KR20240016231A - Novel auristatin prodrug - Google Patents

Abstract

The present invention provides an auristatin prodrug, specifically an auristatin prodrug having a self-immolative group, a method for producing the same, a pharmaceutical composition containing the same, and treatment and/or prevention of diseases. It is about its intended use.

Description

Novel auristatin prodrug}

The present invention relates to an auristatin prodrug, and more specifically, to an auristatin prodrug having a self-immolative group, a method for producing the same, a pharmaceutical composition containing the same, treatment of diseases and/ or its use for prophylaxis.

Auristatin is a major drug used in antibody drug conjugates (ADCs), the most well-known of which are monomethyl auristatin E (MMAE) and monomethyl auristatin F (monomethyl auristatin F). auristatin F, MMAF). Auristatin acts by blocking the polymerization of tubulin, inhibiting mitosis, and interfering with cell proliferation. Like most parenterally administered chemotherapy drugs, auristatin has toxic side effects (e.g. neutropenia, Due to concerns about thrombocytopenia (platelet reduction, etc.), many attempts are being made to use antibody drug conjugates. In the form of an antibody-drug conjugate of auristatin, after the conjugate passes through extracellular fluid, the linker is cleaved by enzymes such as cathepsin in tumor cells, and the drug acts.

As antibody drug conjugates using auristatin have become known, and some antibody drug conjugates using them have been approved by the FDA, many studies using auristatin are being conducted. In this regard, an invention that allows synthesis of a high drug-antibody ratio in ADC by controlling hydrophilicity by modifying the backbone structure of auristatin with azide (Antibody-Drug conjugate Payloads; Study of Auristatin Derivatives, Chem. Pharm. Bull. 68, 201-211 (2020)) has been reported, and a method of treating pancreatic cancer using MMAE prodrug nanoparticles (Light-Activated Monomethyl Auristatin E Prodrug Nanoparticles for Combinational Photo-Chemotherapy of Pancreatic Cancer, Molecules , 27(8) ):2529 (2022)) has been reported, a drug that combines MMAE and albumin to exhibit anti-inflammatory effects (Highly potent monomethyl auristatin E prodrug activated by caspase-3 for the chemoradiotherapy of triple-negative breast cancer, Biomaterials , 192: 109-117, (2019)) has been reported.

Under this technical background, the inventor of the present invention made efforts to develop an auristatin prodrug, and as a result, synthesized an auristatin prodrug that complements the shortcomings of existing drugs due to their high lipophilic properties and has excellent pharmacokinetic properties, cytotoxicity, etc. and confirmed its characteristics and activity to complete the present invention.

The present invention seeks to provide a novel auristatin prodrug with improved hydrophilicity, pharmacokinetic properties, etc.

The present invention provides a compound having a structure represented by the following general formula (I), an isomer thereof, or a pharmaceutically acceptable salt or solvate thereof.

[General formula I]

MMAE-SIG2-SIG1-E

In the general formula I,

MMAE is ego,

SIG2 is *-C(=O)-R ¹ -N(R ² )-**, *-C(=O)OR ¹ -N(R ² )-**, *-C(=O)N( R ² )-R ¹ -N(R ² )-** and *-S(=O) ₂ -R ¹ -N(R ² )-**,

SIG1 is selected from the group consisting of **-C(=O)OR ³ -O-*** and **-C(=O)OR ³ -N(R ⁴ )-***,

E is an organic group derived from the substrate of a cancer cell-specific enzyme,

R ¹ and R ³ are independently in each case a divalent organic group,

R ² and R ⁴ are independently hydrogen or alkyl in each case,

The N at the ** terminal of SIG2 can form an N-containing ring structure with the adjacent carbon atom in R ¹ and R ² ,

The N at the *** terminal of SIG1 can form an N-containing ring structure with the adjacent carbon atom in R ³ and R ⁴ ,

MMAE and SIG2 are connected at the * end;

SIG2 and SIG1 are connected at the ** end;

E is connected to the *** end of SIG1,

The bond between the *** end of SIG1 and E can be broken by the cancer cell-specific enzyme.

In the present invention, in the compound of the structure represented by the general formula I, or an isomer thereof, or a pharmaceutically acceptable salt or solvate thereof, each connecting portion is located in the intracellular environment (e.g., lysosomes or endosomes or caveolae). It can be cut by cutting agents present in caveolea. For example, it may be cleaved by intracellular peptidase or protease enzymes, including but not limited to lysosomal or endosomal proteases. Cleavage agents may include cathepsin B, cathepsin D, and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, releasing the active drug within target cells (e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). For example, it may contain structures that are cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancer tissues (e.g., Phe-Leu, Val-Cit, Phe-Lys, Val-Ala, etc.) .

In addition, structures that can be cleaved by cleavage agents include, for example, malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med) -Chem. 3(10):1299-1304), 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12), beta-glucuronide (β-glucuronide) linker (Jeffery et al., 2006, Bioconjug Chem. 17(3):832-40), or beta-galactosidase linker (Kolodych et al., 2017, Eur J Med Chem. Dec 15;142:376-382).

In the present invention, “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 ( It refers to a chemical moiety covalently bonded to the parent group or fused to the parent group.

In the present invention, “alkyl” refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of an aliphatic or cycloaliphatic, saturated or unsaturated (unsaturated, fully unsaturated) hydrocarbon compound, generally 1 to 12, more specifically. It has 1 to 10 atoms. Examples of saturated alkyl include methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl, and examples of saturated straight-chain alkyl include methyl, ethyl, n-propyl, n-butyl, and n-. Pentyl (amyl), n-hexyl, n-heptyl, etc. Examples of saturated branched alkyl include isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, neopentyl, etc. For example, “C ₁ -C ₅ alkyl” herein refers to a straight or branched chain of 1 to 5 atoms, whether straight or branched, whether carbon atoms or heteroatoms. In the present invention, “alkyl” includes “heteroalkyl” unless otherwise indicated. As used herein, “heteroalkyl” means alkyl in which one or more carbon atoms have been replaced by a heteroatom, such as a nitrogen atom, a sulfur atom, or an oxygen atom.

In the present invention, “alkylene” refers to a divalent moiety obtained by removing a hydrogen atom from a carbon atom of a straight-chain or branched aliphatic or alicyclic, saturated or unsaturated hydrocarbon compound.

In the present invention, “lower” alkylene, arylene, etc. generally refers to alkylene, arylene, etc. having 8 or less carbon atoms.

In the present invention, “aryl” refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound. For example, “C ₆ -C ₁₀ aryl” means a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, where the moiety has 6 to 10 ring atoms, and “C ₅ -C ₁₀ aryl" means a monovalent moiety in which the moiety has 5 to 10 ring atoms and is obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound. Here, the prefix (C ₅ -C ₇ , C ₅ -C ₁₀ , etc.) refers to the number of ring atoms or a range of the number of ring atoms, regardless of whether they are carbon atoms or heteroatoms. For example, “C ₅ -C ₆ aryl” refers to an aryl group having 5 or 6 ring atoms. Here, the ring atoms may be all carbon atoms, as in a “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 containing fused rings, at least one of which is an aromatic ring, include groups derived from indane, indene, isoindene, tetralin, acenaphthene, fluorene, phenalene, acephenanthrene, and aceanthrene, such as It is not limited. Additionally, “aryl” includes “heteroaryl” unless otherwise indicated, where heteroaryl refers to an aromatic ring atom in which the ring atom may contain one or more heteroatoms, examples of which include pyridinylene, It includes, but is not limited to, pyrimidinylene and thiophenylene.

In the present invention, “arylene” refers to a divalent moiety obtained by removing a minority atom from an aromatic ring atom of an aromatic compound.

In the present invention, “alkaryl” and “aralkyl” refer to alkylene-aryl and arylene-alkyl, where “alkarylene” and “aralkyl” refer to alkylene-arylene and arylene. -Means alkylene, and each of alkyl, aryl, alkylene, and arylene is as defined above.

In the present invention, “precursor” or “prodrug” refers to the action of enzymes, etc. under physiological conditions in vivo (e.g., enzymatic oxidation, reduction, and/or hydrolysis, etc.). It refers to a compound that can be converted directly or indirectly into a drug.

In the present invention, the “pharmaceutically acceptable salt” may be an acid addition salt formed from a pharmaceutically acceptable free acid, and the free acid may be an organic acid or an inorganic acid.

The organic acids include, but are not limited to, 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, Includes glutamic acid and aspartic acid. Additionally, the inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid.

For example, if a compound is anionic or has a functional group that may be anionic (e.g., -COOH can be -COO-), a salt can be formed 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 (i.e., NH ₄ ⁺ ) and substituted ammonium ions (e.g., 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 common quaternary ammonium ion is N(CH ₃ ) ₄ ⁺ .

If the compound is cationic or has a functional group that may be cationic (e.g. -NH ₂ may be -NH ₃ ⁺ ), salts can be formed with the 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-acetioxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethane. Disulfonic acid, ethanesulfonic acid, fumaric acid, gluteptonic 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, mucilic 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. Examples include: Examples of suitable polymer organic anions include, but are not limited to, those derived from the following polymer acids: tannic acid, carboxymethyl cellulose, etc.

In the present invention, “solvate” refers to a molecular complex between the compound according to the present invention and solvent molecules. Examples of solvates include water, isopropanol, ethanol, methanol, and dimethyl sulfoxide. It includes, but is not limited to, the compound according to the present invention combined with (dimethylsulfoxide), ethyl acetate, acetic acid, ethanolamine, or a mixed solvent thereof.

It may be convenient or desirable to prepare, purify and/or handle corresponding 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 monohydrate, dihydrate, trihydrate, etc.

In one aspect of the present invention, SIG2, SIG1 and E are each independently a protease cleavable group, an acid-cleavable group, a disulfide, a self-immolative group, malonate, maleimidobenzoyl, 3' -Includes N-amide, beta-glucuronide, or beta-galactoside.

In one aspect of the present invention, SIG2, SIG1, and E are each independently protease cleavage group, self-immolation group, beta-glucuronide, or beta-galactoside. am.

Specifically, SIG 2 and SIG 1 are self-immolative groups and E is a protease cleavable group, beta-glucuronide, or beta-galactoside.

In one aspect of the invention, R ¹ and R ³ are independently in each case substituted or unsubstituted alkylene, substituted or unsubstituted arylene, substituted or unsubstituted alkarylene, or substituted Or it is unsubstituted aralkylene.

In one aspect of the invention, R ¹ is independently in each case substituted or unsubstituted lower alkylene, substituted or unsubstituted lower arylene, substituted or unsubstituted lower alkylene, or substituted or unsubstituted lower aralkylene.

In one aspect of the invention, R ¹ is independently in each case substituted or unsubstituted C ₁ -C ₅ alkylene, substituted or unsubstituted C ₆ -C ₁₀ arylene, substituted or unsubstituted C ₅ -C ₁₀ alkarylene, or substituted or unsubstituted C ₅ -C ₁₀ aralkylene.

In one aspect of the invention, the SIG2 is *-C(=O)-R ¹ -N(R ² )-** or *-C(=O)N(R ² )-R ¹ -N(R ² )-**, where R ¹ and R ² are as defined above.

In one aspect of the present invention, R ² is hydrogen or lower alkyl.

In one aspect of the present invention, R ² is hydrogen or C ₁ -C ₃ alkyl.

In the present invention, each of R ¹ and R ² may be independently selected.

In the present invention, when SIG2 is *-C(=O)N(R ² )-R ¹ -N(R ² )-**, the R ² may be the same or different from each other, for example, * It may be -C(=O)N(CH ₃ )-R ¹ -N(CH ₂ CH ₃ )-**.

In one aspect of the invention, SIG2 is selected from the following group:

, , , , and .

Here, the meanings of * and ** are as described above.

In one aspect of the present invention, R ³ is independently substituted or unsubstituted arylene, substituted or unsubstituted alkarylene, or substituted or unsubstituted aralkylene.

In one aspect of the present invention, R ³ is independently substituted or unsubstituted C ₆ -C ₁₄ arylene, substituted or unsubstituted C ₆ -C ₁₄ alkarylene, or substituted or unsubstituted C It is ₆ -C ₁₄ aralkylene.

In one aspect of the invention, R ³ is independently substituted or unsubstituted C ₆ -C ₁₀ arylene, substituted or unsubstituted C ₆ -C ₁₀ alkarylene, or substituted or unsubstituted C It is ₆ -C ₁₀ aralkylene.

In the present invention, when R ³ is substituted, one or more hydrogens bonded to the carbon atom of arylene, alkarylene, or aralkylene are -C(=O)NH-(CH ₂ ) _n -C _1-3 It is substituted with alkoxy (where n is an integer from 1 to 5).

In one aspect of the invention, R ³ is independently substituted or unsubstituted C ₆ -C ₁₀ alkarylene, or substituted or unsubstituted C ₆ -C ₁₀ aralkylene, and when substituted, carbon One or more hydrogens bonded to the atom are replaced with -C(=O)NH-(CH ₂ ) _n -C _1-3 alkoxy, where n is an integer from 1 to 5.

In one aspect of the present invention, R ⁴ is hydrogen or lower alkyl.

In one aspect of the present invention, R ⁴ is hydrogen or C ₁ -C ₃ alkyl.

In one aspect of the invention, SIG1 is selected from the following group:

and

Here, the meanings of ** and *** are as described above.

In one aspect of the present invention, the cancer cell-specific enzyme is selected from the group consisting of β-glucuronidase, β-galactosidase, and cathepsin B. do.

In one aspect of the present invention, the substrate of the cancer cell-specific enzyme is glucuronic acid, galactoside, or a derivative thereof, an amino acid or a derivative thereof, or a polypeptide or a derivative thereof.

In one specific embodiment of the present invention, the amino acids are alanine, β-alanine, γ-aminobutyric acid, arginine, asparagine, aspartic acid, γ-carboxyglutamic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, At least one selected from the group consisting of lysine, methionine, norleucine, norvaline, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine and derivatives thereof,

Polypeptides include alanine, β-alanine, γ-aminobutyric acid, arginine, asparagine, aspartic acid, γ-carboxyglutamic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norleucine. It is formed from two or more selected from the group consisting of valine, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine and derivatives thereof.

In one aspect of the invention, E is selected from the following group:

, , and a compound having the structure of Formula I below,

[Formula Ⅰ]

,

Here, R ⁵ is -OH, or C _1-4 alkoxy, m is an integer of 1 to 12, and the meaning of *** is as described above.

In one aspect of the invention, E is selected from the following group:

, , , and ,

Here, the meaning of *** is as described above.

In one aspect of the present invention, the compound of general formula I is selected from the following group.

,

,

,

,

.

,

,

.

In another aspect according to the present invention, a ligand-drug conjugate comprising a compound having the structure of General Formula I and a ligand, or a pharmaceutically acceptable salt or solvate thereof is provided.

As used herein, the term “conjugate” refers to a cell binding agent that is covalently linked to one or more molecules of a cytotoxic compound. Here, the “cell binding agent” is a molecule with affinity for a biological target, for example, a ligand, protein, antibody, specifically a monoclonal antibody, protein or antibody fragment, peptide, oligonucleotide, or oligosaccharide. , the binder functions to direct the biologically active compound to the biological target. Here, “biological target” in this specification refers to an antigen located on the surface of a tumor, cancer cell, or extracellular matrix.

In one aspect of the invention, conjugates can be designed to target tumor cells via cell surface antigens. The antigen may be a cell surface antigen that is overexpressed or expressed on abnormal cell types. Specifically, the target antigen may be expressed only on proliferating cells (eg, tumor cells). Target antigens can usually be selected based on differential expression between proliferative and normal tissues. In the present invention, the ligand is coupled to a linker.

As used herein, the term “ligand” refers to a molecule capable of forming a complex with a target biomolecule. An example of a ligand is a molecule that transmits a signal by binding to a specific location on a target protein. For example, a ligand may be an antigen-binding moiety, an antibody, or an antigen-binding fragment, which can bind to a location on a target biomolecule (e.g., a protein) and transmit a signal.

In one aspect of the present invention, the ligand-drug conjugate may refer to a cell binding agent in which a ligand and a drug (eg, a compound having the structure of General Formula I) are covalently bound through a linker. At this time, the ligand may be an antigen-binding moiety, an antibody, or an antigen-binding fragment that can bind to the biomolecule targeted by the cell binding agent, and the ligand-drug conjugate is an antibody-drug conjugate (ADC). Contains the meaning of conjugate). Accordingly, the term ADC described later refers to the ligand-drug conjugate of the present invention.

As used herein, an “antibody” is an immunoglobulin molecule that can specifically bind to a target, such as carbohydrates, polynucleotides, lipids, polypeptides, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term “antibody” refers to an intact polyclonal or monoclonal antibody, as well as any antigen-binding portion of an intact antibody that retains the ability to specifically bind to a given antigen (e.g., “antigen-binding fragment”) or single chains thereof, fusion proteins comprising antibodies, and any other modified arrangement of immunoglobulin molecules comprising an antigen recognition site, including, but not limited to, Fab; Fab'; F(ab')2 Fd fragment; Fv fragment; single domain antibody (dAb) fragment; Isolated complementarity determining region (CDR); Single chain (scFv) and single domain antibodies (e.g., shark and camelid antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv ( See, for example, Hollinger and Hudson, 2005, Nature Biotechnology 23(9): 1126-1136.

Antibodies include antibodies of any class, such as IgG, IgA or IgM (or subclasses thereof), and the antibodies need not be of any particular class. Depending on the amino acid sequence of the constant region of the heavy chain of an antibody, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, several of which are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. can be classified. The heavy chain (HC) constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structure and three-dimensional coordination of different classes of immunoglobulins are well known. The antibody of the present invention can be produced using techniques well known in the related art, such as recombinant technology, phage display technology, synthetic technology, or a combination of the above techniques, or other techniques readily known in the related art.

As used herein, “isolated antibody” refers to an antibody that is substantially free of other antibodies with different antigenic specificities and may be substantially free of other cellular material and/or chemicals.

Antibodies useful in the present invention include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, and heterologous antibodies. Conjugate antibodies, single chain (ScFv), mutants thereof, fusion proteins comprising antibody portions (e.g., domain antibodies), humanized antibodies, and glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. It can encompass any other modified shape of an immunoglobulin molecule containing an antigen recognition site of the required specificity, including. Antibodies may be murine, rat, human or of any other origin (including chimeric or humanized antibodies).

In one aspect of the present invention, the compound and the ligand having the structure of General Formula I may be linked through a linker. The linker is any group or moiety that links, connects, or attaches a ligand described herein, specifically an antibody or antigen binding protein, to a therapeutic moiety, such as a compound having the structure of General Formula I above. In general, suitable binder linkers for the antibody conjugates described herein are those that are stable enough to take advantage of the circulating half-life of the antibody, while simultaneously being able to release the payload after antigen binding and/or antigen-mediated internalization of the conjugate.

The linker included in the ligand-drug conjugate disclosed herein may be cleavable or non-cleavable.

Specifically, the linker is cleavable in vitro and in vivo. Cleavable linkers may include chemically or enzymatically unstable or degradable linkages. Cleavable linkers generally rely on processes inside the cell to liberate the drug, such as reduction in the cytoplasm, exposure to acidic conditions in lysosomes, or cleavage by specific proteases or other enzymes inside the cell. Cleavable linkers generally contain one or more chemical bonds that are chemically or enzymatically cleavable, while the remainder of the linker is non-cleavable.

The linker contains chemically labile groups such as hydrazone and/or disulfide groups. Linkers containing chemically labile groups take advantage of differential properties between plasma and some cytoplasmic compartments. Intracellular conditions to facilitate drug release for hydrazone-containing linkers are the acidic conditions of endosomes and lysosomes, while disulfide-containing linkers are reduced in the cytosol containing high thiol concentrations, such as glutathione. In certain embodiments, the plasma stability of a linker comprising a chemically labile group can be increased by using substituents near the chemically labile group to induce steric hindrance.

Acid-labile groups, such as hydrazone, remain intact during systemic circulation in the neutral pH environment of blood (pH 7.3 to 7.5), and once ADC is transferred to intermediate acidic endosomes (pH 5.0 to 6.5) and lysosomes (pH 4.5 to 5.0) of cells. Once internalized into the compartment, it hydrolyzes and releases the drug. This pH-dependent release mechanism was associated with non-specific release of the drug. To increase the stability of the hydrazone group of the linker, the linker can be altered by chemical modifications, such as substitutions, to achieve more efficient release from the lysosome with minimal loss of circulation.

Cleavable linkers may also include disulfide groups. Disulfides are thermodynamically stable at physiological pH and are designed to release drugs upon internalization inside cells, where the cytosol provides a significantly more reducing environment compared to the extracellular environment. Cleavage of the disulfide bond generally requires the presence of a cytosolic thiol cofactor, such as (reduced) glutathione (GSH), such that the disulfide-containing linker is adequately stable in the circulation, thereby selectively releasing the drug into the cytosol. do. Intracellular enzymes protein disulfide isomerase, or similar enzymes capable of cleaving disulfide bonds, may also contribute to preferential cleavage of disulfide bonds inside cells. GSH is reported to be present in cells in a concentration range of 0.5 to 10 mM compared to the significantly lower concentration of GSH or the most abundant low molecular weight thiol, cysteine, in circulation at approximately 5 μM. Tumor cells, where irregular blood flow causes hypoxia, result in enhanced activity of reductive enzymes and therefore much higher glutathione concentrations. In certain embodiments, the in vivo stability of disulfide-containing linkers can be improved by chemical modification of the linker, such as the use of steric hindrance adjacent to the disulfide bond.

Another type of linker that can be used is a linker that is specifically cleaved by enzymes. Such linkers are typically peptidic or contain a peptide region that acts as a substrate for an enzyme. Peptide-based linkers tend to be more stable in the cytoplasm and extracellular environment than chemically unstable linkers. Peptide bonds generally have good serum stability because lysosomal proteases have very low activity in the blood due to endogenous inhibitors and due to the unfavorably high pH value of blood compared to lysosomes. Release of the drug from the antibody occurs specifically due to the action of lysosomal proteases, such as cathepsins and plasmin. These proteases may be present at elevated levels in certain tumor tissues. In certain embodiments, the linker is cleavable by lysosomal enzymes. In certain embodiments, the linker is cleavable by a lysosomal enzyme, and the lysosomal enzyme is cathepsin B. In certain embodiments, the linker is cleavable by a lysosomal enzyme, and the lysosomal enzyme is β-glucuronidase or β-galactosidase.

A variety of dipeptide-based cleavable linkers useful for linking drugs such as doxorubicin, mitomycin, camptothecin, thalisomycin, and auristatin/aurystatin family members to antibodies have been disclosed (Dubowchik et al. , 1998, J. Org. Chem. 67:1866-1872; Dubowchik et al., 1998, Bioorg. Med. Chem. Lett. 8:3341-3346; Walker et al., 2002, Bioorg 12:217-219; Walker et al., 2004, Bioorg. Med. Chem. Lett. 14:4323-4327; and Francisco et al., 2003, Blood 102: 1458-1465], the contents of each of which are incorporated herein by reference). Any of these dipeptide linkers, or modified variations of these dipeptide linkers, can be used in the ADCs disclosed herein.

Enzymatically cleavable linkers may include self-immolative spacers to spatially space the drug from the enzymatic cleavage site. Direct attachment of a drug to a peptide linker may result in proteolytic release of the drug's amino acid adducts, reducing its activity. The use of a self-immolative spacer allows removal of a fully active, chemically unmodified drug upon amide bond hydrolysis.

One self-immolative spacer is a peptide via an amino group, while an amine-containing drug can be attached to the benzylic hydroxyl group of the linker via a carbamate functional group (giving p-amidobenzylcarbamate, PABC). It is a difunctional para-aminobenzyl alcohol group that is connected to, forming an amide bond. The resulting prodrug is activated upon protease-mediated cleavage, resulting in a 1,6-elimination reaction that releases the remainder of the unmodified drug, carbon dioxide, and linker group.

A variety of truncated β-glucuronic acid-based linkers useful for linking antibodies to drugs such as auristatin, camptothecin and doxorubicin analogs, CBI minor-groove binders, and psymberin have been described. (Jeffrey et al., 2006, Bioconjug. Chem. 17:831-840; Jeffrey et al., Bioorg. Med. Chem. Lett. 17:2278-2280; and Jiang et al. ., 2005, J. Am. Chem. Soc. 127:11254-11255, the contents of each of which are incorporated herein by reference). All of these β-glucuronic acid based linkers can be used in the ADCs disclosed herein. In certain embodiments, the enzymatically cleavable linker is a β-galactoside based linker. While β-galactoside is abundantly present in lysosomes, its enzymatic activity outside the cell is low. Additionally, Bc1-xL inhibitors containing phenol groups can be covalently attached to the linker via the phenolic oxygen. One such linker, disclosed in US Patent Application Publication No. 2009/0318668, is a methodology in which diamino-ethane "SpaceLink" is used in conjugation with a traditional "PABO" based self-immolative group to deliver phenol. It depends.

A cleavable linker may comprise a non-cleavable moiety or segment and/or a cleavable segment or moiety may be included in an otherwise non-cleavable linker to render it cleavable. By way of example only, polyethylene glycol (PEG) and related polymers may contain cleavable groups in the polymer backbone. For example, polyethylene glycol or polymer linkers may contain one or more cleavable groups such as disulfide, hydrazone, or dipeptide.

Other degradable linkages that may be included in the linker include ester linkages formed by the reaction between PEG carboxylic acids or activated PEG carboxylic acids and alcohol groups on the biologically active agent, wherein such ester groups are generally hydrolyzed under physiological conditions. Decomposes to release biologically active agents. Hydrolytically degradable linkages include, but are not limited to, the ends of polymers and the 5' hydroxyl groups of oligonucleotides, including, but not limited to, carbonate linkages; An imine linkage resulting from the reaction of an amine and an aldehyde; a phosphate ester linkage formed by the reaction of an alcohol with a phosphate group; Acetal linkage, a reaction product of an aldehyde and an alcohol; Orthoester linkage, a reaction product of promate and alcohol; and oligonucleotide linkages formed by phosphoramidite groups.

Although cleavable linkers may provide certain advantages, linkers comprising the ADCs disclosed herein need not be cleavable. In the case of non-cleavable linkers, drug release does not depend on differential properties between plasma and some cytoplasmic compartments. Release of the drug is assumed to occur after internalization of the ADC through antigen-mediated endocytosis and delivery to the lysosomal compartment where the antibody is degraded to the level of amino acids through intracellular proteolytic degradation. This process releases the drug derivative formed by the drug, the linker, and the amino acid residues to which the linker is covalently attached. Amino acid drug metabolites from conjugates with non-cleavable linkers are more hydrophilic and generally less membrane permeable, resulting in less bystander effects and less non-specific toxicity compared to conjugates with cleaved linkers. In general, ADCs with non-cleavable linkers have greater stability in circulation than ADCs with cleaved linkers. The non-cleavable linker may be an alkylene chain or may be polymeric in nature, for example based on polyalkylene glycol polymers, amide polymers, or of alkylene chains, polyalkylene glycols and/or amide polymers. Can contain segments. In certain embodiments, the linker comprises polyethylene glycol having 1 to 6 ethylene glycol units.

In another aspect according to the present invention, the present invention provides a pharmaceutically acceptable compound or conjugate, wherein the compound of the general formula I or an isomer thereof, and/or a ligand-drug conjugate containing the same is used as a pharmaceutical. A salt or solvate is provided.

In addition, the present invention provides a pharmaceutically acceptable compound or conjugate, wherein the compound of the general formula I, or an isomer thereof, and/or a ligand-drug conjugate containing the same is used as a pharmaceutical for treating cancer or proliferative diseases. A salt or solvate is provided.

In addition, the present invention provides a pharmaceutical composition containing a compound of the above general formula I, or an isomer thereof, and/or a ligand-drug conjugate containing the same, or a pharmaceutically acceptable salt or solvate thereof as an active ingredient.

In addition, the present invention provides a drug for the treatment of cancer or proliferative disease comprising a compound of the above general formula I, or an isomer thereof, and/or a ligand-drug conjugate containing the same, or a pharmaceutically acceptable salt or solvate thereof as an active ingredient. Pharmaceutical compositions are provided.

In addition, the present invention relates to a compound of the above general formula I, or an isomer thereof, and/or a ligand-drug conjugate containing the same, or a pharmaceutically acceptable salt or solvate thereof; and at least one pharmaceutically acceptable excipient.

As used herein, the term “proliferative disease” refers to unwanted or uncontrolled cell proliferation of undesirable excessive or abnormal cells, such as neoplastic or hyperplastic growth, in vitro or in vivo. The above proliferative diseases include carcinoma, lymphoma, leukemia, blastoma, sarcoma, liposarcoma, neuroendocrinoma, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, squamous cell carcinoma, squamous cell carcinoma, lung cancer, small cell lung cancer, and non-small cell lung cancer. , lung adenocarcinoma, lung squamous carcinoma, peritonoma, hepatocellular carcinoma, gastric carcinoma, gastrointestinal tumor, pancreatic cancer, brain cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrium. Or, it may be selected from the group consisting of uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophageal varices cancer, biliary tract cancer, colon cancer, and head and neck cancer. It is not limited to this.

In one embodiment of the present invention, a treatment method using an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is also provided. In one embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof is provided to a patient. Antibody-drug conjugates, pharmaceutically acceptable salts thereof, or solvates thereof inhibit the metastasis of cancer cells by binding to targets expressed on the surface of cancer cells. In one embodiment, the antibody binds to a target expressed on the surface of cancer cells in a form bound to a cytotoxic agent, thereby specifically delivering the cytotoxic agent bound to the antibody to the cancer cells, thereby inducing death of the cancer cells. In one embodiment, the antibody binds to a target expressed on the surface of cancer cells in the form of an antibody specific for the same target or a different target, thereby increasing the specificity of the polyantibody to cancer cells or forming a link between cancer cells and other types of cells such as immune cells. induces the death of cancer cells.

Also provided is a pharmaceutical composition comprising a therapeutically effective amount of an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant. do. Also included are methods of treating cancer patients, for example, by administering such pharmaceutical compositions. The term “patient” includes human patients.

The pharmaceutical composition may include a pharmaceutically acceptable carrier. The carrier is used to include an excipient, diluent, or auxiliary agent. The carriers include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl chloride. It may be selected from the group consisting of rolidone, water, physiological saline, buffer solutions such as PBS, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, and mineral oil. The composition may include fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, or combinations thereof.

The pharmaceutical composition can be prepared in any dosage form according to conventional methods. The composition may be formulated, for example, as an oral dosage form (e.g., powder, tablet, capsule, syrup, pill, or granule), or a parenteral dosage form (e.g., injection). Additionally, the composition may be prepared as a systemic formulation or a topical formulation.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. It is prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may also be used. Meanwhile, when administered orally, proteins or peptides are digested, so when formulating them in the form of oral compositions, it is necessary to coat the active ingredients or protect them from decomposition in the stomach.

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

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

The pharmaceutical composition is selected from the group consisting of injections, tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. It can have any one formulation.

For intravenous, dermal or subcutaneous injection, etc., the active ingredient may be in the form of an acceptable aqueous solution for parenteral administration that is pyrogen-free and has appropriate pH, isotonicity and stability. Those skilled in the art can prepare appropriate solutions using isotonic vehicles such as, for example, aqueous sodium chloride solution, Ringer's solution, lactated Ringer's solution, etc., and preservatives, stabilizers, buffers, antioxidants or other additives may be included as required. Solid forms suitable for injection can also be prepared as emulsions or in the form of polypeptides encapsulated in liposomes. The conjugate according to the invention may be administered by any device capable of transporting the active substance to the target cell.

The pharmaceutical composition may contain an effective amount of the antibody or antigen-binding fragment thereof, an anticancer agent, or a combination thereof. The term “effective amount” refers to an amount sufficient to produce a prophylactic or therapeutic effect when administered to an individual in need of such prophylaxis or treatment. The effective amount can be appropriately selected by a person skilled in the art depending on the cell or organism selected. Factors including the severity of the disease, the patient's age, weight, health, gender, the patient's sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs combined or used simultaneously with the composition used, and other fields of medicine. It can be determined according to well-known factors.

The dosage of the pharmaceutical composition may be, for example, 10 μg/kg to about 30 mg/kg, optionally 0.1 mg/kg to about 30 mg/kg, or alternatively 0.3 mg/kg to about 20 mg/kg, for an adult. It may be in the range of mg/kg. The administration may be administered once a day, multiple times a day, once a week to four weeks, or once a year to 12 times.

The auristatin prodrug according to the present invention has the advantage that by introducing a self-immolative group, the shortcomings of existing drugs due to high lipophilicity, pharmacokinetic properties, cytotoxicity, etc. are improved, thereby showing excellent drug efficacy. There is.

Figure 1 is a diagram showing the active drug release mechanism of a prodrug according to one embodiment.
Figure 2 is a diagram showing an example of synthesis of an antibody-drug conjugate (ADC) using a prodrug according to an embodiment.
Figure 3 is a graph showing the results of analyzing the hydrophobicity of an antibody-drug conjugate according to an example through HIC-HPLC.

Hereinafter, the present invention will be described in detail through examples and experimental examples.

However, the following examples and experimental examples only illustrate the present invention, and the content of the present invention is not limited to the following examples and experimental examples.

<Example 1> Preparation of Compound 5

Preparation of Compound 1

N,N' -dimethylethylene-1,2-diamine (5 g, 56.72 mmol) was dissolved in tetrahydrofuran (200 mL) and di- t -butyl dicarbonate (4.3 mL, 18.9 mmol) was dissolved at 0°C. was added and the reaction solution was stirred at room temperature for 17 hours. The reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain Compound 1 (1.7 g, 47%). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 3.33 (s, 2H), 2.88 (s, 3H), 2.73 (t, J = 6.6 Hz, 2H) 2.46 (d, J = 0.9 Hz, 3H), 1.46 (s, 9H).

Preparation of Compound 2

Monomethyl auristatin E (MMAE, 500 mg, 0.69 mmol) was dissolved in dichloromethane (10 mL), then fluorenylmethyloxycarbonyl chloride (Fmoc-Cl, 198 mg, 0.77 mmol) and sodium bicarbonate ( 117 mg, 1.39 mmol) was added and the reaction solution was stirred at room temperature for 17 hours. The reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain Compound 2 (642 mg, 98%). EI-MS m/z: [M+H] ⁺ 940.58.

Preparation of Compound 3

Compound 2 (642 mg, 0.68 mmol) was dissolved in dichloromethane (10 mL), then bis(4-nitrophenyl)carbonate (623 mg, 2.05 mmol) and N,N' -diisopropylethylamine (0.47 mL, 2.73 mmol) was added and the reaction solution was stirred at room temperature for 17 hours. The reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain Compound 3 (749 mg, 98%). EI-MS m/z: [M+H] ⁺ 1105.51

Preparation of Compound 4

Compound 3 (749 mg, 0.68 mmol) was dissolved in dichloromethane (5 mL), and then compound 1 (255 mg, 1.36 mmol) and N,N' -diisopropylethylamine (0.35 mL, 2.03 mmol) were added. And the reaction solution was stirred at room temperature for 17 hours. The reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 4 (456 mg, 58%). EI-MS m/z: [M+H] ⁺ 1154.66.

Preparation of Compound 5

Compound 4 (627 mg, 0.54 mmol) was dissolved in dichloromethane (6 mL), then trifluoroacetic acid (2 mL) was added at 0°C under a nitrogen atmosphere, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure to obtain compound 5 (600 mg, crude). EI-MS m/z: [M+H] ⁺ 1054.64.

<Example 2> Preparation of compound 10

Preparation of Compound 7

Compound 6 (600 mg, 1.10 mmol, Compound 6 was prepared by the method described in published patent 10-2018-0110645) was dissolved in N,N-dimethylformamide (10 mL) and then dissolved in bis(4-nitrophenyl). ) Carbonate (370 mg, 1.22 mmol) and N,N-diisopropylethylamine (0.39 mL, 2.22 mmol) were added at 0° C. under nitrogen atmosphere. After stirring the reaction solution at room temperature for 12 hours, distilled water (50 mL) was added to the reaction solution, extracted with ethyl acetate (2 x 50 mL), and the collected organic layer was dried over anhydrous sodium sulfate. After filtration, concentration and purification by column chromatography, compound 7 (567 mg, 72%) was obtained. EI-MS m/z: [M+H] ⁺ 707.38.

Preparation of Compound 8

Compound 5 (600 mg, 0.51 mmol) and Compound 7 (422 mg, 0.59 mmol) were dissolved in dimethylformamide (6 mL) and pyridine (2 mL), and then dissolved in 1-hydroxy-7- in a nitrogen atmosphere at 0°C. Azabenzotriazole (15 mg, 0.11 mmol) and N,N' -diisopropylethylamine (0.2 mL, 1.08 mmol) were sequentially added and stirred at room temperature for 16 hours. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous sodium bicarbonate solution (50 mL), then distilled water (50 mL), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 8 (715 mg, 81%) was obtained. EI-MS m/z: [M+H] ⁺ 1622.57.

Preparation of Compound 9

Compound 8 (65 mg, 0.04 mmol) was dissolved in methanol (0.65 mL) and tetrahydrofuran (0.65 mL), and then lithium hydroxide (5 mg, 0.12 mmol) dissolved in distilled water (0.6 mL) was dissolved in nitrogen atmosphere at -50°C. It was added and stirred at 0°C for 1 hour and 30 minutes. After adjusting the pH to about 4-5 with acetic acid, it was freeze-dried and proceeded with the next reaction without any additional purification process. EI-MS m/z: [M+H] ⁺ 1482.81.

Preparation of Compound 10

Compound 9 (59 mg, 0.04 mmol) was dissolved in dimethylformamide (2 mL), and then piperidine (0.05 mL, 5.05 mmol) was added at 0°C under a nitrogen atmosphere. After stirring at 0°C for 30 minutes, water was added and purified by HPLC to obtain compound 10 (16 mg, 29%). EI-MS m/z: [M+H] ⁺ 1260.67.

<Example 3> Preparation of compound 13

Preparation of Compound 11

2-Carboxybenzaldehyde (5 g, 33.30 mmol) was dissolved in methanol (67 mL), then methylamine (40 wt.% in H ₂ O, 6.8 mL, 66.60 mmol) was added at 0 °C, and the reaction solution was incubated at room temperature for 1 hour. Stirred for an hour. Sodium borohydride (630 mg, 16.65 mmol) was added at 0 °C and stirred for 30 minutes. Acetone was added to the reaction solution and concentrated under reduced pressure. After dissolving the concentrated compound in 1,4-dioxane (100 mL), di- t -butyl dicarbonate (15.3 mL, 66.60 mmol) and 1 N aqueous sodium hydroxide solution (133 mL) were added at 0°C, and the reaction solution was It was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, diluted with ethyl acetate (500 mL), washed with 1 N aqueous hydrochloric acid solution (400 mL), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 11 (4.2 g, 48%) was obtained. ^1H -NMR (400 MHz, CDCl ₃ ) δ 8.08 (d, J = 7.8 Hz, 1H), 7.57 (td, J = 7.6, 1.5 Hz, 1H), 7.36 (td, J = 7.6, 1.2 Hz, 1H) ), 7.30 (d, J = 7.9 Hz, 1H), 4.86 (s, 2H), 2.95 (s, 3H), 1.44 (s, 9H).

Preparation of Compound 12

Compound 11 (1.0 g, 3.82 mmol) was dissolved in dichloromethane (3 mL), then N,N' -diisopropylethylamine (1.1 mL, 6.38 mmol) and bis(2-oxo-3-oxazolidinyl). ) Phosphine chloride (BOP-Cl, 1.0 g, 4.02 mmol) was added and the reaction solution was stirred at room temperature for 2 hours. Compound 2 (600 mg, 0.64 mmol) and 4-dimethylaminopyridine (233 mg, 1.91 mmol) were added to the reaction solution and stirred at room temperature for 17 hours. The reaction solution was diluted with dichloromethane (100 mL), washed with saturated aqueous ammonium chloride solution (50 mL) and distilled water (50 mL), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 12 (633 mg, 83%) was obtained. EI-MS m/z: [M+H] ⁺ 1188.30.

Preparation of Compound 13

Compound 12 (550 mg, 0.46 mmol) was dissolved in dichloromethane (9.5 mL), then trifluoroacetic acid (0.5 mL) was added at 0°C under a nitrogen atmosphere, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure to obtain compound 13 (556 mg, crude). EI-MS m/z: [M+H] ⁺ 1087.26.

<Example 4> Preparation of compound 17

Preparation of Compound 14

Compound 6 (1.0 g, 1.86 mmol) was dissolved in N,N -dimethylformamide (10 mL), and then mixed with bis(pentafluorophenyl)carbonate (881 mg, 2.23 mmol) and N at 0°C under a nitrogen atmosphere. N' -diisopropylethylamine (0.66 mL, 3.72 mmol) was added. After stirring at room temperature for 17 hours, the reaction solution was diluted with ethyl acetate (100 mL), washed with distilled water (2 After filtration, concentration under reduced pressure and purification by column chromatography, compound 14 (1.38 g, 98%) was obtained. EI-MS m/z: [M+H] ⁺ 751.96.

Preparation of Compound 15

Compound 13 (640 mg, 0.53 mmol) and Compound 14 (440 mg, 0.58 mmol) were dissolved in N,N -dimethylformamide (5 mL), then 1-hydroxy-7-azabenzoate was dissolved in 0°C nitrogen atmosphere. Triazole (35 mg, 0.26 mmol) and N,N' -diisopropylethylamine (0.28 mL, 1.60 mmol) were sequentially added and stirred at room temperature for 16 hours. 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, concentration under reduced pressure and purification by column chromatography, compound 15 (775 mg, 87%) was obtained. EI-MS m/z: [M+H] ⁺ 1655.15.

Preparation of Compound 16

Compound 15 (775 mg, 0.47 mmol) was dissolved in N,N -dimethylformamide (10 mL), and then piperidine (0.07 mL, 0.70 mmol) was added at 0°C under a nitrogen atmosphere. After stirring at 0°C for 2 hours, the reaction solution was diluted with ethyl acetate (100 mL), washed with distilled water (50 mL x2), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 16 (602 mg, 89%) was obtained. EI-MS m/z: [M+H] ⁺ 1432.39.

Preparation of Compound 17

Compound 16 (40 mg, 0.028 mmol) was dissolved in 2-propanol (0.5 mL) and tetrahydrofuran (0.5 mL), and then lithium hydroxide (4.7 mg, 0.11 mmol) was dissolved in distilled water (0.75 mL) under nitrogen atmosphere at -50°C. ) and tetramethylammonium hydroxide (1.1 M aqueous solution, 0.02 mL, 0.028 mmol) were sequentially added and stirred at -10°C for 2 hours. After adjusting the pH to about 4-5 with acetic acid, it was purified by HPLC to obtain compound 17 (26 mg, 65%). EI-MS m/z: [M+H] ⁺ 1293.63.

<Example 5> Preparation of compound 22

Preparation of Compound 19

Compound 18 (1.6 g, 2.90 mmol, Compound 18 was prepared by the method described in Korean Patent Publication 10-2018-0110645) was dissolved in N,N -dimethylformamide (10 mL), and then dissolved in nitrogen atmosphere at 0°C. Bis(pentafluorophenyl)carbonate (1.4 g, 3.48 mmol) and N,N' -diisopropylethylamine (1.03 mL, 5.80 mmol) were added under low temperature. The reaction solution was stirred at room temperature for 17 hours, then diluted with ethyl acetate (100 mL), wiped with distilled water (50 mL x 2), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 19 (2.0 g, 90%) was obtained. EI-MS m/z: [M+H] ⁺ 765.89.

Preparation of Compound 20

Compound 13 (50 mg, 0.04 mmol) and Compound 19 (35 mg, 0.05 mmol) were dissolved in dimethylformamide (1.5 mL) and then dissolved in 1-hydroxy-7-azabenzotriazole (1.7%) under nitrogen atmosphere at 0°C. mg, 0.01 mmol) and N,N' -diisopropylethylamine (0.04 mL, 0.21 mmol) were sequentially added and stirred at room temperature for 16 hours. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous sodium bicarbonate solution (50 mL), then distilled water (50 mL), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 21 (63 mg, 90%) was obtained. EI-MS m/z: [M+H] ⁺ 1668.53.

Preparation of Compound 21

Compound 20 (63 mg, 0.04 mmol) was dissolved in dimethylformamide (1.5 mL), and then piperidine (0.006 mL, 0.06 mmol) was added at 0°C under a nitrogen atmosphere. After stirring at 0°C for 2 hours, the reaction solution was diluted with ethyl acetate (100 mL), washed with distilled water (50 mL x2), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 21 (50 mg, 91%) was obtained. EI-MS m/z: [M+H] ⁺ 1447.27.

Preparation of Compound 22

Compound 21 (50 mg, 0.034 mmol) was dissolved in 2-propanol (0.5 mL) and tetrahydrofuran (0.5 mL), and then lithium hydroxide (4.3 mg, 0.10 mmol) was dissolved in distilled water (0.75 mL) under nitrogen atmosphere at -50°C. ) and tetramethylammonium hydroxide (1.1 M aqueous solution, 0.03 mL, 0.035 mmol) were sequentially added and stirred at -10°C for 2 hours. After adjusting the pH to about 4-5 with acetic acid, it was purified by HPLC to obtain Compound 22 (42 mg, 87%). EI-MS m/z: [M+H] ⁺ 1279.27.

<Example 6> Preparation of compound 29

Preparation of Compound 23

3-(2-methoxyethoxy)propanoic acid (1.7 g, 11.74 mmol) was dissolved in dichloromethane (20 mL) and then mixed with N -hydroxysuccinimide (2.0 g, 17.6 mmol) and N at 0°C. -(3-Dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDC·HCl, 3.3 g, 17.6 mmol) was added, and the reaction solution was stirred at room temperature for 3 hours. The reaction solution was diluted with dichloromethane (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 and concentration under reduced pressure, compound 23 (2.9 g, crude) was obtained. ¹ H-NMR (400 MHz, CDCl ₃ ) δ 3.85 (t, J = 6.5 Hz, 2H), 3.65 (dd, J = 5.8, 3.4 Hz, 2H), 3.55 (dd, J = 5.8, 3.4 Hz, 2H ), 3.39 (s, 3H), 2.92 (t, J = 6.5 Hz, 2H), 2.84 (s, 4H).

Preparation of Compound 24

N -( t -butoxycarbonyl)-L-valine (2.0 g, 9.3 mmol) was dissolved in dichloromethane (30 mL) and then N -hydroxysuccinimide (1.6 g, 13.9 mmol) at 0°C. and N -(3-dimethylaminopropyl)- N' -ethylcarbodiimide hydrochloride (EDC·HCl, 2.7 g, 13.9 mmol) were added, and the reaction solution was stirred at room temperature for 3 hours. The reaction solution was diluted with dichloromethane (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 and concentration under reduced pressure, compound 24 (2.89 g, crude) was obtained. ¹ H-NMR (400 MHz, CDCl ₃ ) δ 5.00 (d, J = 9.3 Hz, 1H), 4.60 (dd, J = 9.2, 4.9 Hz, 1H), 2.84 (s, 4H), 2.35-2.28 (m , 1H), 1.46 (s, 9H), 1.08-1.03(m, 6H).

Preparation of Compound 25

Compound 24 (2.89 g) was dissolved in 1,2-dimethoxyethane (20 mL) and tetrahydrofuran (10 mL), and then L-citrulline (1.7 g) dissolved in distilled water (30 mL) under nitrogen atmosphere at 0°C. , 9.7 mmol) and sodium bicarbonate (932 mg, 11.1 mmol) were added and stirred at room temperature for 20 hours. The reaction solution was concentrated under reduced pressure, adjusted to pH 4-5 using an aqueous citric acid solution, diluted with 10% 2-propanol/ethyl acetate solution (3 x 100 mL), and extracted. The collected organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain Compound 25 (3.7 g, crude). EI-MS m/z: [M+H] ⁺ 375.28.

Preparation of Compound 26

Compound 25 (3.7 g) was dissolved in dichloromethane (50 mL) and methanol (25 mL), then dissolved in 4-aminobenzyl alcohol (1.4 g, 11.1 mmol) and 2-ethoxyl alcohol at 0°C, protected from light, under a nitrogen atmosphere. -2H -quinoline-1-carboxylic acid ethyl ester (5.0 g, 20.2 mmol) was sequentially added and stirred at room temperature for 20 hours. The reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 26 (1.9 g, 39%). EI-MS m/z: [M+H] ⁺ 480.25.

Preparation of Compound 27

Compound 26 (1.3 g, 2.75 mmol) was dissolved in dichloromethane (9 mL), then trifluoroacetic acid (3 mL) was added at 0°C under a nitrogen atmosphere, and the mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure to obtain compound 27 (1.3 g, crude). EI-MS m/z: [M+H] ⁺ 380.23.

Preparation of Compound 28

Compound 27 (1.3 g) was dissolved in dichloromethane (10 mL), and N,N' -diisopropylethylamine (2.9 mL, 1.65 mmol) and compound 24 (810 mg, 3.30 mmol) were added at 0 °C. And the reaction solution was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 28 (830 mg, 59%). EI-MS m/z: [M+H] ⁺ 510.22.

Preparation of Compound 29

Compound 28 (830 mg, 1.6 mmol) was dissolved in dimethylformamide (10 mL), then mixed with bis(pentafluorophenyl)carbonate (770 mg, 1.9 mmol) and N,N' -dialyte at 0°C under nitrogen atmosphere. Isopropylethylamine (0.56 mL, 3.26 mmol) was added. After stirring at room temperature for 17 hours, the reaction solution was diluted with ethyl acetate (100 mL), washed with distilled water (2 After filtration, concentration under reduced pressure and purification by column chromatography, compound 29 (680 mg, 58%) was obtained. EI-MS m/z: [M+H] ⁺ 720.11.

<Example 7> Preparation of compound 31

Preparation of Compound 30

Compound 13 (556 mg, 0.46 mmol) and compound 29 (366 mg, 0.51 mmol) were dissolved in N,N -dimethylformamide (7 mL), and then dissolved in 1-hydroxy-7-azabenzoate under nitrogen atmosphere at 0°C. Triazole (19 mg, 0.14 mmol) and N,N' -diisopropylethylamine (0.4 mL, 2.32 mmol) were sequentially added and stirred at room temperature for 20 hours. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (2 After filtration, concentration under reduced pressure and purification by column chromatography, compound 30 (620 mg, 82%) was obtained. EI-MS m/z: [M+H] ⁺ 1623.22.

Preparation of Compound 31

Compound 30 (60 mg, 0.02 mmol) was dissolved in N,N -dimethylformamide (1.5 mL), and then piperidine (0.006 mL, 0.06 mmol) was added at 0°C under a nitrogen atmosphere. After stirring at 0°C for 2 hours, the reaction solution was diluted with ethyl acetate (100 mL), washed with distilled water (2 After filtration, concentration under reduced pressure and purification by HPLC, compound 31 (19 mg, 67%) was obtained. EI-MS m/z: [M+H] ⁺ 1401.38.

<Example 8> Preparation of compound 36

Preparation of Compound 32

Hexaethylene glycol (3 g, 10.84 mmol) was dissolved in dichloromethane (100 mL) and then triisopropylsilyl chloride (1.2 mL, 5.42 mmol) and imidazole (443 mg, 6.50 mmol) under nitrogen atmosphere at 0°C. was added. The reaction solution was stirred at room temperature for 17 hours, concentrated, and purified by column chromatography to obtain compound 32 (1.1 g, 46%). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 3.84 (t, J = 5.7 Hz, 2H), 3.75 - 3.70 (m, 2H), 3.70 - 3.55 (m, 20H), 1.67 (s, 3H), 1.13 - 1.05 (m, 18H).

Preparation of Compound 33

Compound 32 (1.1 g, 2.51 mmol) was dissolved in acetonitrile (20 mL), then ethyl propiolate (0.51 mL, 5.01 mmol) and N -methylmorpholine (0.05 mL, 0.05 mmol) at 0°C under nitrogen atmosphere. was added. The reaction solution was stirred at room temperature for 17 hours, concentrated, and purified by column chromatography to obtain compound 33 (1.2 g, 93%). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.60 (d, J = 12.3 Hz, 1H), 5.23 - 5.19 (d, J = 12.6, 1H), 4.18 - 4.14 (m, 2H), 3.99 (s, 2H), 3.84 (t, J = 5.8 Hz, 2H), 3.76 (t, J = 4.7 Hz, 2H), 3.65 (t, J = 3.2 Hz, 16H), 3.63 - 3.55 (m, 2H), 1.57 ( t, J = 1.6 Hz, 3H), 1.27 (td, J = 7.1, 1.8 Hz, 3H), 1.13 - 1.05 (m, 18H).

Preparation of Compound 34

Compound 33 (1.26 g, 2.35 mmol) was dissolved in ethanol (20 mL), and then palladium/charcoal (10 wt.%, 200 mg) was added. After stirring for 5 hours under a hydrogen atmosphere at room temperature, the reaction solution was filtered through Celite and concentrated under reduced pressure. Compound 34 (1.24 g, 98%) was obtained without further purification. ¹ H-NMR (400 MHz, CDCl ₃ ) δ 4.14 (dd, J = 8.2, 6.4 Hz, 2H), 3.84 (t, J = 5.8 Hz, 2H), 3.76 (dd, J = 7.6, 5.6 Hz, 2H ), 3.70 - 3.55 (m, 22H), 2.63 - 2.55 (m, 2H), 1.61 (s, 3H), 1.30 - 1.22 (m, 3H), 1.13 - 1.05 (m, 18H).

Preparation of Compound 35

Compound 34 (1.24 g, 2.30 mmol) was dissolved in tetrahydrofuran (20 mL), then lithium hydroxide (144 mg, 3.45 mmol) dissolved in distilled water (10 mL) was added under a nitrogen atmosphere at 0°C and incubated at room temperature for 4 hours. It was stirred for a while. The reaction solution was adjusted to pH 4-5 with 1 N aqueous hydrochloric acid, diluted with ethyl acetate (200 mL), washed with distilled water (100 mL), and dried with anhydrous sodium sulfate. After filtration and concentration under reduced pressure, compound 35 (1.16 g, crude) was obtained without further purification. ¹ H-NMR (400 MHz, CDCl ₃ ) δ 3.84 - 3.79 (m, 4H), 3.70 - 3.64 (m, 20H), 3.60 (d, J = 5.9 Hz, 2H), 2.61 (t, J = 6.0 Hz) , 2H), 1.13 - 1.05 (m, 21H).

Preparation of Compound 36

Compound 35 (1.16 g, 2.27 mmol) was dissolved in dichloromethane (20 mL) and then mixed with N -hydroxysuccinimide (392 mg, 3.41 mmol) and N -(3-dimethylaminopropyl)- at 0°C. N' -ethylcarbodiimide hydrochloride (EDC·HCl, 653 mg, 3.41 mmol) was added, and the reaction solution was stirred at room temperature for 17 hours. The reaction solution was diluted with dichloromethane (100 mL), washed with saturated aqueous sodium hydrogen carbonate solution (2 x 50 mL), and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, compound 36 (1.41 g, crude) was obtained. ¹ H-NMR (400 MHz, CDCl ₃ ) δ 3.86 - 3.80 (m, 4H), 3.65 (d, J = 2.1 Hz, 20H), 3.58 (t, J = 5.8 Hz, 2H), 2.90 (dd, J = 7.6, 5.5 Hz, 2H), 2.84 (s, 4H), 1.13 - 1.05 (m, 21H).

<Example 9> Preparation of compound 41

Preparation of Compound 37

Compound 36 (1.41 g, 2.32 mmol) was dissolved in dichloromethane (30 mL) and tetrahydrofuran (10 mL), then triethylamine (2.0 mL, 13.9 mmol) and compound 27 (1.14 g, 2.32 mmol) were dissolved in dichloromethane (30 mL) and tetrahydrofuran (10 mL). mmol) was added, and the reaction solution was stirred at room temperature for 19 hours. The reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 37 (1.35 g, 66%). EI-MS m/z: [M+H] ⁺ 872.42, [M+Na] ⁺ 894.43.

Preparation of Compound 38

Compound 37 (1.35 g, 1.55 mmol) was dissolved in N,N -dimethylformamide (10 mL), and then mixed with bis(pentafluorophenyl)carbonate (793 mg, 2.01 mmol) and N at 0°C under a nitrogen atmosphere. N' -diisopropylethylamine (0.54 mL, 3.09 mmol) was added. After stirring at room temperature for 17 hours, the reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous sodium hydrogen carbonate solution (50 mL) and distilled water (2 After filtration, concentration under reduced pressure and purification by column chromatography, compound 38 (1.26 g, 75%) was obtained. EI-MS m/z: [M+H] ⁺ 1082.43.

Preparation of Compound 39

Compound 13 (445 mg, 0.37 mmol) and compound 38 (440 mg, 0.41 mmol) were dissolved in N,N -dimethylformamide (5 mL), and then 1-hydroxy-7-azabenzoate under nitrogen atmosphere at 0°C. Triazole (15 mg, 0.11 mmol) and N,N' -diisopropylethylamine (0.74 mL, 2.59 mmol) were sequentially added and stirred at room temperature for 4 hours. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (2 After filtration, concentration under reduced pressure and purification by column chromatography, compound 39 (668 mg, 82%) was obtained. EI-MS m/z: [M+H] ⁺ 1986.72, 1/2[M+H] ⁺ 993.44.

Preparation of Compound 40

Compound 39 (668 mg, 0.34 mmol) was dissolved in N,N -dimethylformamide (5 mL), and then piperidine (0.05 mL, 0.50 mmol) was added at 0°C under a nitrogen atmosphere. After stirring at 0°C for 2 hours, the reaction solution was diluted with ethyl acetate (100 mL), washed with distilled water (2 After filtration, concentration under reduced pressure and purification by column chromatography, compound 40 (485 mg, 81%) was obtained. EI-MS m/z: [M+H] ⁺ 1762.82, 1/2[M+H] ⁺ 882.45.

Preparation of Compound 41

Compound 40 (40 mg, 0.02 mmol) was dissolved in dichloromethane (1 mL), and then trifluoroacetic acid (0.1 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 41 (27 mg, 70%). EI-MS m/z: [M+H] ⁺ 1606.67, 1/2[M+H] ⁺ 804.27.

<Example 10> Preparation of compound 45

Preparation of Compound 43

Compound 16 (150 mg, 0.10 mmol) and Compound 42 (118 mg, 0.13 mmol, Compound 42 was prepared by the method described in Korean Patent Application No. 10-2022-0046444) were mixed with N,N -dimethylformamide (3 mL) ), then 1-hydroxy-7-azabenzotriazole (7.1 mg, 0.05 mmol) and N,N' -diisopropylethylamine (0.06 mL, 0.31 mmol) were added sequentially at 0°C under nitrogen atmosphere. and stirred at room temperature for 20 hours. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (2 After filtration, concentration under reduced pressure and purification by column chromatography, compound 43 (205 mg, 89%) was obtained. EI-MS m/z: [M+H] ⁺ 2190.03, 1/2[M+H] ⁺ 1095.15.

Preparation of Compound 44

Compound 43 (205 mg, 0.065 mmol) was dissolved in 2-propanol (2 mL) and tetrahydrofuran (2 mL), and then lithium hydroxide (23.6 mg, dissolved in distilled water (2.5 mL) at -50°C under a nitrogen atmosphere. 0.56 mmol) and tetramethylammonium hydroxide (10 wt% aqueous solution, 0.16 mL) were sequentially added and stirred at -5°C for 3 hours. After adjusting the pH to about 4-5 with acetic acid, it was purified by HPLC to obtain compound 44 (165 mg, 90%). EI-MS m/z: [M+H] ⁺ 1908.92, 1/2[M+H] ⁺ 955.14.

Preparation of Compound 45

Compound 44 (85 mg, 0.045 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 2 hours, concentrated, and purified by HPLC to obtain compound 45 (63 mg, 78%). EI-MS m/z: [M+H] ⁺ 1807.98, 1/2[M+H] ⁺ 905.00.

<Example 11> Preparation of compound 51

Preparation of Compound 46

2-(2-(2-Chloroethoxy)ethoxy)ethanol (5.0 g, 29.6 mmol) was dissolved in acetone (30 mL), sodium iodide (13.3 mg, 88.9 mmol) was added, and the reaction solution was dissolved in 12 It was refluxed and stirred for an hour. The reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 46 (7.0 g, 91%). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 3.80-3.73 (m, 4H), 3.72-3.65 (m, 4H), 3.63-3.61 (m, 2H), 3.27 (t, J = 6.4 Hz, 2H) .

Preparation of Compound 47

Compound 46 (7.0 g, 26.9 mmol) was dissolved in acetone (200 mL), then Jones reagent (20 mL) was added at 0°C under a nitrogen atmosphere, and the mixture was stirred at 0°C for 15 hours. The reaction solution was filtered, concentrated under reduced pressure, diluted with ethyl acetate (200 mL), washed with distilled water (150 mL), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 47 (7.0 g, 94%) was obtained. ¹ H-NMR (400 MHz, CDCl ₃ ) δ 4.22 (s, 2H), 3.85-3.70 (m, 6H), 3.35-3.25 (m, 2H).

Preparation of Compound 48

Compound 47 (7.0 g, 25.5 mmol) was dissolved in methanol (70 mL) at 0°C under a nitrogen atmosphere, and then oxalyl chloride (3.2 mL, 38.3 mmol) was added at 0°C. After stirring the reaction solution at 0°C for 16 hours, the reaction solution was filtered, concentrated under reduced pressure, and purified by column chromatography to obtain compound 48 (5.7 g, 77%). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 4.19 (s, 2H), 3.80-3.67 (m, 9H), 3.27 (t, J = 6.8 Hz, 2H).

Preparation of Compound 49

Compound 48 (2.5 g, 8.67 mmol) was dissolved in N,N -dimethylformamide (30 mL) and then dissolved in t -butyl( t -butoxycarbonyl)(hydroxy)carbamate ( 2.6 g, 11.2 mmol) and sodium hydride (60% in oil, 454 mg, 0.4 mmol) were added. The reaction solution was stirred at 0°C for 16 hours and then concentrated under reduced pressure. The concentrated solution was diluted with ethyl acetate (200 mL), washed with distilled water (3 After filtration, concentration under reduced pressure and purification by column chromatography, compound 49 (1.87 g, 73%) was obtained. ¹ H-NMR (400 MHz, CDCl ₃ ) δ 4.17 (s, 2H), 4.08 (m, 2H), 3.78-3.65 (m, 9H), 1.53 (s, 18H).

Preparation of Compound 50

Compound 49 (1.87 g, 6.38 mmol) was dissolved in methanol (15 mL) and tetrahydrofuran (45 mL), and then sodium hydroxide (600 mg, 15.9 mmol) dissolved in distilled water (15 mL) was dissolved in nitrogen atmosphere at 0°C. It was added and stirred at room temperature for 3 hours. The reaction solution was adjusted to pH 4-5 with 1 N aqueous hydrochloric acid, diluted with ethyl acetate (200 mL), washed with distilled water (100 mL), and dried with anhydrous sodium sulfate. After filtration and concentration under reduced pressure, compound 50 (1.6 g, 90%) was obtained without further purification. ¹ H-NMR (400 MHz, CDCl ₃ ) δ 4.17 (s, 2H), 4.08-4.02 (m, 2H), 3.80-3.67 (m, 6H), 1.48 (s, 9H).

Preparation of Compound 51

Compound 50 (500 mg, 1.79 mmol) was dissolved in dichloromethane (20 mL) and then mixed with N -hydroxysuccinimide (309 mg, 2.68 mmol) and N -(3-dimethylaminopropyl)- at 0°C. N' -ethylcarbodiimide hydrochloride (EDC·HCl, 515 mg, 2.68 mmol) was added, and the reaction solution was stirred at room temperature for 2 hours. The reaction solution was diluted with dichloromethane (30 mL), washed with saturated aqueous sodium hydrogen carbonate solution (2 x 30 mL), and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, compound 51 (669 mg, crude) was obtained without further purification. EI-MS m/z : [M+Na] ⁺ 399.19, : [M-Boc] ⁺ 277.21.

<Example 12> Preparation of compound 54

Preparation of Compound 52

Triethylene glycol (30 g, 199.8 mmol) was dissolved in dichloromethane (20 mL), and then 4-toluenesulfonyl chloride (76 g, 399.6 mmol) and triethylamine (67 mL, 479.5 mmol) were dissolved in dichloromethane (20 mL) at 0°C. mmol) was added and the reaction solution was stirred at room temperature for 16 hours. The reaction solution was diluted with dichloromethane (500 mL), washed with saturated aqueous ammonium chloride solution (300 mL) and brine (200 mL) in that order, and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, compound 52 (92 g, crude) was obtained without further purification. ¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.79 (d, 4H), 7.34 (d, 4H), 4.13 (t, 4H), 3.65 (t, 4H), 3.85 (s, 4H), 2.44 (s , 6H).

Preparation of Compound 53

Compound 52 (92 g, crude) was dissolved in N,N -dimethylformamide (500 mL), and then sodium azide (39 g, 599.3 mmol) was added under a nitrogen atmosphere. The reaction solution was stirred at 50°C for 16 hours and concentrated under reduced pressure. The concentrated solution was diluted with ethyl acetate (500 mL), washed with distilled water (300 mL), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 53 (36 g, 90%) was obtained. ¹ H-NMR (400 MHz, CDCl ₃ ) δ 3.70-3.68 (m, 8H), 3.39 (t, 4H).

Preparation of Compound 54

Compound 53 (32 g, 159.8 mmol) was dissolved in ethyl acetate (50 mL), and then 1 N aqueous hydrochloric acid solution (150 mL) was added at 0°C under a nitrogen atmosphere. After 5 minutes, triphenylphosphine (42 g, 159.8 mmol) dissolved in ethyl acetate (150 mL) was slowly added dropwise to the reaction solution and stirred at room temperature for 16 hours. The reaction solution was adjusted to pH 1-2 with a 6 N aqueous hydrochloric acid solution, diluted with ethyl acetate (200 mL), and washed with distilled water (200 mL). The aqueous solution layer was adjusted to pH 13 with sodium hydroxide, extracted with ethyl acetate (2 x 300 mL), and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, compound 54 (23 g, 82%) was obtained without further purification. ¹ H-NMR (400 MHz, CDCl ₃ ) δ 3.76-3.65 (m, 6H), 3.52 (t, 2H), 3.40 (t, 2H), 2.87 (t, 2H).

<Example 13> Preparation of compound 62

Preparation of Compound 55

3-Aminopentanedioic acid hydrochloride (5.0 g, 27.2 mmol) was dissolved in distilled water/1,4-dioxane (6 mL/44 mL) and then mixed with aqueous sodium hydroxide solution (4 M, 20.4 mL, 81.6 mmol) and di- t . -Butyl dicarbonate (6.87mL, 29.9 mmol) was added. The reaction solution was stirred at room temperature for 15 hours, then adjusted to pH 2 with an aqueous potassium hydrogen sulfate solution and extracted with ethyl acetate (3 x 50 mL). The collected organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 55 (4.0 g, 60%). ¹ H-NMR (400 MHz, CD ₃ OD) δ 4.25 (t, J = 6.2 Hz, 1H), 2.56 (d, J = 5.6 Hz, 4H), 1.43 (s, 9H). EI-MS m/z: [M+Na] ⁺ 270.3.

Preparation of Compound 56

-메틸 에스터 (H-D-Glu(OMe)-OH, 40 g, 249.8 mmol)을 1,4-다이옥세인 (80 mL)과 증류수(40 mL)에 녹인 후 0 ℃, 질소 대기 하에서 다이-t-부틸 다이카보네이트 (71 g, 324.7 mmol)와 탄산수소 나트륨 (63 g, 749.3 mmol)을 첨가하였다. 반응 용액을 5 시간 동안 상온에서 교반한 후 1 N 염산 수용액으로 pH 4 정도로 맞춘 후, 에틸 아세테이트 (200 mL)로 묽히고, 증류수 (100 mL)로 닦아 주고 무수 황산 나트륨으로 건조하였다. 여과 후 감압 농축하고 N,N-다이메틸폼아마이드 (20 mL)에 녹인 후 0 ℃, 질소 대기 하에서 탄산 칼륨 (46 g, 328.4 mmol)과 벤질 브로마이드(60 mL, 505.2 mmol)를 첨가하였다. 반응 용액을 17 시간 동안 상온에서 교반하고 에틸 아세테이트 (200 mL)로 묽힌 후, 증류수 (100 mL)로 닦아 주고 무수 황산 나트륨으로 건조하였다. 여과 후 감압 농축하고 농축하고 컬럼 크로마토그래피로 정제하여 화합물 56 (80 g, 90%)을 수득하였다. ¹H-NMR (400 MHz, CDCl₃) δ 7.39-7.31 (m, 5H), 5.17 (s, 2H), 4.38-4.37 (m, 1H), 2.43-2.31 (m, 2H), 2.23-2.17 (m, 1H), 1.98-1.91 (m, 1H), 1.43 (s, 9H). EI-MS m/z : [M+Na]⁺ 374.36, : [M-Boc]⁺ 252.38.D-glutamic acid

-Methyl ester (HD-Glu(OMe)-OH, 40 g, 249.8 mmol) was dissolved in 1,4-dioxane (80 mL) and distilled water (40 mL), and then di- t -butyl was dissolved in nitrogen atmosphere at 0°C. Dicarbonate (71 g, 324.7 mmol) and sodium bicarbonate (63 g, 749.3 mmol) were added. The reaction solution was stirred at room temperature for 5 hours, adjusted to pH 4 with 1 N aqueous hydrochloric acid solution, diluted with ethyl acetate (200 mL), washed with distilled water (100 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure, dissolved in N,N -dimethylformamide (20 mL), and potassium carbonate (46 g, 328.4 mmol) and benzyl bromide (60 mL, 505.2 mmol) were added under a nitrogen atmosphere at 0°C. The reaction solution was stirred at room temperature for 17 hours, diluted with ethyl acetate (200 mL), washed with distilled water (100 mL), and dried over anhydrous sodium sulfate. After filtration, the mixture was concentrated under reduced pressure, concentrated, and purified by column chromatography to obtain compound 56 (80 g, 90%). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.39-7.31 (m, 5H), 5.17 (s, 2H), 4.38-4.37 (m, 1H), 2.43-2.31 (m, 2H), 2.23-2.17 ( m, 1H), 1.98-1.91 (m, 1H), 1.43 (s, 9H). EI-MS m/z : [M+Na] ⁺ 374.36, : [M-Boc] ⁺ 252.38.

Preparation of Compound 57

Compound 56 (48 g, 136.6 mmol) was dissolved in dichloromethane (160 mL), then hydrochloric acid (4 M 1,4-dioxane solution, 80 mL, 320 mmol) was added at 0°C under a nitrogen atmosphere and incubated for 5 hours. It was stirred for a while. After concentrating the reaction solution, compound 57 (43 g, crude) was obtained without further purification. EI-MS m/z: [M+H] ⁺ 252.31.

Preparation of Compound 58

Compound 55 (16.7 g, 67.5 mmol) was dissolved in dichloromethane (150 mL) and then dissolved in 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl) at 0°C under nitrogen atmosphere. , 32 g, 168.9 mmol), 1-hydroxybenzotriazole (23 g, 168.8 mmol), compound 57 (43 g, 148.6 mmol) and N -methylmorpholine (33 mL, 303.9 mmol) were added sequentially and incubated at room temperature. It was stirred for 6 hours. The reaction solution was diluted with dichloromethane (100 mL), washed with saturated aqueous sodium bicarbonate solution (50 mL), then distilled water (z50 mL), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 58 (46 g, 95%) was obtained. EI-MS m/z: [M+H] ⁺ 714.33.

Preparation of Compound 59

Compound 58 (46 g, 64.4 mmol) was dissolved in methanol (600 mL) and tetrahydrofuran (144 mL), and then palladium/charcoal (10 wt.%, 7.6 g) was added. The reaction solution was stirred at room temperature under a hydrogen atmosphere for 3 hours. The reaction solution was filtered using Celite and concentrated under reduced pressure to obtain compound 59 (43 g, crude) without further purification. EI-MS m/z: [M+H] ⁺ 534.16.

Preparation of Compound 60

Compound 59 (20 g, 37.5 mmol) and compound 54 (16 g, 93.7 mmol) were dissolved in N,N -dimethylformamide (150 mL) and then N,N,N',N' at 0°C under nitrogen atmosphere. -tetramethyl- O -(1 H -benzotriazol-1-yl)uronium hexafluorophosphate (HBTU, 35 g, 93.7 mmol) and N,N' -diisopropylethylamine (26 mL, 149.9 mmol) ) were sequentially added and stirred at room temperature for 22 hours. The reaction solution was diluted with ethyl acetate (500 mL), washed with saturated aqueous sodium bicarbonate solution (300 mL), then distilled water (300 mL), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 60 (13 g, 41%) was obtained. EI-MS m/z: [M+H] ⁺ 846.21.

Preparation of Compound 61

Compound 60 (13 g, 15.4 mmol) was dissolved in dichloromethane (10 mL), then hydrochloric acid (4 M 1,4-dioxane solution, 9.5 mL, 38.4 mmol) was added at 0°C under a nitrogen atmosphere and incubated for 5 hours. It was stirred for a while. After concentrating the reaction solution, compound 61 (11.6 g, crude) was obtained without further purification. EI-MS m/z: [M+H] ⁺ 746.18.

Preparation of Compound 62

Compound 61 (11.5 g, 14.7 mmol) and compound 51 (5.6 g, 14.9 mmol) were dissolved in dichloromethane (150 mL) and then dissolved in N,N' -diisopropylethylamine (7.7 mL) at 0°C under nitrogen atmosphere. , 44.2 mmol) was added and stirred at room temperature for 3 hours. The reaction solution was diluted with dichloromethane (300 mL), washed with saturated aqueous ammonium chloride solution (100 mL) and brine (100 mL) in that order, and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 62 (8.4 g 57%) was obtained. EI-MS m/z: [M+H] ⁺ 1007.52.

<Example 14> Preparation of compound 66

Preparation of Compound 63

Compound 62 (3 g, 2.98 mmol) was dissolved in tetrahydrofuran (100 mL), and then triphenylphosphine (1.7 g, 6.55 mmol) was added at 0°C under a nitrogen atmosphere. After stirring at room temperature for 30 minutes, distilled water (0.54 mL, 29.8 mmol) was added and the mixture was refluxed and stirred for 17 hours. The reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 63 (2.2 g, 77%). EI-MS m/z: [M+H] ⁺ 955.18.

Preparation of Compound 65

Compound 63 (2.0 g, 2.13 mmol) and Compound 64 (2.3 g, 4.70 mmol, Compound 64 was prepared by the method described in published patent WO2017089895A) were dissolved in N,N -dimethylformamide (15 mL) and then dissolved in 0 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 2.0 g, 5.34°C) under nitrogen atmosphere. mmol) and N,N' -diisopropyl-ethylamine (1.9 mL, 10.7 mmol) were sequentially added and stirred at room temperature for 22 hours. The reaction solution was diluted with ethyl acetate (200 mL), washed with saturated aqueous ammonium chloride solution (100 mL), saturated aqueous sodium bicarbonate solution (100 mL), and distilled water (100 mL), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 65 (3.2 g, 79%) was obtained. EI-MS m/z: [M+H] ⁺ 1888.39, 1/2[M+H] ⁺ 944.62.

Preparation of Compound 66

Compound 65 (1.0 g, 0.53 mmol) was dissolved in dichloromethane (15 mL), then mixed with bis(pentafluorophenyl)carbonate (626 mg, 1.59 mmol) and pyridine (0.17 ml, 2.12 ml) at 0°C under nitrogen atmosphere. mmol) was added. After stirring at room temperature for 17 hours, the reaction solution was diluted with dichloromethane (100 mL), washed with saturated aqueous sodium bicarbonate solution (50 mL), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 66 (835 mg, 68%) was obtained. EI-MS m/z: [M+H] ⁺ 2308.11, 1/2[M+H] ⁺ 1154.66.

<Example 15> Preparation of compound 69

Preparation of Compound 67

Compound 16 (297 mg, 0.19 mmol) and compound 66 (200 mg, 0.087 mmol) were dissolved in N,N -dimethylformamide (4 mL) and pyridine (1 mL), and then incubated at 0°C under nitrogen atmosphere. Hydroxy-7-azabenzotriazole (6 mg, 0.04 mmol) and N,N' -diiso-propylethylamine (0.06 mL, 0.36 mmol) were sequentially added and stirred at room temperature for 20 hours. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (2 After filtration, concentration under reduced pressure and purification by column chromatography, compound 67 (415 mg, 72%) was obtained. EI-MS m/z: 1/2[M+H] ⁺ 2402.01, 1/3[M+H] ⁺ 1602.09, 1/4[M+H] ⁺ 1201.92.

Preparation of Compound 68

Compound 67 (360 mg, 0.075 mmol) was dissolved in 2-propanol (2 mL) and tetrahydrofuran (2 mL), and then lithium hydroxide (41 mg, dissolved in distilled water (2.5 mL) at -50°C under a nitrogen atmosphere. 0.98 mmol) and tetramethylammonium hydroxide (10 wt% aqueous solution, 0.2 mL) were sequentially added and stirred at -5°C for 2 hours. After adjusting the pH to about 4~5 with acetic acid, it was purified by HPLC to obtain compound 68 (124 mg, 38%). EI-MS m/z: 1/2[M+H] ⁺ 2109.79, 1/3[M+H ] ⁺ 1406.41, 1/4[M+H] ⁺ 1055.15.

Preparation of Compound 69

Compound 68 (124 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 2 hours, concentrated, and purified by HPLC to obtain compound 69 (76 mg, 63%). EI-MS m/z: 1/2[M+H] ⁺ 2058.79, 1/3[M+H] ⁺ 1372.72, 1/4[M+H] ⁺ 1029.89.

<Example 16> Preparation of compound 72

Preparation of Compound 70

Compound 21 (105 mg, 0.07 mmol) and compound 66 (70 mg, 0.03 mmol) were dissolved in N,N -dimethylformamide (4 mL) and pyridine (1 mL), and then incubated at 0°C under nitrogen atmosphere. Hydroxy-7-azabenzotriazole (2 mg, 0.02 mmol) and N,N' -diisopropyl-ethylamine (0.02 mL, 0.16 mmol) were sequentially added and stirred at room temperature for 20 hours. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (2 After filtration, concentration under reduced pressure and purification by column chromatography, compound 70 (131 mg, 68%) was obtained. EI-MS m/z: 1/2[M+H] ⁺ 2415.76, 1/3[M+H] ⁺ 1610.92, 1/4[M+H] ⁺ 1208.46.

Preparation of Compound 71

Compound 70 (117 mg, 0.02 mmol) was dissolved in 2-propanol (1 mL) and tetrahydrofuran (1 mL), and then lithium hydroxide (10 mg, 0.23 mg) dissolved in distilled water (1.5 mL) at -50°C under a nitrogen atmosphere. mmol) and tetramethylammonium hydroxide (10 wt% aqueous solution, 0.07 mL) were sequentially added and stirred at -5°C for 2 hours. After adjusting the pH to about 4-5 with acetic acid, it was purified by HPLC to obtain compound 71 (29 mg, 35%). EI-MS m/z: 1/2[M+H] ⁺ 2094.31, 1/3[M+H] ⁺ 1396.42, 1/4[M+H] ⁺ 1047.45.

Preparation of Compound 72

Compound 71 (29 mg, 0.007 mmol) was dissolved in dichloromethane (1.5 mL), and then trifluoroacetic acid (0.07 mL) was added at 0°C under a nitrogen atmosphere. The reaction solution was stirred at 0° C. for 2 hours, concentrated, and purified by HPLC to obtain compound 72 (18 mg, 64%). EI-MS m/z: 1/2[M+H] ⁺ 2044.48, 1/3[M+H] ⁺ 1363.00, 1/4[M+H] ⁺ 1022.41.

<Example 17> Preparation of compound 75

Preparation of Compound 73

Compound 31 (267 mg, 0.19 mmol) and compound 66 (200 mg, 0.087 mmol) were dissolved in N,N -dimethylformamide (4 mL) and pyridine (1 mL), and then incubated at 0°C under nitrogen atmosphere. Hydroxy-7-azabenzotriazole (5.9 mg, 0.04 mmol) and N,N' -diisopropyl-ethylamine (0.09 mL, 0.52 mmol) were sequentially added and stirred at room temperature for 20 hours. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous sodium hydrogen carbonate solution (50 mL) and distilled water (2 After filtration, concentration under reduced pressure and purification by column chromatography, compound 73 (309 mg, 75%) was obtained. EI-MS m/z: 1/2[M+H] ⁺ 2371.82, 1/3[M+H] ⁺ 1581.40, 1/4[M+H] ⁺ 1186.30.

Preparation of Compound 74

Compound 73 (309 mg, 0.065 mmol) was dissolved in 2-propanol (2 mL) and tetrahydrofuran (2 mL), and then lithium hydroxide (21.9 mg, 0.52 mg) dissolved in distilled water (3 mL) at -50°C under a nitrogen atmosphere. mmol) and tetramethylammonium hydroxide (10 wt% aqueous solution, 0.12 mL) were sequentially added and stirred at -5°C for 2 hours. After adjusting the pH to about 4-5 with acetic acid, it was purified by HPLC to obtain compound 74 (123 mg, 42%). EI-MS m/z: 1/2[M+H] ⁺ 2217.15, 1/3[M+H] ⁺ 1478.62, 1/4[M+H] ⁺ 1109.27.

Preparation of Compound 75

Compound 74 (123 mg, 0.028 mmol) was dissolved in dichloromethane (2 mL), and then trifluoroacetic acid (0.1 mL) was added at 0°C under a nitrogen atmosphere. The reaction solution was stirred at 0° C. for 2 hours, concentrated, and purified by HPLC to obtain compound 75 (116 mg, 90%). EI-MS m/z: 1/2[M+H] ⁺ 2167.27, 1/3[M+H] ⁺ 1445.29, 1/4[M+H] ⁺ 1084.27.

<Example 18> Preparation of compound 78

Preparation of Compound 76

Compound 40 (218 mg, 0.12 mmol) and compound 66 (130 mg, 0.056 mmol) were dissolved in N,N -dimethylformamide (4 mL) and pyridine (1 mL), and then incubated at 0°C under nitrogen atmosphere. Hydroxy-7-azabenzotriazole (3.8 mg, 0.03 mmol) and N,N' -diisopropyl-ethylamine (0.05 mL, 0.39 mmol) were sequentially added and stirred at room temperature for 20 hours. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (2 After filtration, concentration under reduced pressure and purification by column chromatography, compound 76 (272 mg, 88%) was obtained. EI-MS m/z: 1/2[M+H] ⁺ 2733.04, 1/3[M+H] ⁺ 1822.44, 1/4[M+H] ⁺ 1367.08.

Preparation of Compound 77

Compound 76 (272 mg, 0.05 mmol) was dissolved in 2-propanol (1 mL) and tetrahydrofuran (1 mL), and then lithium hydroxide (16.7 mg, dissolved in distilled water (1.5 mL) at -50°C under a nitrogen atmosphere. 0.40 mmol) and tetramethylammonium hydroxide (10 wt% aqueous solution, 0.13 mL) were sequentially added and stirred at -5°C for 3 hours. After adjusting the pH to about 4-5 with acetic acid, it was purified by HPLC to obtain compound 77 (105 mg, 43%). EI-MS m/z: 1/3[M+H] ⁺ 1615.57, 1/4[M+H] ⁺ 1211.97.

Preparation of Compound 78

Compound 77 (105 mg, 0.022 mmol) was dissolved in dichloromethane (3 mL), and then trifluoroacetic acid (0.1 mL) was added at 0°C under a nitrogen atmosphere. The reaction solution was stirred at 0° C. for 2 hours, concentrated, and then purified by HPLC to obtain compound 78 (49 mg, 46%). EI-MS m/z: 1/2[M+H] ⁺ 2372.25, 1/3[M+H] ⁺ 1582.21, 1/4[M+H] ⁺ 1186.89.

<Example 19> Preparation of compound 81

Preparation of Compound 80

Compound 79 (Compound 79 was prepared by the method described in Korean Patent Application No. 10-2023-0032106), 1.5 g, 0.67 mmol) was dissolved in dichloromethane (24 mL) and then dissolved in pyridine (0.22 mL, 2.68 mmol). Bis(pentafluorophenyl)carbonate (0.79 g, 2.01 mmol) was added at room temperature under a nitrogen atmosphere and stirred for 16 hours. The reaction solution was diluted with ethyl acetate (80 mL), washed with saturated aqueous sodium bicarbonate solution (50 mL x 2) and brine (50 mL), and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, compound 80 (2.06 g) was used in the next reaction without further purification. EI-MS m/z: [M+H] ⁺ 2670.3, [½M+H] ⁺ 1335.7, [⅓M+H] ⁺ 890.8.

Preparation of Compound 81

Compound 81 was synthesized from compound 16 and compound 80 in the same manner as compound 69. EI-MS m/z: 1/2[M+H] ⁺ 2199.20, 1/3[M+H] ⁺ 1466.60, 1/4[M+H] ⁺ 1100.23.

<Example 20> Preparation of compound 82

Preparation of Compound 82

Compound 82 was synthesized from compound 31 and compound 80 in the same manner as compound 75. EI-MS m/z: 1/2[M+H] ⁺ 2306.26, 1/3[M+H] ⁺ 1538.12, 1/4[M+H] ⁺ 1153.86.

<Example 21> Preparation of compound 84

Preparation of Compound 83

Compound 8 (304 mg, 0.19 mmol) was dissolved in N,N -dimethylformamide (5 mL), and then piperidine (0.03 mL, 0.28 mmol) was added at 0°C under a nitrogen atmosphere. After stirring at 0°C for 2 hours, the reaction solution was diluted with ethyl acetate (100 mL), washed with distilled water (50 mL x2), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 83 (97 mg, 36%) was obtained. EI-MS m/z: [M+H] ⁺ 1400.63.

Preparation of Compound 84

Compound 84 was synthesized from compound 83 and compound 80 in the same manner as compound 69. Compound 84 (12 mg, 46%). EI-MS m/z: 1/2[M+H] ⁺ 2164.98.

<Example 22> Preparation of compound 88

Preparation of Compound 86

Compound 85 (754 mg, 1.12 mmol, Compound 85 was prepared by the method described in published patent WO2020222573A1) and Compound 64 (1.3 g, 2.70 mmol) were dissolved in N,N -dimethylformamide (15 mL) and then dissolved in 0 N,N,N',N' -tetramethyl- O- (1 H -benzotriazol-1-yl)uronium hexafluorophosphate (HBTU, 1.4 g, 3.72 mmol) and N under nitrogen atmosphere at ℃. ,N' -Diisopropyl-ethylamine (1 mL, 5.63 mmol) was sequentially added and stirred at room temperature for 22 hours. The reaction solution was diluted with ethyl acetate (200 mL), washed with saturated aqueous ammonium chloride solution (100 mL), saturated aqueous sodium bicarbonate solution (100 mL), and distilled water (100 mL), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 65 (347 mg, 19%) was obtained. EI-MS m/z: 1602.55.

Preparation of Compound 87

Compound 86 (347 mg, 2.16 mmol) was dissolved in dichloromethane (20 mL), then pyridine (0.08 mL, 0.93 mmol) and bis(4-nitrophenyl)carbonate (188 mg, 0.93 mmol) were added at room temperature. , was added under a nitrogen atmosphere and stirred for 16 hours. The mixture was concentrated under reduced pressure and subjected to column chromatography to obtain compound 87 (330 mg, 79%). EI-MS m/z: 1932.44

Preparation of Compound 88

Compound 88 was synthesized from compound 16 and compound 87 in the same manner as compound 69. EI-MS m/z: 1/2[M+H] ⁺ 1929.64, 1/3[M+H] ⁺ 1286.96.

<Example 23> Preparation of compound 89

Preparation of Compound 89

Compound 89 was synthesized from compound 83 and compound 87 in the same manner as compound 69. EI-MS m/z: 1/2[M+H] ⁺ 1896.88, 1/3[M+H] ⁺ 1264.82, 1/4[M+H] ⁺ 948.95.

<Example 24> Preparation of Compound 98

Preparation of Compound 90

Dissolve 1,3-benzenedimethanol (10 g, 72.38 mmol) in N,N -dimethylformamide (40 mL), then imidazole (24.638 g, 361.9 mmol) and t -butyldimethylsilyl chloride at 0°C. (27.273 g, 180.95 mmol) was sequentially added and stirred at room temperature for 16 hours. The reaction solution was diluted with ethyl acetate (100 mL), washed sequentially with saturated aqueous ammonium chloride solution (70 mL) and distilled water (70 mL), and then dried over anhydrous sodium sulfate. After filtration and concentration, the product was purified by column chromatography to obtain compound 90 (21.49 g, 81%). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.35-7.15 (m, 4H), 4.74 (s, 4H), 0.95 (s, 18 H), 0.10 (s, 12H).

Preparation of Compound 91

Acetic anhydride (100 mL) was added at 0°C, nitric acid (12.1 mL, 60%) was slowly added, and stirred at room temperature for 20 minutes. Compound 90 (21.49 g, 58.605 mmol) was mixed with acetic anhydride (45 mL) and slowly added dropwise at -20°C. After stirring at 0°C for 30 minutes, diluted with ethyl acetate (150 mL), washed with distilled water (80 mL), saturated aqueous sodium bicarbonate solution (3 did. After filtration and concentration, the product was purified by column chromatography to obtain compound 91 (13.05 g, 54%). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.09 (d, J = 8.4 Hz, 1H), s, 7.88 (s, 1H), 7.36 (d, J = 6.0 Hz, 1H), 5.11 (s, 2H) ), 4.82 (s, 2H), 0.96 (d, J = 7.2 Hz, 18H), 0.14 (d, J = 8.8 Hz, 12H).

Preparation of Compound 92

Compound 91 (4 g, 9.716 mmol) was dissolved in methanol/tetrahydrofuran (40 mL/40 mL), then palladium/charcoal (5 wt.%, 0.2 g) and ammonium formate (0.98 g, 15.55 mmol) were added. did. The reaction solution was stirred at room temperature under a hydrogen atmosphere for 2 hours, then filtered through Celite, and the filtrate was concentrated and purified by column chromatography to obtain compound 92 (3.58 g, 96%). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.02-6.99 (m, 2H), 6.63 (d, J = 8.0 Hz, 1H), 4.70 (s, 2H), 4.61 (s, 2H), 0.90 (d) , J = 9.6 Hz, 18H), 0.07 (s, 12H). EI-MS m/z: [M+H]+ 382.42.

Preparation of Compound 94

Compound 93 (892 mg, 0.93 mmol, Compound 93 was prepared by the method described in published patent WO2017089895A1) and Compound 64 (539 mg, 1.11 mmol) were dissolved in N,N -dimethylformamide (5 mL) and then dissolved in 0 ℃, under nitrogen atmosphere 1-[bis(dimethylamino)methylene]-1 H -1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 458 mg, 1.21 mmol) and N,N' -diisopropylethylamine (0.48 mL, 2.78 mmol) were sequentially added and stirred at room temperature for 22 hours. The reaction solution was concentrated under reduced pressure and diluted with dichloromethane (100 mL) and methanol (10 mL), followed by saturated aqueous ammonium chloride solution (50 mL), saturated aqueous sodium bicarbonate solution (50 mL), and distilled water (50 mL). It was wiped and dried with anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 94 (885 mg, 66%) was obtained. EI-MS m/z: [M+H] ⁺ 1429.31, 1/2[M+H] ⁺ 715.05.

Preparation of Compound 95

Compound 94 (885 mg, 0.62 mmol) was dissolved in dichloromethane (10 mL), then mixed with bis(pentafluorophenyl)carbonate (293 mg, 0.74 mmol) and pyridine (0.1 ml, 1.24 mmol) at 0°C under nitrogen atmosphere. mmol) was added. After stirring at room temperature for 17 hours, the reaction solution was diluted with dichloromethane (100 mL) and added with saturated aqueous sodium bicarbonate solution (50 mL), 0.5 N aqueous hydrochloric acid solution (50 mL) and 5% aqueous sodium hydroxide solution (50 mL). ) and then dried with anhydrous sodium sulfate. After filtration and concentration under reduced pressure, compound 95 (989 mg, 97%) was obtained without further purification. EI-MS m/z: [M+H] ⁺ 1639.08, 1/2[M+H] ⁺ 819.96.

Preparation of Compound 96

Compound 95 (188 mg, 0.11 mol) and compound 92 (57 mg, 0.15 mmol) were dissolved in N,N -dimethylformamide (2 mL) and then 1-hydroxy-7-azabenzoate at 0° C. under nitrogen atmosphere. Triazole (8 mg, 0.06 mmol) and N,N' -diisopropylethylamine (0.06 mL, 0.34 mmol) were sequentially added and stirred at room temperature for 17 hours. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (2 After filtration, concentration under reduced pressure and purification by column chromatography, compound 96 (126 mg, 60%) was obtained. EI-MS m/z: [M+H] ⁺ 1837.22., 1/2[M+H] ⁺ 919.03.

Preparation of Compound 97

Compound 96 (126 mg, 0.07 mol) was dissolved in methanol (4 mL), then (1R)-(-)-10-camphorsulfonic acid (6.4 mg, 0.027 mmol) was added at 0°C under nitrogen atmosphere and incubated at 0°C for 2 hours. It was stirred for a while. The reaction solution was neutralized with triethylamine, concentrated under reduced pressure, and purified by column chromatography to obtain compound 97 (84 mg, 76%). EI-MS m/z: [M+H] ⁺ 1608.82, [M+Na] ⁺ 1629.76, 1/2[M+H] ⁺ 804.68.

Preparation of Compound 98

After dissolving Compound 97 (84 mg, 0.05 mmol) in dichloromethane (5 mL), bis(pentafluorophenyl)carbonate (62 mg, 0.16 mmol) and pyridine (0.017 ml, 0.21 ml) were dissolved in dichloromethane (5 mL) at 0°C. mmol) was added. The reaction solution was stirred at room temperature for 17 hours, then diluted with dichloromethane (100 mL), washed with saturated aqueous sodium bicarbonate solution (50 mL), and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, compound 98 (117 mg, crude) was obtained without further purification. EI-MS m/z: [M+H] ⁺ 2027.45, [M+Na] ⁺ 2049.73, 1/2[M+H] ⁺ 1014.77.

<Example 25> Preparation of compound 101

Preparation of Compound 99

Compound 16 (163 mg, 0.05 mmol) and compound 98 (105 mg, 0.11 mmol) were dissolved in N,N -dimethylformamide (4 mL) and pyridine (1 mL), and then incubated at 0°C under nitrogen atmosphere. Hydroxy-7-azabenzotriazole (3 mg, 0.03 mmol) and N,N' -diisopropyl-ethylamine (0.05 mL, 0.26 mmol) were sequentially added and stirred at room temperature for 48 hours. The reaction solution was diluted with ethyl acetate (100 mL), washed with saturated aqueous sodium bicarbonate solution (50 mL) and distilled water (2 After filtration, concentration under reduced pressure and purification by column chromatography, compound 99 (60 mg, 23%) was obtained. EI-MS m/z: 1/2[M+H] ⁺ 2263.24, 1/3[M+H] ⁺ 1509.29, 1/4[M+H] ⁺ 1132.19.

Preparation of Compound 100

Compound 99 (60 mg, 0.013 mmol) was dissolved in 2-propanol (1 mL) and tetrahydrofuran (1 mL), and then lithium hydroxide (5 mg, 0.13 mg) dissolved in distilled water (1.5 mL) at -50°C under a nitrogen atmosphere. mmol) and tetramethylammonium hydroxide (10 wt% aqueous solution, 0.03 mL) were sequentially added and stirred at -5°C for 2 hours. After adjusting the pH to about 4-5 with acetic acid, it was purified by HPLC to obtain compound 100 (19 mg, 19%). EI-MS m/z: 1/2[M+H] ⁺ 2053.20, 1/3[M+H] ⁺ 1369.5, 1/4[M+H] ⁺ 1027.02.

Preparation of Compound 101

Compound 100 (19 mg, 0.005 mmol) was dissolved in dichloromethane (1 mL), and then trifluoroacetic acid (0.025 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 101 (14 mg, 71%). EI-MS m/z: 1/2[M+H] ⁺ 2002.60, 1/3[M+H] ⁺ 1335.78, 1/4[M+H] ⁺ 1002.11.

<Example 26> Preparation of compound 109

Preparation of Compound 102

4-Hydroxyisophthalic acid (2 g, 10.98 mmol) was dissolved in methanol (30 mL), then thionyl chloride (2.4 mL, 32.94 mmol) was added at 0°C under a nitrogen atmosphere, and the mixture was stirred at room temperature for 48 hours. The reaction solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 102 (2.1 g, 91%). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 11.20 (s, 1H), 8.57 (d, J = 2.6 Hz, 1H), 8.14 (t, J = 9.3 Hz, 1H), 7.02 (d, J = 8.9 Hz, 1H), 3.99 (d, J = 1.9 Hz, 3H), 3.91 (d, J = 1.9 Hz, 3H).

Preparation of Compound 103

Compound 102 (2.1 g, 9.99 mmol) was dissolved in dichloromethane (30 mL), then triisopropylsilyl chloride (2.7 mL, 12.99 mmol) and imidazole (1.4 g, 19.98 mmol) were dissolved in nitrogen atmosphere at 0°C. Added. After stirring and concentrating the reaction solution at room temperature for 20 hours, the reaction solution was diluted with dichloromethane (100 mL), washed with saturated aqueous ammonium chloride solution (50 mL) and distilled water (50 mL), and dried over anhydrous sodium sulfate. did. It was filtered, concentrated under reduced pressure, and purified by column chromatography to obtain compound 103 (2.8 g, 75%). ¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.43 (d, J = 2.4 Hz, 1H), 8.02 (dd, J = 8.6, 2.4 Hz, 1H), 6.89 (d, J = 8.7 Hz, 1H), 3.89 (d, J = 6.0 Hz, 6H), 1.39 - 1.27 (m, 3H), 1.13 - 1.10 (m, 18H)

Preparation of Compound 104

Compound 103 (2.8 g, 7.56 mmol) was dissolved in tetrahydrofuran (100 mL), and then lithium aluminum hydride (LAH, 1.0 M in THF, 15 mL, 15.1 mmol) was slowly added dropwise under a nitrogen atmosphere at 0°C. The reaction solution was stirred at room temperature for 20 hours, then distilled water (2 mL) was added at 0°C and concentrated. The concentrated solution was diluted with dichloromethane (300 mL), washed with 1 N aqueous hydrochloric acid (100 mL) and distilled water (100 mL), and dried over anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 104 (1.3 g, 55%) was obtained. ¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.32 (s, 1H), 7.16 (d, J = 8.6 Hz, 1H), 6.81 (d, J = 8.2 Hz, 1H), 4.72 (d, J = 6.2 Hz, 2H), 4.62 (d, J = 5.6 Hz, 2H), 2.06 (t, J = 6.5 Hz, 1H), 1.39 - 1.27 (m, 3H), 1.12 (d, J = 7.5 Hz, 18H).

Preparation of Compound 105

Compound 104 (1.3 g, 4.19 mmol) was dissolved in N,N -dimethylformamide (10 mL), and then t -butyldimethylsilyl chloride (1.4 g, 9.21 mmol) and imidazole (855) were dissolved in nitrogen atmosphere at 0°C. mg, 12.56 mmol) was added. The reaction solution was stirred at room temperature for 20 hours, diluted with ethyl acetate (100 mL), washed with saturated aqueous ammonium chloride solution (50 mL) and distilled water (2 After filtration and concentration under reduced pressure, compound 105 (2.0 g, crude) was obtained without further purification. EI-MS m/z: [M+H] ⁺ 539.87.

Preparation of Compound 106

Compound 105 (2.0 g, crude) was dissolved in N,N -dimethylformamide (10 mL) and distilled water (1 mL), and then sodium acetate (446 mg, 5.44 mmol) was added at 0°C under a nitrogen atmosphere. The reaction solution was stirred at 60°C for 20 hours, diluted with ethyl acetate (100 mL), washed with saturated aqueous ammonium chloride solution (50 mL) and distilled water (2 After filtration and concentration under reduced pressure, the concentrated filtrate was dissolved in N,N -dimethylformamide (10 mL), and t -butyldimethylsilyl chloride (419 mg, 2.78 mmol) and imidazole (189) were added under nitrogen atmosphere at 0°C. mg, 2.78 mmol) was added. The reaction solution was stirred at room temperature for 1 hour, diluted with ethyl acetate (100 mL), washed with saturated aqueous ammonium chloride solution (50 mL) and distilled water (2 After filtration, concentration under reduced pressure and purification by column chromatography, compound 106 (818 mg, 77%) was obtained. ^1H -NMR (400 MHz, CDCl ₃ ) δ 7.97 (d, J = 2.2 Hz, 1H), 7.12 (d, J = 8.3 Hz, 1H), 6.91 (s, 1H), 6.83 (d, J = 8.4 Hz, 1H), 4.90 (s, 2H), 4.63 (s, 2H), 0.93 (s, 18H), 0.14 (d, J = 2.2 Hz, 6H), 0.08 (d, J = 2.2 Hz, 6H).

Preparation of Compound 107

Compound 106 (818 mg, 2.14 mmol) was dissolved in ethyl acetate (10 mL) and then added to bis(4-nitrophenyl)carbonate (975 mg, 3.21 mmol) and 4-(dimethylamino)pyridine at 0°C under nitrogen atmosphere. (26 mg, 0.21 mmol) and triethylamine (0.9 mL, 6.41 mmol) were added. The reaction solution was stirred at room temperature for 3 hours, diluted with ethyl acetate (100 mL), washed with 0.1 N aqueous hydrochloric acid (50 mL) and distilled water (2 After filtration, concentration under reduced pressure and purification by column chromatography, compound 107 (1.1 g, 94%) was obtained. ¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.33 (d, J = 8.9 Hz, 2H), 7.54 - 7.47 (m, 3H), 7.20 (d, J = 8.3 Hz, 1H), 4.78 (d, J = 10.7 Hz, 4H), 0.95 (dt, J = 4.7, 2.4 Hz, 18H), 0.17 - 0.07 (m, 12H).

Preparation of Compound 108

Compound 107 (1.1 g, 2.00 mmol) was dissolved in N,N -dimethylformamide (5 mL), then triethylamine (0.28 mL, 2.00 mmol) and t -butyl methyl (2-( Methylamino)ethyl)carbamate (453 mg, 2.41 mmol) was added. The reaction solution was stirred at room temperature for 17 hours, diluted with ethyl acetate (100 mL), washed with 5% aqueous sodium hydroxide solution (50 mL) and distilled water (2 After filtration, concentration under reduced pressure and purification by column chromatography, compound 108 (1.1 g, 89%) was obtained. ¹ H-NMR (400 MHz, CDCl ₃ ) δ 7.47 (s, 1H), 7.20 (d, J = 8.3 Hz, 1H), 7.02 (t, J = 7.2 Hz, 1H), 4.71 (d, J = 11.8) Hz, 4H), 3.47 (s, 3H), 3.13 (s, 2H), 3.03 (s, 2H), 2.92 (d, J = 8.8 Hz, 3H), 1.46 (d, J = 9.5 Hz, 9H), 0.93 (s, 16H), 0.09 (d, J = 4.4 Hz, 12H).

Preparation of Compound 109

Compound 108 (240 mg, 0.40 mmol) was dissolved in dichloromethane (4 mL) and methanol (2 mL) and then dissolved in hydrochloric acid (4 M 1,4-dioxane solution, 1.2 mL, 4.8 mmol) under nitrogen atmosphere at 0°C. ) was added and stirred for 5 hours. After concentrating the reaction solution, compound 109 (125 mg, crude) was obtained without further purification. EI-MS m/z: [M+H] ⁺ 269.26.

<Example 27> Preparation of compound 111

Preparation of Compound 110

Compound 95 (330 mg, 0.20 mol) and compound 109 (122 mg, 0.40 mmol) were dissolved in N,N -dimethylformamide (3 mL), and then dissolved in N,N' -diisopropyl at 0°C under nitrogen atmosphere. Ethylamine (0.18 mL, 1.01 mmol) was added and stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure and diluted with dichloromethane (100 mL) and methanol (10 mL), followed by saturated aqueous ammonium chloride solution (50 mL), saturated aqueous sodium bicarbonate solution (50 mL), and distilled water (50 mL). It was wiped and dried with anhydrous sodium sulfate. After filtration, concentration under reduced pressure and purification by column chromatography, compound 110 (225 mg, 66%) was obtained. EI-MS m/z: [M+H] ⁺ 1722.95, 1/2[M+H] ⁺ 862.04.

Preparation of Compound 111

Compound 111 was synthesized in the same manner as compound 98. EI-MS m/z: [M+H] ⁺ 2142.97, 1/2[M+H] ⁺ 1072.07.

<Example 28> Preparation of compound 112

Preparation of Compound 112

Compound 112 was synthesized from compound 16 and compound 111 in the same manner as compound 101. EI-MS m/z: 1/2[M+H] ⁺ 2059.34, 1/3[M+H] ⁺ 1373.34, 1/4[M+H] ⁺ 1030.28.

<Example 29> Preparation of Comparative Compound 113

Comparative compound 113 was prepared by the method described in published patent WO2020180121A1.

<Example 30> Preparation of ADC

The ADC was manufactured through the following two steps, and the commonly used LCB14-0606 was manufactured by the method described in Korean Application No. 10-2013-7032628. The above document is incorporated herein by reference in its entirety. The structural formula of LCB14-0606 is as follows:

Step 1: Preparation of prenylated antibody

<Manufacture of prenylated antibody using LCB14-0606>

A prenylation reaction mixture of the DLK1 antibody 18A5-CaaX described in published patent WO2020180121A1 was prepared and reacted at 30°C for 16 hours. The reaction mixture contained a total of 1000 mg of 48 μM antibody, 400 nM FTase (Genscript), and 288 μM LCB14-0606 in buffer solution (50 mM Tris-HCl (pH 7.4), 5 mM MgCl ₂ , 10 μM ZnCl ₂ , 0.5 μM ZnCl 2 ). It consisted of (mM DTT). After completion of the reaction, the prenylated antibody was decontaminated through ultrafiltration (Vivaspin 20, Satorius) using a PBS buffer solution. As a result, a total of 906 mg of prenylated antibody was prepared.

Step 2: Drug-conjugation method

<Conjugation by oxime bond formation of ADC1>

The oxime bond formation reaction between the prenylated antibody and the linker-drug was performed using a total of 50.0 mg of 100 μM Na-acetate buffer pH 5.2, 10% DMSO, 48 μM prenylated antibody and 6 equivalents of the linker-drug (in house, Example 29). Compound No. 113) was mixed and stirred at 500 to 600 rpm at 30°C for 24 hours. After the reaction, excess low-molecular-weight compounds were removed through ultrafiltration (Vivaspin 20, Satorius) using a PBS buffer solution, and the protein fraction was collected and concentrated. As a result, a total of 34.4 mg of ADC1 was obtained.

<Conjugation by oxime bond formation of ADC2>

The oxime bond formation reaction between the prenylated antibody and the linker-drug was performed using a total of 50.0 mg of 100 μM Na-acetate buffer pH 5.2, 10% DMSO, 48 μM prenylated antibody and 6 equivalents of the linker-drug (in house, Example 15). Compound No. 69) was mixed and stirred at 500 to 600 rpm at 30°C for 24 hours. After the reaction, excess low-molecular-weight compounds were removed through ultrafiltration (Vivaspin 20, Satorius) using a PBS buffer solution, and the protein fraction was collected and concentrated. As a result, a total of 39.9 mg of ADC2 was obtained.

<Conjugation by oxime bond formation of ADC3>

The oxime bond formation reaction mixture between the prenylated antibody and the linker-drug was 100 μM Na-acetate buffer pH 5.2, 10% DMSO, 48 μM a total of 20.1 mg of prenylated antibody and 6 equivalents of the linker-drug (in house, Example 10) Compound No. 45) was mixed and stirred at 500 to 600 rpm at 30°C for 24 hours. After the reaction, excess low-molecular-weight compounds were removed through ultrafiltration (Vivaspin 20, Satorius) using a PBS buffer solution, and the protein fraction was collected and concentrated. As a result, a total of 11.2 mg of ADC3 was obtained.

In order to confirm the characteristics of the three types of ADCs prepared above, they were analyzed using a mobile phase prepared from ammonium sulfate, acetonitrile, and potassium phosphate in HPLC (high performance liquid chromatography) equipment using a HIC (Hydrophobic interaction chromatography) column, and the results were is shown in Figure 3.

As shown in Figure 3, ADC1, ADC2, and ADC3 all have different hydrophobicities, and the hydrophobicity was confirmed to be greater in the order ADC2 > ADC1 > ADC3, and the purity in terms of DAR (drug-antibody ratio) was confirmed to be more than 96%. did.

The ADC manufactured through the above two steps is summarized in Table 1 below.

ADC Antibody Linker-toxin ADC1
18A5-CaaX Compound 113 ADC2 Compound 69 ADC3 Compound 45

<Experimental Example 1> In vitro cytotoxicity evaluation of new auristatin prodrug

The cell proliferation inhibitory ability of the compounds listed in Table 2 below was measured in cancer cell lines and hematopoietic stem cells. MIA-PaCa2, a human pancreatic cancer cell, was used as a cancer cell line, and commercially available CD34-positive hematopoietic stem cells (Stem cells, Cat No. 70002.2) were used as hematopoietic stem cells. In a 96-well plate, 3,000 MIA-PaCa2 and 2,000 CD34 positive hematopoietic stem cells were seeded per well. After 24 hours, cancer cell lines were treated with the compounds at a concentration of 0.256 pM~100 nM (5-fold serial dilution), and hematopoietic stem cells were treated at a concentration of 12.8 pM~5 μM (5-fold serial dilution). After 144 hours of culture, the number of viable cancer cells was measured using SRB (Sulforhodamine B) assay, and hematopoietic stem cells were measured using CTG (Cell titer glo, Promega) assay.

Test samples CC ₅₀ (nM) MIA-PaCa2 hematopoietic stem cells MMAE 2.70 1.01 Compound 17 >100 176.6 Compound 22 >100 38.11 Compound 31 >100 189.7

CC ₅₀ in cancer cell lines was measured at MMAE of 2.70 nM, and the remaining three compounds could not be measured because they all showed cell viability of more than 95% even at the highest treatment concentration. When CC ₅₀ was measured in CD34-positive hematopoietic stem cells, which are normal cells, MMAE was measured at 1.01 nM, Compound 17 at 176.6 nM, Compound 22 at 38.11 nM, and Compound 31 at 189.7 nM. MMAE showed the strongest cytotoxicity, and Compound 17 and Compound 31 were measured at 189.7 nM. Compound 31 was more than 100 times less toxic, confirming its superior safety as an MMAE prodrug.

<Experimental Example 2> In vitro cytotoxicity evaluation of ADC

The cell proliferation inhibitory ability of the ADCs listed in Table 3 below was measured in cancer cell lines and hematopoietic stem cells. MIA-PaCa2_DLK1+, a human pancreatic cancer cell that overexpresses DLK1, was used as a cancer cell line, HaCaT was used as a normal skin cell, Fa2N4 was used as a normal liver cell, and hPBMC was used as a peripheral blood mononuclear cell. In a 96-well plate, 3,000 MIA-PaCa2_DLK1+, 1000 HaCaT, 15,000 Fa2N4, and 60,000 hPBMC were seeded per well. After 24 hours, the compounds were treated at a concentration of 0.256 pM ~ 100 nM (5-fold serial dilution) in cancer cell lines, 2.56 pM ~ 1000 nM (5-fold serial dilution) in HaCaT and Fa2N4, and 12.8 pM ~ 5-fold serial dilution in hPBMC. Treated at μM (5-fold serial dilution) concentration. After 144 hours of culture, the number of viable cancer cells, HaCaT, and Fa2N4 were measured using SRB (Sulforhodamine B) assay, and hPBMC was measured using CTG (Cell titer glo, Promega) assay.

Test samples CC ₅₀ (nM) MIA-PaCa2_DLK1+
(DLK1+ pancreatic cancer cells) HaCaT
(Normal skin cells) Fa2N4
(normal liver cells) hPBMCs
(Peripheral blood mononuclear cells) ADC1 0.18 43.9 >1000 ^* (64.5%) 57.8 ADC2 0.24 258.7 >1000 ^* (99.3%) 2568.0 ADC3 0.85 501.8 >1000 ^* (99.1%) 1746.0

^* : Cell viability at highest concentration

The CC50 in cancer cell lines was measured to be 0.18, 0.24, and 0.85 nM for ADC1, ADC2, and ADC3, respectively. The CC50 in normal skin cells, HaCaT, for ADC1, ADC2, and ADC3 were measured to be 43.9, 258.7, and 501.8 nM, respectively. CC50 in normal liver cells Fa2N4 could not be measured because ADC1, ADC2, and ADC3 all showed cell viability of more than 50% at the highest treatment concentration, but the viability was measured at 64.5, 99.3, and 99.1%, respectively. The CC50 in hPBMC, a peripheral blood mononuclear cell, was measured to be 57.8, 2568.0, and 1746.0 nM for ADC1, ADC2, and ADC3, respectively. ADC2 and ADC3 introduced with MMAE prodrug were confirmed to have superior safety in normal cells compared to ADC1 introduced with MMAE.

The drug therapeutic indices for HaCaT and hPBMC are listed in Table 4 below. The drug therapeutic index was calculated by dividing the CC50 in normal cells by the CC50 in cancer cells.

Test samples TI (drug therapeutic index, times) CC50 HaCaT /
CC50 MIA-PaCa2_DLK1+ CC50hPBMC/
CC50 MIA-PaCa2_DLK1+ ADC1 247 325 ADC2 1087 10786 ADC3 590 2054

The pharmacotherapy index of ADC1 was calculated as 247 and 325, respectively, the pharmacotherapy index of ADC2 was calculated as 1087 and 10786, respectively, and the pharmacotherapy index of ADC3 was calculated as 590 and 2054, respectively. ADC2 and ADC3, which introduced MMAE prodrugs, showed a higher drug therapeutic index compared to ADC1, which introduced MMAE.

Claims (14)

A compound having the structure of general formula I, or an isomer thereof, or a pharmaceutically acceptable salt or solvate thereof:
[General Formula I]
MMAE-SIG2-SIG1-E
In the general formula I,
MMAE is ego,
SIG2 is *-C(=O)-R ¹ -N(R ² )-**, *-C(=O)OR ¹ -N(R ² )-**, *-C(=O)N( R ² )-R ¹ -N(R ² )-** and *-S(=O) ₂ -R ¹ -N(R ² )-**,
SIG1 is selected from the group consisting of **-C(=O)OR ³ -O-*** and **-C(=O)OR ³ -N(R ⁴ )-***,
E is an organic group derived from the substrate of a cancer cell-specific enzyme,
R ¹ and R ³ are independently in each case a divalent organic group,
R ² and R ⁴ are independently hydrogen or C _1-8 alkyl in each case,
The N at the ** terminal of SIG2 can form an N-containing ring structure with the adjacent carbon atom in R ¹ and R ² ,
The N at the *** terminal of SIG1 can form an N-containing ring structure with the adjacent carbon atom in R ³ and R ⁴ ,
MMAE and SIG2 are connected at the * end;
SIG2 and SIG1 are connected at the ** end;
E is connected to the *** end of SIG1,
The bond between the *** end of SIG1 and E can be broken by the cancer cell-specific enzyme.
According to paragraph 1,
R ¹ and R ³ at each occurrence are independently substituted or unsubstituted alkylene, substituted or unsubstituted arylene, substituted or unsubstituted alkarylene, or substituted or unsubstituted aralkylene. ,
A compound or an isomer thereof, or a pharmaceutically acceptable salt or solvate thereof.
According to paragraph 1,
SIG2 is *-C(=O)-R ¹ -N(R ² )-** or *-C(=O)N(R ² )-R ¹ -N(R ² )-**,
where R ¹ and R ² are as defined in clause 1,
A compound or an isomer thereof, or a pharmaceutically acceptable salt or solvate thereof.
According to paragraph 1,
SIG2 is selected from the group consisting of:
Compound or isomer thereof, or pharmaceutically acceptable salt or solvate thereof:
, , , , and ,
Here, the meanings of * and ** are as described in paragraph 1.
According to paragraph 1,
SIG1 is selected from the group consisting of:
Compound or isomer thereof, or pharmaceutically acceptable salt or solvate thereof:
, and ,
Here, the meanings of ** and *** are as described in paragraph 1.
According to paragraph 1,
The cancer cell-specific enzyme is selected from the group consisting of β-glucuronidase, β-galactosidase, and cathepsin B,
A compound or an isomer thereof, or a pharmaceutically acceptable salt or solvate thereof.
According to paragraph 1,
The substrate of the cancer cell-specific enzyme is glucuronic acid, galactoside, or a derivative thereof, an amino acid or a derivative thereof, or a polypeptide or a derivative thereof,
A compound or an isomer thereof, or a pharmaceutically acceptable salt or solvate thereof.
In clause 7,
Amino acids are alanine, β-alanine, γ-aminobutyric acid, arginine, asparagine, aspartic acid, γ-carboxyglutamic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, and norvaline. , ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine and derivatives thereof,
Polypeptides include alanine, β-alanine, γ-aminobutyric acid, arginine, asparagine, aspartic acid, γ-carboxyglutamic acid, citrulline, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, norleucine, norleucine. Formed from two or more selected from the group consisting of valine, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine and derivatives thereof,
A compound or an isomer thereof, or a pharmaceutically acceptable salt or solvate thereof.
According to paragraph 1,
E is selected from the group consisting of:
Compound or isomer thereof, or pharmaceutically acceptable salt or solvate thereof:
, , and a compound having the structure of Formula I below,
[Formula Ⅰ]

From here,
R ⁵ is -OH, or C _1-4 alkoxy,
m is an integer from 1 to 12,
The meaning of *** is as described above.
According to paragraph 1,
Any one compound or isomer thereof, or a pharmaceutically acceptable salt or solvate thereof, selected from the group consisting of:
,
,
,
,
.
,
, and
.
A ligand-drug conjugate comprising the compound of any one of claims 1 to 10 and a ligand, or a pharmaceutically acceptable salt or solvate thereof.
Containing the compound of claim 1 or an isomer thereof, or the ligand-drug conjugate of claim 11 or a pharmaceutically acceptable salt or solvate thereof as an active ingredient,
Pharmaceutical composition for preventing or treating proliferative diseases.
According to clause 12,
A pharmaceutical composition for preventing or treating proliferative diseases, further comprising a pharmaceutically acceptable excipient.
According to clause 12,
The above proliferative diseases include carcinoma, lymphoma, leukemia, blastoma, sarcoma, liposarcoma, neuroendocrinoma, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, squamous cell carcinoma, squamous cell carcinoma, lung cancer, small cell lung cancer, and non-small cell lung cancer. , lung adenocarcinoma, lung squamous carcinoma, peritonoma, hepatocellular carcinoma, gastric carcinoma, gastrointestinal tumor, pancreatic cancer, brain cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrium. or any one selected from the group consisting of uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophageal varices cancer, biliary tract cancer, colon cancer, and head and neck cancer. Pharmaceutical composition for preventing or treating phosphorus and proliferative diseases.