.48 (s, 18H).

Preparation of compound 39

Compound 38 (3.0 g, 6.19 mmol, compound 38 was prepared by the method described in WO2017/089890) and compound 37 (2.71 g, 7.43 mmol) were dissolved in N , N -dimethylformamide (50 mL) After N,N,N',N' -tetramethyl- O- (1 H -benzotriazol-1-yl)uronium hexafluorophosphate (HBTU, 3.52 g, 9.29 mmol) and N , N -diiso Propylethylamine (2.2 mL, 12.4 mmol) was added at 0 ^° C, under nitrogen atmosphere. After the reaction solution was stirred at room temperature for 2 hours, a saturated aqueous ammonium chloride solution (100 mL) was added to the reaction solution, followed by extraction with ethyl acetate (2 x 100 mL). The collected organic layers were washed with brine (200 mL) and dried over anhydrous sodium sulfate. After filtration, concentration and purification by column chromatography, compound 39 (2.14 g, 42%) was obtained. EI-MS m/z: [M+H] ⁺ 831.5, [M-Boc+H] ⁺ 731.4, [M-2Boc+H] ⁺ 631.3.

Preparation of compound 40

Compound 39 (346 mg, 0.42 mmol) was dissolved in dry tetrahydrofuran (5 mL) and then N,N' -diisopropylethylamine (0.12 mL, 0.69 mmol) and triphosgene (15% toluene solution) at -45 ° C. , 0.32 mL, 0.45 mmol) were added sequentially, and stirred for 1 hour under a nitrogen atmosphere. Compound 35 (100 mg, 0.347 mmol) and N,N' -diisopropylethylamine (0.06 mL, 0.347 mmol) were dissolved in dry tetrahydrofuran (2 mL) under a nitrogen atmosphere, and this solution was added to the reaction solution. . The reaction temperature was slowly raised to -15 °C and stirred for 1.5 hours. The reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 40 (99 mg, 25%). EI-MS m/z: [M+Na] ⁺ 1167.7.

Preparation of compound 41

Compound 40 (255 mg, 0.09 mmol) was dissolved in dimethylformamide (3 mL) and then tetrakis(triphenylphosphine)palladium(0) (10.3 mg, 0.089 mmol) and sodium-2-ethylhexa Noate (44.4 mg, 0.267 mmol) was sequentially added and stirred at room temperature for 1 hour. The reaction solution was diluted with ethyl acetate (30 mL), washed with distilled water (50 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 41 (90 mg, 98%). EI-MS m/z: [M+H] ⁺ 1061.8.

Preparation of compound 42

Compound 41 (103 mg, 0.97 mmol) was dissolved in dry tetrahydrofuran (3 mL) and then N,N' -diisopropylethylamine (0.03 mL, 0.19 mmol) and triphosgene (15 % toluene solution) at -45 ° C. , 0.01 ml, 0.097 mmol) were added sequentially and stirred for 1 hour under a nitrogen atmosphere. Compound 8 (41 mg, 0.019 mmol) and N,N' -diisopropylethylamine (0.01 mL, 0.016 mmol) were dissolved in dry tetrahydrofuran (1 mL) under a nitrogen atmosphere, and then added to the reaction solution. After stirring at -15 °C for 1.5 hours, the reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 42 (85 mg, 64%). EI-MS m/z: [M+H] ⁺ 1360.9, [M + Na] ⁺ 1383.7.

Preparation of compound 43

Compound 42 (51 mg, 0.038 mmol) was dissolved in 2-propanol (0.5 mL) and tetrahydrofuran (0.5 mL), and then lithium hydroxide (6.4 mg, 0.15) dissolved in distilled water (0.5 mL) at -50 °C under a nitrogen atmosphere. mmol) and tetramethylammonium hydroxide (1.1 M aqueous solution, 0.038 mL, 0.038 mmol) were sequentially added and stirred at -10 °C for 3 hours. After adjusting the pH to about 4-5 with acetic acid, it was freeze-dried to obtain compound 43 (crude). EI-MS m/z: [M+H] ⁺ 1220.8, [M+Na] ⁺ 1242.4.

Preparation of compound 44

Compound 43 (Crude) obtained above was dissolved in dichloromethane (1 mL), and then trifluoroacetic acid (1 mL) was added at 0 °C under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 3 hours, concentrated, and purified by HPLC to obtain compound 44 (10 mg). EI-MS m/z: [M+H] ⁺ 1020.7, [M + Na] ⁺ 1042.6.

<Example 10> Preparation of compound 48

Preparation of compound 45

Compound 2-iodo-4-nitrotoluene (600 mg, 2.28 mmol) was dissolved in toluene (5 mL), followed by palladium acetate (20.5 mg, 0.09 mmol), Xpos (XPhos, 131 mg, 0.27 mmol), cesium carbonate ( 2.22 g, 6.84 mmol), and n -propylamine (0.28 mL, 3.42 mmol) were added and reacted at 130 ^o C for 2 hours in a microwave reactor. The reaction solution was purified by column chromatography to obtain compound 45 (111 mg, 25%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.49 (dd, 1H), 7.38 (d, 1H), 7.13 (d, 1H), 3.71 (br, 1H), 3.20 (q, 2H), 2.19 (s, 3H), 1.75-1.70 (m, 2H), 1.05 (t, 3H). EI-MS m/z: [M+H] ⁺ 195.1.

Preparation of compound 46

Compound 45 (111 mg, 0.57 mmol) was dissolved in methanol (3 mL) and ethyl acetate (6 mL), and palladium/charcoal (10% w/w, 50 mg) was added thereto. The reaction solution was stirred at room temperature under a hydrogen atmosphere for 18 hours. The reaction solution was filtered through celite and concentrated to obtain compound 46 (Crude 93 mg). EI-MS m/z: [M+H] ⁺ 165.2.

Preparation of compound 47

After dissolving compound 46 (20 mg, 0.12 mmol) in dichloromethane (1 mL), bis (pentafluorophenyl) carbonate (53 mg, 0.13 mmol) and pyridine (0.01 ml, 0.16 mmol) were added thereto, and 5 at room temperature time was stirred. The reaction solution was diluted with dichloromethane (10 mL), washed with distilled water (10 mL * 2), and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, compound 47 (crude 40 mg) was obtained. EI-MS m/z: [M+H] ⁺ 375.3.

Preparation of compound 48

Compound 47 (40 mg, 0.107 mmol) was dissolved in dichloromethane (2 mL) at 0 ° C. with N,N' -diisopropylethylamine (0.057 mL, 0.32 mmol) and compound 8 (42 mg, 0.107 mmol) was added. The reaction solution was stirred at room temperature for 2 hours, concentrated and purified by HPLC to obtain compound 48 (43 mg, 68%). EI-MS m/z: [M+H] ⁺ 464.3.

<Example 11> Preparation of compound 52

Preparation of compound 49

After dissolving 6-nitroindoline (500 mg, 3.04 mmol) in dichloromethane (10 mL) at 0 ° C, under a nitrogen atmosphere, di- t -butyl dicarbonate (798 mg, 3.65 mmol), 4-dimethyl Aminopyridine (74 mg, 0.61 mmol), and triethylamine (0.85 mL, 6.09 mmol) were added. The reaction solution was stirred at room temperature for 17 hours, diluted with dichloromethane (100 mL), washed with distilled water (50 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 49 (673 mg, 83%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.82 (d, 1H), 7.25 (d, 1H), 4.08 (t, 2H), 3.17 (t, 2H), 1.59 (s, 9H).

Preparation of compound 50

Compound 49 (50 mg, 0.19 mmol) was dissolved in methanol (5 mL), and palladium/charcoal (10 % w/w, 10 mg) was added thereto. The reaction solution was stirred at room temperature under a hydrogen atmosphere for 3 hours. The reaction solution was filtered through celite and concentrated to obtain compound 50 (45 mg, quant.). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 6.89 (d, 1H), 6.27 (dd, 1H), 3.93 (t, 2H), 2.96 (t, 2H), 1.55 (s, 9H).

Preparation of compound 51

Compound 50 (21 mg, 0.09 mmol) was dissolved in dichloromethane (3 mL) and then bis(pentafluorophenyl)carbonate (41 mg, 0.11 mmol) and pyridine (0.01 mL, 0.15 mmol) were dissolved in dichloromethane (3 mL) at 0 °C under a nitrogen atmosphere. ) was added. After the reaction solution was stirred at room temperature for 3 hours, compound 8 (29 mg, 0.08 mmol) and N,N' -diisopropylethylamine (0.03 mL, 0.15 mmol) were dissolved in dimethylformamide (1 mL) and added. and stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure, diluted with ethyl acetate (50 mL), washed with saturated aqueous ammonium chloride solution (50 mL), saturated aqueous sodium hydrogen carbonate solution (50 mL), and distilled water (50 mL) in that order, and dried over anhydrous sodium sulfate. . After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 51 (22 mg, 55%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.98 (s, 1H), 8.60 (m, 1H), 7.86 (m, 1H), 7.69 (d, 1H), 7.51 (s, 1H), 7.44 (d, 1H), 7.00 (m, 2H), 6.64 (m, 1H), 5.12-5.08 (dd, 1H), 4.47-4.28 (m, 4H), 3.87 (t, 2H) 2.97-2.91 (m, 3H), 2.62-2.57 (m, 1H), 2.43-2.37 (m, 1H), 2.09-1.84 (m, 1H), 1.49 (s, 9H).

Preparation of compound 52

Compound 51 (22 mg, 0.04 mmol) was dissolved in dichloromethane (1.5 mL), and then trifluoroacetic acid (0.5 mL) was added at 0° C. under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 1.5 hours, concentrated and purified by HPLC to obtain compound 52 (14 mg, 62%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.99 (s, 1H), 8.76 (s, 1H), 7.69 (d, 1H), 7.51-7.43 (m, 3H), 7.21-7.08 (m, 1H), 6.95-6.90 (m, 1H), 6.77 (t, 1H), 5.12-5.08 (dd, 1H), 4.46-4.28 (m, 4H), 3.59 (t, 2H), 3.00 (t, 2H) , 2.98-2.88(m, 1H) 2.67-2.57 (m, 1H), 2.40-2.36 (m, 1H), 2.00-1.90 (m, 1H). EI-MS m/z: [M+H] ⁺ 434.42.

<Example 12> Preparation of compound 59

Preparation of compound 53

Compound 50 (261 mg, 1.11 mmol) was dissolved in dichloromethane (10 mL), and pyridine (0.13 ml, 1.67 mmol) and allyl chloroformate (0.13 ml, 1.22 mmol) were added at -78 ^o C, Stirred under nitrogen atmosphere for 17 h. The reaction solution was concentrated and purified by column chromatography to obtain compound 53 (268 mg, 75%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.63 (s, 1H), 7.06 (d, 1H), 6.54 (br, 1H), 5.99-5.92 (m, 1H), 5.37 (d, 1H), 5.26 (d, 1H), 4.65 (d, 2H), 3.97 (t, 2H), 3.03 (t, 2H). 1.5 (s, 9H).

Preparation of compound 54

Compound 53 (145 mg, 0.455 mmol) was dissolved in dichloromethane (4 mL), and then trifluoroacetic acid (1 mL) was added at 0 °C under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 3 hours and concentrated to obtain compound 54 (140 mg). EI-MS m/z: [M+H] ⁺ 219.2.

Preparation of compound 55

Compound 39 (203 mg, 0.24 mmol) was dissolved in dry tetrahydrofuran (5 mL) and then N,N' -diisopropylethylamine (0.07 mL, 0.41 mmol) and triphosgene (15% toluene solution) at -45 ° C. , 0.18 mL, 0.26 mmol) were added sequentially, and stirred for 1 hour under a nitrogen atmosphere. Compound 54 (67 mg, 0.20 mmol) and N,N' -diisopropylethylamine (0.04 mL, 0.20 mmol) were dissolved in dry tetrahydrofuran (2 mL) under a nitrogen atmosphere, and then added to the reaction solution. The reaction temperature was slowly raised to -15 °C and stirred for 1.5 hours. The reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 55 (138 mg, 63%). EI-MS m/z: [M+Na] ⁺ 1076.7.

Preparation of compound 56

Compound 55 (138 mg, 0.13 mmol) was dissolved in dimethylformamide (3 mL) and then tetrakis(triphenylphosphine)palladium(0) (15.0 mg, 0.01 mmol) and sodium-2-ethylhexa Noate (64.0 mg, 0.39 mmol) was sequentially added and stirred at room temperature for 1 hour. The reaction solution was diluted with ethyl acetate (10 mL), washed with distilled water (20 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 56 (105 mg, 83%). EI-MS m/z: [M+Na] ⁺ 991.7.

Preparation of compound 57

Compound 56 (40 mg, 0.04 mmol) was dissolved in dry tetrahydrofuran (2 mL) and then N,N' -diisopropylethylamine (0.01 mL, 0.08 mmol) and triphosgene (15% toluene solution) at -45 ° C. , 0.03 ml, 0.03 mmol) were added sequentially, and stirred under a nitrogen atmosphere for 1 hour. Compound 8 (17.2 mg, 0.04 mmol) and N,N' -diisopropylethylamine (0.01 mL, 0.08 mmol) were dissolved in dry tetrahydrofuran (1 mL) under a nitrogen atmosphere, and then added to the reaction solution. After stirring at -15 °C for 1.5 hours, the reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 57 (32 mg, 60%). EI-MS m/z: [M+H] ⁺ 1291.6.

Preparation of compound 58

Compound 57 (32 mg, 0.02 mmol) was dissolved in 2-propanol (0.5 mL) and tetrahydrofuran (0.5 mL), followed by lithium hydroxide (4.2 mg, 0.10) dissolved in distilled water (0.5 mL) at -50 ° C under nitrogen atmosphere. mmol) and tetramethylammonium hydroxide (1.1 M aqueous solution, 0.02 mL, 0.02 mmol) were sequentially added and stirred at -10 °C for 3 hours. After adjusting the pH to about 4-5 with acetic acid, it was freeze-dried to obtain compound 58 (crude). EI-MS m/z: [M+H] ⁺ 1150.7.

Preparation of compound 59

Compound 58 (crude) obtained above was dissolved in dichloromethane (1 mL), and then trifluoroacetic acid (1 mL) was added at 0 °C under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 3 hours, concentrated, and purified by HPLC to obtain compound 59 (14 mg). EI-MS m/z: [M+H] ⁺ 950.5.

<Example 13> Preparation of compound 64

Preparation of compound 60

Dissolve 5-nitro- 1H -indazol-3( 2H )-one (4.46 g, 24.9 mmol) in distilled water (60 mL), add sodium bicarbonate until it becomes a weak base (pH 9), and then 40 stirred for minutes. Di- t -butyl dicarbonate (10 mL, 49.8 mmol) was added to the reaction solution and stirred at room temperature. After 4 hours, sodium hydrogen carbonate (10 g, 119 mmol) and di- t -butyl dicarbonate (5 mL, 24.9 mmol) were added, and the mixture was stirred at room temperature for 16 hours. The reaction solution was diluted with ethyl acetate (100 mL), washed with distilled water (30 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 60 (700 mg, 10%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 12.62 (br s, 1H), 8.61 (d, 1H), 8.44-8.41 (m, 1H), 8.17 (d, 1H), 1.62 (s, 9H) .

Preparation of compound 61

Compound 60 (200 mg, 0.72 mmol) was dissolved in tetrahydrofuran (10 mL), cesium carbonate (257 mg, 0.79 mmol) and methyl iodide (0.07 mL, 1.07 mmol) were added thereto, followed by stirring at room temperature for 12 hours. . The reaction solution was diluted with ethyl acetate (40 mL), washed with distilled water (20 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 61 (85 mg, 40%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 8.57 (m, 1H), 8.51 (d, 1H), 8.04 (d, 1H), 3.57 (s, 3H), 1.63 (s, 9H).

Preparation of compound 62

Compound 61 (85 mg, 0.29 mmol) was dissolved in tetrahydrofuran (2 mL) and methanol (2 mL), and palladium/charcoal (46 mg) was added thereto, followed by stirring under a hydrogen atmosphere for 2 hours. The reaction solution was filtered through Celite and concentrated under reduced pressure to obtain compound 62 (75 mg, 99%).

Preparation of compound 63

After dissolving bis(pentafluorophenyl)carbonate (63 mg, 0.16 mmol) in dichloromethane (3 mL), compound 8 (35 mg, 0.13 mmol) and pyridine (0.03 mL, 0.40 mmol) were added to dichloromethane A solution in phosphorus (2 mL) was added to the reaction solution. After 30 minutes of reaction, a solution of compound 62 (57 mg, 0.15 mmol) in dimethylformamide (2 mL) was added to the reaction solution and stirred at room temperature for 1 hour. The reaction solution was diluted with ethyl acetate (30 mL), washed with distilled water (15 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 63 (9.4 mg, 12%). EI-MS m/z: [M+H] ⁺ 563.5.

Preparation of compound 64

Compound 63 (9.4 mg, 0.017 mmol) was dissolved in dichloromethane (2 mL), and then trifluoroacetic acid (0.5 mL) was added at 0° C. under a nitrogen atmosphere, followed by stirring at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure and purified by HPLC to obtain compound 64 (2 mg). EI-MS m/z: [M+H] ⁺ 463.4.

<Example 14> Preparation of compound 69

Preparation of compound 65

After dissolving 2-iodo-4-nitrotoluene (800 mg, 3.04 mmol) in toluene (14 mL), palladium acetate (27.3 mg, 0.12 mmol), Xpos (XPhos, 174 mg, 0.36 mmol), cesium carbonate ( 2.97 g, 9.12 mmol), and 1-( t -butoxycarbonyl)piperazine (1.7 g, 9.12 mmol) were added thereto and reacted for 2 hours at 130 ^o C in a microwave reactor. The reaction solution was purified by column chromatography to obtain compound 65 (315 mg, 32%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.88-7.82 (m, 2H), 7.32 (d, 1H), 3.61-3.59 (m, 4H), 2.91-2.89 (m, 4H), 2.40 (s, 3H) ), 1.49 (s, 9H).

Preparation of compound 66

Compound 65 (315 mg, 0.98 mmol) was dissolved in methanol (5 mL) and ethyl acetate (2 mL), and palladium/charcoal (10 % w/w, 65 mg) was added thereto. The reaction solution was stirred at room temperature under a hydrogen atmosphere for 18 hours. The reaction solution was filtered through celite and concentrated to obtain compound 66 (crude 280 mg). ¹ H NMR (400 MHz, CDCl ₃ ): δ 6.95 (d, 1H), 6.40-6.35 (m, 2H), 3.56-3.53 (m, 4H), 2.83-2.17 (m, 4H), 2.18 (s, 3H), 1.48 (s, 9H).

Preparation of compound 67

After dissolving compound 66 (70 mg, 0.24 mmol) in dichloromethane (1.5 mL), bis(pentafluorophenyl)carbonate (104 mg, 0.26 mmol) and pyridine (0.02 ml, 0.31 mmol) were added thereto and at room temperature 3 time was stirred. After concentration of the reaction solution, compound 67 obtained was directly used in the next reaction.

Preparation of compound 68

Compound 8 (92 mg, 0.24 mmol) was dissolved in N,N -dimethylformamide (2 mL), and N,N' -diisopropylethylamine (0.13 mL, 0.72 mmol) was added thereto. Compound 67 (crude) was added to the reaction solution and stirred at room temperature for 16 hours. The reaction solution was diluted with ethyl acetate (20 mL), washed with distilled water (20 mL X 2), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 68 (93 mg, 65%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.25 (s, 1H), 7.68 (d, 1H), 7.44 (s, 1H), 7.32-7.30 (d, 1H), 7.10-7.08 (d, 1H) , 7.01 (s, 1H), 6.95-6.93 (d, 1H), 6.90 (s, 1H), 5.56-5.52 (t, 1H), 5.15-5.10 (m, 1H), 4.56-4.44 (m, 2H) , 4.44-4.24 (m, 2H), 4.16-4.08 (m, 1H), 3.55-3.52 (m, 4H), 2.92-2.72 (m, 6H), 2.40-2.16 (m, 5H), 1.48 (s, 9H).

Preparation of compound 69

Compound 68 (9.4 mg, 0.017 mmol) was dissolved in dichloromethane (2 mL), and then trifluoroacetic acid (0.5 mL) was added at 0 °C under a nitrogen atmosphere and stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure and purified by HPLC to obtain compound 69 (2 mg). ¹ H NMR (400 MHz, MeOH-d ₄ ): δ 7.77 (s, 1H), 7.55 (s, 1H), 7.50 (d, 1H), 7.43 (d, 1H), 7.09 (d, 1H), 6.81 ( d, 1H), 5.15 (d, 1H), 4.54-4.43 (m, 4H), 3.37-3.31 (m, 4H), 3.13-3.10 (m, 4H), 2.91-2.86 (m, 1H), 2.81- 2.79 (m, 1H), 2.51-2.47 (m, 1H), 2.25 (s, 3H), 2.20-2.12 (m, 1H).

<Example 15> Preparation of compound 74

Preparation of compound 71

After dissolving compound 70 (2.0 g, 2.74 mmol, compound 70 was prepared by the method described in WO2017/089890) in dichloromethane (14 mL), 0 ° C., under a nitrogen atmosphere, bis(pentafluorophenyl) ) carbonate (1.2 g, 3.01 mmol) and pyridine (0.44 ml, 5.47 mmol) were added. After stirring at room temperature for 5 hours, the reaction solution was diluted with dichloromethane (20 mL), washed with distilled water (30 mL X 2), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 71 (2 g, 77 %).

Preparation of compound 72

Compound 69 (40 mg, 0.066 mmol) was dissolved in N,N -dimethylformamide (2 mL), and N,N' -diisopropylethylamine (0.24 mL, 0.13 mmol) was dissolved at 0 °C under a nitrogen atmosphere and the compound 71 (68 mg, 0.073 mmol) was added sequentially, followed by stirring at room temperature for 2 hours. The reaction solution was diluted with ethyl acetate (30 mL), washed with brine (20 mL X 2), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 72 (62 mg, 75%). EI-MS m/z: [M+H] ⁺ 1247.90.

Preparation of compound 73

Compound 72 (62 mg, 0.049 mmol) was dissolved in 2-propanol (1 mL) and tetrahydrofuran (1 mL), and lithium hydroxide (6.2 mg, 0.15) dissolved in distilled water (1 mL) at -50 ° C under a nitrogen atmosphere. mmol) and tetramethylammonium hydroxide (1.1 M aqueous solution, 0.05 mmol) were sequentially added and stirred at -10 °C for 3 hours. After adjusting the pH to about 4-5 with acetic acid, compound 73 (crude) was obtained by concentration. EI-MS m/z: [M+H] ⁺ 1107.76.

Preparation of compound 74

Compound 73 (crude) obtained above was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (1 mL) was added at 0 °C under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 1.5 hours, concentrated, and purified by HPLC to obtain compound 74 (18 mg). EI-MS m/z: [M+H] ⁺ 1007.68, 1/2 [M+H] ⁺ 504.60.

<Example 16> Preparation of compound 80

Preparation of compound 75

Ethyl bromodifluoroacetate (2.02 g, 9.98 mmol), 4-iodo-1-methyl-2-nitro-benzene (2.6 g, 9.98 mmol), and copper powder (1.6 g, 25.95 mmol) were placed in a nitrogen atmosphere. It was dissolved in dimethyl sulfoxide (15 mL) under the conditions and stirred at 60 °C for 12 hours. After lowering the temperature to room temperature, the mixture was diluted with ethyl acetate (50 mL), filtered through celite, and the filtrate was washed sequentially with 1 N aqueous hydrochloric acid (15 mL) and brine (2 X 15 mL), followed by anhydrous sodium sulfate. dried. Filtration, concentration, and purification by column chromatography gave compound 75 (1.07 g, 41%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.21 (s, 1H), 7.75 (d, 1H), 7.48 (d, 1H), 4.31 (q, 2H), 2.67 (s, 3H), 1.32 (t) , 3H).

Preparation of compound 76

Compound 75 (1.07 g, 4.12 mmol) was dissolved in ethanol (35 mL), and palladium/charcoal (10 % w/w, 100 mg) was added thereto. The reaction solution was stirred at room temperature under a hydrogen atmosphere for 18 hours. The reaction solution was filtered through celite and concentrated to obtain compound 76 (0.92 g, 98%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.07 (d, 1H), 6.92 (d, 1H), 6.89 (s, 1H), 4.31 (q, 2H), 2.18 (s, 3H), 1.32 (t) , 3H).

Preparation of compound 77

Compound 76 (444 mg, 1.94 mmol) was dissolved in toluene (10 mL), di- t -butyl dicarbonate (0.53 mL, 2.33 mmol) was added, and the reaction solution was stirred at 100° C. under a nitrogen atmosphere for 16 hours. The reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 77 (624 mg, 98%). EI-MS m/z: [M+H] ⁺ 484.45

Preparation of compound 78

Compound 77 (200 mg, 0.61 mmol) was dissolved in tetrahydrofuran (1 mL), methanol (2.5 mL), and distilled water (2 mL), and potassium carbonate (120 mg, 0.87 mmol) was added thereto and at room temperature for 3 hours. stirred. The reaction solution was concentrated under reduced pressure, adjusted to pH 3 with 0.1 N aqueous hydrochloric acid solution, extracted with ethyl acetate (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 78 (163 mg, crude). ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.98 (br, 1H), 7.26-7.21 (m, 3H), 2.28 (s, 3H), 1.52 (s, 9H).

Preparation of compound 79

After dissolving compound 78 (163 mg, 0.54 mmol) and compound 8 (167 mg, 0.54 mmol) in dimethylformamide (3 mL), 0 ° C. under a nitrogen atmosphere, N,N' -diisopropylethylamine (0.29 mL) , 1.62 mmol) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-bi]pyridinium 3-oxide hexafluorophosphate (HATU, 226mg , 0.595) mmol) and stirred at room temperature for 16 h. The reaction solution was diluted with ethyl acetate (10 mL), washed with saturated aqueous sodium hydrogen carbonate solution (10 mL) and distilled water (10 mL) in that order, and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 79 (72 mg, 23%). EI-MS m/z: [M+H] ⁺ 557.3.

Preparation of compound 80

Compound 79 (72 mg, 0.13 mmol) was dissolved in dichloromethane (1 mL), and then trifluoroacetic acid (1 mL) was added at 0° C. under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 3 hours, concentrated, and purified by HPLC to obtain compound 80 (53 mg). EI-MS m/z: [M+H] ⁺ 457.2.

<Example 17> Preparation of compound 83

Preparation of compound 81

Compound 39 (130 mg, 0.160 mmol) was dissolved in dry tetrahydrofuran (3 mL), followed by phosgene (15% toluene solution, 0.02 mL, 0.18 mmol) and N,N' -diisopropylethylamine (0.047 mL, 0.13) mmol) was added at -40 °C under a nitrogen atmosphere and stirred for 40 min. Compound 80 (76 mg, 0.13 mmol) dissolved in dry tetrahydrofuran (1 mL) was added to the reaction solution at 0° C. under a nitrogen atmosphere. After stirring for 12 hours, the reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 81 (70 mg, 40%). EI-MS m/z: [M+Na] ⁺ 1335.8.

Preparation of compound 82

Compound 81 (32 mg, 0.025 mmol) was dissolved in 2-propanol (1 mL) and tetrahydrofuran (1 mL), followed by lithium hydroxide (4.2 mg, 0.099) dissolved in distilled water (1 mL) at -50 ° C under a nitrogen atmosphere. mmol) and tetramethylammonium hydroxide (1.1 M aqueous solution, 0.025 mmol) were sequentially added and stirred at -10 °C for 3 hours. After adjusting the pH to about 4-5 with acetic acid, it was concentrated to obtain compound 82 (crude) EI-MS m/z: [M+H] ⁺ 1173.7.

Preparation of compound 83

Compound 82 (crude 21 mg) obtained above was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (1 mL) was added at 0 °C under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 1.5 hours, concentrated, and purified by HPLC to obtain compound 83 (17 mg). EI-MS m/z: [M+H] ⁺ 973.7.

<Example 18> Preparation of compound 88

Preparation of compound 84

Diethyl azodicarboxylate (40% toluene solution, 273 mg, 1.57 mmol) and t -butyl 4-hydroxypiperidine-1-carboxylate (289 mg, 1.44 mmol) in toluene (10 mL) After dissolution, triphenylphosphine (411 mg, 1.57 mmol) and 2-methyl-5-nitrophenol (200 mg, 1.31 mmol) were added under a nitrogen atmosphere at room temperature. After stirring for 4 hours, it was diluted with ethyl acetate (50 mL), washed with distilled water (50 mL), and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, it was purified by column chromatography to obtain compound 84 (331 mg, 75%). ¹ H-NMR (400 MHz, CDCl ₃ )δ 7.76-7.74 (m, 1H), 7.66 (s, 1H), 7.27 (d, 1H), 4.64-4.62 (m, 2H), 3.67-3.61 (m, 2H), 3.49-3.43 (m, 2H), 2.30 (s, 3H), 1.99-1.94 (m, 2H), 1.83-1.81 (m, 2H), 1.48 (s, 9H).

Preparation of compound 85

Compound 84 (350 mg, 1.04 mmol) was dissolved in methanol (10 mL), and palladium/charcoal (10 % w/w, 70 mg) was added thereto. The reaction solution was stirred at room temperature under a hydrogen atmosphere for 18 hours. The reaction solution was filtered through celite and concentrated to obtain compound 85 (325 mg). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 6.74 (d, 1H), 6.23 (s, 1H), 6.06 (d, 1H), 4.78 (s, 2H), 4.33 (s, 1H), 3.60- 3.50 (m, 2H), 3.30-3.25 (m, 2H), 1.99 (s, 3H), 1.90-1.80 (m, 2H), 1.60-1.45 (m, 2H), 1.40 (s, 9H).

Preparation of compound 86

Compound 85 (70 mg, 0.23 mmol) was dissolved in dichloromethane (2 mL), bis (pentafluorophenyl) carbonate (99 mg, 0.25 mmol) and pyridine (0.024 ml, 0.29 mmol) were added at room temperature. Stirred for 3 hours. After the reaction solution was concentrated under reduced pressure, compound 86 obtained was immediately used in the next reaction.

Preparation of compound 87

Compound 8 (85 mg, 0.22 mmol) was dissolved in N,N -dimethylformamide (1.5 mL), and N,N′ -diisopropylethylamine (0.12 mL, 0.66 mmol) was sequentially added thereto. After adding the compound 86 obtained above, the mixture was stirred at room temperature for 16 hours. The reaction solution was diluted with dichloromethane (10 mL), washed with distilled water (2 X 10 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 87 (65 mg, 49%).

Preparation of compound 88

Compound 87 (65 mg, 0.11 mmol) was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (1 mL) was added at 0° C. under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 3 hours, concentrated, and purified by HPLC to obtain compound 88 (40 mg). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.98 (s, 1H), 8.61 (s, 1H), 7.69 (d, 1H), 7.50 (s, 1H), 7.43 (d, 1H), 7.32 (s, 1H), 6.79 (t, 1H), 6.73 (d, 1H), 5.13-5.08 (m, 1H), 4.60-4.50 (m, 1H), 4.47-4.28 (m, 4H), 2.96-2.87 (m, 1H), 2.62-2.50 (m, 1H), 2.45-2.36 (m, 1H), 2.09-1.96 (m, 5H), 1.87-1.82 (m, 2H).

<Example 19> Preparation of compound 89

Compound 89 was synthesized from compound 88 and compound 71 in a similar manner to compound 74. EI-MS m/z: [M+H] ⁺ 1022.03,1/2 [M+H] ⁺ 512.25.

<Example 20> Preparation of compound 91

Preparation of compound 90

After dissolving compound 10 (100 mg, 0.19 mmol) in N,N -dimethylformamide (5 mL) at 0 °C, under a nitrogen atmosphere, N- ( t -butoxycarbonyl) -N -methylglycine (53 mg, 0.28 mmol), N,N,N',N' -tetramethyl- O- (1 H -benzotriazol-1-yl)uronium hexafluorophosphate (HBTU, 127 mg, 0.34 mmol), and N, N' -diisopropylethylamine (0.1 mL, 0.56 mmol) was sequentially added and stirred at room temperature for 2 hours. The reaction solution was diluted with ethyl acetate (50 mL), washed with saturated aqueous sodium hydrogen carbonate solution (50 mL), and then distilled water (50 mL) in that order, and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 90 (64 mg, 58%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.99 (s, 1H), 9.23-9.20 (m, 1H), 8.59 (s, 1H), 7.68 (d, 1H), 7.51-7.48 (m, 2H), 7.43 (d, 1H), 7.16 (d, 1H), 7.03 (d, 1H), 6.64 (m, 1H), 5.12-5.08 (m, 1H), 4.46-4.28 (m, 4H), 3.96 (m, 2H), 2.94-2.86 (m, 4H), 2.61-2.57 (m, 1H), 2.40-2.35 (m, 1H), 2.10 (s, 3H), 2.00-1.97 (m, 1H), 1.41 -1.34 (m, 9H).

Preparation of compound 91

Compound 90 (13 mg, 0.02 mmol) was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (1 mL) was added at 0° C. under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 2 hours, concentrated and purified by HPLC to obtain compound 91 (8 mg, 60%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.97 (s, 1H), 9.78 (s, 1H), 8.74 (br, 2H), 8.65 (s, 1H) 7.69 (d, 1H), 7.64 (s) , 1H), 7.51 (s, 1H), 7.44 (d, 1H), 7.08 (s, 2H), 6.72 (t, 1H), 5.13-5.08 (m, 1H), 4.46-4.28 (m, 4H), 3.94 (t, 2H), 2.96-2.87 (m, 1H), 2.67-2.57 (m, 4H), 2.40-2.36 (m, 1H), 2.14 (s, 3H), 2.01-1.98 (m, 1H). EI-MS m/z: [M+H] ⁺ 493.48.

<Example 21> Preparation of compound 92

Compound 92 was synthesized from compound 91 and compound 71 in a similar manner to compound 74. EI-MS m/z: [M+H] ⁺ 1009.65.

<Example 22> Preparation of compound 94

Preparation of compound 93

Compound 10 (164 mg, 0.31 mmol) was dissolved in N,N -dimethylformamide (3 mL) and then 1-( t -butoxycarbonyl)azetidine-3-carboxylic acid ( 92 mg, 0.46 mmol), N,N,N',N' -tetramethyl- O- (1 H -benzotriazol-1-yl)uronium hexafluorophosphate (HBTU, 209 mg, 0.55 mmol), Then , N,N' -diisopropylethylamine (0.16 mL, 0.91 mmol) was sequentially added and stirred at room temperature for 2 hours. The reaction solution was diluted with ethyl acetate (50 mL), washed with saturated aqueous sodium hydrogen carbonate solution (50 mL), and then distilled water (50 mL) in that order, and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 93 (117 mg, 63%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.98 (s, 1H), 9.35 (s, 1H), 8.60 (s, 1H), 7.69 (d, 1H), 7.51-7.50 (m, 2H) , 7.44 (d, 1H), 7.16 (d, 1H), 7.04 (d, 1H), 6.67 (t, 1H), 5.12-5.08 (m, 1H), 4.47-4.28 (m, 4H), 4.00-3.91 (m, 4H), 3.55-3.50 (m, 1H), 2.94-2.86 (m, 1H), 2.61-2.57 (m, 1H), 2.40-2.32 (m, 1H), 2.08 (s, 3H), 2.00 -1.97 (m, 1H), 1.38-1.35 (m, 9H). EI-MS m/z: [M+H] ⁺ 605.58.

Preparation of compound 94

Compound 93 (13 mg, 0.02 mmol) was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (1 mL) was added at 0° C. under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 2 hours, concentrated and purified by HPLC to obtain compound 94 (8 mg, 60%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.98 (s, 1H), 9.45 (s, 1H), 8.74-8.68 (m, 3H), 7.69 (d, 1H), 7.61 (s, 1H), 7.51 (s, 1H), 7.44 (d, 1H), 7.08 (s, 2H), 6.71 (t, 1H), 5.13-5.08 (m, 1H), 4.46-4.28 (m, 4H), 4.00-3.91 ( m, 4H), 3.81-3.71 (m, 1H), 2.94-2.86 (m, 1H), 2.67-2.58 (m, 1H), 2.40-2.36 (m, 1H), 2.09 (s, 3H), 2.01- 1.98 (m, 1H). EI-MS m/z: [M+H] ⁺ 505.52.

<Example 23> Preparation of compound 95

Compound 95 was synthesized from compound 94 and compound 71 in a similar manner to compound 74. ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.98 (s, 1H), 9.38 (s, 1H) 8.60 (s, 1H), 8.32 (t, 1H), 7.78 (d, 1H), 7.68 ( d, 1H), 7.51-7.49 (m, 3H), 7.43 (d, 1H), 7.25 (d, 2H), 7.16 (d, 1H), 7.04 (d, 1H) 6.67 (t, 1H), 5.15 5.02 (m, 4H), 4.47-4.28 (m, 4H), 4.10-3.96 (m, 7H), 3.65-3.52 (m, 12H) 2.95-2.86 (m, 1H), 2.67-2.57 (m, 1H) , 2.40-2.32 (m, 1H), 2.07 (s, 3H), 2.03-1.98 (m, 1H). EI-MS m/z: [M+H] ⁺ 1021.72.

<Example 24> Preparation of compound 100

Preparation of compound 96

Diethyl azodicarboxylate (40% toluene solution, 297 mg, 1.47 mmol) and t -butyl (trans-4-hydroxycyclohexyl) carbamate (230 mg, 1.08 mmol) in tetrahydrofuran (5 mL) After dissolving, triphenylphosphine (385 mg, 1.47 mmol) and 2-methyl-5-nitrophenol (150 mg, 0.98 mmol) were added at room temperature and under a nitrogen atmosphere. After stirring under reflux for 16 hours, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 96, which was used in the next reaction without purification. ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.73 (d, 1H), 7.64 (s, 1H), 7.25 (d, 1H), 4.45 (s, 1H), 4.35-4.25 (m, 1H), 3.70 ( s, 1H), 2.27 (s, 3H), 2.17-2.09 (t, 4H), 1.67-1.55 (m, 2H), 1.46 (s, 9H), 1.38-1.25 (m, 2H).

Preparation of compound 97

Compound 96 obtained above was dissolved in methanol (3 mL) and ethyl acetate (2 mL), and palladium/charcoal (10 % w/w, 35 mg) was added thereto. The reaction solution was stirred at room temperature under a hydrogen atmosphere for 1 hour. The reaction solution was filtered through celite, concentrated, and purified by column chromatography to obtain compound 97 (90 mg, 28%, 2 steps).

Preparation of compound 98

Compound 97 (85 mg, 0.27 mmol) was dissolved in dichloromethane (2 mL), bis (pentafluorophenyl) carbonate (115 mg, 0.29 mmol) and pyridine (0.028 ml, 0.35 mmol) were added at room temperature. Stirred for 3 hours. After the reaction solution was concentrated under reduced pressure, the obtained compound 98 was directly used in the next reaction.

Preparation of compound 99

Compound 8 (103 mg, 0.27 mmol) was dissolved in N,N -dimethylformamide (1.5 mL), and N,N′ -diisopropylethylamine (0.14 mL, 0.80 mmol) was sequentially added thereto. After adding compound 98 to the reaction solution, the mixture was stirred at room temperature for 16 hours. The reaction solution was diluted with ethyl acetate (20 mL), washed with distilled water (2 X 10 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 99 (147 mg, 88%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.99 (s, 1H), 7.69 (d, 1H), 7.51 (s, 1H), 7.43 (d, 1H), 7.18 (s, 1H), 6.94 (d, 1H), 6.81-6.75 (m, 2H), 6.70-6.65 (m, 1H), 5.13-5.08 (m, 1H), 4.50-4.29 (m, 4H), 4.09 (s, 1H), 2.99 -2.85 (m, 1H), 2.62-2.36 (m, 2H), 2.10-1.98 (m, 5H), 1.85-1.75 (d, 2H), 1.47-1.15 (m, 15H).

Preparation of compound 100

Compound 99 (147 mg, 0.23 mmol) was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (1 mL) was added at 0° C. under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 3 hours, concentrated and purified by HPLC to obtain compound 100 (75 mg). ¹ HNMR (400 MHz, DMSO-d ₆ ): δ 10.99 (s, 1H), 8.58 (s, 1H), 7.80 (s, 2H), 7.70 (d, 1H), 7.51 (s, 1H), 7.43 ( d, 1H), 7.29 (s, 1H), 6.95 (d, 1H), 6.76-6.71 (m, 2H), 5.13-5.08 (m, 1H), 4.09 (s, 1H), 3.09 (s, 1H) , 2.93-2.88 (m, 1H), 2.62-2.36 (m, 2H), 2.10-1.98 (m, 8H), 1.49-1.41 (m, 4H). EI-MS m/z: [M+H] ⁺ 520.56.

<Example 25> Preparation of compound 101

Compound 101 was synthesized from Compound 100 and Compound 71 in a similar manner to Compound 74. EI-MS m/z: [M+H] ⁺ 1036.75,1/2 [M+H] ⁺ 519.16.

<Example 26> Preparation of compound 105

Preparation of compound 102

After dissolving 2-(methylamino)ethanol (1 g, 13.3 mmol) in dichloromethane (40 mL), di- t -butyl dicarbonate (3.0 mL, 13.3 mmol) was added, and the reaction solution was cooled to room temperature, nitrogen atmosphere. under stirring for 1 hour. The reaction solution was washed with brine, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain compound 102 (2.29 g, 98%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 3.75 (q, 2H), 3.40 (t, 2H), 2.92 (s, 3H), 1.46 (s, 9H).

Preparation of compound 103

After dissolving diethyl azodicarboxylate (40% toluene solution, 3.92 mmol) and compound 102 (629 mg, 3.59 mmol) in toluene (10 mL) at room temperature, under a nitrogen atmosphere, triphenylpostine (1.03 g, 3.92 mmol) and 2-methyl-5-nitrophenol (500 mg, 3.26 mmol) were added. After stirring at room temperature for 18 hours, distilled water (50 mL) was added, extraction was performed with ethyl acetate (50 mL), and the organic layer was dried over anhydrous sodium sulfate. Filtration, concentration under reduced pressure, and purification by column chromatography gave compound 103 (780 mg, 77%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.78 (d, 1H), 7.64 (s, 1H), 7.27 (s, 1H), 4.15-4.13 (m, 2H), 3.69-3.65 (m, 2H) , 3.02 (d, 3H), 2.30 (s, 3H), 1.46 (s, 9H).

Preparation of compound 104

Compound 104 was synthesized from compound 103 in a manner similar to that of compound 87. EI-MS m/z: [M+H] ⁺ 580.6.

Preparation of compound 105

Compound 104 (145 mg, 0.455 mmol) was dissolved in dichloromethane (4 mL), and then trifluoroacetic acid (1 mL) was added at 0° C. under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 3 hours, concentrated and purified by HPLC to obtain compound 105 (150 mg, 56 %). EI-MS m/z: [M+H] ⁺ 480.4.

<Example 27> Preparation of compound 109

Preparation of compound 106

Compound 39 (150 mg, 0.18 mmol) was dissolved in dichloromethane (3 mL) and then bis(pentafluorophenyl)carbonate (78 mg, 0.19 mmol) and pyridine (0.019 mL, 0.235 mmol) at 0 °C under a nitrogen atmosphere ) was added. The reaction solution was stirred at room temperature for 3 hours and then concentrated to obtain compound 106 (180 mg). EI-MS m/z: [M+H] ⁺ 1042.1.

Preparation of compound 107

Compound 106 (175 mg, 0.168 mmol) and compound 105 (100 mg, 0.168 mmol) were dissolved in dimethylformamide (2 mL) and N,N' -diisopropylethylamine (0.07 mL, 0.50 mmol) was added thereto The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, diluted with ethyl acetate (50 mL), washed with saturated aqueous ammonium chloride solution (50 mL), saturated aqueous sodium hydrogen carbonate solution (50 mL), and distilled water (50 mL) in that order, and dried over anhydrous sodium sulfate. . After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 107 (181 mg, 80%). EI-MS m/z: [M+H] ⁺ 1337.0.

Preparation of compound 108

After dissolving compound 107 (181 mg, 0.13 mmol) in 2-propanol (2 mL) and tetrahydrofuran (2 mL), lithium hydroxide (23 mg, 0.54 mmol) aqueous solution and tetramethylammonium hydroxide (1.1 M aqueous solution, 0.13 mL, 0.13 mmol) were sequentially added and stirred at -10 °C for 3 hours. After adjusting the pH to about 4-5 with acetic acid, compound 108 (crude) was obtained by concentration. EI-MS m/z: [M+H] ⁺ 1196.8.

Preparation of compound 109

Compound 108 (crude) obtained above was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (1 mL) was added at 0 °C under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 1.5 hours, concentrated and purified by HPLC to obtain compound 109 (70.4 mg). EI-MS m/z: [M+H] ⁺ 996.7.

<Example 28> Preparation of compound 117

Preparation of compound 110

Ethyl 2-methyl-6-oxo-1 H -pyridine-3-carboxylate (1.48 g, 8.17 mmol) was dissolved in phosphoryl chloride (15 mL) at 0° C. under a nitrogen atmosphere. The reaction temperature was raised to 100 °C and stirred for 2 hours under nitrogen atmosphere. After cooling to 0 °C, ice water (50 mL) was slowly added dropwise. The pH was adjusted to 7 using an aqueous ammonia solution and filtered to obtain compound 110 (1.52 g, 93%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.16 (d, 1H), 7.23 (d, 1H), 4.38 (q, 2H), 2.82 (s, 3H), 1.40 (t, 3H). EI-MS m/z: [M+H] ⁺ 200.25.

Preparation of compound 111

After dissolving compound 110 (1.52 g, 7.61 mmol) in benzene (20 mL) under nitrogen atmosphere, N -bromosuccinimide (NBS, 2.17 g, 12.18 mmol) and azobisisobutyronitrile (AIBN, 1.62 g) , 9.90 mmol) were added sequentially and stirred under reflux for 17 hours. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous sodium hydrogen carbonate solution (50 mL), distilled water (50 mL) in that order, and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 111 (1.18 g, 56%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 8.24 (d, 1H), 7.35 (d, 1H), 4.96 (s, 2H), 4.43 (q, 2H), 1.44 (t, 3H).

Preparation of compound 112

Compound 111 (1.18 g, 4.24 mmol) and 3-aminopiperidine-2,6-dione hydrochloride (836 mg, 5.08 mmol) were dissolved in dimethylformamide (20 mL) at 0 ° C under a nitrogen atmosphere. N,N' -diisopropylethylamine (2.2 mL, 12.71 mmol) was added and stirred at 80 °C for 16 h. After concentration under reduced pressure and dilution with dichloromethane (100 mL), the resulting solid was filtered and compound 112 (970 mg, 82%) was obtained. ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 11.03 (s, 1H), 8.20 (d, 2H), 7.68 (d, 1H), 5.19-5.15 (m, 1H), 4.57-4.36 (m, 2H), 2.92-2.87 (m, 1H), 2.58-2.56 (m, 1H), 2.47-2.33 (m, 1H), 2.05-1.99 (m, 1H). EI-MS m/z: [M+H] ⁺ 280.32.

Preparation of compound 113

Compound 112 (970 mg, 3.47 mmol) was dissolved in dimethylformamide (10 mL), and zinc cyanide (Zn(CN) ₂ , 2.03 g, 17.34 mmol) was added under nitrogen atmosphere. After bubbling with nitrogen for 20 minutes, tetrakis(triphenylphosphine)palladium(0) (801 mg, 0.69 mmol) was added. The reaction solution was stirred at 120 °C for 17 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, diluted with dichloromethane (5 mL) and diethyl ether (20 mL), and the resulting solid was filtered and compound 113 was filtered. (603 mg, 82%) was obtained. ¹ H-NMR (400 MHz, DMSO-d ₆ ): δ 11.06 (s, 1H), 8.42 (d, 2H), 8.21 (d, 1H), 5.24-5.19 (m, 1H), 4.66-4.45 (m, 2H), 2.96-2.87 (m, 1H), 2.59-2.56 (m, 1H), 2.46-2.40 (m, 1H), 2.06-2.02 (m, 1H). EI-MS m/z: [M+H] ⁺ 271.35.

Preparation of compound 114

Compound 113 (603 mg, 2.23 mmol) was dissolved in dimethylformamide (15 mL) and methanol (5 mL), and then Raney Ni (2 g) and di- t -butyl dicarbonate (0.61 mL, 2.67 mmol) ) were sequentially added, and the reaction solution was stirred at room temperature under a hydrogen atmosphere for 48 hours. The reaction solution was filtered through Celite, concentrated under reduced pressure, and purified by column chromatography to obtain compound 114 (209 mg, 25%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 11.00 (s, 1H), 8.13 (d, 1H), 7.57 (t, 1H), 7.41 (d, 1H) 5.19-5.14 (m, 1H), 4.45 -4.29 (m, 4H), 2.95-2.87 (m, 1H), 2.61-2.58 (m, 1H), 2.47-2.40 (m, 1H), 2.03-1.98 (m, 1H), 1.40 (s, 9H) . EI-MS m/z: [M+H] ⁺ 375.43.

Preparation of compound 115

Compound 114 (209 mg, 0.55 mmol) was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (1 mL) was added at 0° C. under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 1.5 hours and concentrated under reduced pressure to obtain compound 115 (210 mg, crude). EI-MS m/z: [M+H] ⁺ 275.40.

Preparation of compound 116

Compound 2 (156 mg, 0.70 mmol) was dissolved in dichloromethane (5 mL) and then bis(pentafluorophenyl)carbonate (319 mg, 0.81 mmol) and pyridine (0.09 mL, 1.08 mmol) were dissolved in dichloromethane (5 mL) at 0 °C under a nitrogen atmosphere. ) was added. After the reaction solution was stirred at room temperature for 3 hours, compound 115 (210 mg, 0.54 mmol) and N,N' -diisopropylethylamine (0.29 mL, 1.62 mmol) were dissolved in dimethylformamide (1 mL) and added. and stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, diluted with ethyl acetate (50 mL), washed with saturated aqueous ammonium chloride solution (50 mL), saturated aqueous sodium hydrogen carbonate solution (50 mL), and distilled water (50 mL) in that order, and dried over anhydrous sodium sulfate. . After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 116 (192 mg, 67%). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 10.99 (s, 1H), 8.73 (s, 1H), 8.39 (s, 1H), 8.12-8.09 (m, 1H), 7.48-7.46 (m, 1H) ), 7.39 (s, 1H), 7.12-7.11 (m, 1H), 6.98-6.96 (m, 1H), 6.74-6.72 (m, 1H), 5.17-5.14 (m, 1H), 4.49-4.30 (m) , 4H), 3.01-2.86 (m, 1H), 2.71-2.56 (m, 1H), 2.33-2.31 (m, 1H) 2.08-1.96 (m, 4H), 1.43 (s, 9H). EI-MS m/z: [M+H] ⁺ 523.55.

Preparation of compound 117

Compound 116 (17 mg, 0.03 mmol) was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (1 mL) was added at 0° C. under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 1.5 hours and concentrated under reduced pressure to obtain compound 117 (12 mg). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 11.01 (s, 1H), 8.83 (s, 1H), 8.13 (d, 1H), 7.48 (d, 1H), 7.42 (br, 1H), 7.04 ( d, 1H), 6.93-6.85 (m, 2H), 5.20-5.15 (m, 1H), 4.52-4.32 (m, 4H), 2.97-2.88 (m, 1H), 2.67-2.59 (m, 1H), 2.45-2.42 (m, 1H), 2.13 (s, 3H), 2.04-2.01 (m, 1H). EI-MS m/z: [M+H] ⁺ 423.49.

<Example 29> Preparation of compound 120

Preparation of compound 118

Compound 117 (76 mg, 0.14 mmol) and compound 71 (146 mg, 0.16 mmol) were dissolved in dimethylformamide (3 mL), and then 1-hydroxy-7-azabenzotriazole ( 9.5 mg, 0.07 mmol) and N,N' -diisopropylethylamine (0.08 mL, 0.42 mmol) were sequentially added and stirred at room temperature for 3 hours. The reaction solution was diluted with ethyl acetate (50 mL), washed with saturated aqueous ammonium chloride solution (50 mL), saturated aqueous sodium hydrogen carbonate solution (50 mL), and distilled water (50 mL) in that order, and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 118 (154 mg, 92%). EI-MS m/z: [M+H] ⁺ 1179.76.

Preparation of compound 119

After dissolving compound 118 (154 mg, 0.13 mmol) in 2-propanol (1 mL) and tetrahydrofuran (1 mL), lithium hydroxide (27 mg, 0.39 mmol) aqueous solution and tetramethylammonium hydroxide (1.1 M aqueous solution, 0.12 mL, 0.13 mmol) were sequentially added, and the mixture was stirred at -10 °C for 3 hours. After adjusting the pH to about 4-5 with acetic acid, it was concentrated to obtain compound 119 (130 mg, crude). EI-MS m/z: [M+H] ⁺ 1039.71.

Preparation of compound 120

Compound 119 (130 mg, crude) was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (1 mL) was added at 0 ℃ under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 1.5 hours, concentrated and purified by HPLC to obtain compound 120 (56 mg). ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 11.01 (s, 1H), 8.88 (s, 1H), 8.76 (s, 1H), 8.31 (t, 1H), 8.31 (d, 1H), 7.92 ( d, 1H), 7.61-7.45 (m, 3H), 7.27 (d, 1H), 7.15 (d, 1H), 7.00 (d, 1H), 6.78 (t, 1H), 5.19-5.10 (m, 4H) , 4.53-4.05 (m, 4H), 3.98-3.97 (m, 2H), 3.72-3.63 (m, 2H), 3.50-3.36 (m, 8H), 2.97-2.87 (m, 1H), 2.67-2.58 ( m, 1H), 2.49-2.44 (m, 1H), 2.04 (s, 3H), 2.04-1.90 (m, 1H). EI-MS m/z: [M+H] ⁺ 939.66.

<Example 30> Preparation of compound 122

Preparation of compound 121

Methyl 2-(2-(2-aminoethoxy)ethoxy)acetate (573 mg, 3.23 mmol) and 2-( t -butoxycarboxylamino)oxy-2-methyl-propionic acid (779 mg, 3.56 mmol) were mixed After dissolving in dry dimethylformamide (10 mL) under a nitrogen atmosphere, N,N,N',N' -tetramethyl- O- (1 H -benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) , 1.35 g, 3.56 mmol) was added. The reaction solution was stirred at room temperature for 5 hours. The reaction solution was diluted with ethyl acetate (50 mL), washed with saturated aqueous ammonium chloride solution (50 mL) and distilled water (50 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 121 (510 mg, 42%). ¹ HNMR (400 MHz, DMSO-d ₆ ): δ 4.13 (s, 1H), 3.64 (s, 3H), 3.45-3.20 (m, 6H), 1.49 (q, 2H), 1.42 (s, 3H), 1.39 (s, 9H), 1.30 (s, 3H)

Preparation of compound 122

After dissolving compound 121 (150 mg, 0.40 mmol) in methanol (1 mL) and tetrahydrofuran (3 mL), sodium hydroxide (24 mg, 0.60 mmol) dissolved in distilled water (1 mL) at 0 ° C under a nitrogen atmosphere Aqueous solution was added and stirred at room temperature for 3 hours. After adjusting the pH to about 4-5 with 1 N aqueous hydrochloric acid, lyophilization was performed to obtain compound 122 (135 mg, 92%). EI-MS m/z: [M+H] ⁺ 364.50 ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 9.38 (brs, 1H), 4.01 (s, 1H), 3.62-3.48 (m, 4H), 3.42 (t, 2H), 3.23 (t, 2H), 1.42 (s, 3H), 1.39 (s, 9H), 1.30 (s, 3H),

<Example 31> Preparation of compound 125

Preparation of compound 124

After dissolving compound 123 (149 mg, 0.30 mmol) and compound 122 (110 mg, 0.30 mmol) in dry dimethylformamide (3 mL) under a nitrogen atmosphere, N,N,N',N' -tetramethyl- O - (1 H -Benzotriazol-1-yl)uronium hexafluorophosphate (HBTU, 126 mg, 0.33 mmol) and N,N′ -diisopropylethylamine (0.16 mL, 0.91 mmol) were added. The reaction solution was stirred at room temperature for 7 hours. The reaction solution was diluted with ethyl acetate (50 mL), washed with saturated aqueous ammonium chloride solution (50 mL) and distilled water (50 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 124 (100 mg, 48%). EI-MS m/z: [M+H] ⁺ 806.63.

Preparation of compound 125

Compound 124 (100 mg, 0.30 mmol) was dissolved in tetrahydrofuran (5 mL) and water (1 mL), then triphenylphosphine (78 mg, 0.30 mmol) was added at room temperature and stirred at 60 °C for 7 hours. . The reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 125 (82 mg, 88%). EI-MS m/z: [M+H] ⁺ 754.62.

<Example 32> Preparation of compound 131

Preparation of compound 127

Compound 126 (478 mg, 0.82 mmol) was dissolved in dry tetrahydrofuran (9 mL), followed by phosgene (15% toluene solution, 0.089 mL, 0.82 mmol) and N,N' -diisopropylethylamine (0.36 mL, 1.04) mmol) was added at -40 °C under a nitrogen atmosphere and stirred for 40 min. After 40 minutes, compound 35 (200 mg, 0.68 mmol) dissolved in dry tetrahydrofuran (5 mL) was added to the reaction solution at 0° C. under a nitrogen atmosphere. After stirring for 12 hours, the reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 127 (320 mg, 50%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.52 (brs, 1H), 7.42-7.30 (m, 6H), 7.15-7.02 (m, 3H), 6.02-5.90 (m, 1H), 5.40-5.24 ( m, 5H), 5.20-5.10 (m, 2H), 5.06-4.92 (m, 1H), 4.72-4.60 (m, 2H), 4.20-4.16 (m, 1H), 3.71 (s, 3H), 2.12- 1.96 (m, 12H), 1.80-1.62 (m, 3H), 1.44-1.28 (m, 3H), 0.92-1.26 (m, 2H).

Preparation of compound 128

After dissolving compound 127 (140 mg, 0.16 mmol) in dry dimethylformamide (4 mL) at 0 ° C under a nitrogen atmosphere, tetrakis (triphenylphosphine) palladium (0) (9 mg, 0.008 mmol) and pyrroly Dean (0.04 mL, 0.48 mmol) was sequentially added and the temperature was slowly raised to room temperature, followed by stirring for 5 hours. The reaction solution was diluted with ethyl acetate (50 mL), washed with saturated aqueous ammonium chloride solution (50 mL) and distilled water (50 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 128 (70 mg, 55%). ¹ H NMR (400 MHz, CDCl ₃ ): δ 7.55 (brs, 1H), 7.46-7.30 (m, 5H), 8.10-7.04 (m, 1H), 6.88-6.96 (m, 1H), 6.56-6.50 (m , 1H), 6.38 (s, 1H), 5.38-5.24 (m, 5H), 5.18-5.08 (m, 1H), 5.04-4.92 (m, 2H), 4.18-4.14 (m, 1H), 3.72 (s) , 3H), 3.53 (s, 2H), 2.08-2.00 (m, 9H), 1.96-1.92 (m, 2H), 1.78-1.62 (m, 3H), 1.36-1.24 (s, 2H), 1.10-0.92 (m, 2H), EI-MS m/z: [M+H] ⁺ 805.56.

Preparation of compound 129

Compound 128 (70 mg, 0.087 mmol) was dissolved in dry tetrahydrofuran (9 mL) and then phosgene (15% toluene solution, 0.009 mL, 0.087 mmol) and N,N' -diisopropyl at -45 ° C under a nitrogen atmosphere Ethylamine (0.046 mL, 0.26 mmol) was added sequentially and stirred for 30 min. After 30 minutes, compound 8 (36 mg, 0.096 mmol) dissolved in dry tetrahydrofuran (5 mL) was added to the reaction solution at -45°C. The reaction solution was slowly raised to room temperature and stirred for 5 hours. The reaction solution was diluted with ethyl acetate (50 mL), washed with saturated aqueous ammonium chloride solution (50 mL) and distilled water (50 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 129 (84 mg, 87%). EI-MS m/z: [M+H] ⁺ 1104.83.

Preparation of compound 130

Compound 129 (84 mg, 0.087 mmol) was dissolved in methanol (3 mL) and ethyl acetate (2 mL), and palladium/charcoal (5 % w/w, 10 mg) was added thereto. The reaction solution was stirred at room temperature under a hydrogen atmosphere for 5 hours. The reaction solution was filtered through celite and concentrated to obtain compound 130 (60 mg, 78%).

Preparation of compound 131

After dissolving compound 130 (135 mg, 0.134 mmol) and compound 125 (42 mg, 0.056 mmol) in dry dimethylformamide (5 mL) under nitrogen atmosphere, N,N,N',N' -tetramethyl- O - (1 H -Benzotriazol-1-yl)uronium hexafluorophosphate (HBTU, 50 mg, 0.134 mmol) and N,N′ -diisopropylethylamine (30 uL, 0.17 mmol) were added. The reaction solution was stirred at room temperature for 7 hours. The reaction solution was diluted with ethyl acetate (50 mL), washed with saturated aqueous ammonium chloride solution (50 mL) and distilled water (50 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 131 (58 mg, 38%). EI-MS m/z: [M+2H] ⁺ 2747.39, 1/2 [M+H] ⁺ 1374.09.

<Example 33> Preparation of compound 133

Preparation of compound 132

Compound 131 (58 mg, 0.02 mmol) was dissolved in 2-propanol (1 mL) and tetrahydrofuran (1 mL), followed by lithium hydroxide (6.2 mg, 0.15) dissolved in distilled water (1.5 mL) at -50 ° C under a nitrogen atmosphere. mmol) aqueous solution and tetramethylammonium hydroxide (1.1 M aqueous solution, 0.04 mL, 0.042 mmol) were sequentially added, and the mixture was stirred at -10 °C for 3 hours. After adjusting the pH to about 4-5 with acetic acid, it was freeze-dried to obtain compound 132 (27 mg, crude). EI-MS m/z: 1/2 [M+H] ⁺ 1234.46.

Preparation of compound 133

Compound 132 (27 mg, crude) obtained above was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (0.5 mL) was added at 0 ℃ under a nitrogen atmosphere. The reaction solution was stirred at room temperature for 1.5 hours, concentrated and purified by HPLC to obtain compound 133 (14 mg). EI-MS m/z: [M+NH ₄ ] ⁺ 2368.37, 1/2 [M+H] ⁺ 1184.10.

<Example 34> Preparation of ADC

ADC was prepared through the following two steps, and commonly used isoprenoids LCB14-0511, 0512 and 0606 were prepared by the method described in Korean Patent Application Laid-Open No. 10-2014-0035393. The structural formulas of LCB14-0511, 0512 and 0606 are as follows.

Step 1: Preparation of prenylated antibody

In the present invention, the prenylation reaction of the antibody is performed in a buffer solution containing 24 μM antibody, 600 nM FTase and 0.144 mM or 0.250 mM isoprenoid (LCB14-0511, 0512 or 0606) {50 mM Tris hydrochloride (Tris-HCl) , pH 7.4), 5 mM magnesium chloride (MgCl ₂ ), 10 μM zinc chloride (ZnCl ₂ ), 0.250 mM dithiothreitol (DTT)} was used. After completion of the reaction, the prenylated antibody was decontaminated with a G25 Sepharose column (AKTA purifier, GE healthcare) equilibrated with PBS buffer.

Step 2: Drug-conjugation reaction

ADC using oxime binding reaction is prepared by mixing 300 mM sodium acetate (NaOAc) buffer (pH 5.2), 10% dimethyl sulfoxide (DMSO), 24 uM prenylated antibody and 240 uM linker-drug. and gently stirred at 30 °C. After 6 or 24 hours of reaction, excess low molecular weight compounds were removed using a G25 Sepharose column, and protein fractions were collected and concentrated. A typical ADC reaction pathway using an oxime binding reaction is shown in FIG. 2 .

For ADC using copper-free click binding reaction, a reaction mixture is prepared by mixing PBS buffer (pH 7.4), 1% dimethyl sulfoxide (DMSO), 10 uM prenylation antibody and 100 uM linker-drug. After 6 hours of reaction at 25 °C, excess low molecular weight compounds were removed using a G25 Sepharose column, and protein fractions were collected and concentrated.

ADC using copper click-bonding reaction is 20 uM prenylation antibody, 200 uM linker-drug, 10% dimethyl sulfoxide (DMSO), 1 mM copper sulfate pentahydrate, 2 mM (BimC4A)3 (Sigma-Aldrich) , 10 mM sodium ascorbate (sodium ascorbate) and 10 mM aminoguanidine hydrochloride were mixed and reacted at 25 °C for 3 hours, then treated with 20 mM EDTA and reacted for 30 minutes. After completion of the reaction, excess low molecular weight compounds were removed using a G25 Sepharose column, and protein fractions were collected and concentrated. A typical ADC reaction pathway using a click-bonding reaction is shown in FIG. 3 .

Anti-EGFR-ADC Manufacturing List ADC number Drug number* Linker-Drug Number* ADC1 10 14 ADC2 21 24 ADC3 34 44 ADC4 52 59 ADC5 69 74 ADC6 80 83 ADC7 88 89 ADC8 91 92 ADC9 94 95 ADC10 100 101 ADC11 105 109 ADC12 117 120 ADC13 27 30 ADC14** 34 133

* Example compound number, ** ADC with four molecules of compound introduced

<Example 35> in vitro Cytotoxicity Assessment

The anti-EGFR-ADC and the drug itself prepared in Example 34 were measured for cell proliferation inhibitory activity against cancer cells. For this purpose, commercially available cancer cell lines (HCC827 (NSCLC, non-small cell lung cancer cell line), MDA-MB-468 (TNBC, triple negative breast cancer cell line)) were used. Each cancer cell line was inoculated into a 96-well plate by 1500-5000 per well, incubated for 24 hours, and then the ADC and drug samples were serially diluted and processed. After 72 hours, the number of viable cells was quantified using SRB assay. The results of cytotoxicity against cancer cells are shown in Table 2.

Comparison of cytotoxicity of ADCs and drugs in antigen-expressing cancer cell lines ADC
number IC ₅₀ (nM) drug number IC ₅₀ (nM) HCC827
(NSCLC) MDA-MB-468
(TNBC) HCC827
(NSCLC) MDA-MB-468
(TNBC) ADC1 One 0.7 compound 10 850 35.3 ADC2 >200 >200 compound 21 >2000 >2000 ADC3 0.5 3.4 compound 34 10.2 0.8 ADC4 0.4 10.8 compound 52 1700 25.6 ADC5 0.3 15.0 compound 69 >2000 1200 ADC6 7.7 >200 compound 80 >2000 >2000 ADC7 0.6 58.9 compound 88 >2000 >2000 ADC8 0.7 80.8 compound 91 >2000 >2000 ADC9 0.5 0.7 compound 94 >2000 >2000 ADC10 0.3 1.1 compound 100 389 200 ADC11 0.4 4.3 compound 105 >2000 >2000 ADC12 1.7 10.7 compound 117 >2000 >2000 ADC13 865 >200 compound 27 450 238 ADC14 0.09 0.1 compound 34 10.2 0.8

In the antigen-overexpressed cancer cell line, ADC1 showed IC ₅₀ values of 1 nM (HCC827) and 0.7 nM (MDA-MB-468), whereas Compound 10 of the drug itself showed 850 nM (HCC827), 35.3 nM (MDA-MB-) 468) showed cytotoxicity. The above results showed that ADC1 derived according to the present invention was about 50 to 850 times superior to the cytotoxicity of the drug itself. In addition, compared to compounds 69, 88, 91, 94, 105, and 117, which have very weak drug activity, the cytotoxicity of these ADCs showed hundreds of times better activity than the compound itself. Four molecules of compound 34 were introduced In the cytotoxicity of ADC14, significantly improved activity (5.5-34-fold improvement) was observed compared to ADC3 in which two molecules of compound 34 were introduced.

On the other hand, ADC13 to which Compound 27 having a different scaffold structure was applied, which is a control material, showed a marked decrease in activity to hundreds of nM or more.

Claims (54)

A conjugate represented by the following general formula (I) or a pharmaceutically acceptable salt thereof:
[General Formula I]
Ab - [Linker - (B) _m ] _n
In the above formula,
Ab is an antigen specific binding moiety;
Linker is a linker;
B is a protein degrader moiety;
m and n are each independently an integer selected from 1 to 20.
The method of claim 1,
The antigen-specific binding moiety is an antibody construct or an antibody-like protein, a conjugate or a pharmaceutically acceptable salt thereof.
3. The method of claim 2,
The antibody construct comprises an antigen-binding domain and an Fc domain that binds to an antigen, a conjugate or a pharmaceutically acceptable salt thereof.
3. The method of claim 2,
The antibody construct further comprises an effector antigen-binding domain that binds to an antigen presented by an antigen-presenting cell, a conjugate or a pharmaceutically acceptable salt thereof.
3. The method of claim 2,
The antibody construct may be a monoclonal antibody, a domain antibody (dAb), a single chain antibody (scAb), a Fab fragment, a F(ab') ₂ fragment, a single chain variable antibody (single). chain variable fragment; scFv), scFv-Fc fragment, single domain heavy chain antibody, single domain light chain antibody, antibody variant (variant antibody), multimeric antibody and a bispecific antibody (bispecific antibody), the conjugate or a pharmaceutically acceptable salt thereof.
The method of claim 1,
The linker is cleavable, a conjugate or a pharmaceutically acceptable salt thereof.
The method of claim 1,
The linker is a protease cleavable linker, acid-cleavable linker, disulfide linker, self-immolative linker (self-immolative linker), self-stabilizing linker (self stabilizing linker), is selected from the group consisting of a malonate linker, a maleimidobenzoyl linker, a 3'-N-amide analog, a beta-glucuronide linker and a beta-galactoside linker,
wherein the protease cleavable linker comprises a thiol-reactive maleimidocaproyl spacer, valine-citrulline dipeptide or p-amino-benzyloxycarbonyl spacer,
The acid-cleavable linker is a hydrazine linker or a quaternary ammonium linker, a conjugate or a pharmaceutically acceptable salt thereof.
The conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the linker has the structure of Formula II:
[General Formula Ⅱ]

In the above formula,
The tilde indicates a site linked to Ab, * indicates a site linked to a proteolytic agent;
G is a glucuronic acid moiety or

, wherein R ³ is hydrogen or a carboxyl protecting group, and each R ⁴ is independently hydrogen or a hydroxyl protecting group;
R ¹ and R ² are each independently hydrogen, C _1-8 alkyl or C _3-8 cycloalkyl;
W is -C(O)-, -C(O)NR'-, -C(O)O-, -SO ₂ NR'-, -P(O)R''NR'-, -SONR'- or -PO ₂ NR'-, wherein C, S or P is directly bonded to a phenyl ring, and R' and R'' are each independently hydrogen, C _1-8 alkyl, C _3-8 cycloalkyl, C _1-8 alkoxy, C _1-8 alkylthio, mono- or di- C _1-8 alkylamino, C _3-20 heteroaryl or C _6-20 aryl;
Z is independently C _1-8 alkyl, halogen, cyano or nitro;
n is an integer from 0 to 3;
L is an unbranched linker or a branched linker.
9. The method of claim 8,
where G is

and R ³ is hydrogen or a carboxyl protecting group, and each R ⁴ is independently hydrogen or a hydroxyl protecting group, a conjugate or a pharmaceutically acceptable salt thereof.
9. The method of claim 8,
wherein R ¹ and R ² are both hydrogen;
n is 0;
wherein W is -C(O)NR'-, wherein C is directly bonded to a phenyl ring, and R' is each independently hydrogen, C _1-8 alkyl, C _3-8 cycloalkyl, C _1-8 alkoxy, C _1-8 alkylthio, mono- or di- C _1-8 alkylamino, C _3-20 heteroaryl or C _6-20 aryl, and NR′ is bonded to L. or a pharmaceutically acceptable salt thereof.
9. The method of claim 8,
wherein L is covalently bonded to Ab through a thioether bond,
The thioether bond comprises the sulfur atom of the cysteine of Ab, a conjugate or a pharmaceutically acceptable salt thereof.
The method of claim 1,
wherein the Ab contains an amino acid motif recognizable by isoprenoid transferase at the C-terminus of the Ab;
The thioether bond comprises a sulfur atom of cysteine of the amino acid motif, a conjugate or a pharmaceutically acceptable salt thereof.
13. The method of claim 12,
wherein the amino acid motif is a CYYX sequence, C is cysteine, Y is an aliphatic amino acid, and X is any one selected from the group consisting of glutamine, glutamate, serine, cysteine, methionine, alanine and leucine; and
The thioether bond comprises a sulfur atom of cysteine of the amino acid motif, a conjugate or a pharmaceutically acceptable salt thereof.
13. The method of claim 12,
wherein the amino acid motif is a CYYX sequence, and Y is any one selected from the group consisting of alanine, isoleucine, leucine, methionine and valine; or
CVIM or CVLL sequence, the conjugate or a pharmaceutically acceptable salt thereof.
13. The method of claim 12,
At least one of 1 to 20 amino acids preceding the amino acid motif is glycine, or a conjugate or a pharmaceutically acceptable salt thereof.
9. The method of claim 8,
Wherein the Ab comprises the amino acid sequence GGGGGGGCVIM at the C-terminus, or a pharmaceutically acceptable salt thereof.
9. The method of claim 8,
Wherein L is 3 to 50 heteroalkylene including oxime,
The oxygen atom of the oxime is on the side of L connected to W,
the carbon atom of the oxime is on the side of L linked to Ab; or
the carbon atom of the oxime is on the side of L connected to W,
The conjugate or a pharmaceutically acceptable salt thereof, wherein the oxygen atom of the oxime is on the side of L linked to Ab.
9. The method of claim 8,
wherein L comprises an oxime, and at least one isoprenyl unit covalently bonds the oxime to Ab.
9. The method of claim 8,
Wherein L is further comprising a linking unit represented by the general formula VIII or IX, a conjugate or a pharmaceutically acceptable salt thereof:
[General Formula VIII]
-(CH ₂ ) _r (V(CH ₂ ) _p ) _q -
[General Formula IX]
-(CH ₂ CH ₂ X) _w -
V is a single bond, -O-, -S-, -NR ²¹ -, -C(O)NR ²² -, -NR ²³ C(O)-, -NR ²⁴ SO ₂ - or -SO ₂ NR ²⁵ - ego;
X is —O—, C _1-8 alkylene or —NR ²¹ —;
R ²¹ to R ²⁵ are each independently hydrogen, C _1-6 alkyl, C _1-6 alkyl-C _6-20 aryl or C _1-6 alkyl-C _3-20 heteroaryl;
r is an integer from 0 to 10;
p is an integer from 0 to 10;
q is an integer from 1 to 20; and
w is an integer from 1 to 20;
20. The method of claim 19,
wherein q is an integer from 1 to 10;
r and p are each 1 or 2;
V is -O-, a conjugate or a pharmaceutically acceptable salt thereof.
20. The method of claim 19,
wherein X is -O-;
w is an integer from 1 to 10, a conjugate or a pharmaceutically acceptable salt thereof.
20. The method of claim 19,
The L is

or

A conjugate or a pharmaceutically acceptable salt thereof, comprising at least one polyethylene glycol unit represented by
9. The method of claim 8,
Wherein L is a conjugate or a pharmaceutically acceptable salt thereof, further comprising a bonding unit formed by the reaction of an alkynyl group with an azide, or an aldehyde or ketone group with a hydrazine or hydroxylamine.
9. The method of claim 8,
Wherein L is further comprising a binding unit represented by the following general formula IV _a , IV _b , IV _c , IV _d or IV _e , a conjugate or a pharmaceutically acceptable salt thereof:
[General formula Ⅳ _a ]

[General formula Ⅳ _b ]

[General formula Ⅳ _c ]

[General formula Ⅳ _d ]

[General formula IVe]

In the above formula,
wherein L ₁ and L ₂ are each independently a single bond or C _1-30 alkylene;
R ₁₁ is hydrogen or C _1-10 alkyl.
25. The method of claim 24,
The L ₁ and L ₂ are each independently a single bond; or C ₁₁ alkylene; Or C ₁₂ alkylene, a conjugate or a pharmaceutically acceptable salt thereof.
9. The method of claim 8,
The isoprenoid transferase is a farnesyl protein transferase (FTase) or a geranylgeranyl transferase (GGTase), a conjugate or a pharmaceutically acceptable salt thereof.
The method of claim 1, wherein the Linker comprises one or more branched linkers covalently bonded to the Ab,
i) each branched linker comprises a branched unit covalently linked to the Ab by a first linker (PL);
ii) each branched linker comprises a first branch (B1) wherein the first proteolytic agent is covalently linked to the branched unit by a second linker (SL) and a cleavage group (CG); and
iii) each branched linker comprises: a) a second branch (B2) in which a second proteolytic agent is covalently bound to the branched unit by a second linker (SL) and a cleavage group (CG); or b) a second branch in which a polyethylene glycol moiety is covalently bonded to the branched unit,
The conjugate or a pharmaceutically acceptable salt thereof, wherein each cleaving group is hydrolyzed to release a proteolytic agent from the conjugate.
28. The method of claim 27,
The linker is a nitrogen-containing 1-100 membered heteroalkylene,
the linker comprises two or more atoms of a hydrophilic amino acid;
Wherein the nitrogen forms a peptide bond with the carbonyl of a hydrophilic amino acid, a conjugate or a pharmaceutically acceptable salt thereof.
28. The method of claim 27,
The branched unit is

,

,

or

ego,
The L ² , L ³ , L ⁴ are each independently a direct bond or -C _n H _2n -, wherein n is an integer from 1 to 30,
The G ¹ , G ² , G ³ are independently a direct bond,

,

,

or

is,
wherein R ³⁰ is hydrogen or C _1-30 alkyl;
wherein R ⁴⁰ is hydrogen or L ⁵ -COOR ₆ ;
L ⁵ is a direct bond or -C _n' H _2n' -, wherein n' is an integer from 1 to 10, and R ₆ is hydrogen or C _1-30 alkyl, a conjugate or a pharmaceutically acceptable salt thereof. .
28. The method of claim 27,
the cleavage group is cleavable in the target cell; or a conjugate, or a pharmaceutically acceptable salt thereof, capable of releasing one or more active agents.
28. The method of claim 27,
the conjugate comprises an Ab;
1, 2, 3 or 4 or more branched linkers covalently attached to the Ab;
A conjugate or a pharmaceutically acceptable salt thereof, wherein the branched linker comprises one or two or more proteolytic agents.
The method of claim 1,
Wherein the proteolytic agent induces target protein degradation or modulation of the target protein, a conjugate or a pharmaceutically acceptable salt thereof.
28. The method of claim 27,
The proteolytic agent does not have a heterobifunctional (heterobifunctional), the conjugate or a pharmaceutically acceptable salt thereof.
The method of claim 1,
Wherein the proteolytic agent is a monovalent degrader or a molecular glue degrader, a conjugate or a pharmaceutically acceptable salt thereof.
35. The method of claim 34,
The monovalent degradation agent binds only to a target protein to induce target protein degradation, a conjugate or a pharmaceutically acceptable salt thereof.
35. The method of claim 34,
The molecular adhesive degrading agent binds to E3 ubiquitin ligase and a target protein to induce target protein degradation;
or binds to E3 ubiquitin ligase or a target protein to induce target protein degradation,
wherein the target protein is a native substrate or a non-native substrate of E3 ubiquitin ligase, a conjugate or a pharmaceutically acceptable salt thereof.
37. The method of claim 36,
The E3 ubiquitin ligase is VHL (von Hippel-Lindau), cereblon, XIAP, XIAP/cIAP, E3A, MDM2. APC (Anaphase-promoting complex), APC/cdc20, APC/cdh1, CRL (cullin ring ubiquitin ligase), UBR5 (EDD1), beta-TrCP, SOCS/BCbox/eloBC/CUL5/RING, LNXp80, LNX1, CBX4, CBL , CBLL1, C6orf157, CHFR, DTL, E6-AP, HACE1, HECTD1, HECTD2, HECTD3, HECW1, HECW2, HERC1, HERC2, HERC3, HERC4, HERC5, HUWE1, HYD, ITCH, mahogunin, MARCH-II, MARCH-II , MARCH-III, MARCH-IV, MARCH-VI, MARCH-VII, MARCH-VIII, MARCH-X, MEKK1, MIB1, MIB2, MycBP2, NEDD4, NEDD4L, PELI1, Pirh2, PJA1, PJA2, PPIL2, PRPF19, PIAS1 , PIAS2, PIAS3, PIAS4, RANBP2, RFFL, RFWD2, Rictor, RNF4, RNF5, RNF8, RNF19, RNF190, RNF20, RNF34, RNF40, RNF125, RNF128, RNF138, RNF12, RBX1, SMURF1, SMURF2, STUB1, TOPORS, STUB , UBE3A, UBE3B, UBE3C, UBE4A, UBE4B, UBOX5, UBR5, WWP1, WWP2, Parkin, A20/TNFAIP3, AMFR/gp78, ARA54, beta-TrCP1/BTRC, SCF/beta-TrCP, BRCA1, CHIP, CHIP/STUB1 , E6;E6AP/UBE3A, F-box protein 15/FBXO15, FBXW7/Cdc4, SCF/FBW7, GRAIL/RNF128, HOIP/RNF31, cIAP-1/HIAP-2, cIAP-2/HIAP-1, cIAP (pan ), ITCH/AIP4, KAP1, MARCH8, Mind Bomb 1/MIB1, Mind Bomb 2/MIB2, MuRF1/TRIM63, NDFIP1, NleL, Parkin, RNF2, RNF4, RNF8, RNF168, RNF43, SART1, Skp2, SCF/Skp2, SPRH, SIAH1, SIAH2, SMURF1, SMURF2, TOPORS, TRAF-1, TRAF 2, TRAF-3, TRAF-4, TRAF-5, TRAF-6, TRAF-7, TRIM5, TRIM21, TRIM32, TRIM63, UBE3B, UBE3C, UBR1, UBR2, UHRF2, WWP1, WWP2, ZNRF1 and ZNRF3. Which is selected from, a conjugate or a pharmaceutically acceptable salt thereof.
37. The method of claim 36,
The E3 ubiquitin ligase is cereblon, a conjugate or a pharmaceutically acceptable salt thereof.
37. The method of claim 36,
The native substrate or non-native substrate of the E3 ubiquitin ligase is NKX3.1, β-Catenin, aiolos, Akt1, Aurora A, B7-2, Bim, Caspase-3, Caspase-8, Caspase-10, CD147 , Cdc20, Cdc25A, CDH1, CHK1, CHK2, c-Jun, CK1-α, Claspin, C-Myc, CRY2, Cyclin A, Cyclin B, Cyclin E, CDK12/Cyclin K, Delta, Jagged, Dlg, DR4, DVL1 , ELF, ErbB4, Erk, Fbxo45, Foxo1, Foxp3, gp190, GSPT1, GSPT2, H2A,H2AX, HIF-1α, HIF-2α, HIPK2, Histone H2A, Histone H2A.X, Histone H2B, HSF1, HSP70/90, ikaros, IKZF1/3, IL-1β, IL-6, iNOS, IRAK, IRS1 IBα, IκBα, JAMP, KAI1, KLFs, LRRK2, Mad2, Mcl-1, MDM2, MED13, MEDL, MEIS2, MHC class I, MHC class II, MKK4, MTA1, MyBP-C, MyLC1/2, NEMO, NF-κB, N-Myc, Nrf2, NOTCH, NUMB, p21, p27, p53, p73, P-gp, paxillin, PCNA, PCNP, PHD1 /3, PLK1, Rbm39, Rbm23, RIP2, RhoA, RNF8, Runx1, SALL4, Securin, Skp2, Skp2-CKS1, SGK1, SMAC, Smad2, Smads, SOD1 and its variants, TCF/LEF, TNF-α, TopBP1, TP53, TRAF2, TRB3, TRIP, Troponin I, TSC2, UCK1, Weel, WNK1 and WNK4, the conjugate or a pharmaceutically acceptable salt thereof selected from the group consisting of.
37. The method of claim 36,
The native substrate or non-native substrate of the E3 ubiquitin ligase is GSPT1 or GSPT2, a conjugate or a pharmaceutically acceptable salt thereof.
35. The method of claim 34,
The molecular adhesive disintegrant is a compound represented by the following general formula X1, a conjugate or a pharmaceutically acceptable salt thereof:
[General formula X1]

In the above formula,
D ^x1 is C(=O) or CH ₂ ;
D ^x2 is O, NC≡N or NH;
a1 is an integer of 0, 1, 2 or 3;
a2 is an integer of 0 or 1;
a3 is an integer of 0, 1, or 2;
A is C, N, or O;
R ^xa is hydrogen, halo or C _1-6 alkyl;
R ^xb and R ^xc are each independently hydrogen, halo, C _1-10 alkyl, CF ₃ , or R ^x10 -(R ^x11 ) _a -R ^x12 ;
or together with the moiety to which R ^xb and R ^xc are attached form C _5-8 cycloalkyl, C _4-8 heterocycloalkyl, C _6-10 aryl, or C _6-10 heteroaryl;
From here,
R ^x10 is a direct bond, -NH-, or -O-,
R ^x11 is each independently a direct bond, -C(O)-, C _1-5 alkylene, C _2-5 alkenylene, C _6-10 arylene, C _6-10 heteroarylene, C _3-8 cyclo selected from the group consisting of alkylene, or C _3-8 heterocycloalkylene,
R ^x12 is OH, NH ₂ , NH-R ^x12' , C _6-10 aryl, C _6-10 heteroaryl, C _3-8 cycloalkyl, or C _3-8 heterocycloalkyl;
wherein R ^x12′ is C _1-5 alkyl, C _2-5 alkenyl, C _6-10 aryl, C _6-10 heteroaryl, C _3-8 cycloalkyl, or C _3-8 heterocycloalkyl;
a is an integer from 1 to 5,
Any one of R ^xb or R ^xc forms a monovalent moiety and is capable of binding to Linker,
However, when a2 is 0, NH binds directly to the Linker.
42. The method of claim 41,
wherein D ^x1 is CH ₂ ; D ^x2 is O, a conjugate or a pharmaceutically acceptable salt thereof.
42. The method of claim 41,
wherein R ^xa is hydrogen or halo; A is C, or N, or a conjugate or a pharmaceutically acceptable salt thereof.
42. The method of claim 41,
Any one or more of R ^xb and R ^xc is C _1-10 alkyl, a conjugate or a pharmaceutically acceptable salt thereof.
42. The method of claim 41,
Any one or more of R ^xb and R ^xc is R ^x10 -(R ^x11 ) _a -R ^x12 ,
a is an integer of 1 to 3, a conjugate or a pharmaceutically acceptable salt thereof.
42. The method of claim 41,
Any one or more of R ^xb and R ^xc is R ^x10 -(R ^x11 ) _a -R ^x12 ,
R ^x10 and R ^x11 are a direct bond, a conjugate or a pharmaceutically acceptable salt thereof.
42. The method of claim 41,
Any one or more of R ^xb and R ^xc is R ^x10 -(R ^x11 ) _a -R ^x12 ,
Wherein R ^x11 is each independently selected from the group consisting of a direct bond, -C(O)-, C _1-5 alkylene, or C _2-5 alkenylene,
a is an integer of 1 to 3, a conjugate or a pharmaceutically acceptable salt thereof.
42. The method of claim 41,
Any one or more of R ^xb and R ^xc is R ^x10 -(R ^x11 ) _a -R ^x12 ,
wherein R ^x12 is NH ₂ ; C _6-10 heteroaryl containing one or more N atoms or C _3-8 heterocycloalkyl containing one or more N atoms, a conjugate or a pharmaceutically acceptable salt thereof.
42. The method of claim 41,
Wherein R ^xb and R ^xc are together with the moiety to which R ^xb and R ^xc are bonded to form C _4-8 heterocycloalkyl or C _6-10 heteroaryl.
42. The method of claim 41, wherein the molecular adhesive disintegrant is

,

,

,

,

,

,

,

,

,

,

, or

Phosphorus, a conjugate, or a pharmaceutically acceptable salt thereof.
The method of claim 1,
The [Linker - (B) _m ] is

,

,,

,

,

,

,

,

,

,

,

,

,
or

Phosphorus, a conjugate, or a pharmaceutically acceptable salt thereof.
A pharmaceutical composition for the prevention or treatment of hyperproliferative, cancer or angiogenic diseases, containing the conjugate according to claim 1 as an active ingredient.
53. The pharmaceutical composition of claim 52, further comprising a pharmaceutically effective amount of a chemotherapeutic agent.
53. The method of claim 52,
The cancer is lung cancer, small cell lung cancer, gastrointestinal cancer, colorectal cancer, bowel cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma and the group consisting of melanoma Any one selected from the pharmaceutical composition.

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