Tetrafunctional chemical probe and method for identifying target membrane proteins from living cells or tissues using said probe

Abstract

This tetra-functional compound is formed by linking, via a spacer as needed, a ligand binding site (A) or a ligand, a reactive site (D), a potential cleavage site (E), and a biotin site (B), wherein: the ligand binding site (A) is an active functional group such as an amine reactive group or a reactive functional group such as -COOH; the reactive site (D) is a group having a structure of a compound such as 2-aryl-5-carboxy-tetrazole; the potential cleavage site (E) is a group having a structure of a compound such as 1-(4 4-dimethyl-2 6-dioxocyclohex-1-ylidene)ethyl; and the spacer is a group obtained by substitution with a cross-linking group such as a linear or branched alkylene group having 1 or more carbon atoms.

Description

[Technical Field]

[0001] This paper relates to a probe that can directly and unbiasedly and comprehensively identify target proteins of specific ligands from living cells and living tissues that reflect phenotypes using chemoproteomics, and an identification method using the probe.In particular, the present invention relates to a probe that can detect various target membrane proteins, including ion channels, GPCRs, and multi-transmembrane and multimeric receptors, from living cells and living tissues such as brain slices, and a method for identifying target membrane proteins using the probe. [Background technology]

[0002] In recent years, in the research field of chemical biology, molecular biology and organic chemistry techniques have been utilized to detect and quantify trace amounts of biological molecules, such as proteins or biologically active substances, and to elucidate the functions or reactions of these molecules. As part of these efforts, chemical probes, or compounds that specifically interact with biological molecules, have been developed to elucidate the functions or reactions of biological molecules.

[0003] In this research field, chemoproteomics is a method for elucidating the structure and function of proteins using organic chemical techniques.

[0004] Chemoproteomics is a method that enables unbiased and comprehensive direct identification of target proteins from living cells or tissues that reflect the phenotype, and is thought to make a significant contribution to pharmaceutical development, such as identifying unknown target proteins of drug candidate compounds discovered through phenotypic screening and identifying antigens of unknown antibodies.

[0005] Known conventional techniques for detecting membrane proteins include a method of labeling living cells using photoaffinity reactive groups (Non-Patent Document 1), a method of identifying membrane proteins from cell membrane fractions of living cells by proteomics (Non-Patent Document 2), a method of detecting target membrane proteins from overexpressing cells using chemical probes (Non-Patent Documents 3 and 4), a method of detecting target membrane proteins from cultured cells and primary cultured cells using chemical probes (Non-Patent Document 5), a method of detecting target proteins within cells and on the membrane of cultured cells using chemical probes (Non-Patent Document 6), a method of fluorescent imaging of target membrane proteins on brain slices using chemical probes (Non-Patent Document 7), and a method of identifying target membrane proteins using proteins such as growth factor EGF and FGFR antibodies (Non-Patent Document 8).

[0006] To identify such target proteins, a probe having a site (ligand site) that specifically binds to the target protein is used. For example, a method for identifying target proteins is known, using a compound probe with biotin attached to one end and a ligand with affinity for the target protein attached to the other end (e.g., Non-Patent Documents 1 and 9). With this probe, the target protein is bound via the ligand, and then a biotin-avidin complex is formed via biotin. Using this as an indicator, the target protein can be isolated, purified, and identified. However, it is not easy to identify membrane proteins that are expressed in low amounts.

[0007] Specifically, Non-Patent Document 1 discloses a method for detecting a target protein that has affinity for a ligand using a probe in which tetrazole is placed near the ligand. In this method, after the target protein is coordinated to the ligand, a covalent bond is formed between the tetrazole and the protein by irradiating the probe with ultraviolet light, and therefore, the protein coordinated to the ligand does not detach during processes such as purification.

[0008] Non-Patent Document 9 discloses a probe in which a cleavage site is introduced at an appropriate position between the ligand and biotin in order to remove non-specific proteins including avidin under mild conditions.

[0009] Furthermore, Patent Document 1 discloses a trifunctional cross-linking reagent as a probe for identifying a target glycoprotein present on a living cell or in a biological fluid, which carries (i) a ligand-reactive group for conjugation with a target ligand having at least one binding site on the target glycoprotein receptor, (ii) a hydrazone group for capturing the oxidized receptor-glycoprotein, and (iii) an affinity group selected from azide and alkyne for detecting, isolating, and purifying the captured glycoprotein.

[0010] However, none of the above methods are highly reproducible and practical for identifying target membrane proteins from living cells or tissues that accurately reflect the phenotype. This is because many proteins on cell membranes are expressed at low levels, and the reactive groups commonly used in known chemoproteomic methods have low capture efficiency for target membrane proteins, making it difficult to specifically and sensitively detect true target membrane proteins. Furthermore, there is the problem of nonspecific proteins contaminating the purified sample due to heat treatment of avidin beads or protein digestion on the beads. For these reasons, no examples of identifying target membrane proteins from living tissues using chemoproteomics have been reported. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] International Publication No. 2017 / 081069 [Non-patent literature]

[0012] [Non-Patent Document 1] J. Am. Chem. Soc., 138, 14609-14615 (2016) [Non-patent document 2] Cell Chemical Biology 2017, 24, 3-8 [Non-patent document 3] ChemBioChem 2017, 18, 1639-1649 [Non-patent document 4] Mol. Pharmacol. 2019, 95, 196-209 [Non-Patent Document 5] J. Am. Chem. Soc. 2018, 140, 6067-6075 [Non-patent document 6] J. Am. Chem. Soc. 2018, 140, 4259-4268 [Non-Patent Document 7] Nature Communications, 8, 14850 (2017) [Non-patent document 8] Nature Communications, 9, 1519 (2018) [Non-Patent Document 9] Chem Commun, 49, 5366 (2013) Summary of the Invention [Problem to be solved by the invention]

[0013] An object of the present invention is to provide a novel compound that can be used as a probe for identifying proteins expressed in living cells or living tissues. More preferably, an object of the present invention is to provide a novel compound that can be used as a probe capable of identifying even membrane proteins expressed in low amounts. Furthermore, the present invention provides a method for identifying a target protein, preferably a target membrane protein, present in living cells or living tissues using the compound as a probe. [Means for solving the problem]

[0014] To achieve the above objective, we designed a tetrafunctional compound with four functions: 1) a ligand-binding moiety for conjugating a ligand with affinity for a target protein, 2) a reactive moiety for capturing the target protein, 3) a cleavable moiety for selectively eluting the target protein captured on avidin beads, and 4) a biotin tag useful for concentrating or purifying the captured target protein. We found that by conjugating a small molecule or antibody with affinity for the target protein as a ligand to the tetrafunctional compound and using it as a tetrafunctional chemical probe, target membrane proteins can be detected in living cells or brain slices.

[0015] The present invention was completed based on these findings and through further investigation, and includes the following aspects. Item 1. A tetrafunctional compound comprising a ligand binding site (A), a reactive site (D), a cleavable site (E), and a biotin site (B) linked together, optionally via a spacer, the ligand binding site (A) is at least one activated functional group selected from the group consisting of an amine-reactive group, a hydroxy-reactive group, a thiol-reactive group, an aldehyde- or keto-reactive group, an alkyl- or aryl-halide-reactive group, an alkyl- or aryl-sulfonate-reactive group, an amide-reactive group, a sulfonamide-reactive group, an aryl-reactive group, a diol-reactive group, and a carboxy-reactive group, or at least one reactive functional group selected from the group consisting of -COOH, -NH, -OH, -SH, -CH=CH-, -(C=O)-CH=CH-, an alkyl-, vinyl-, or aryl-halide, an alkyl-, vinyl-, or aryl-sulfonate, an alkynyl, an azide, an epoxy, and a click tag; the reactive site (D) is a group having a structure of at least one compound selected from the group consisting of 2-aryl-5-carbonyltetrazole, phenyl azide, diazirine, α-ketoamide, 4-hydroxybenzene, phthalhydrazide, and benzophenone; the cleavable site (E) is a group having the structure of at least one compound selected from the group consisting of 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl, levulinoyl ester, vicinal diol, diazobenzene, bisarylhydrazone, dialkoxydiphenylsilane, disulfide, a peptide consisting of the sequence ENLYFQG (SEQ ID NO: 1), and a peptide consisting of the sequence ENLYFQS (SEQ ID NO: 2); The biotin moiety (B) is a group represented by the following formula (1): [ka] (wherein * means a bond to an adjacent group); The spacer is a linear or branched alkylene group having 1 or more carbon atoms, or a linear or branched alkylene group having 3 or more carbon atoms, in which non-adjacent —CH— groups are independently —O—, —OCH—, —CHO—, —CO—, or —NR a -[R a means a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. The same applies hereinafter.], -CO-NR a -, -NR a A tetrafunctional compound, which is a group substituted with at least one crosslinking group selected from the group consisting of -CO-, a group represented by the following formula (1-1), and a group represented by the following formula (1-2): [ka] (In the formula, m represents 0 or 1, and n represents 1 or 2.)

[0016] Item 2. The reactive site (D) is a group represented by the following formula (2) or (2'): [ka] (In the formula, * represents a bond to an adjacent group, and n represents 0 or 1.) or / and, The cleavable site (E) is a group represented by the following formula (3): [ka] (In the formula, R1, R2, R3, R4, R5, and R6 each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and * represents a bond to an adjacent group.) Item 1. A tetrafunctional compound according to item 1.

[0017] Item 3. The tetrafunctional compound according to Item 1 or 2, which is a compound represented by the following formula (I) or (II): [ka] (A, B, C, D, and E represent the moieties described in item 1, and S1, S2, S3, S4, S5, S6, and S7 each independently represent a spacer.) Item 4. The tetrafunctional compound according to any one of Items 1 to 3, wherein the ligand binding site (A) is an N-hydroxysuccinimide ester group. Item 5. The tetrafunctional compound according to Item 4, which is a compound represented by the following formula (i), (ii), or (iii): [ka] (In the formula, p represents 2.) Item 6. A tetrafunctional chemical probe comprising the tetrafunctional compound according to any one of items 1 to 5, wherein a ligand is bound to the ligand binding site (A). Item 7. The tetrafunctional chemical probe according to Item 6, wherein the ligand is any one selected from the group consisting of proteins, peptides, lipids, sugars, and low-molecular-weight compounds that have affinity for membrane proteins derived from living cells or living tissues. Item 8. The tetrafunctional chemical probe according to item 6 or 7, represented by formula (iv): [ka] (In the formula, S1 represents a spacer, and p represents 2.)

[0018] Item 9. A method for detecting a target protein in a cell or tissue using the tetrafunctional chemical probe according to any one of items 6 to 8, (1) reacting a tetrafunctional chemical probe with cells or tissues to bind a ligand in the tetrafunctional chemical probe to a target protein; (2) forming a covalent bond between the reactive site (D) in the tetrafunctional chemical probe and the target protein; (3) purifying the fraction containing the target protein bound to the tetrafunctional chemical probe; (4) binding the biotin moiety in the tetrafunctional chemical probe with avidin to form a tetrafunctional chemical probe-avidin complex; (5) cleaving the formed tetrafunctional chemical probe-avidin complex at the cleavable site in the tetrafunctional chemical probe; and (6) detecting fractions containing the target protein A method for detecting a target protein, comprising the steps of: [Effects of the Invention]

[0019] The tetrafunctional compound of the present invention is a compound comprising a ligand-binding site (A), a reactive site (D), a cleavable site (E), and a biotin site (B) linked, optionally via a spacer, and can be used as a tetrafunctional chemical probe by binding a ligand having affinity (binding ability) for a target protein to be detected or identified to the ligand-binding site.

[0020] The tetrafunctional chemical probe of the present invention, based on the structure of the tetrafunctional compound, has a reactive site that exhibits high reactivity and high selectivity and a cleavable site that cleaves under mild conditions. This structure is characterized by excellent recovery yield, detection sensitivity, and reproducibility of membrane proteins. Therefore, by using the tetrafunctional chemical probe of the present invention, target membrane proteins can be identified in living cells with higher sensitivity than conventional techniques. Furthermore, it is also useful for identifying target membrane proteins in living tissues, which have a more advanced and complex physiological environment. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 shows the results of Test Example 1 in which a target molecule was isolated from living cells using a compound (CPP-127). [Figure 2] FIG. 1 shows the results of Test Example 2 in which a target molecule was isolated from living tissue using a compound (CPF-224). [Figure 3] FIG. 1 shows the results of Test Example 2 in which a target molecule was isolated from living tissue using a compound (CPF-202). [Figure 4] FIG. 1 shows the results of Test Example 2 in which a target molecule was isolated from living tissue using a compound (CPF-242). [Figure 5] FIG. 1 shows the results of Test Example 3 in which a cell surface antigen was captured using a labeled antibody (CPA-321-labeled anti-CD71 antibody). [Figure 6] 1 is a graph showing the results of detecting the amount of CD71, an antigen, in THP-1 cells using anti-CD71 antibodies labeled with compounds having different linker lengths. [Figure 7] 1 is a graph showing the results of detecting the amount of CD71, an antigen, in A431 cells using anti-CD71 antibodies labeled with probes having different cross-linking groups. [Figure 8] 1 is a graph showing the results of detecting the amount of EGF receptor, an antigen, in MDA-MB-231 cells using His-Tag EGF labeled with different probes. [Figure 9]1 is a graph showing the results of detecting the amount of CD71, an antigen, in A431 cells using anti-CD71 antibodies labeled by different methods. [Figure 10] 1 is a graph showing the results of detecting the amount of CD71, an antigen, in THP-1 cells using the anti-CD71 antibody DF1513 labeled by the secondary antibody method. DETAILED DESCRIPTION OF THE INVENTION

[0022] (I) Explanation of terms used in this specification Unless otherwise specified, the terms and phrases used in this specification have the meanings explained below.

[0023] "Tetrafunctional" means having four functional groups. As mentioned above, one of the four functional groups is a functional group constituting the ligand binding site or a ligand site formed at that site, another is a functional group constituting the reactive site, another is a functional group constituting the cleavable site, and the remaining is a biotin residue capable of binding to avidin. Biotin is a water-soluble vitamin (5-[(3aS,4S,6aR)-2-oxahexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoic acid) that is classified as a vitamin B group.

[0024] As used herein, "alkylene" refers to a divalent radical derived from a hydrocarbon and represented by the formula "-(CHR)n-" where R is a hydrogen atom or an optional substituent. The alkylene group preferably has about 1 to 30 (n=1 to 30), and more preferably about 1 to 10, carbon atoms.

[0025] In this specification, "alkyl" refers to an alkyl group having 1 to 6 carbon atoms (C 1-6) means a straight-chain or branched-chain lower alkyl. Specific examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 3-methylpentyl, etc. A straight-chain lower alkyl having 1 to 3 carbon atoms is preferred. 1-6 "Alkyl" also includes lower alkyl in which one to three hydrogen atoms are replaced by deuterium atoms.

[0026] As used herein, "protein" and "peptide" have the same meaning and refer to amino acid polymers of any length (peptides are typically referred to as fragments of proteins). The polymers may be linear, branched, or cyclic. The amino acids may be natural or unnatural amino acids, or mutant amino acids.

[0027] In the present invention, proteins may be naturally occurring or synthetic. They may also be naturally occurring proteins with synthetically engineered sequences. Proteins may be intracellular proteins, cell surface proteins (i.e., proteins bound to the surface of a cell), or solution proteins (i.e., proteins secreted into the medium). Proteins may also be glycoproteins or membrane proteins. Proteins of interest in the present invention may be any pharmaceutically or commercially relevant protein with useful biological or chemical activity, such as receptors, antibodies, enzymes, hormones, regulatory factors, antigens, or binding agents. The proteins listed below that may be used in the methods of the present invention are merely exemplary and are not intended to be limiting. Those skilled in the art will appreciate that any protein may be a target protein in the probes and methods of the present invention.

[0028] As used herein, the term "target protein" refers to a protein to which one or more specific types of ligands can bind. Such target proteins are preferably, but not limited to, those present in biological fluids, on cells, or in tissues derived from mammals, including humans and non-human animals. More preferably, they are proteins expressed and present on living cells or living tissues. Thus, if the target protein is, for example, a membrane protein expressed on the cell surface, it will be associated with the plasma membrane of a living cell and will sometimes have at least one amino acid exposed to the extracellular space, making it available for binding to one or more ligands.

[0029] The target protein of the present invention is not particularly limited, but is preferably a membrane protein. A membrane protein refers to a protein that can directly or indirectly interact with a lipid membrane, particularly a lipid bilayer membrane, and in which one or more of the amino acid molecules that make up the protein are present in the extracellular environment. Examples of such proteins include G protein-coupled receptors, seven-transmembrane receptors, receptor tyrosine kinases, the immunoglobulin superfamily and its related proteins, scavenger receptors, and other receptors; transporters, ion channels, solution transporters, active transporters, auxiliary transporter proteins, enzymes, and others.

[0030] More specifically, examples of G protein-coupled receptors and seven-transmembrane receptors include Frizzled receptors, nucleic acid receptors, adenosine receptors, adrenergic receptors, angiotensin receptors, apelin receptors, vasopressin receptors, bradykinin receptors, bombesin receptors, chemokine receptors, cholecystokinin receptors, muscarinic acetylcholine receptors, cannabinoid receptors, cysteinyl leukotriene receptors, dopamine receptors, sphingolipid receptors, lysophosphatidin receptors, sphingosine monophosphate receptors, endothelin receptors, protease-activated receptors, free lipid receptors, galanin receptors, growth hormone secretagogue receptors, gonadotropin receptors, bile acid receptors, nicotinic acid receptors, lysophosphatidic acid receptors, anaphylaxis receptors, and the like. Examples of such receptors include ceratoxin chemotactic receptors, gastrin-releasing peptide receptors, orexin receptors, histamine receptors, serotonin receptors, interleukin receptors, leucine-rich repeat-containing G protein-coupled receptors, leukotriene receptors, adrenocorticotropic hormone receptors, melanocortin receptors, melanin-concentrating hormone receptors, melatonin receptors, neuromedin receptors, neuropeptide receptors, neurotensin receptors, opioid receptors, nociceptin receptors, oxoglutarate receptors, oxytocin receptors, P2Y purinergic receptors, prostaglandin receptors, rhodopsin, relaxin receptors, somatostatin receptors, succinate receptors, substance P receptors, substance K receptors, thromboxane receptors, urotensin receptors, calcitonin receptors, taste receptors, metabotropic glutamate receptors, and olfactory receptors.

[0031] Examples of receptor tyrosine kinases include activin receptors, bone morphogenetic protein receptors, TNF-β receptors, AXL receptors, epidermal growth factor receptors, ephrin receptors, insulin receptors, nerve growth factor receptors, discoidin domain receptors, vascular endothelial growth factor receptors, leukocyte receptors, hepatocyte growth factor receptors, macrophage-stimulating protein receptors, platelet-derived growth factor receptors, and enterotoxin receptors, as well as their precursors.

[0032] Examples of the immunoglobulin superfamily and its associated proteins include immunoglobulin receptors, killer cell immunoglobulin-like receptors, leukocyte immunoglobulin-like receptors, netrin receptors, T cell receptors, various cytokine receptors, various fragments of the T cell surface glycoprotein CD, superfamily members, and their precursors. Other receptors include, for example, adiponectin receptors, progestin receptors, contactin-associated proteins, delrin, integrins and their precursors, neurexins, neuropilins, Notch receptors, plexins and their precursors, receptor tyrosine phosphatases, selectins and their precursors, syndecan receptors, tumor necrosis factor (TNF) receptors, Toll-like receptors, transferrin receptors, and sortilin and its precursors.

[0033] Examples of transporters include aquaporins, chloride ion channels, bestrophin, ryanodine receptors, voltage-gated potassium channels, cyclic nucleotide-gated channels, calcium-activated potassium channels, transient receptor potential channels (TRP channels), voltage-gated sodium channels, voltage-gated calcium channels, other voltage-gated channels, serotonin receptors, acetylcholine receptors, gamma-aminobutyric acid receptors, glycine receptors, ionotropic glutamate receptors, and P2X purinergic receptors. Examples of solution transporters include various SLC family proteins, and examples of active transporters include ion-transporting ATPases and ABC transporters. Further enzymes include NADH-ubiquinone oxidoreductase, cytochrome c, flavin-containing monooxygenase, cytochrome P450, and other oxidoreductases; acyltransferases, glucosetransferases, sulfotransferases, and other transferases; ligases, tyrosine phosphatases, phosphodiester hydrolases, glycosylases, serine peptide endohydrolases, metalloendopeptidases, nucleoside diphosphate phosphatases, and other hydrolases.

[0034] As used herein, "sample" or "biological sample" refers to any living cell or any solid or fluid sample obtained from an organism (biological sample). These include, for example, tissue cultures, bioreactors, human or non-human animal tissues, plants (including fruits and vegetables), single-celled microorganisms (e.g., bacteria and yeast), and multicellular organisms. Biological samples can also be, for example, blood, plasma, serum, urine, bile, semen, cerebrospinal fluid, aqueous humor, or vitreous humor, or any bodily secretion, transudate, or exudate (e.g., fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (e.g., a normal joint or a joint affected by a disease such as rheumatoid arthritis, osteoarthritis, gout, or septic arthritis). Biological samples can also be, for example, samples obtained from any organ or tissue (including biopsy or autopsy specimens). Cells also include primary cells and cultured cells.

[0035] Preferably, the living cells and tissues are derived from humans or non-human animals. The living cells that can be used in the present invention are not particularly limited, as long as they express a protein, preferably a membrane protein, that can be detected using a ligand. Examples include, but are not limited to, CHO cells, MDCK cells, 3T3-L1 cells, 293 cells, MCF7 cells, A431 cells, 3T3 cells, CV-I cells, HeLa cells, L cells, BHK21 cells, HL-60 cells, U937 cells, HaK cells, Jurkat cells, THP-1 cells, and other transformed cell lines, as well as cell lines derived from in vitro cultures of blood cells, primary tissues, and primary grafts isolated from living organisms. In addition to two-dimensional cultures, these also include those cultured in three-dimensional culture systems, such as organoid cultures. Furthermore, cells that use these as hosts and have a gene encoding a specific protein introduced via a vector or the like may also be used. Examples include, but are not limited to, CHO-K1 cells that stably express the dopamine D2 receptor.

[0036] The tissues that can be used in the present invention are not particularly limited as long as they express a protein, preferably a membrane protein, that can be detected using a ligand, such as, but not limited to, tissue slices and organ slice cultures of the brain, liver, kidney, etc.

[0037] As used herein, "living cells" refers to mammalian (human or non-human) derived cells established as cell lines, or cells derived from non-mammals that have been cultured and not subjected to denaturing treatment. It also includes cells isolated under non-denaturing conditions from any organ or tissue (e.g., blood cells, organ-forming cells, primary cultured cells, etc.). This includes not only two-dimensional cultures but also those cultured in three-dimensional culture systems, such as organoid cultures. Furthermore, as used herein, "living tissue" refers to tissue slices or tissue culture samples (e.g., tissue slices or organ slice cultures of the brain, liver, or kidney) isolated under non-denaturing conditions from any organ or tissue of a mammal (human or non-human animal).

[0038] In the present invention, the term "ligand" refers to a compound that has affinity for a target protein and can bind thereto. It is not limited to, but preferably refers to any compound that can bind to a cell membrane and interact with or bind to a target membrane protein expressed and present on the cell surface. Examples of ligands include peptides, polypeptides, proteins including glycoproteins or phosphoproteins, carbohydrates, glycolipids, phospholipids, oligonucleotides, polynucleotides, aptamers, vitamins, antigens and fragments thereof, haptens, receptor agonists, partial agonists, mixed agonists, antagonists, drugs, chemokines, hormones (e.g., LH, FSH, TRH, TSH, ACTH, CRH, PRH, MRH, MSH, glucagon, and prolactin; transferrin; lactoferrin; angiotensin; histamine; insulin; lectins), transmitters, autacoids, growth factors (e.g., PDGF, VEGF, EGF, TGFα, T These include, but are not limited to, natural or synthetic organic molecules capable of specifically binding to receptors, including BFβ, GM-CSF, G-CSF, M-CSF, FGF, IGF, bombesin, thrombopoietin, erythropoietin, oncostatin, and endothelin 1), interleukins (e.g., interleukins 1-15), lymphokines and cell signaling molecules, cytokines (including, for example, tumor necrosis factors (tumor necrosis factors α and β) and interferons (e.g., interferons α, β, and γ)), prosthetic groups, coenzymes, cofactors, regulatory factors, and receptors, as well as fragments, analogs, and other derivatives thereof that maintain the same binding properties.

[0039] The ligand may be an antibody. An antibody has the ability to specifically bind to a specific antigen. The antibody may be a monoclonal antibody or a polyclonal antibody. Preferred antibodies are monoclonal antibodies, particularly therapeutic antibodies such as trastuzumab and bevacizumab. Furthermore, the antibody may be a humanized antibody.

[0040] The method may be either a primary antibody method or a secondary antibody method. The primary antibody method is a method in which an antibody (primary antibody) having affinity for the target protein is used as a ligand, while the secondary antibody method is a method in which a secondary antibody is integrated with the ligand binding site of a probe, and the primary antibody (ligand) forms a complex with the secondary antibody (ligand binding site). Even if an antibody whose affinity with the target protein cannot be detected with high sensitivity using the primary antibody method, it may be possible to detect it with high sensitivity using the secondary antibody method.

[0041] The ligand may also be an engineered affinity binder, such as ankyrin repeat binders, particularly His-tag binders, affinity binders generated by phage display, or oligonucleic acid or peptide aptamers. The ligand may also be a protein, such as a protein receptor, or a domain of a cell surface protein, such as a cell surface protein receptor. Furthermore, the ligand may be a microorganism or a virus.

[0042] According to the present invention, a ligand interacts with its target protein through its binding site. The binding site is a specific peptide fragment of the target protein, e.g., a particular amino acid sequence or three-dimensional structure of that fragment of the target protein, and is referred to as the binding site. The term "interaction" with respect to a ligand binding to a binding site of its (cell surface or secreted) target protein includes transient or permanent, direct or indirect contact between the cell surface or secreted target protein and the ligand, and can be characterized by its binding affinity, i.e., dissociation equilibrium constant, Kd. Typical binding affinities of a ligand to its target protein are at least 10 -5 M, preferably 10 -6 M or more, for example, about 10 -7 M~about 10 -12 It can be M.

[0043] Thus, the ligand is not particularly limited as long as it has affinity for the target protein, but is preferably a compound (pharmaceutical compound) used as an active ingredient in a drug for treating a disease in a human or non-human animal. The pharmaceutical compound may be any of a low molecular weight compound, a medium molecular weight compound, a natural product, and a protein including an antibody. Furthermore, the pharmaceutical compound may be modified to a degree that does not affect the affinity for the target protein in order to bind to the ligand-binding site of the tetrafunctional compound of the present invention. In the tetrafunctional chemical probe of the present invention, the ligand and the reactive site can be bound via a spacer.

[0044] As used herein, the terms "alkylation reaction, hydrolysis reaction, amination reaction, esterification reaction, amidation reaction, etherification reaction, nucleophilic substitution reaction, addition reaction, oxidation reaction, and reduction reaction" refer to methods known per se. Examples of such methods include those described in "Experimental Chemistry Lectures" (5th ed., edited by the Chemical Society of Japan, Maruzen Co., Ltd.), "ORGANIC FUNCTIONAL GROUP PREPARATIONS" 2nd ed., Academic Press, Inc., 1989, "Comprehensive Organic Transformations" (VCH Publishers Inc., 1989), and "Greene's Protective Groups in Organic Synthesis" by P.G. M. Uts and T.W. Greene, 4th ed., 2006.

[0045] In this specification, the term "palladium compound" is not particularly limited, but includes, for example, tetravalent palladium catalysts such as sodium hexachloropalladate(IV) tetrahydrate and potassium hexachloropalladate(IV); [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride dichloromethane adduct (Pd(dppf)Cl2·CHCl2), (2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) methanesulfonate (XPhos Pd Divalent palladium catalysts such as palladium(II) chloride, palladium(II) bromide, palladium(II) acetate, palladium acetylacetonate(II), dichlorobis(benzonitrile)palladium(II), dichlorobis(acetonitrile)palladium(II), dichlorobis(triphenylphosphine)palladium(II), dichlorotetraamminepalladium(II), dichloro(cycloocta-1,5-diene)palladium(II), palladium trifluoroacetate(II), and 1,1'-bis(diphenylphosphino)ferrocenedichloropalladium(II)-dichloromethane complex; and zero-valent palladium catalysts such as tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3), tris(dibenzylideneacetone)dipalladium chloroform complex(0), and tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4). These palladium compounds may be used singly or in combination of two or more.

[0046] As used herein, examples of the "carbodiimide" include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC), 3-ethyl-1-(3-dimethylaminopropyl)carbodiimide (WSC), N-[3-(Dimethylamino)propyl]-N'-ethylcarbodiimide, N-[3-(Dimethylamino)propyl]-N'-ethylcarbodiimide methiodide, N-tert-Butyl-N'-ethylcarbodiimide, N-Cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate, N,N'-Di-tert-butylcarbodiimide, and N,N'-Di-p-tolylcarbodiimide.

[0047] As used herein, the term "protecting group" includes, for example, a t-butoxycarbonyl (BOC) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a 2,2,2-trichloroethoxycarbonyl (Troc) group, a benzyloxycarbonyl (Z) group, an allyloxycarbonyl (Alloc) group, a trifluoroacetyl group, a phthaloyl group, a paratoluenesulfonyl (Ts) group, and a 2-nitrobenzenesulfonyl (Ns) group.

[0048] As used herein, the term "solvent" refers to a solvent inert to the reaction, and examples thereof include water, ethers (e.g., dioxane, tetrahydrofuran, diethyl ether, 1,2-dimethoxyethane, diethylene glycol dimethyl ether, ethylene glycol dimethyl ether), halohydrocarbons (e.g., methylene chloride, chloroform, 1,2-dichloroethane, carbon tetrachloride), aromatic hydrocarbons (e.g., benzene, toluene, xylene), lower alcohols (e.g., methanol, ethanol, isopropanol), and polar solvents (e.g., N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), hexamethylphosphoric triamide, acetonitrile). These solvents may be used alone or in combination.

[0049] As used herein, the term "amine" includes trialkylamines (for example, trimethylamine, triethylamine, N,N-diisopropylethylamine), dialkylamines (for example, diethylamine, diisopropylamine), and the like.

[0050] As used herein, the term "base" includes, for example, inorganic bases, organic bases, etc. Examples of the "inorganic base" include alkali metal hydroxides (e.g., lithium hydroxide, sodium hydroxide, potassium hydroxide), alkaline earth metal hydroxides (e.g., magnesium hydroxide, calcium hydroxide, barium hydroxide), alkali metal carbonates (e.g., sodium carbonate, potassium carbonate, cesium carbonate), alkaline earth metal carbonates (e.g., magnesium carbonate, calcium carbonate, barium carbonate), alkali metal bicarbonates (e.g., sodium bicarbonate, potassium bicarbonate), alkali metal phosphates (e.g., sodium phosphate, potassium phosphate, cesium phosphate), alkaline earth metal phosphates (e.g., magnesium phosphate, calcium phosphate), alkali metal alkoxides (e.g., sodium methoxide, sodium ethoxide, potassium tert-butoxide), alkali metal hydrides (e.g., sodium hydride, potassium hydride), etc. Examples of the "organic base" include trialkylamines (e.g., trimethylamine, triethylamine (TEA), N,N-diisopropylethylamine (DIPEA)), dialkylamines (e.g., diethylamine, diisopropylamine), 4-dimethylaminopyridine (DMAP), N-methylmorpholine, picoline, 1,5-diazabicyclo[4.3.0]non-5-ene, 1,4-diazabicyclo[2.2.2]octane, and 1,8-diazabicyclo[5.4.0]-7-undecene (DBU). One or more of these may be appropriately selected and mixed for use.

[0051] As used herein, examples of the "condensing agent" include 1-hydroxybenzotriazole (HOBt), 3-hydroxy-3,4-dihydro-1,2,3-benzotriazin-4-one (HOOBt), N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC), 2-(1H-7-azabenztriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 3,4-dihydro-1,2,3-benzotriazin-4-one-3-oxytetramethyluronium hexafluorophosphate (HDTU), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yloxytris-(pyrrolidino)-phosphonium hexafluorophosphate (PyBop), (3,4-dihydro-1,2,3-benzotriazin-4-one-3-oxy)diethylphosphate (DEPBt), 3,4-dihydro-1,2,3-benzotriazin-4-one-3-oxytris-(pyrrolidino)-phosphonium hexafluorophosphate (PDOP), 2-(benzotriazol-1-yloxy)-1,3 -dimethyl-2-pyrrolidin-1-yl-1,3,2-diazaphosphoridinium hexafluorophosphate (BOMP), 5-(1H-7-azabenzotriazol-1-yloxy)-3,4-dihydro-1-methyl 2H-pyrrolium hexachloroantimonate (AOMP), (1H-7-azabenzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (AOP), 5-(1H-benzotriazol-1-yl)-3,4-Dihydro-1-methyl 2H-pyrrolium hexachloroantimonate: N-oxide (BDMP), 2-bromo-3-ethyl-4-methylthiazolium tetrafluoroborate (BEMT), 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP), 2-bromo-1-ethylpyridinium hexachloroantimonate (BEPH), benzotriazol-1-yloxy-N,N-dimethylmethaniminium hexachloroantimonate (BOMI), N,N' -Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl), 1-(1H-benzotriazol-1-yloxy)phenylmethylenepyrrolidinium hexachloroantimonate (BPMP), 1,1,3,3-bis(tetramethylene)fluorouronium hexafluorophosphate (BTFFH), 4-(chloro-4-morpholinylmethylene)morpholinium hexafluorophosphate (CMMM), 2-chloro-1,3-dimethyl-1H-benzimidazolium hexafluorophosphate (CMBI), 2-fluoro-1-ethylpyridinium tetrafluoroborate (FEP), 2-fluoro-1-ethylpyridinium hexachloroantimonate (FEPH), 1-(1-pyrrolidinyl-1H-1,2,3-triazolium) O-(1H-benzotriazol-1-yl)-N,N,N',N'-bis-(pentamethylene)uronium hexafluorophosphate (HBPipU), O-(1H-benzotriazol-1-yl)N,N,N0,N0-bis(tetramethylene)urinium hexafluorophosphate (HBPyU), (1H-7-azabenzotriazol-1-yloxy)tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP), bromo-tripyrrolidinophosphonium hexafluorophosphate (PyBrOp), chloro-tripyrrolidinophosphonium hexafluorophosphate (PyClOP), 1,1,3,3-Bis(tetramethylene)chlorouronium hexafluorophosphate (PyClU), tetramethylfluoro-manmidinium hexafluorophosphate (TFFH), triphosgene, triazine-based reagents [cyanuric chloride, cyanuric fluoride, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT)], bis(2-chlorophenyl)phosphorochloridate, diphenylphosphorochloridate, diphenylphosphoroazide (DPPA), N-[1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino(morpholino)]uronium Examples include hexafluorophosphate (COMU), N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU), etc.

[0052] As used herein, the term "deprotecting agent" is not particularly limited as long as it can remove a protecting group, and examples thereof include bases such as piperidine and lithium diisopropylamide, zinc, acids such as TFA, nucleophiles in the presence of a zero-valent palladium catalyst, basic aqueous solutions, methylamine, hydrazine, one-electron reducing agents, and thiols.

[0053] (II) Tetrafunctional compounds and tetrafunctional chemical probes The tetrafunctional compound of the present invention is a compound having a structure in which a ligand binding moiety (A), a reactive moiety (D), a cleavable moiety (E), and a biotin tag (B) are linked, optionally via a spacer. By binding a ligand having affinity (binding ability) for a target protein to the ligand binding moiety of this compound, a tetrafunctional chemical probe useful for detecting, identifying, or purifying the target protein can be prepared. In other words, the tetrafunctional chemical probe is a compound having a structure in which a ligand moiety (A), a reactive moiety (D), a cleavable moiety (E), and a biotin tag (B) are linked, with or without a spacer.

[0054] The four functional group moieties possessed by the tetrafunctional compound and tetrafunctional chemical probe of the present invention, and the spacer connecting them will be described below.

[0055] (ligand binding site) In the tetrafunctional compound of the present invention, the ligand binding site (A) is a site (coupling site) for binding a ligand to the tetrafunctional compound, and therefore can be a reactive or activated functional group (collectively referred to as a "ligand reactive group") that reacts with a reactive counterpart group present on the ligand.

[0056] "Activated functional group" refers to a reactive functional group that has been activated by standard chemical techniques to yield the corresponding activated functional group. In certain embodiments, the activated functional group is selected from the group consisting of an amine-reactive group, a hydroxyl-reactive group, a thiol-reactive group, an aldehyde- or keto-reactive group, an alkyl- or aryl-halide-reactive group, an alkyl- or aryl-sulfonate-reactive group, an amide-reactive group, a sulfonamide-reactive group, an aryl-reactive group, a diol-reactive group, and a carboxy-reactive group.

[0057] An "amine-reactive group" is an activated functional group that reacts with primary or secondary amines. Typical amine-reactive groups include aryl or alkyl-activated carboxylic acid esters -COOR, such as N-hydroxysuccinimide esters or derivatives thereof (e.g., sulfo-N-hydroxysuccinimide esters), phenol esters or derivatives thereof (e.g., R is phenol, p-nitrophenol, or tetrafluorophenol). Other amine-reactive groups include acyl chlorides (-COCl), aryl or alkyl imidates (-C(NH)OMe), alkyl or aryl isocyanates (-NCO), isothiocyanates (-NCS), aldehydes, particularly 2-pyridinecarboxaldehyde, carbonyls, epoxys, α,β-unsaturated carbonyls, alkyl or aryl halides, and alkyl or aryl sulfonates.

[0058] A "hydroxyl-reactive group" is an activated functional group that reacts with hydroxyl. Typical hydroxyl-reactive groups include, for example, alkyl or aryl isocyanates -NCO, aryl or alkyl activated carboxylic acid esters -COOR, epoxy, α,β unsaturated carbonyl, alkyl or aryl halide, alkyl or aryl sulfonate.

[0059] A "thiol-reactive group" is an activated functional group that reacts with thiols. Typical thiol-reactive groups include, for example, maleimide, α-haloamide (—NH—CO—CH2-Hal), epoxy, α,β-unsaturated carbonyl, alkyl or aryl halide, and alkyl or aryl sulfonate.

[0060] An "aldehyde- or keto-reactive group" is an activated functional group that reacts with an aldehyde or ketone. Typical aldehyde- or keto-reactive groups include, for example, aryl or alkylamines, aryl or alkylhydrazines (-NHNH), aryl or alkylacylhydrazines (-CO-NHNH), alkyl or arylhydroxylamines (-ONH), and alkyl or aryl magnesium halides.

[0061] An "alkyl or aryl halide reactive group" is an activated reactive group that reacts with an alkyl or aryl halide. Typical alkyl or aryl halide activated reactive groups include amines, alcohols, mercaptos, amides, sulfonamides, carboxylic acids, alkenes, alkynes, boronic acids, boronic esters, and alkyltins.

[0062] An "alkyl or aryl sulfonate-reactive group" is an activated reactive group that reacts with an alkyl or aryl sulfonate. Typical alkyl or aryl sulfonate-activated reactive groups include amines, alcohols, mercaptos, amides, sulfonamides, carboxylic acids, alkenes, alkynes, boronic acids, boronic esters, and alkyltins.

[0063] An "amide reactive group" is an activated reactive group that reacts with unsubstituted or substituted amides. Typical amide reactive groups include epoxy, α,β unsaturated carbonyl, alkyl or aryl halide, and alkyl or aryl sulfonate.

[0064] A "sulfonamide reactive group" is an activated reactive group that reacts with unsubstituted or substituted sulfonamides. Typical sulfonamide reactive groups include epoxy, α,β-unsaturated carbonyl, alkyl or aryl halide, and alkyl or aryl sulfonate.

[0065] An "aryl-reactive group" is an activated functional group that reacts with aryl groups. Typical aryl-reactive groups include, for example, 4-hydroxybenzene and phthalhydrazide.

[0066] A "diol-reactive group" is an activated functional group that reacts with a diol group. Typical diol-reactive groups include, for example, boronic acids.

[0067] A "carboxy-reactive group" is an activated functional group that reacts with a carboxyl group. Typical carboxy-reactive groups include, for example, halogen, alkyl or aryl sulfonate, hydroxyl, epoxy, mercapto, amino, isocyanato, and carbodiimide.

[0068] The term "reactive functional group" refers to a free, unprotected functional group unless otherwise specified. Specifically, the reactive functional group can be selected from the group consisting of -COOH, -NH, -OH, -SH, -CH=CH-, -(C=O)-CH=CH-, alkyl, vinyl or aryl halide, alkyl, vinyl or aryl sulfonate, alkynyl, azide, epoxy, and click tag.

[0069] Examples of activating reagents used to activate reactive functional groups include 1-hydroxybenzotriazole (HOBt), 3-hydroxy-3,4-dihydro-1,2,3-benzotriazin-4-one (HOOBt), N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC), 2-(1H-7-azabenztriazole ...). 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 3,4-dihydro-1,2,3-benzotriazin-4-one-3-oxytetramethyluronium hexafluorophosphate (HDTU), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate phosphate (BOP), benzotriazol-1-yloxytris-(pyrrolidino)-phosphonium hexafluorophosphate (PyBop), (3,4-dihydro-1,2,3-benzotriazin-4-one-3-oxy)diethyl phosphate (DEPBt), 3,4-dihydro-1,2,3-benzotriazin-4-one-3-oxytris-(pyrrolidino)-phosphonium hexafluorophosphate (PDOP), 2-(benzotriazol-1-yloxy)-1 ,3-dimethyl-2-pyrrolidin-1-yl-1,3,2-diazaphosphoridinium hexafluorophosphate (BOMP), 5-(1H-7-azabenzotriazol-1-yloxy)-3,4-dihydro-1-methyl 2H-pyrrolium hexachloroantimonate (AOMP), (1H-7-azabenzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (AOP), 5-(1H-benzotriazol-1-yl)-3,4-Dihydro-1-methyl 2H-pyrrolium hexachloroantimonate: N-oxide (BDMP), 2-bromo-3-ethyl-4-methylthiazolium tetrafluoroborate (BEMT), 2-bromo-1-ethylpyridinium tetrafluoroborate (BEP), 2-bromo-1-ethylpyridinium hexachloroantimonate (BEPH), benzotriazol-1-yloxy-N,N-dimethylmethaniminium hexachloroantimonate (BOMI), N,N' -Bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl), 1-(1H-benzotriazol-1-yloxy)phenylmethylenepyrrolidinium hexachloroantimonate (BPMP), 1,1,3,3-bis(tetramethylene)fluorouronium hexafluorophosphate (BTFFH), 4-(chloro-4-morpholinylmethylene)morpholinium hexafluorophosphate (CMMM), 2-chloro-1,3-dimethyl-1H-benzimidazolium hexafluorophosphate (CMBI), 2-fluoro-1-ethylpyridinium tetrafluoroborate (FEP), 2-fluoro-1-ethylpyridinium hexachloroantimonate (FEPH), 1-(1-pyrrolidinyl-1H-1,2,3-triazolium) O-(1H-benzotriazol-1-yl)-N,N,N',N'-bis-(pentamethylene)uronium hexafluorophosphate (HBPipU), O-(1H-benzotriazol-1-yl)N,N,N0,N0-bis(tetramethylene)urinium hexafluorophosphate (HBPyU), (1H-7-azabenzotriazol-1-yloxy)tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP), bromo-tripyrrolidinophosphonium hexafluorophosphate (PyBrOp), chloro-tripyrrolidinophosphonium hexafluorophosphate (PyClOP), 1,1,3,3-bis(tetramethylene)chlorouronium hexafluorophosphate (PyClU), tetramethylfluoro-manmidinium hexafluorophosphate (TFFH), triphosgene, triazine-based reagents [cyanuric chloride, cyanuric fluoride, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT)], bis(2-chlorophenyl)phosphorochloridate, diphenylphosphorochloridate, diphenylphosphoroazide (DPPA), N-[1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino(morpholino)]uronium hexafluorophosphate (COMU), These include, but are not limited to, bases, Lewis acids, palladium acetate, 1,1'-bis(diphenylphosphino)ferrocene, copper sulfate, (tris(2-carboxyethyl)phosphine (TCEP), sodium ascorbate, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), tris(2-benzimidazolylmethyl)amine, 3,3',3''-(4,4',4''-(nitrilotris(methylene))tris(1H-1,2,3-triazol-4,1-diyl))tris(propan-1-ol), and any combination thereof.

[0070] Those skilled in the art can easily understand which ligand-reactive group should be selected for coupling with a selected ligand, and can appropriately select and use one from the above.

[0071] In a preferred embodiment, the ligand binding site (A) is an activated functional group selected from the group consisting of an amine-reactive group, a hydroxyl-reactive group, a thiol-reactive group, an aldehyde- or keto-reactive group, an alkyl- or aryl halide-reactive group, an alkyl- or arylsulfonate-reactive group, an amide-reactive group, a sulfonamide-reactive group, an aryl-reactive group, a diol-reactive group, and a carboxy-reactive group. Particularly preferred is an aryl- or alkyl-activated carboxylic acid ester -COOR, such as the N-hydroxysuccinimide ester of the formula: [ka] (In the formula, * indicates a bonding site with an adjacent group.)

[0072] In another preferred embodiment, the ligand binding site (A) is a reactive functional group selected from the group consisting of -COOH, -NH, -OH, -SH, -CH=CH-, -(C=O)-CH=CH-, alkyl, vinyl, or aryl halides, alkyl, vinyl, or aryl sulfonates, alkynyl, azide, epoxy, and click tags. Click tags are functional groups used in bioorthogonal reactions, such as diphenylphosphinylphenyl, 1,2,4,5-tetrazin-3-yl, trans-cyclooctenyl, norbornenyl, cyclopropenyl, boronovinyl, boronoaryl, and aryl halides.

[0073] (Reactive site: D) The reactive moiety (D), another functional moiety in the tetrafunctional compound of the present invention, is a group capable of forming a covalent bond with a target protein. Such a group is not particularly limited as long as it has the above-mentioned function, but is preferably a group that forms a covalent bond under simple reaction conditions. Examples of such a group include a group having the structure (moiety) of at least one compound selected from the group consisting of 2-aryl-5-carbonyltetrazole (ACT), phenyl azide, diazirine, α-ketoamide, 4-hydroxybenzene, phthalhydrazide, and benzophenone. ACT can covalently bond primarily to carboxyl groups of proteins, while phenyl azide can covalently bond primarily to amino groups of proteins. 4-hydroxybenzene and phthalhydrazide can covalently bond primarily to 4-hydroxyphenyl groups of proteins. Diazirine, α-ketoamide, and benzophenone can nonspecifically form covalent bonds with proteins.

[0074] A preferred group is a group having the structure ACT shown in formula (2):

[0075] [ka] (In the formula, * means a bonding site with an adjacent group. n means 0 or 1. When n is 0, it means that there is no group coordinated to the benzene ring, that is, ACT is located at the end of the tetrafunctional compound of the present invention. When n is 1, the bond with the adjacent group may be at any of the ortho-, meta-, or para-position relative to the tetrazole group coordinated to the benzene ring, but is preferably at the para-position.)

[0076] In the above, a simple reaction can be exemplified by ultraviolet irradiation. The wavelength and irradiation time of the ultraviolet light to be irradiated are not particularly limited as long as they are conditions that allow a covalent bond to be formed between the reactive site of the tetrafunctional compound of the present invention and the target protein. A preferred wavelength is 254 to 365 nm, more preferably 302 nm. A preferred irradiation time is 1 second to 10 minutes, more preferably 30 seconds to 5 minutes, and even more preferably 1 minute.

[0077] Another preferred group is a group having the diazirine structure shown in formula (2'):

[0078] [ka] (In the formula, * indicates a bonding site to an adjacent group.)

[0079] In the above, a simple reaction can be exemplified by ultraviolet irradiation. The wavelength and irradiation time of the ultraviolet light to be irradiated are not particularly limited as long as they are conditions that allow a covalent bond to be formed between the reactive site of the tetrafunctional compound of the present invention and the target protein. A preferred wavelength is 254 to 380 nm, more preferably 365 nm. A preferred irradiation time is 5 to 30 minutes, more preferably 10 to 20 minutes, and even more preferably 15 minutes.

[0080] (Cuttable part: E) The cleavable moiety (E), another functional moiety in the tetrafunctional compound of the present invention, is a group that can be cleaved under specific conditions. Such a group is not particularly limited as long as it has the above-mentioned function, but is preferably a group that cleaves under mild reaction conditions. Examples of such a group include a group having the structure (moiety) of at least one compound selected from the group consisting of 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde), levulinoyl ester, vicinal diol, diazobenzene, bisarylhydrazone, dialkoxydiphenylsilane, disulfide, a peptide consisting of the sequence ENLYFQG (SEQ ID NO: 1), and a peptide consisting of the sequence ENLYFQS (SEQ ID NO: 2), or a disulfide group. Preferably, it is a group having the structure of Dde represented by the following formula (3):

[0081] [ka] (In the formula, R1, R2, R3, R4, R5, and R6 each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. A preferred embodiment is one in which R1 and R2 are alkyl groups, and R3, R4, R5, and R6 are hydrogen atoms. Here, the alkyl group is preferably an alkyl group having 1 to 3 carbon atoms, more preferably a methyl group. * represents a bond to an adjacent group.)

[0082] The mild reaction conditions mentioned above are conditions under which the captured protein is not decomposed by the treatment and contaminating proteins nonspecifically bound to the avidin beads are not eluted. Specific examples include conditions under which the cleavable site (E) is cleaved chemically or biochemically, such as treatment with a dilute aqueous hydrazine solution, a dilute aqueous sodium periodate solution, a dilute aqueous sodium dithionite solution, a dilute formic acid solution, a tobacco etch virus (TEV) protease solution, a dilute aqueous tris(2-carboxyethyl)phosphine solution, and a dilute aqueous dithiothreitol solution. A dilute aqueous hydrazine solution is more preferred. Here, "dilute" refers to a concentration of 5% by mass or less, preferably 3% by mass or less, although not limited thereto.

[0083] (Biotin site: B) The biotin moiety (E), which is another functional moiety in the tetrafunctional compound of the present invention, is a group having the structure of biotin represented by the following formula (1):

[0084] [ka] (In the formula, * means a bond to an adjacent group.)

[0085] The biotin moiety is a tag that can specifically bind to avidin- or streptavidin-bound solid supports (e.g., magnetic particles, beads, plates, filters, membranes, chromatography resins, etc.) and functions effectively in detecting, identifying, or purifying target proteins.

[0086] (Spacer) The tetrafunctional compound of the present invention is formed by linking the four functional group moieties described above via spacers that are independent of each other. Here, the spacer that links the four functional group moieties is a linear or branched alkylene group having one or more carbon atoms, and an alkylene chain consisting of a linear or branched alkylene group having three or more carbon atoms, in which non-adjacent -CH2- groups are independently -O-, -OCH2-, -CHO-, -CO-, -NR a -[R a means a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. The same applies hereinafter.], -CO-NR a -, -NR a Examples of suitable bridging groups include -CO-, a group represented by the following formula (1-1), and a group represented by the following formula (1-2). The bridging group can replace a -CH2- group within the alkylene chain or a -CH2- group at the end of the alkylene chain. R a is preferably a hydrogen atom.

[0087] [ka] (In the formula, m represents 0 or 1, and n represents 1 or 2.)

[0088] Specific examples of the group represented by the above formula (1-1) or the group represented by the formula (1-2) include the following formulae (1-3) to (1-16). [ka]

[0089] The type and length of the spacer can be selected so as to minimize steric congestion and not impair the effects of the four functional groups described above. To this extent, the number of carbon atoms in the alkylene group (alkylene chain length) is not limited, but is preferably 1 to 20 carbon atoms, and more preferably 2 to 10 carbon atoms, for example.

[0090] The tetrafunctional compounds of the present invention include linear compounds represented by the following formula (I) and branched compounds represented by the following formula (II). [ka]

[0091] (II-1) Linear tetrafunctional compounds and tetrafunctional chemical probes In formula (I), the ligand binding site represented by A, the reactive site represented by D, the cleavable site represented by E, and the biotin site represented by B are as described above. In formula (I), the sites represented by S1, S2, and S3 are spacer groups independent of each other.

[0092] Examples of the spacer group represented by S1 include groups represented by the following formula (5). [ka] (In the formula, a is an integer of 0 to 10, and b, c, d, e, f, g, and h are each independently an integer selected from 0 to 6.)

[0093] One embodiment of S1 is a linear alkylene group having 1 or more carbon atoms. This corresponds to the case where, in formula (5), g, c, and h are 0, and a is an integer of 1 or more. a may be any integer of 1 or more, and is preferably 1 to 30, and more preferably 1 to 10.

[0094] In another embodiment of S1, in an alkylene chain consisting of a linear alkylene group having 3 or more carbon atoms, non-adjacent -CH2- groups are independently -O-, -OCH2-, -CHO-, -CO-, -NR a -[R a means a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. The same applies hereinafter.], -CO-NR a - and -NR aExamples include a group substituted with at least one bridging group selected from the group consisting of -CO-, preferably at least one bridging group selected from the group consisting of -O-, -CO-, and -NH-CO-. Examples of the number of carbon atoms include, without limitation, 3 to 40, preferably 10 to 35. Specific examples include a case where, in formula (5), g is 0, a, b, d, and e are 2, c and f are 4, and h is 1, and a case where g, c, and f are 0, a is 3, d is 4, and h is 1.

[0095] The spacer groups represented by S2 and S3 may each independently be groups represented by the following formula (6): [ka] (In the formula, a, b, c, d, e, f, g, and h are each independently an integer selected from 0 to 6. Also, i is 0 or 1. However, a, c, h, and i cannot all be 0 at the same time.)

[0096] In one embodiment of S2, in an alkylene chain consisting of a linear alkylene group having one or more carbon atoms, non-adjacent -CH2- groups are independently -O-, -OCH2-, -CHO-, -CO-, -NR a -[R a means a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. The same applies hereinafter.], -CO-NR a - and -NR a Examples of the group include a group substituted with at least one bridging group selected from the group consisting of -CO-, preferably at least one bridging group selected from the group consisting of -O-, -NH-, and -NH-CO-. Examples of the number of carbon atoms include, without limitation, 1 to 20, preferably 1 to 5. Specifically, for example, in formula (6), a and c are 0, d is 3, f and g are 0, and i is 1.

[0097] In one embodiment of S3, in an alkylene chain consisting of a linear alkylene group having 3 or more carbon atoms, non-adjacent -CH2- groups are independently -O-, -OCH2-, -CHO-, -CO-, -NR a -[R a means a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. The same applies hereinafter.], -CO-NR a - and -NR a Examples of the group include a group substituted with at least one bridging group selected from the group consisting of -CO-, preferably at least one bridging group selected from the group consisting of -O- and -NH-. Examples of the number of carbon atoms include, without limitation, 3 to 20, preferably 5 to 15. Specifically, for example, in formula (6), a and b are 2, c is 4, and h and i are 0.

[0098] The molecular weight of the linear tetrafunctional compound is not limited as long as it can appropriately coordinate proteins with high affinity for the ligand when bound to the ligand-binding site (i.e., as a tetrafunctional chemical probe). It is typically 500 to 3000, but is preferably 800 to 2000, and more preferably 1000 to 1500. Having such a molecular weight can reduce membrane permeability. Furthermore, according to a literature review discussing the cell membrane permeability of various ligands (Methods Mol Biol. 2015; 1266: 29-53), it is believed that in order to reduce membrane permeability, the number of hydrogen bond donor groups should be 6 or more, the number of hydrogen bond acceptor groups should be 15 or more, and the polar surface area should be 140 Å or more, more preferably 300 Å or more. The tetrafunctional compounds and tetrafunctional chemical probes of the present invention described below have 6 to 9 hydrogen bond donor groups, 15 to 28 hydrogen bond acceptor groups, and a polar surface area of ​​300 Å or more. In recent years, membrane permeability has been considered to depend more on molecular size than molecular weight. Measurements have been conducted with cyclic peptides, and it is known that membrane permeability is reduced at molecular sizes of 800 Å or greater and significantly reduced at molecular sizes of 1000 Å or less. The tetrafunctional compounds and tetrafunctional chemical probes of the present invention, described below, have molecular sizes of 900 Å or greater. Furthermore, the aforementioned literature suggests that membrane permeability is good when the number of rotatable bonds is 10 or less. In other words, the number of rotatable bonds required to suppress membrane permeability is considered to be 10 or greater, and more preferably 30 or greater. The number of rotatable bonds in the tetrafunctional compounds and tetrafunctional chemical probes of the present invention is 33 to 60. The tetrafunctional chemical probes of the present invention, designed in this way, have reduced cell membrane permeability and are unable to penetrate membranes, allowing them to efficiently coordinate with membrane proteins.

[0099] A method for producing a linear tetrafunctional compound whose ligand binding site is an N-hydroxysuccinimide ester group (NHS ester group) will be described below using the compound (CPA-306) (see Production Example 15) as an example. Note that, when a compound whose ligand binding site is an NHS ester group is used, an antibody probe capable of recognizing and binding to a specific antigen as a target protein can be prepared by binding an antibody to the ligand binding site.

[0100] [Method for producing a linear tetrafunctional compound in which the ligand binding site is an NHS ester group] (Example) [ka] [ka] (In the formula, each q independently represents an integer of 0 to 6.)

[0101] (first step) This step can be carried out by stirring PEG-NH2 and allyl alcohol in the presence of thionyl chloride, either without a solvent or in a reaction-inert solvent, typically for 0.1 hours to 5 days at a temperature ranging from 0°C to reflux.

[0102] (Second process) This step can be carried out by stirring the compound obtained in the first step and 2-aryl-5-carboxytetrazole (ACT) in the presence of N-[1-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino(morpholino)]uronium hexafluorophosphate (COMU) and an amine in a reaction inert reaction solvent, usually for 0.1 hours to 5 days, under temperature conditions ranging from 0°C to reflux.

[0103] (Third step) Deprotection This step is carried out by stirring the compound obtained in the second step in the presence of morpholine and a palladium compound in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, under temperature conditions ranging from 0°C to reflux.

[0104] (Fourth step) Side chain extension This step can be carried out by stirring the compounds obtained in the first and third steps in the presence of COMU and an amine in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, at a temperature ranging from 0°C to reflux.

[0105] (Fifth step) Deprotection This step can be carried out by stirring the compound obtained in the fourth step and a deprotecting agent in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, under temperature conditions ranging from 0°C to reflux.

[0106] (Sixth step) This step can be carried out by stirring the compound obtained in the fifth step and the deprotected compound in the presence of an amine in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, under temperature conditions ranging from 0°C to reflux.

[0107] (Seventh step) Deprotection This step can be carried out by stirring the compound obtained in the sixth step in the presence of 1,3-dimethylbarbituric acid and a palladium compound in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, under temperature conditions ranging from 0°C to reflux.

[0108] (Eighth process) This step can be carried out by stirring the compound obtained in the seventh step and N-hydroxysuccinimide in the presence of carbodiimide (WSC) in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, under temperature conditions ranging from 0°C to reflux.

[0109] The desired antibody can be bound to the ligand binding site (NHS ester group) of the linear tetrafunctional compound thus prepared according to a known method, thereby producing the linear tetrafunctional chemical probe of the present invention. Examples of the method for binding the desired antibody to the NHS ester group include, but are not limited to, the following methods.

[0110] (Method for labeling antibodies with tetrafunctional compounds) A desired antibody having affinity for the target protein is adjusted to a certain concentration using a conventional NHS labeling buffer, and then the tetrafunctional compound of the present invention is added and reacted for approximately 5 to 60 minutes at a temperature of 0 to 37°C. Next, a lysine solution is added and reacted for approximately 5 to 60 minutes at 0 to 37°C. In this way, a linear tetrafunctional chemical probe (labeled antibody) can be obtained in which the desired antibody is bound to the ligand binding site (NHS ester group) of the tetrafunctional compound of the present invention.

[0111] On the other hand, linear tetrafunctional compounds in which the ligand-binding site is a ligand-reactive group other than an NHS ester group can be produced by directly binding a desired ligand to the ligand-binding site, i.e., as a tetrafunctional chemical probe. An example of the production method is described below.

[0112] [Method for producing linear tetrafunctional chemical probes] (Example) [First step] [ka] (In the formula, r represents an integer of 1 to 6. R1 and R2 are as defined above.)

[0113] This step can be carried out by stirring compound 1-1, carbodiimide, and cyclohexane-1,3-dione in the presence of N,N-dimethyl-4-aminopyridine (DMAP) in a reaction-inert reaction solvent, typically for 0.1 hours to 5 days, at a temperature ranging from 0°C to reflux.

[0114] [Second and third steps] [ka] (In the formula, m represents an integer of 1 to 6.)

[0115] (Second process) This step can be carried out by stirring Compound 1-3 and Compound 1-4 in the presence of COMU and an amine in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, at a temperature ranging from 0°C to reflux.

[0116] (Third step) This step can be carried out by stirring compound 1-5 in the presence of a base in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, at a temperature ranging from 0°C to reflux.

[0117] (Fourth step) [ka] (wherein s is an integer of 1 to 10, and m is the same as above). This step can be carried out by stirring compound 1-7 and compound 1-6 obtained in the third step in the presence of HATU and an amine in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, under temperature conditions ranging from 0°C to reflux.

[0118] (Fifth step) [ka] (wherein m, r, s, R1 and R2 are the same as above) This step can be carried out by stirring and concentrating compound 1-8 obtained in step 4 in the presence of a weak acid in a reaction-inert reaction solvent, typically for 0.1 hours to 5 days, at temperatures ranging from 0°C to reflux, followed by stirring compound 1-2 obtained in step 1 and an amine in a reaction-inert reaction solvent, typically for 0.1 hours to 5 days, at temperatures ranging from 0°C to reflux. Examples of "weak acids" that can be used in this reaction include formic acid, acetic acid, trifluoroacetic acid, citric acid, and oxalic acid. Examples of "amines" that can be used in this reaction include diisopropylethylamine and triethylamine.

[0119] The compounds 1-9 thus prepared have the desired ligand bound to the ligand-binding site of the tetrafunctional compound of the present invention, and can be used as tetrafunctional chemical probes. The ligands used here include, without limitation, the aforementioned compounds, excluding antibodies.

[0120] (II-2) Branched-chain tetrafunctional compounds and tetrafunctional chemical probes In the formula (II), the ligand binding site represented by A, the reactive site represented by D, the cleavable site represented by E, and the biotin site represented by B are as described above. In addition, in the formula (II), the sites represented by S4, S5, S6, and S7 are spacer groups independent of each other.

[0121] Examples of the spacer group represented by S4 include a group represented by the following formula (7). [ka] In the formula, a, b, c, d, e, f, g, h, and i are each independently an integer selected from 0 to 6, provided that a, c, and i are not all 0 at the same time, and d, f, and g are not all 0 at the same time.

[0122] In one embodiment of S4, in an alkylene chain consisting of a linear alkylene group having 3 or more carbon atoms, non-adjacent -CH2- groups are independently -O-, -OCH2-, -CHO-, -CO-, -NR a -[R a means a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. The same applies hereinafter.], -CO-NR a - and -NR a Examples include groups substituted with at least one crosslinking group selected from the group consisting of -CO-, preferably at least one crosslinking group selected from the group consisting of -O- and -NH-. Examples of the number of carbon atoms include, without limitation, 1 to 20, preferably 1 to 15, and more preferably 3 to 10. Specific examples include those in which, in the above formula, a is 2, b is 2, c is 2, h is 0, and i is 0.

[0123] Examples of the spacer group represented by S5 include groups represented by the following formulae (8) to (10). [ka] (In the formula, w, x, y, and z are each independently an integer selected from 0 to 6, provided that x and y are not 0 at the same time.)

[0124] In one embodiment of S5, in an alkylene chain consisting of a linear alkylene group having 3 or more carbon atoms (having a substituent), non-adjacent -CH2- groups are independently -O-, -OCH2-, -CHO-, -CO-, -NR a -[R a means a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. The same applies hereinafter.], -CO-NR a - and -NR aExamples of the group include a group substituted with at least one bridging group selected from the group consisting of -CO-, preferably at least one bridging group selected from the group consisting of -CO-, -NH-, -CO-NH-, and -NH-CO-. Examples of the number of carbon atoms include, without limitation, 3 to 20, preferably 3 to 15, and more preferably 5 to 15. Specifically, for example, in the above formula (8), a case where x and y are 4 can be exemplified.

[0125] Examples of the spacer group represented by S6 include groups represented by the following formula: [ka] (In the formula, a, b, c, and d are each independently an integer selected from 0 to 6, provided that a and c cannot be 0 at the same time.)

[0126] In one embodiment of S6, in an alkylene chain consisting of a linear alkylene group having 3 or more carbon atoms, non-adjacent -CH2- groups are independently -O-, -OCH2-, -CHO-, -CO-, -NR a -[R a means a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. The same applies hereinafter.], -CO-NR a - and -NR a Examples of the group include a group substituted with at least one bridging group selected from the group consisting of -CO-, preferably at least one bridging group selected from the group consisting of -O- and -NH-. Examples of the number of carbon atoms include, without limitation, 3 to 20, preferably 5 to 15. Specifically, for example, in the above formula (11), a is 3, b is 2, c is 3, and d is 1.

[0127] Examples of the spacer group represented by S7 include groups represented by the following formula: [ka] (In the formula, a, b, c, d, e, f, g, and h are each independently an integer selected from 0 to 6. However, a and c cannot be 0 at the same time, and a, c, g, and h cannot be 0 at the same time.)

[0128] In one embodiment of S7, in an alkylene chain consisting of a linear alkylene group having 3 or more carbon atoms, non-adjacent -CH2- groups are independently -O-, -OCH2-, -CHO-, -CO-, -NR a -[R a means a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. The same applies hereinafter.], -CO-NR a - and -NR a and -CO-, preferably a group substituted with at least one crosslinking group selected from the group consisting of -O-, -CO-, and -NH-CO-. Examples of the number of carbon atoms include, without limitation, 3 to 40, preferably 3 to 30, and more preferably 3 to 20. Specific examples include those in which, in the above formula, g is 0, a is 5, c is 0, d is 2, f is 0, and h is 1.

[0129] Another embodiment of the spacer group represented by S7 is a group represented by the following formula: [ka] (In the formula, a, b, c, d, e, f, and g are each independently an integer selected from 0 to 6. However, a and c cannot be 0 at the same time, and a, c, and h cannot be 0 at the same time.)

[0130] In one embodiment of S7, in an alkylene chain consisting of a linear alkylene group having 3 or more carbon atoms, non-adjacent -CH2- groups are independently -O-, -OCH2-, -CHO-, -CO-, -NR a -[R a means a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. The same applies hereinafter.], -CO-NR a -, -NR aExamples of the substituted group include at least one bridging group selected from the group consisting of -CO-, a group represented by the following formula (1-1), and a group represented by the following formula (1-2), preferably -O-, -CO-, -NH-CO-, a group represented by the following formula (1-3), a group represented by the following formula (1-7), and a group represented by the following formula (1-15). Examples of the number of carbon atoms include, without limitation, 3 to 40, preferably 3 to 30, and more preferably 3 to 20. Specifically, for example, in the above formula (13), g is 0, a is 5, c is 0, d is 2, f is 0, and h is 1.

[0131] [ka] (In the formula, m represents 0 or 1, and n represents 1 or 2.)

[0132] [ka]

[0133] The molecular weight of the branched tetrafunctional compound is not limited, as long as it can appropriately coordinate proteins with high affinity for the ligand when bound to the ligand-binding site (i.e., as a tetrafunctional chemical probe). It is typically 500 to 3000, but is preferably 800 to 2000, and more preferably 1000 to 1500. Having such a molecular weight can reduce membrane permeability. Furthermore, according to a literature review discussing the cell membrane permeability of various ligands (Methods Mol Biol. 2015; 1266: 29-53), in order to reduce membrane permeability, it is desirable for the compound to have 6 or more hydrogen bond donor groups, 15 or more hydrogen bond acceptor groups, or a polar surface area of ​​140 Å or more, more preferably 300 Å or more. The tetrafunctional compounds and tetrafunctional chemical probes of the present invention described below have 6 to 9 hydrogen bond donor groups, 15 to 28 hydrogen bond acceptor groups, and a polar surface area of ​​300 Å or more. In recent years, membrane permeability has been considered to depend more on molecular size than molecular weight. Measurements have been conducted with cyclic peptides, and it is known that membrane permeability is reduced at molecular sizes of 800 Å or greater and significantly reduced at molecular sizes of 1000 Å or less. The tetrafunctional compounds and tetrafunctional chemical probes of the present invention, described below, have molecular sizes of 900 Å or greater. Furthermore, the aforementioned literature suggests that membrane permeability is good when the number of rotatable bonds is 10 or less. In other words, the number of rotatable bonds required to suppress membrane permeability is considered to be 10 or greater, and more preferably 30 or greater. The number of rotatable bonds in the tetrafunctional compounds and tetrafunctional chemical probes of the present invention is 33 to 60. The tetrafunctional chemical probes of the present invention, designed in this way, have reduced cell membrane permeability and are unable to penetrate membranes, allowing them to efficiently coordinate with membrane proteins.

[0134] A method for producing a branched tetrafunctional compound having an N-hydroxysuccinimide ester group (NHS ester group) as the ligand binding site will be described below using the compound (CPA-306) (see Production Example 15) as an example. Note that when a compound having an NHS ester group as the ligand binding site is used, an antibody probe capable of recognizing and binding to a specific antigen as a target protein can be prepared by binding an antibody to the ligand binding site.

[0135] [Method for producing branched tetrafunctional compounds in which the ligand binding site is an NHS ester group] (Example)

[0136] For convenience of explanation, the reaction process is divided into five steps: [Reaction Step I] [ka] (In the formula, t is an integer of 0 to 6, and v is an integer of 1 to 5.)

[0137] (first step) This step can be carried out by stirring the diamine and the acid chloride in a reaction inert solvent usually for 0.1 hours to 5 days at a temperature ranging from 0° C. to reflux.

[0138] (Second process) This step can be carried out by stirring the diamine to which the protecting group has been attached in the first step and the linker having a branched amine in the presence of carbodiimide (WSC) and 1-hydroxybenzotriazole (HOBt) in a reaction inert reaction solvent, usually for 0.1 hours to 5 days, under temperature conditions ranging from 0°C to reflux.

[0139] (Third step) This step can be carried out by stirring the compound obtained in the second step in the presence of 1,3-dimethylbarbituric acid and a palladium compound in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, under temperature conditions ranging from 0°C to reflux.

[0140] (Fourth step) This step can be carried out by stirring the compound obtained in the third step and a biotin compound in the presence of an amine in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, under temperature conditions ranging from 0°C to reflux.

[0141] (Fifth step) This step can be carried out by stirring the compound obtained in the third step in the presence of piperidine in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, under temperature conditions ranging from 0°C to reflux.

[0142] [Reaction Step II] [ka] (In the formula, w represents an integer of 0 to 6.)

[0143] (first step) This step can be carried out by stirring a 1-aminoalkylcarboxylic acid and an allyl alcohol in the presence of thionyl chloride in a reaction inert solvent, usually for 0.1 hours to 5 days, at a temperature ranging from 0°C to reflux.

[0144] (Second process) This step can be carried out by stirring the 1-aminoalkylcarboxylic acid obtained in the first step and an alkyldicarboxylic anhydride in the presence of an amine in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, at a temperature ranging from 0°C to reflux. The alkyldicarboxylic anhydride used in the above reaction is not limited, but examples thereof include succinic anhydride, glutaric anhydride, adipic anhydride, etc.

[0145] [Reaction Step III] [ka] (In the formula, c and d each represent an integer of 0 to 6.)

[0146] (first step) This step can be carried out by stirring the BOC-protected diamine and tetrazole-containing carboxylic acid in the presence of an amine and HATU in a reaction inert solvent, typically for 0.1 hours to 5 days, at a temperature ranging from 0°C to reflux.

[0147] (Second process) This step can be carried out by stirring the compound obtained in the first step and a deprotecting agent in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, under temperature conditions ranging from 0°C to reflux.

[0148] (Third step) This step and the fourth step are optional steps for further extending the linker length. They can be performed by stirring the compound obtained in the second step and an aminocarboxylic acid having a BOC-protected amino group in the presence of an amine and COMU in a reaction-inert reaction solvent, usually for 0.1 hours to 5 days, at a temperature ranging from 0°C to reflux.

[0149] (Fourth step) This step can be carried out by stirring the compound obtained in the third step and a deprotecting agent in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, under temperature conditions ranging from 0°C to reflux.

[0150] [Reaction Step IV] [ka] (In the formula, t and u are each independently an integer of 0 to 6, and v, w, R1, and R2 are as defined above.)

[0151] (first step) This step can be carried out by stirring the compounds obtained in Reaction Steps II and III in the presence of water-soluble carbodiimide (WSC) and 1-hydroxybenzotriazole (HOBt) in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, at a temperature ranging from 0°C to reflux.

[0152] (Second process) This step can be carried out by stirring the compound obtained in the first step and a deprotecting agent in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, under temperature conditions ranging from 0°C to reflux.

[0153] (Third step) This step can be carried out by stirring the compound obtained in the second step and an alkyldicarboxylic anhydride in the presence of an amine in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, under temperature conditions ranging from 0°C to reflux.

[0154] (Fourth step) This step can be carried out by stirring the compound obtained in the third step and cyclohexanedione in the presence of water-soluble carbodiimide (WSC) and N,N-dimethyl-4-aminopyridine (DMAP) in a reaction inert solvent, usually for 0.1 hours to 5 days, under temperature conditions ranging from 0°C to reflux.

[0155] [Reaction Step V] [ka] (In the formula, C, u, v, w, t, R1, and R2 are as defined above.) (first step) This step can be carried out by stirring the compounds obtained in Steps III and IV, respectively, in the presence of an amine in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, at a temperature ranging from 0°C to reflux.

[0156] (Second process) This step can be carried out by stirring the compound obtained in the first step in the presence of a palladium compound and 1,3-dimethylbarbituric acid in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, under temperature conditions ranging from 0°C to reflux.

[0157] (Third step) This step can be carried out by stirring the compound obtained in the second step and N-hydroxysuccinimide in the presence of carbodiimide (WSC) in a reaction solvent inert to the reaction, usually for 0.1 hours to 5 days, under temperature conditions ranging from 0°C to reflux.

[0158] (Fourth step) This step is an optional step for converting the ligand-binding site, and can be carried out by stirring the compound obtained in the third step and 6-(piperazin-1-ylmethyl)picolinaldehyde in the presence of an amine (DIPEA) in a reaction-inert reaction solvent, usually for 0.1 hours to 5 days, at a temperature ranging from 0°C to reflux.

[0159] A desired antibody can be bound to the ligand binding site (NHS ester group) of the branched tetrafunctional compound thus prepared according to a known method, thereby producing the branched tetrafunctional chemical probe of the present invention. An example of a method for binding a desired antibody to the NHS ester group is the antibody labeling method using a tetrafunctional compound described above, although it is not limited thereto.

[0160] In each reaction in each of the above-described reaction schemes, the product can be used in the next reaction either as the reaction mixture itself or as a crude product, or it can be isolated from the reaction mixture in a conventional manner or easily purified by conventional separation means, such as recrystallization, distillation, and chromatography.

[0161] The starting compounds, intermediate compounds, and target compounds (tetrafunctional compounds and tetrafunctional chemical probes of the present invention) in each step include geometric isomers, stereoisomers, optical isomers, and tautomers. Various isomers can be separated by common optical resolution methods. They can also be produced from appropriate optically active starting compounds.

[0162] The tetrafunctional compound and tetrafunctional chemical probe of the present invention are not limited to the synthesis methods shown in the above reaction schemes, and can be produced by methods similar to those methods or other methods. Furthermore, unless otherwise specified, the starting compounds used in the production may be commercially available compounds, or compounds produced by known methods or methods similar thereto.

[0163] The starting compounds and target compounds in each step can be used in the form of suitable salts. These salts include, but are not limited to, acid addition salts and, depending on the type of substituent, salts with bases. Examples of acids that form such acid addition salts include inorganic acids (e.g., hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc.) and organic acids (e.g., methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, citric acid, tartaric acid, maleic acid, fumaric acid, malic acid, lactic acid, etc.). Examples of bases that form salts include inorganic bases (e.g., sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, etc.) and organic bases (e.g., methylamine, diethylamine, trimethylamine, triethylamine, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, tris(hydroxymethyl)methylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, guanidine, pyridine, picoline, choline, etc.) and ammonium salts. Furthermore, it may form a salt with an amino acid such as lysine, arginine, aspartic acid, or glutamic acid.

[0164] (II) Method for detecting target protein The above-described tetrafunctional chemical probe of the present invention can be used to detect a target protein in a sample. Detection of a target protein using the tetrafunctional chemical probe of the present invention can be carried out by a method including, but not limited to, the following steps: (1) reacting a tetrafunctional chemical probe with cells or tissues in a sample to bind a ligand in the tetrafunctional chemical probe to a target protein; (2) forming a covalent bond between the reactive site (D) in the tetrafunctional chemical probe and the target protein; (3) purifying the fraction containing the target protein bound to the tetrafunctional chemical probe; (4) binding the biotin moiety in the tetrafunctional chemical probe with avidin to form a tetrafunctional chemical probe-avidin complex; (5) cleaving the formed tetrafunctional chemical probe-avidin complex at the cleavable site in the tetrafunctional chemical probe; (6) detecting or identifying the target protein.

[0165] Each of these steps is explained below: (1) A step of binding a ligand to a protein This step can be carried out, without limitation, by culturing cells expressing a target protein at 37°C in a 5% CO2 atmosphere, and after confirming that the cells have become confluent, adding the tetrafunctional chemical probe of the present invention prepared in DMSO to a desired concentration, and allowing the cells to stand for approximately 30 to 60 minutes at 37°C in a 5% CO2 atmosphere.

[0166] (2) forming a covalent bond between the reactive site (D) in the tetrafunctional chemical probe and the target protein; This step can be carried out by, but is not limited to, using a UV crosslinker and irradiating with light at a wavelength of 254 to 365 nm, preferably 302 nm, for 1 second to 10 minutes, preferably 30 seconds to 5 minutes, and more preferably 1 minute.

[0167] (3) Step of purifying a fraction containing a target protein bound to a tetrafunctional chemical probe Although not limited to this step, the following method can be used, for example. First, Tris homogenate buffer is added to the cells, followed by homogenization, and then centrifugation. The precipitate is separated, and sodium carbonate buffer is added, followed by homogenization, and then incubation. By centrifuging and separating the precipitate, a fraction (e.g., a membrane fraction) containing a target protein bound to a tetrafunctional chemical probe can be purified and recovered.

[0168] (4) A step of binding the biotin moiety in the tetrafunctional chemical probe with avidin to form a tetrafunctional chemical probe-avidin complex. This step is not limited to, but includes, for example, adding 8M urea-containing RIPA buffer to the fraction collected above and dissolving the fraction in the buffer using an ultrasonic grinder. The supernatant is separated by centrifugation, and streptavidin magnetic particles, for example, are added. Thus, the biotin moiety in the chemical probe binds to avidin to form a tetrafunctional chemical probe-avidin complex. Furthermore, magnetic separation is performed according to standard methods, and the supernatant is discarded, allowing the tetrafunctional chemical probe-avidin complex to be purified and recovered.

[0169] (5) Cleavage of the formed tetrafunctional chemical probe-avidin complex at the cleavable site in the tetrafunctional chemical probe. This step can be performed, for example and without limitation, by adding SDS (sodium dodecyl sulfate) and hydrazine aqueous solution to the tetrafunctional chemical probe-avidin complex (magnetic particle complex) recovered in the previous step and allowing it to stand at room temperature (1-30°C) for 30 minutes. This reaction cleaves the cleavable site in the tetrafunctional chemical probe, separating the tetrafunctional chemical probe-avidin complex into the target protein binding domain and the biotin-avidin binding domain. The reaction solution is then magnetically separated to obtain the supernatant, allowing the target protein binding domain fragment to be recovered.

[0170] (6) detecting or identifying the target protein For detecting or identifying a target protein, the previously collected binding region fragment of the target protein may be subjected to purification or hydrolysis, if necessary. Specifically, but not limited to, for example, dithiothreitol is added to the obtained supernatant and stirred at 70°C for 15 minutes. Next, iodoacetamide is added and the mixture is left to stand at room temperature for 30 minutes. SP3 beads are added to the resulting solution, followed by MeCN and incubation for 18 minutes. After incubation, magnetic separation is performed and the supernatant is discarded. For example, peptide degradation treatment is performed by adding a trypsin solution to the collected residue and incubating at 37°C for 12 to 16 hours. MeCN is added, followed by incubation for 18 minutes, magnetic separation is performed, and the supernatant is discarded. Aqueous TFA solution is added to the residue and the mixture is incubated for 3 minutes. Magnetic separation is performed to obtain the supernatant.

[0171] The peptides thus prepared are desalted and resuspended in an appropriate buffer solution for analysis using a high-mass-accuracy mass spectrometer. The mass spectrometer is operated in a data-dependent acquisition mode, where ion signals above a predetermined threshold automatically trigger the instrument to switch from MS to MS / MS mode to generate collision-induced dissociation (CID) or higher-energy collision dissociation (HCD) spectra of the peptides. All MS / MS spectra are searched against a standard protein database.

[0172] The peptides thus obtained are analyzed. Any method for analyzing such compounds available in the field can be used, and the preferred method is mass spectrometry. Mass spectrometry methods are well known to those skilled in the art (see, for example, Yates, J. Mass Spect. 33:1-19 (1998); Kinter and Sherman, Protein Sequencing and Identification Using Tandem Mass Spectrometry, John Wiley and Sons, New York (2000); Aebersold and Goodlett, Chem. Rev. 101:269-295 (2001)). For high-resolution polypeptide fragment separation, liquid chromatography ESI-MS / MS or automated LC-MS / MS can be used, using capillary reversed-phase chromatography as a separation method (Yates et al., Methods Mol. Biol. 112:553-569 (1999)). Preferably, data-dependent collision-induced dissociation (CID) or high-energy collision-induced dissociation (HCD) with dynamic exclusion is used as the mass spectrometry method of choice (Goodlett et al., Anal. Chem. 72:1112-1118 (2000)). For such analyses, mass spectrometers are typically operated in a data-dependent acquisition mode, in which ion signals above a predetermined threshold automatically trigger the instrument to switch from MS to MS / MS mode to generate a collision-induced dissociation (CID) spectrum or a high-energy collision-induced dissociation (HCD) spectrum of the peptide.

[0173] For protein identification, all MS / MS spectra are searched against standard protein databases using standard algorithms (SEQUEST, Mascot, X!tandem, MS Aanda, etc.), with typical filtering to limit the false-positive protein identification rate to less than 1%.

[0174] In one embodiment, the concentration of membrane proteins in a sample can be quantitatively compared with that of a control sample. This allows for the detection of specific enrichment of target membrane protein receptors. For this non-labeled mass analysis, reversed-phase chromatography immediately prior to mass analysis can be displayed as an MS feature map, plotting retention time features against mass / charge ratio. As detected by the mass spectrometer, peptides in such a map appear with distinct isotopic patterns over a given time period and with a defined ion current intensity depending on their abundance in the sample. Once peptides are identified through fragmentation and MS / MS analysis, this information can be assigned to specific peptide features in the MS map and combined with semi-quantitative data analysis using open-source or commercial algorithms such as MaxQuant (Cox et al., Nature Biotechnology (2008) vol. 26 pp. 1367-1372) or Proteome Discoverer (Thermo Fischer Scientific). MS feature maps of different samples (e.g., sample vs. control) can then be overlaid and compared to obtain peptide abundance ratios. For stochastically labeled peptides derived from membrane proteins, these ratios should be approximately 1, with membrane protein peptides that are specifically captured based on the ligand obtaining higher values ​​compared to the control sample.

[0175] In other embodiments, alternative mass spectrometry-based quantification methods can be used, such as single reaction monitoring (SRM), stable isotope labeling with amino acids in cell culture (SILAC; see, e.g., Nilsson et al., Nat Methods (2010) vol. 7 (9) pp. 681-5), data-independent acquisition of mass spectra (SWATH MS; see, e.g., Gillet et al., (Targeted Data Extraction of the MS / MS Spectra Generated by Data-independent Acquisition: A New Concept for Consistent and Accurate Proteome Analysis)), tandem mass tags (TMT; see, e.g., Dayon et al., (Relative Quantification of Proteins in Human Cerebrospinal Fluids by MS / MS using 6-Plex Isobaric Tags)), etc.

[0176] It is contemplated that MS analysis may be substituted with other analytical methods, and such other methods are included as part of the present method.

[0177] By using the chemical probe of the present invention to identify the peptide sequence to which a portion of the probe structure has been added by mass spectrometry, the binding site between the target protein and the ligand can be determined. This allows the binding site with the ligand to be recognized, thereby making it possible to more accurately identify the target protein. Furthermore, it is possible to design probes with improved detection capabilities for the target protein.

[0178] The disclosures of all patent and non-patent literature cited herein are hereby incorporated by reference in their entirety. [Example]

[0179] The present invention will be further explained in detail by the following test examples and production examples, but these do not limit the present invention and may be modified within the scope of the present invention. The following abbreviations may be used in this specification:

[0180] [Table 1]

[0181] In the following examples, "room temperature" generally refers to about 10° C. to about 35° C. Ratios shown for mixed solvents are by volume unless otherwise specified. % refers to mass % unless otherwise specified.

[0182] 1 H NMR (proton nuclear magnetic resonance) spectra were measured using a Fourier transform NMR (either a Bruker AVANCE III 400 (400 MHz) or a Bruker AVANCE III HD (500 MHz)) using tetramethylsilane as the standard. Spectral splitting patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet or more overlapping signals (m), and broadened signals (br). The solvent was indicated in parentheses.

[0183] Mass spectra were measured using a Waters ACQUITY H-Class and SQD Mass spectrometer in combination with electrospray ionization (ESI). High-resolution mass spectrometry (HRMS) was measured using a Waters Xevo G2-XS QTof spectrometer in combination with electrospray ionization (ESI). [M+H] + means monoisotopic molecular weight.

[0184] In the examples, Yamazen Corporation's Hi-Flash column or Biotage's SNAP Ultra HP-Sphere 25 μm was used for silica gel column chromatography. Yamazen Corporation's Octadecyl C18 or Biotage's SNAP Ultra C18 was used for ODS column chromatography.

[0185] The anti-CD71 antibody OKT9 used in the test examples was manufactured by Thermo Fisher Scientific (Cat. # 16-0719-85), the anti-CD71 antibody DF1513 was manufactured by Abcam (ab212863), His-Tag EGF was manufactured by Oriental Yeast Co., Ltd. (47061000), and Goat anti-mouse IgG was manufactured by SouthernBiotech (Cat. 1030-01).

[0186] Production Example 1: Production of Compound (CPI-003) Compound (CPI-003) was produced according to the method shown in the following scheme.

[0187] [ka]

[0188] (1) Preparation of Compound (CPI-002) N-(tert-butoxycarbonyl)-4-aminobutyric acid (0.24 g), the compound represented by the formula (CPI-001) (0.31 g), COMU (0.55 g), and DIPEA (0.44 ml) were dissolved in DMF (2 ml) and stirred at room temperature for 17 hours. The reaction mixture was then concentrated. The residue was purified by silica gel chromatography (ODS, water / MeCN, containing 0.1% AcOH) to obtain the target product (CPI-002) (0.45 g). 1H NMR (500 MHz, DMSO-d6) δ10.31(s, 1H), 8.08 (d, J = 9.0 Hz, 2H), 7.89 (d, J = 9.0 Hz, 2H), 6.86 (t, J = 5.5 Hz, 1H), 4.47 (q, J = 7.0 Hz, 2H), 2.98 (q, J = 6.5 Hz, 2H), 2.36 (t, J = 7.5 Hz, 2H), 1.71 (m, J = 7.2 Hz, 2H), 1.38 (t, J = 7.1 Hz, 12H); HRMS (ESI) calculation for C 19 H 27 N6O5419.2043 [M+H] + , found 419.2054.

[0189] (2) Preparation of Compound (CPI-003) A solution of the synthesized CPI-002 (0.45 g) in MeOH (15 ml) and 0.2 M aqueous LiOH solution (5.9 mL) was added under ice cooling and stirred for 30 minutes. AcOH (0.30 μL) was added to the reaction solution, and the solvent was removed. The residue was purified by silica gel chromatography (ODS, water / MeCN, containing 0.1% AcOH) to obtain the target product (0.45 g). 1H NMR (500 MHz, DMSO-d6) δ 10.30 (s, 1H), 8.06 (d, J = 9.1 Hz, 2H), 7.89 (d, J = 9.1 Hz, 2H), 6.86 (t, J = 5.5 Hz, 1H), 2.98 (q, J = 6.6 HRMS (ESI) calcd for C 17 H 23 N6O5391.1730 [M+H] + , found 391.1724.

[0190] Production Example 2: Production of Compound (CPI-005) Compound (CPI-005) was produced according to the method shown in the following scheme.

[0191] [ka]

[0192] A mixture of dimedone (63 mg), the compound (CPI-004) shown above (0.20 g), WSC hydrochloride (82 mg), DMAP (50 mg), and DMF (3 mL) was stirred at room temperature for 14 hours, and the reaction mixture was concentrated. The residue was purified by silica gel chromatography (ODS, water / MeCN, containing 0.1% AcOH) to give the target product (0.14 g). 1H NMR (500 MHz, DMSO-d6) δ 7.82 (t, J = 5.6 Hz, 1H), 6.41 (br, 1H), 6.35 (br, 1H ), 4.30 (dd, J = 7.9, 5.0 Hz, 1H), 4.14-4.11 (m, 1H), 3.68 (t, J = 6.4 Hz, 2H), 3.52-3.49 (m, 12H), 3.39 (t, J = 5.9 Hz, 2H), 3.23 (t, J = 6.4 Hz, 2H), 3.18 (q, J = 5.7 Hz, 2H), 3.11-3.07 (m, 1H), 2.82 (dd, J = 12.5, 5.1 Hz, 1H), 2.63 (s, 2H), 2.57 (d, J = 12.5 Hz, 1H), 2.33 (s, 2H), 2.06 (t, J = 7.5 Hz, 2H), 1.64-1.57 (m, 1H), 1.54-1.42 (m, 3H), 1.36-1.24 (m, 2H), 1.00 (s, 6H); HRMS (ESI) calcd for C 29 H 48 N3O9S 614.3111 [M+H] + , found 614.3112.

[0193] Production Example 3: Production of Compound (CPI-104) The following formula shows the method and method for producing the compound (CPI-104).

[0194]

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[0195] (1) Production of compound (CPI-102) A mixture of the compound (CPI-101) (0.21 g), 1-bromo-3-chloropropane (5.0 mL), and NMP (5 mL) was reacted at 120 °C for 6 hours using a microwave reactor. Water was added to the reaction mixture, which was then extracted with AcOEt. The organic layer was washed with saturated brine and dried over Na2SO4, and the solvent was evaporated. The residue was purified by column chromatography (silica gel, hexane / AcOEt) to obtain the intermediate (0.18 g). A suspension of the intermediate (0.18 g), phthalimide (0.20 g), potassium carbonate (0.19 g), and sodium iodide (14 mg) in DMF (9 mL) was stirred at 70 °C for 10 hours. After evaporation of the solvent, water was added and the mixture was extracted with AcOEt. The organic layer was washed with saturated brine and dried over Na2SO4, and the solvent was evaporated. The residue was purified by column chromatography (silica gel, hexane / AcOEt) to obtain the compound (0.24 g). The obtained compound (0.24 g) was dissolved in DCM (9 mL), and chloroacetyl chloride (0.15 mL) and triethylamine (0.25 mL) were added and stirred at room temperature for 2 hours. Water was added to the reaction mixture, which was then extracted with DCM. The organic layer was washed with saturated brine, dried over Na2SO4, and the solvent was evaporated. The residue was purified by column chromatography (silica gel, hexane / AcOEt) to obtain the desired product (0.18 g). 1H NMR (400 MHz, CDCl3) δ 7.83-7.69 (m, 6H), 7.63-7.59 (m, 2H), 7.55 (d, J = 8.4 Hz, 1H), 7.49-7.45 (m, 3H), 4.15-4.05 (m, 1H), 3.99 (d, J = 13.0 Hz, 1H), 3.87 (d, J = 13.0 Hz, 1H), 3.64 (t, J = 7.5 Hz, 2H), 3.02-2.95 (m, 1H), 1.82-1.75 (m, 2H); LC-MS: [M+H] + = 495.04.

[0196] (2) Preparation of Compound (CPI-103) The compound (CPI-102) (0.18 g) synthesized above was dissolved in EtOH (5 mL), and hexamethylenetetramine (0.10 g) and AcONH4 (56 mg) were added. The mixture was heated to reflux for 9 hours. Hexamethylenetetramine (51 mg) and AcONH4 (28 mg) were added, and the mixture was heated to reflux for 9 hours. Water was added to the reaction mixture, and the mixture was extracted with AcOEt. The organic layer was washed with saturated brine, dried over Na2SO4, and the solvent was evaporated. The residue was purified by column chromatography (silica gel, hexane / AcOEt) to obtain the target product (0.14 g). 1H NMR (400 MHz, CDCl3) δ 7.83-7.79 (m, 2H), 7.73-7.68 (m, 2H), 7.67-7.64 (m, 2H), 7.50-7.40 (m, 4H), 7.31-7.29 (m, 2H), 4.80 (d, J = 10.6 Hz, 1H), 4.38 (dt, J = 14.4, 7.9 Hz, 1H), 3.74 (d, J = 10.6 Hz, 1H), 3.71-3.52 (m, 3H), 1.93 (tt, J = 7.2, 7.2 Hz, 2H); LC-MS: [M+2H] 2+ = 458.08.

[0197] (3) Preparation of Compound (CPI-104) To a solution (0.5 mL) of the compound (CPI-103) (10 mg) synthesized above in MeOH, methylamine and 40% MeOH solution (22 μL) were added and stirred at room temperature for 2 hours. A 40% MeOH solution (22 μL) of methylamine was added and stirred at room temperature for 2 hours. Water was added to the reaction mixture, which was then extracted with AcOEt. The organic layer was washed with saturated brine, dried over Na2SO4, and the solvent was evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (1.6 mg). 1H NMR (400 MHz, CD3OD) δ 7.66 (d, J = 1.4 Hz, 2H), 7.58-7.45 (m, 6H), 7.24 (t, J = 1.4 Hz, 1H), 4.64 (d, J = 10.6 Hz, 1H), 4.53-4.43 (m, LC-MS: [M+H] + = 328.17.

[0198] Production Example 4: Production of Compound (CPP-112) Compound (CPP-112) was produced according to the method shown in the following scheme. [ka]

[0199] A mixture of the compound (CPP-111) (0.44 g), hydroxylamine hydrochloride (0.18 g), pyridine (1.0 ml), and EtOH (20 mL) was heated to reflux for 2 hours. The solvent was evaporated, and the residue was acidified with 2 M hydrochloric acid and extracted with DCM. The organic layer was washed with saturated brine and dried over Na2SO4. The solvent was evaporated to give a colorless oily crude product (0.51 g). To a solution of DMAP (0.26 g) in DCM (15 mL), thionyl chloride (0.31 ml) was added under ice cooling and stirred for 30 minutes. Under ice cooling, a DCM solution (5 mL) of the crude product (0.51 g) obtained in the previous step and DMAP (0.39 g) were added, warmed to room temperature, and stirred for 3 days. Water was added to the reaction mixture, followed by extraction with DCM. The organic layer was washed with saturated brine and dried over Na2SO4, and the solvent was evaporated. The product was purified by column chromatography (silica gel, hexane / AcOEt) to give the desired product (0.30 g). 1H NMR (400 MHz, CDCl3)δ 7.94-7.89 (m, 1H), 7.87 (s, 1H), 7.42-7.37 (m, 2H), 3.92 (s, 3H), 2.84 (t, J = 7.3 Hz, 2H), 2.34 (t, J = 7.3 Hz, 2H), 2.02 (tt, J = 7.3, 7.3 Hz, 2H); LC-MS: [M+H] + = 204.02.

[0200] Production Example 5: Production of compound (CPP-117) Compound (CPP-117) was produced according to the method shown in the following scheme. [ka]

[0201] (1) Production of CPP-114 To a mixture of the compound (CPP-113) (100 mg) represented by the formula above and THF (3 mL), 1.6 M n-butyllithium in n-hexane (0.53 mL) was added at -78°C and stirred for 1 hour. A THF solution (2 mL) of the compound (CPP-112) (84 mg) prepared in Preparation Example 4 was added, and the mixture was stirred at -78°C for 5 hours. The mixture was warmed to room temperature over 3 hours and stirred at room temperature for 13 hours. Water was added to the reaction mixture, which was then extracted with AcOEt. The organic layer was washed with saturated brine, dried over Na2SO4, and the solvent was evaporated. The residue was purified by column chromatography (silica gel, hexane / AcOEt) to obtain the target product (48 mg). 1H NMR (400 MHz, CDCl3) δ 7.66-7.36 (m, 6H), 7.31-7.13 (brm, 1H), 3.14-2.92 (brm, 3H), 2.90-2.77 (brs, 2H), 2.34 (t, J = 6.9 Hz, 2H), 2.00 (tt, J = 6.9, 6.9 Hz, 2H), 1.26 (s, 9H); LC-MS: [M+Na] + = 434.94.

[0202] (2) Manufacturing of CPP-115 To a solution of the compound (CPP-114) (48 mg) prepared above in DCM (2 mL) was added TFA (0.45 mL) and stirred at room temperature for 2 hours, after which the solvent was evaporated. The residue was diluted with DCM (2 mL), and TEA (49 μL) and chloroacetyl chloride (28 μL) were added. The mixture was stirred at room temperature for 3 hours. Water was added to the reaction mixture, which was then extracted with AcOEt. The organic layer was washed with saturated brine, dried over Na2SO4, and the solvent was evaporated. The residue was purified by column chromatography (silica gel, hexane / AcOEt). A mixture of the purified product (38 mg), hexamethylenetetramine (33 mg), AcONH4 (18 mg), and EtOH (2 mL) was heated to reflux for 6 hours. The solvent was evaporated, and the mixture was purified by column chromatography (silica gel, hexane / AcOEt) to obtain the target product (28 mg). 1H NMR (400 MHz, CDCl3) δ 7.53 (dd, J = 8.8, 2.5 Hz, 1H), 7.50 (s, 1H), 7.41-7.30 (m, 4H), 7.27 (d, J = 2.5 Hz, 1H), 4.84 (d, J = 10.8 Hz, 1H), 3.77 (d, J = 10.8 Hz, 1H), 3.40 (s, 3H), 2.88-2.77 (m, 2H), 2.36 (t, J = 7.0 Hz, 2H), 2.05-1.97 (m, 2H); LC-MS: [M+H] + = 352.08.

[0203] (3) Manufacturing of CPP-116 A mixture of the compound (CPP-115) (28 mg), 30% aqueous hydrogen peroxide (0.16 mL), K2CO3 (11 mg), and DMSO (1 mL) was stirred at room temperature for 2 hours. Aqueous sodium thiosulfate was added and the mixture was stirred. The mixture was then extracted with AcOEt. The organic layer was washed with saturated brine, dried over Na2SO4, and the solvent was evaporated. The residue was purified by column chromatography (silica gel, AcOEt / MeOH) to obtain the desired product (28 mg). 1H NMR (400 MHz, CDCl3) δ 7.52 (dd, J = 8.8, 2.5 Hz, 1H), 7.50-7.46 (brm, 1H), 7.37-7.28 (m, 5H), 5.60-5.18 (brm, 2H), 4.82 (d, J = 10.8 Hz, 1H), 3.77 (d, J = 10.8 Hz, 1H), 3.40 (s, 3H), 2.79-2.65 (m, 2H), 2.23 (t, J = 7.5 Hz, 2H), 2.08-1.91 (m, 2H); LC-MS: [M+H] + = 370.00.

[0204] (4) Manufacturing of CPP-117 A mixture of the compound (CPP-116) (28 mg), lead tetraacetate (40 mg), and t-BuOH (0.72 mL) prepared above was stirred at 80°C for 3 hours. Lead tetraacetate (40 mg) was added, and the mixture was stirred at 80°C for 3 hours. After that, water / AcOEt was added and the mixture was filtered through Celite. The filtrate was extracted with AcOEt, and the organic layer was washed with saturated brine, dried over Na2SO4, and the solvent was evaporated. The residue was purified by column chromatography (silica gel, hexane / AcOEt) to obtain the target product (19 mg). 1H NMR (400 MHz, CDCl3) δ 7.53-7.49 (m, 2H), 7.33-7.28 (m, 5H), 4.83 (d, J = 10.8 Hz, 1H), 4.62-4.47 (brs, 1H), 3.77 (d, J = 10.8 Hz, 1H), 3.40 (s, 3H), 3.22-3.10 (brm, 2H), 2.68 (t, J = 7.6 Hz, 2H), 1.82 (tt, J = 7.6, 7.6 Hz, 2H), 1.44 (s, 9H); LC-MS: [M+H] + = 442.11.

[0205] Production Example 6: Production of compound (CPP-124) Compound (CPP-124) was produced according to the method shown in the following scheme.

[0206] [ka]

[0207] (1) Preparation of Compound (CPP-122) A mixture of the compound (CPP-121) (0.51 g), 60% NaH (72 mg), and DMF (50 mL) was stirred at room temperature for 30 minutes. 1-Bromo-3-chloropropane (0.18 mL) was added to the reaction mixture, and the mixture was stirred at room temperature for 60 hours. Water was added to the reaction mixture, and the mixture was concentrated. AcOEt was added to the residue, and the mixture was washed with water, dried over Na2SO4, and the solvent was evaporated. The residue was purified by column chromatography (silica gel, AcOEt / MeOH) to obtain the target product (0.45 g). 1H NMR (500 MHz, CDCl3) δ 7.61 (d, J = 2.7 Hz, 1H), 7.55 (d, J = 8.1 Hz, 1H), 7.42 (d, J = 5.5 Hz, 1H), 7.38 (d, J = 5.5 Hz, 1H), 7.27 (t, J = 7.8 Hz, 1H), 7.09 (d, J = 8.3 Hz, 1H), 6.99 (dd, J = 8.3, 2.7 Hz, 1H), 6.90 (d, J = 7.6 Hz, 1H), 4.11 (t, J = 6.3 Hz, 2H), 3.70 (t, J = 6.8 Hz, 2H), 3.63 (t, J = 6.4 Hz, 2H), 3.59 (t, J = 6.7 Hz, 2H), 3.20 (br, 4H), 2.94 (t, J = 6.6 Hz, 2H), 2.73 (br, 4H), 2.64 (t, J = 7.3 Hz, 2H), 2.15 (m, J = 6.7 Hz, 2H), 2.04 (m, J = 6.5 Hz, 2H); HRMS (ESI) calcd for C 27 H 33 N3O2S 498.1982 [M+H] + , found 498.1979.

[0208] (2) Preparation of Compound (CPP-123) A suspension of the compound (CPP-122) (0.45 g), phthalimide (0.16 mg), K2CO3 (0.15 g), and NaI (41 mg) in DMF (10 mL) was stirred at 70 °C for 17 hours. The reaction mixture was concentrated, and water was added to the residue. The mixture was then extracted with EtOAc. The organic layer was dried over Na2SO4, and the solvent was evaporated. The residue was purified by column chromatography (silica gel, AcOEt / MeOH) to give the desired product (0.43 g). 1H NMR (500 MHz, CDCl3) δ 7.84 (d, J = 3.0 Hz, 1H) 7.83 (d, J = 3.0 Hz, 1H), 7.60 (d, J = 2.7 Hz, 1H), 7.54 (d, J = 8.1 Hz, 1H), 7.41 (dd, J = 5.5, 0.6 Hz, 1H), 7.38 (d, J = 5.5 Hz, 1H), 7.27 (t, J = 7.8 Hz, 1H), 7.09 (d, J = 8.3 Hz, 1H), 6.98 (dd, J = 8.3, 2.8 Hz, 1H), 6.90 (dd, J = 7.7, 0.6 Hz, 1H), 4.10 (t, J = 6.4 Hz, 2H), 3.77 (t, J = 7.3 Hz, 2H), 3.66 (t, J = 7.1 Hz, 2H), 3.57 (t, J = 6.7 Hz, 2H), 3.20 (br, 4H), 2.96 (t, J = 6.6 Hz, 2H), 2.75 (br, 4H), 2.65 (t, J = 7.4 Hz, 2H), 2.07- 2.01 (m, 4H); HRMS (ESI) calculation for C 35 H 37 N4O4S 609.2535 [M+H] + , found 609.2538.

[0209] (3) Preparation of Compound (CPP-124) A mixture of the compound prepared above (CPP-123 (0.43 g)), hydrazine monohydrate (0.069 mL), and EtOH (12 mL) was heated to reflux for 8 hours. The reaction mixture was concentrated, and 1 M aqueous NaOH solution (50 mL) was added to the residue, followed by extraction with AcOEt. The organic layer was washed with saturated brine, dried over Na2SO4, and the solvent was evaporated. The residue was purified by column chromatography (silica gel, DCM / MeOH). The purified product was dissolved in MeOH (10 mL), and 2 M aqueous HCl solution (2.1 mL) was added dropwise. The mixture was stirred at room temperature for 15 hours, and the solvent was evaporated. AcOEt was added to the residue, and the mixture was stirred. The resulting solid was collected by filtration to obtain the target product (0.35 g). 1H NMR (500 MHz, CDCl3) δ 11.4 (br, 1H), 7.98 (br, 3H), 7.78 (d, J = 5.5 Hz, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.50 (d, J = 5.5 Hz, 1H), 7.43 (d, J = 2.7 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.26 (d, J = 8.4 Hz, 1H), 7.10 (dd, J = 8.3 , 2.7 Hz, 1H), 6.98 (d, J = 7.6 Hz, 1H), 4.13 (t, J = 5.9 Hz, 2H), 3.66 (br, 2H), 3.57-3.53 (m, 6H), 3.37 (br, under the water signal), 3.27 (t, J = 11.9 Hz, 2H), 2.92 (t, J = 6.5 Hz, 2H), 2.82-2.76 (m, 2H), 2.30-2.25 (m, 2H), 1.88 (m, J = 7.3 Hz, 2H); HRMS (ESI) calculation for C 27 H 35 N4O2S 479.2481 [M+H] + , found 479.2482.

[0210] Production Example 7: Production of compound (CPP-125) Compound (CPP-125) was produced according to the method shown in the following scheme.

[0211] [ka]

[0212] A mixture of the compound (CPP-124) (20 mg) prepared in Preparation Example 6, Boc-5-amino-n-valeric acid (7.9 mg), COMU (17 mg), DIPEA (27 μL), and DMF (1 ml) was stirred at room temperature for 5 hours, and the reaction mixture was concentrated. The residue was purified by silica gel chromatography (ODS, water / MeCN, containing 0.1% AcOH) to obtain the target product (15 mg). 1H NMR (500 MH, DMSO-d6) δ 7.78 (t, J = 5.5 Hz, 1H), 7.69 (d, J = 5.6 Hz, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.40 (d, J = 5.6 Hz, 1H), 7.39 (d, J = 2.7 Hz, 1H), 7.27 (t, J = 7.8 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.05 (dd, J = 8.3, 2.6 Hz, 1H), 6.90 (d, J = 7.7 Hz, 1H), 6.77 (t, J = 5.4 Hz, 1H), 4.06 (t, J = 6.3 Hz, 2H), 3.50 (t, J = 7.0 Hz, under the water signal), 3.46 (t, J = 6.6 Hz, under the water signal), 3.08-3.03 (m, 6H), 2.91-2.87 (m, 4H), 2.64 (br, 4H), 2.54 (t, J = 7.1 Hz, 2H), 2.04 (t, J = 7.3 Hz, 2H), 1.93 (m, J = 6.7 Hz, 2H), 1.82 (s, 3H), 1.67 (m, J = 7.0 Hz, 2H), 1.46 (m, J = 7.5 Hz, 2H), 1.36-1.31 (m, 11H) ;HRMS (ESI) calcd for C 37 H 52 N5O5S 678.3689 [M+H] + , found 678.3693.

[0213] Production Example 8: Production of compound (CPF-202) The following formula shows the method and method for producing the compound (CPF-202).

[0214]

change

[0215] (1) Manufacturing of CPF-201 A mixture of compound (CPI-104) (9.0 mg) prepared in Preparation Example 3, compound (CPI-003) (11 mg) prepared in Preparation Example 1, COMU (13 mg), DIPEA (11 μL), and DMF (1 mL) was stirred at room temperature for 2 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN, containing 0.1% AcOH) to obtain the target product (10 mg). 1H NMR (400 MHz, CDCl3) δ 9.57 (s, 1H), 8.13 (dt, J = 9.1, 2.4 Hz, 2H), 7.87 (d, J = 8.9 Hz, 2H), 7.64-7.59 (m, 3H), 7.54-7.47 (m, 2H), 7.45-7.39 (m, 3H), 7.30 (d, J = 2.4 Hz), 4.88-4.83 (m, 2H), 4.54-4.47 (m, 1H), 3.81-3.71 (m, 2H), 3.64-3.56 (m, 1H), 3.29 (dt, J = 6.1, 6.1 Hz, 2H), 3.11-3.03 (m, 1H), 2.44-2.41 (m, 2H), 2.00-1.86 (m, 3H), 1.77-1.65 (m, 1H), 1.50 (s, 9H); LC-MS: [M+H] + = 700.29.

[0216] (2) Manufacturing of CPF-202 A mixture of the compound (CPF-201) (10 mg) prepared above, TFA (0.11 mL), and DCM (1 mL) was stirred at room temperature for 2 hours, and then the solvent was evaporated. A mixture of the residue, the compound (CPI-005) (10 mg) prepared in Preparation Example 2, DIPEA (10 μL), and DMF (1 mL) was stirred at 60° C. for 3 hours, and then the solvent was evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (11 mg). 1H NMR (400 MHz, CDCl3) δ 13.69-13.66 (m, 1H), 9.41 (s, 1H), 8.09 (dt, J = 9.0, 2.4 Hz, 2H), 7.88-7.86 (m, 3H), 7.61-7.59 (m, 2H), 7.54-7.48 (m, 2H), 7.45-7.39 (m, 3H), 7.30 (d, J = 2.4 Hz, 1H), 6.44-6.39 (m, 1H), 5.61-5.58 (m, 1H), 4.85 (d, J = 10.5 Hz, 1H), 4.68 (d, J = 7.4 Hz, 1H), 4.53-4.46 (m, 2H), 4.35-4.32 (m, 1H), 3.81-3.48 (m, 20H), 3.37-3.29 (m, 4H), 3.17-3.05 (m, 2H), 2.95-2.90 (m, 1H), 2.72 (dd, J = 12.8, 4.0 Hz, 1H), 2.57 (t, J = 7.1 Hz, 2H), 2.34 (s, 4H), 2.14 (dd, J = 7.1, 7.1 Hz, 4H), 1.99-1.91 (m, 1H), 1.77-1.52 (m, 6H), 1.47-1.41 (m, 2H), 1.00 (s, 6H); LC-MS: [M+H] + = 1195.3.

[0217] Production Example 9: Production of compound (CPF-212)

Chem.

[0218] (1) Preparation of CPF-211 TFA (0.17 mL) was added dropwise to a DCM (1 mL) solution of the compound (CPP-117) (19 mg) produced in Production Example 5, and the mixture was stirred at room temperature for 2 hours. The solvent was then evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the intermediate compound (13 mg). The intermediate compound (13 mg) and the compound (CPI-003) (16.78 mg) produced in Production Example 1 were dissolved in DMF (1 mL), and COMU (18 mg) and DIPEA (0.23 mL) were added. The mixture was stirred at room temperature for 3 hours. The solvent was then evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (18 mg). 1H NMR (400 MHz, CDCl3) δ 9.62 (s, 1H), 8.14 (dt, J = 9.1, 2.4 Hz, 2H), 7.90-7.87 (m, 2H), 7.53-7.50 (m, 2H), 7.38-7.33 (m, 3H), 7.30 (dd, J = 5.6, 3.1 Hz, 2H), 7.26-7.22 (m, 1H), 4.84-4.82 (m, 2H), 3.77 (d, J = 10.8 Hz, 1H), 3.60 (dt, J = 6.8, 6.8 Hz, 2H), 3.40 (s, 3H), 3.29 (dt, J = 6.1, 6.1 Hz, 2H), 2.79 (t, J = 7.7 Hz, 2H), 2.44-2.41 (m, 2H), 2.07-2.00 (m, 2H), 1.92-1.86 (m, 2H), 1.50 (s, 9H); LC-MS: [M+H] + = 714.08.

[0219] (2) Manufacturing of CPF-212 TFA (97 μL) was added dropwise to a DCM (1 mL) solution of the compound (CPF-211) (18 mg) prepared above, and the mixture was stirred at room temperature for 1.5 hours. The solvent was then evaporated. DIPEA (18 μL) was added to a DMF (1 mL) solution of the residue and the compound (CPI-005) (22 mg) prepared in Preparation Example 2, and the mixture was stirred at 60°C for 3 hours. DIPEA (18 μL) was added, and the mixture was stirred at 60°C for an additional 3 hours. The solvent was then evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (13 mg).1H NMR (400 MHz, DMSO-d6) δ 13.53 (t, J = 5.1 Hz, 1H), 10.37 (s, 1H), 9.19 (t, J = 5.7 Hz, 1H), 8.06 (dt, J = 9.1, 2.3 Hz, 2H), 7.89 (dt, J = 9.1, 2.3 Hz, 2H), 7.81 (t, J = 5.6 Hz, 1H), 7.70 (dd, J = 8.9, 2.5 Hz, 1H), 7.60 (d, J = 8.9 Hz, 1H), 7.49 (s, 1H), 7.42-7.36 (m, 2H), 7.30 (dt, J = 7.4, 1.6 Hz, 1H), 7.22 (d, J = 2.5 Hz, 1H), 6.40 (s, 1H), 6.35 (s, 1H), 4.57 (d, J = 10.7 Hz, 1H), 4.31-4.28 (m, 1H), 4.13-4.10 (m, 1H), 3.78 (d, J = 10.7 Hz, 1H), 3.63-3.58 (m, 3H), 3.47-3.46 (m, 9H), 3.38-3.05 (m, under the water signal), 2.80 (dd, J = 12.4, 5.1 Hz, 1H), 2.71-2.66 (m, 3H), 2.58-2.49 (m, overlapped with the DMSO signal), 2.28 (s, 4H), 2.05 (t, J = 7.4 Hz, 2H), 1.97-1.84 (m, 4H), 1.64-1.55 (m, 1H), 1.52-1.40 (m, 3H), 1.32-1.24 (m, 2H), 0.94 (s, 6H); LC-MS: [M+H]. + = 1209.12.

[0220] Production Example 10: Production of compound (CPF-224) The following formula shows the method and method for producing the compound (CPF-224).

[0221]

change

[0222] (1) Preparation of Compound (CPF-222) The compound prepared in Preparation Example 1 (CPI-003, 20 mg) and the compound represented by the formula above (CPF-221, 23 mg) were dissolved in DMF (2 mL), COMU (24 mg) and DIPEA (0.020 mL) were added, and the mixture was stirred for 1 hour. The solvent was then evaporated. The residue was purified by silica gel chromatography (ODS, water / MeCN, containing 0.1% AcOH) to obtain the target product (33 mg). 1H NMR (400 MHz, DMSO-d6) δ 12.46 (br, 1H), 10.30 (s, 1H), 9.43 (t, J = 6.1 Hz, 1H), 8.06 (d, J = 9.2 Hz, 2H), 7.89 (d, J = 9.1 Hz, 2H), 7.60 (s, 1H), 7.52 (br, 1H), 6.88-6.83 (m, 3H), 6.28 (dd, J = 2.9, 1.7 Hz, 1H), 5.00 (s, 2H), 4.41 (d, J = 6.0 Hz, 2H), 4.13 (q, J = 7.1 Hz, 2H), 2.98 (q, J = 6.6 Hz, 2H), 2.36 (t, J = 7.3 Hz, 2H), 1.71 (m, J = 7.3 Hz, 2H), 1.38 (s, 9H), 1.17 (t, J = 7.1 Hz, 3H); LC-MS: [M+H] + = 783.31.

[0223] (2) Preparation of Compound (CPF-223) To a solution of the compound (CPF-222) (33 mg) synthesized above in MeOH (2 mL), 1M aqueous LiOH solution (0.13 mL) and water (0.36 mL) were added and stirred for 21 hours. The reaction mixture was neutralized with 0.1% aqueous AcOH, and the solution was concentrated. The residue was purified by silica gel chromatography (ODS, water / MeCN, containing 0.1% AcOH) to obtain the target product (30 mg). 1H NMR (400 MHz, DMSO-d6) δ 12.46 (s, 1H), 10.30 (s, 1H), 9.43 (t, J = 5.9 Hz, 1H), 8.06 (d, J = 9.1 Hz, 2H), 7.88 (d, J = 9.1 Hz, 2H), 7.61 (s, 1H), 7.47 (br, 1H), 6.90-6.83 (m, 3H), 6.28 (dd, J = 2.9, 1.7 Hz, 1H), 4.92 (s, 2H), 4.41 (d, J = 5.9 Hz, 2H), 2.98 (q, J = 6.5 Hz, 2H), 2.36 (t, J = 7.4 Hz, 2H), 1.71 (m, J = 7.2 Hz, 2H), 1.38 (s, 9H); LC-MS: [M+H] + = 754.99.

[0224] (3) Preparation of Compound (CPF-224) The compound (CPF-223) (29 mg) synthesized above was dissolved in DCM (3 mL), and TFA (0.29 mL) was added dropwise. The mixture was stirred at room temperature for 2 hours, and then the solution was concentrated. DIPEA (27 μL), the compound (CBI-455) (39 mg) prepared in Preparation Example 2, and DMF (2 mL) were added to the residue, and the mixture was stirred at 60° C. for 5 hours. The reaction mixture was then concentrated. The residue was purified by silica gel chromatography (ODS, water / MeCN, containing 0.1% TFA) to obtain the target product (17 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.53 (t, J = 5.5 Hz, 1H), 12.47 (s, 1H), 10.38 (s, 1H), 9.44 (t, J = 6.2 Hz, 1H), 8.07 (d, J = 9.1 Hz, 2H), 7.89 (d, J = 9.1 Hz, 2H), 7.82 (t, J = 5.7 Hz, 1H), 7.61 (s, 1H), 7.49 (br, 1H), 6.90 (br, 1H), 6.84 (t, J = 2.4 Hz, 1H), 6.41 (br, 1H), 6.35 (br, 1H), 6.28 (dd, J = 2.8, 1.7 Hz, 1H), 4.95 (s, 2H), 4.41 (d, J = 5.9 Hz, 2H), 4.30 (dd, J = 7.8, 4.4 Hz, 1H), 4.12 (dd, J = 7.6, 4.4 Hz, 1H), 3.61 (t, J = 6.3 Hz, 4H), 3.49-3.46 (m, under the water signal), 3.30 -3.25 (m, 4H), 3.17 (q, J = 5.7 Hz, 2H), 3.10-3.06 (m, 1H), 2.81 (dd, J = 12.5, 5.1 Hz, 1H), 2.57 (d, J = 12.5 Hz, 1H), 2.47 (t, overlapped with the DMSO signal), 2.28 (s, 4H), 2.05 (t, J = 7.4 Hz, 2H), 1.93 (m, J = 6.9 Hz, 2H), 1.60-1.55 (m, 1H), 1.53-1.40 (m, 3H), 1.33-1.23 (m, 2H)), 0.94 (s, 6H) ; HRMS (ESI) calcd for C 57 H 71 N 13 O 14 F3S 1250.4916 [M+H] + , found 1250.4919..

[0225] Production Example 11: Production of compound (CPF-232) Compound (CPF-232) was produced according to the method shown in the following scheme.

[0226] [ka]

[0227] (1) Preparation of compound (CPF-231) The compound (CPP-124, 40 mg) prepared in Preparation Example 6 and the compound (CPI-003, 26 mg) prepared in Preparation Example 1 were diluted with DMF (2 ml), and HATU (26 mg) and DIPEA (0.036 ml) were added. The mixture was stirred for 5 hours, and the reaction mixture was concentrated. The residue was purified by silica gel chromatography (ODS, water / MeCN, containing 0.1% TFA) to obtain the target product (50 mg). 1H NMR (500 MHz, DMSO-d6) δ 11.96 (br, 1H), 10.30 (s, 1H), 9.16 (t, J = 5.8 Hz, 1H), 8.06 (d, J = 9.1 Hz, 2H), 7.89 (d, J = 9.1 Hz, 2H), 7.74 (d, J = 5.1 Hz, 1H), 7.67 (d, J = 7.7 Hz, 1H), 7.46 (d, J = 4.1 Hz, 1H), 7.43 (d, J = 2.3 Hz, 1H), 7.30 (t, J = 7.8 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.07 (dd, J = 8.3, 2.7 Hz, 1H), 6.95 (d, J = 7.4 Hz, 1H), 6.86 (t, J = 5.4 Hz, 1H), 4.10 (t, J = 5.4 Hz, 2H), 3.62-3.51 (m, 6H), 3.38-3.35 (m, under the water signal), 3.10 (br, 2H), 2.98 (q, J = 6.5 Hz, 2H), 2.92 (t, J = 6.5 Hz, 2H), 2.36 (t, J = 7.4 Hz, 2H), 2.08 (br, 2H), 1.91 (s, 3H), 1.86 (m, J = 7.0 Hz, 2H), 1.72 (m, J = 7.2 Hz, 2H); LC-MS: [M+H] + = 609.52

[0228] (2) Preparation of Compound (CPF-232) The compound (CPF-231) (19 mg) prepared above was dissolved in DCM (2 mL), and TFA (0.16 mL) was added dropwise. The mixture was stirred at room temperature for 1 hour, and then the reaction mixture was concentrated. The compound (CPI-005 (15 mg)) prepared in Preparation Example 2, DIPEA (17 μL), and DMF (2 mL) were added to the residue, and the mixture was stirred at 60° C. for 2 hours. The reaction mixture was then concentrated. The residue was purified by silica gel chromatography (ODS, water / MeCN, containing 0.1% TFA) to give (8.7 mg). 1H NMR (500 MHz, DMSO-d6) δ 13.53 (t, J = 5.3 Hz, 1H), 10.38 (s, 1H), 9.51 (br, 1H ), 9.17 (t, J = 5.9 Hz, 1H), 8.06 (d, J = 9.1 Hz, 2H), 7.89 (d, J = 9.1 Hz, 2H), 7.81 (t, J = 5.5 Hz, 1H), 7.78 (d, J = 5.5 Hz, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.51 (d, J = 5.6 Hz, 1H), 7.45 (d, J = 2.7 Hz, 1H), 7.33 (t, J = 7.9 Hz, 1H), 7.25 (d, J = 8.5 Hz, 1H), 7.08 (dd, J = 8.3, 2.7 Hz, 1H), 6.99 (d, J = 7.7 Hz, 1H), 6.41 (br, 1H), 6.36 (br, 1H ), 4.29 (dd, J = 7.8, 4.9 Hz, 1H), 4.14-4.10 (m, 3H), 3.70 (br, 2H), 3.62-3.55 (m, under the water signal), 3.41-3.34 (m, under the water signal), 3.27 (t, J = 6.2 Hz, 2H), 3.16 (q, J = 5.8 Hz, 2H), 3.10-3.06 (m, 3H), 2.92 (t, J = 6.6 Hz, 2H), 2.80 (dd, J = 12.3, 5.1 Hz, 1H), 2.57 (d, J = 12.4 Hz, 1H), 2.28 (s, 4H), 2.22-2.17 (m, 2H), 2.05 (t, J = 7.4 Hz, 2H), 1.93 (m, J = 7.1 Hz, 2H), 1.86 (m, J = 6.8 Hz, 2H), 1.63-1.56 (m, 1H), 1.51-1.40 (m, 3H), 1.33-1.22 (m, 2H), 0.94 (s, 6H) ; HRMS (ESI) calcd for C 68 H 92 N 13 O 12S21346.6429 [M+H] + , found 1346.6445.

[0229] Production Example 12: Production of compound (CPP-127) Compound (CPP-127) was produced according to the method shown in the following scheme.

[0230] [ka]

[0231] (1) Preparation of Compound (CPP-126) The compound prepared in Preparation Example 7 (CPP-125, 15 mg) was dissolved in DCM (3 mL), and TFA (0.15 μL) was added dropwise. The mixture was stirred at room temperature for 2 hours, and then the reaction mixture was concentrated. The residue was diluted with the compound prepared in Preparation Example 1 (CPI-003 (9.1 mg)) and DMF (2 mL). COMU (9.35 mg) and DIPEA (15 μL) were added, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was then concentrated. The residue was purified by silica gel chromatography (ODS, water / MeCN, containing 0.1% AcOH) to obtain the target product (16 mg). 1H NMR (500 MHz, DMSO-d6) δ 11.94 (br, 1H), 10.29 (s, 1H), 9.14 (t, J = 5.9 Hz, 1H), 8.04 (d, J = 9.1 Hz, 2H), 7.87 (d, J = 9.1 Hz, 2H), 7.81 (t, J = 5.5 Hz, 1H), 7.69 (br, 1H), 7.61 (br, 1H ), 7.41-7.38 (m, 2H), 7.28 (t, J = 7.6 Hz, 1H), 7.19 (d, J = 8.6 Hz, 1H), 7.04 (dd, J = 8.5, 2.7 Hz, 1H), 6.90 (br, 1H), 6.86 (t, J = 5.4 Hz, 1H), 4.05 (br, 2H), 3.50 (t, J = 6.5 Hz, 2H), 3.46 (t, J = 7.1 Hz, 2H), 3.09-3.04 (m, 6H), 2.98 (q, J = 6.5 Hz, 2H), 2.87 (t, J = 6.7 Hz, 2H), 2.64(br,4H),2.36 (t, J = 7.4 Hz, 2H), 2.10 (br, 2H), 1.94 (br, 2H), 1.91 (s, 3H), 1.71 (m, J = 7.2 Hz, 2H), 1.67 (m, J = 7.0 Hz, 2H), 1.55 (br, 4H), 1.37 (s, 9H); HRMS (ESI) calculation for C 49 H 64 N 11 O7S 950.4711 [M+H] +, found 950.4713.

[0232] (2) Preparation of Compound (CPP-127) The compound (CPP-126) (14 mg) prepared above was dissolved in DCM (5 mL), and TFA (0.23 mL) was added dropwise. The mixture was stirred at room temperature for 12 hours, and then the reaction mixture was concentrated. The compound (CPI-005 (9.2 mg)) prepared in Preparation Example 2, DIPEA (12 μL), and DMF (2 mL) were added to the residue, and the mixture was stirred at 60°C for 2 hours. The reaction mixture was then concentrated. The residue was purified by silica gel chromatography (ODS, water / MeCN, containing 0.1% TFA) to obtain the target product (13 mg). 1H NMR (500 MHz, DMSO-d6) δ 13.53 (t, J = 5.4 Hz, 1H), 10.38 (s, 1H), 9.63 (br, 1H), 9.15 (t, J = 5.9 Hz, 1H), 8.05 (d, J = 9.1 Hz, 2H), 7.88 (d, J = 9.1 Hz, 2H), 7.84-7.81 (m, 2H), 7.78 (d, J = 5.6 Hz, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.51 (t, J = 5.5 Hz, 1H), 7.43 (d, J = 2.8 Hz, 1H), 7.33 (t, J = 7.9 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.06 (dd, J = 8.4, 2.8 Hz, 1H), 6.99 (d, J = 7.3 Hz, 1H), 6.41 (br, 1H), 6.36 (br, 1H), 4.29 (dd, J = 7.9, 4.9 Hz, 1H), 4.13-4.10 (m, under the water signal), 3.70 (br, 2H), 3.62-3.58 (m, 6H), 3.52-3.49 (m, 4H), 3.47-3.46 (m, 12H), 3.41 (br, 4H), 3.37 (t, J = 5.9 Hz, 2H), 3.32 (br, 2H), 3.27 (t, J = 6.1 Hz, 2H), 3.17 (q, J = 5.8 Hz, 2H), 3.11-3.04 (m, 5H), 2.88 (t, J = 6.4 Hz, 2H), 2.81 (dd, J = 12.4, 5.1 Hz, 1H), 2.57 (d, J = 12.3 Hz, 1H), 2.48 (d, overlapped with the DMSO signal), 2.28 (s, 4H), 2.22-2.17 (m, 2H), 2.11 (br, 2H), 2.05 (t, J = 7.3 Hz, 2H), 1.93 (m, J = 7.0 Hz, 2H), 1.68 (m, J = 6.9 Hz, 2H), 1.62-1.41 (m, 8H), 1.33-1.23 (m, 2H), 0.94 (s, 6H); HRMS (ESI) calcd for C. 73 H 101 N 14 O 13 S21445.7114 [M+H] + , found 1445.7126.

[0233] Production Example 13: Production of compound (CPP-133) Compound (CPP-133) was produced according to the method shown in the following scheme.

[0234] [ka]

[0235] (1) Manufacturing of CPP-132 Compound (CPP-131) (15 mg) and compound (CPI-003) (16 mg) prepared in Preparation Example 1 were dissolved in DMF (1 mL), and COMU (19 mg) and DIPEA (15 μL) were added. The mixture was stirred at room temperature for 20 hours, and the solvent was then evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (22 mg). 1H NMR (400 MHz, CDCl3) δ 9.30 (s, 1H), 9.11 (s, 1H), 8.10 (dt, J = 9.1, 2.4 Hz, 2H), 7.85 (d, J = 9.1 Hz, 2H), 7.31 - 7.25 (m, 2H), 7.05 (d, J = 2.4 Hz, 1H), 7.01 (td, J = 7.5, 0.9 Hz, 1H), 6.86 (dd, J = 7.7, 0.7 Hz, 1H), 6.82 (dd, J = 8.3, 2.4 Hz, 1H), 6.65 (d, J = 8.3 Hz, 1H), 5.21 (s, 1H), 4.87 - 4.78 (brm, 1H), 3.70 (dt, J = 5.6, 5.6 Hz, 2H), 3.66 - 3.50 (brs, 4H), 3.28 (dt, J = 6.0, 6.0 Hz, 2H), 2.67 - 2.64 (m, 6H), 2.43 - 2.40 (m, 2H), 1.93 - 1.84 (m, 4H), 1.48 (s, 9H); LC-MS: [M+H] + = 742.29.

[0236] (2) Preparation of CPP-133 The compound (CPP-132) (22 mg) prepared above was diluted in DCM (1 mL), TFA (0.11 mL) was added dropwise, and the mixture was stirred at room temperature for 20 hours. The solvent was then evaporated. The compound (CPI-005) (18 mg) prepared in Preparation Example 2, DIPEA (21 μL), and DMF (1.5 mL) were added to the residue, and the mixture was stirred at 60°C for 3 hours. DIPEA (26 μL) was added, and the mixture was stirred at 60°C for an additional 1 hour. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN, containing 0.1% AcOH) to obtain the target product (16 mg). 1H NMR (400 MHz, CDCl3) δ 13.73-13.66 (brm, 1H), 9.28-9.16 (brm, 2H), 8.06 (dt, J = 9.1, 2.4 Hz, 2H), 7.86 (d, J = 9.1 Hz, 2H), 7.31-7.26 (m, 2H), 7.04 (d, J = 2.4 Hz, 1H), 7.00 (td, J = 7.5, 0.9 Hz, 1H), 6.87 (d, J = 7.9 Hz, 1H), 6.81 (dd, J = 8.3, 2.4 Hz, 1H), 6.68 (d, J = 8.3 Hz, 1H), 6.49-6.39 (brs, 1H), 5.58 (s, 1H), 5.39 (s, 1H), 4.65 (s, 1H), 4.52-4.49 (m, 1H), 4.34-4.31 (m, 1H), 3.77 (t, J = 11.4 Hz, 2H), 3.72-3.48 (m, 23H), 3.38-3.28 (m, 4H), 3.16-3.12 (m, 1H), 2.92 (dd, J = 12.9, 5.0 Hz, 1H), 2.72-2.54 (m, 8H), 2.35 (s, 4H), 2.19-2.09 (m, 4H), 1.90-1.82 (m, 2H), 1.73-1.41 (m, 6H), 1.01 (s, 6H); LC-MS: [M+H] + = 1237.14.

[0237] Production Example 14: Production of compound (CPA-302) Compound (CPA-302) was produced according to the method shown in the following scheme. [ka]

[0238] (1) Manufacturing of CPA-301 To a mixture of 3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propionic acid (22 mg) and allyl alcohol (78 μL), thionyl chloride (0.17 mL) was added and stirred at room temperature for 1 hour. Water was added, and the mixture was concentrated under reduced pressure. The residue and the compound (CPI-003) (30 mg) prepared in Preparation Example 1 were dissolved in DMF (2 mL), DIPEA (40 μL) and COMU (36 mg) were added, and the mixture was stirred at room temperature for 3 hours. The solvent was then evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (37 mg). 1H NMR (400 MHz, CDCl3) δ 9.60 (s, 1H), 8.14 (dt, J = 9.0, 2.5 Hz, 2H), 7.88 (d, J = 9.0 Hz, 2H), 7.71 (t, J = 5.4 Hz, 1H), 5.95-5.86 (m, 1H), 5.31 (ddt, J = 17.2, 1.4, 1.4 Hz, 1H), 5.22 (ddt, J = 10.4, 1.4, 1.4 Hz, 1H), 4.86-4.82 (m, 1H), 4.59 (dt, J = 5.6, 1.4 Hz, 2H), 3.78-3.60 (m, 18H), 3.32-3.27 (m, 2H), 2.63 (t, J = 6.5 Hz, 2H), 2.44-2.41 (m, 2H), 1.92-1.86 (m, 2H), 1.50 (s, 9H); LC-MS: [M+H] + = 678.27.

[0239] (2) Manufacturing of CPA-302 A mixture of the compound (CPA-301) (36 mg), morpholine (9.3 μL), Pd(PPh3)4 (3.1 mg), and THF (1 mL) was stirred at room temperature for 6 hours. Morpholine (4.6 μL) and Pd(PPh3)4 (3.1 mg) were added, and the mixture was stirred at room temperature for 1 hour. The solvent was then evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the desired product (27 mg). 1H NMR (400 MHz, DMSO-d6) δ 10.54 (s, 1H), 9.09 (t, J = 5.7 Hz, 1H), 8.05 (dt, J = 9.2, 2.4 Hz, 2H), 7.91 (dt, J = 9.2, 2.3 Hz, 2H), 6.91 (t, J = 5.2 Hz, 1H), 3.60-3.42 (m, 18H), 2.98 (dt, J = 6.4, 6.4 Hz, 2H), 2.66 (t, J = 6.4 Hz, 1H), 2.37 (t, J = 7.4 Hz, 2H), 2.29 (t, J = 6.4 Hz, 2H), 1.71 (tt, J = 6.4, 6.4 Hz, 2H), 1.37 (s, 9H); LC-MS: [M+H] + = 638.23.

[0240] Production Example 15: Production of compound (CPA-306) Compound (CPA-306) was produced according to the method shown in the following scheme. [ka]

[0241] (1) Manufacturing of CPA-303 A mixture of 3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propionic acid (11 mg), thionyl chloride (8.8 μL), and allyl alcohol (35 μL) was stirred at room temperature for 1 hour. Water was added, and the mixture was concentrated under reduced pressure. The residue and the compound (CPA-302) (22 mg) prepared in Preparation Example 14 were dissolved in DMF (1 mL), and COMU (16 mg) and DIPEA (18 μL) were added. The mixture was stirred at room temperature for 5 hours, and the solvent was then evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the target compound (17 mg). 1H NMR (400 MHz, CDCl3) δ 9.63 (s, 1H), 8.13 (dt, J = 9.0, 2.3 Hz, 2H), 7.89 (d, J = 9.0 Hz, 2H), 7.78 (t, J = 5.3 Hz, 1H), 6.72 (s, 1H), 5.96-5.86 (m, 1H), 5.31 (ddt, J = 17.2, 1.4, 1.4 Hz, 1H), 5.23 (ddt, J = 10.4, 1.4 , 1.4 Hz, 1H), 4.89 (s, 1H), 4.59 (dt, J = 5.7, 1.4Hz, 2H), 3.78-3.60 (m, 32H), 3.54 (t, J = 5.3 Hz, 2H), 3.43 (dt, J = 5.3, 5.3 Hz, 2H), 3.28 (dt, J = 5.9 Hz, 2H), 2.62 (t, J = 6.5 Hz, 2H), 2.48-2.42 (m, 4H), 1.93-1.87 (m, 2H), 1.49 (s, 9H); LC-MS: [M+H] + = 925.27.

[0242] (2) Manufacturing of CPA-304 The compound (CPA-303) (17 mg) prepared above was dissolved in DCM (1 mL), and TFA (0.14 mL) was added dropwise. The mixture was stirred at room temperature for 2 hours, and then the solvent was evaporated. DIPEA (13 μL) was added to a DMF solution (1 mL) of the residue and the compound (CPI-005) (17 mg) prepared in Preparation Example 2. The mixture was stirred at 60°C for 4 hours, and then the solvent was evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (22 mg). 1H NMR (400 MHz, CDCl3) δ 13.68 (t, J = 5.0 Hz, 1H), 9.50 (s, 1H), 8.10 (d, J = 9.0 Hz, 2H), 7.89-7.87 (m, 3H), 6.79-6.76 (m, 1H), 6.66-6.63 (m, 1H), 5.96-5.86 (m, 2H), 5.31 (ddt, J = 17.2, 1.4, 1.4 Hz, 1H), 5.23 (ddt, J = 10.4, 1.4, 1.4 Hz, 1H), 5.11-5.06 (m, 1H), 4.59 (dt, J = 5.6, 1.4 Hz, 2H), 4.54-4.51 (m, 1H), 4.34-4.31 (m, 1H), 3.78-3.50 (m, 50H), 3.43 (dt, J = 5.4, 5.4 Hz, 2H), 3.38 (dt, J = 5.3, 5.3 Hz, 2H), 3.33 (dt, J = 5.5, 5.5 Hz, 2H), 3.17-3.12 (m, 1H), 2.92 (dd, J = 12.8, 4.9 Hz, 1H), 2.74 (d, J = 12.8 Hz, 1H), 2.63 (t, J = 6.5 Hz, 2H), 2.58 (t, J = 7.1 Hz, 2H), 2.47 (t, J = 6.1 Hz, 2H), 2.34 (s, 4H), 2.18 (t, J = 7.2 Hz, 2H), 2.12 (t, J = 6.9 Hz, 2H), 1.82-1.60 (m, 6H), 1.47-1.40 (m, 2H), 1.01 (s, 6H); LC-MS: [M+H] + = 1420.69.

[0243] (3) Manufacturing of CPA-305 A mixture of the compound (CPA-304) (10 mg), 1,3-dimethylbarbituric acid (2.2 mg), Pd(PPh3)4 (0.81 mg), and AcOEt / DCM (1:1, 0.5 mL) was stirred at room temperature for 1 hour, and the solvent was evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to give the target product (9.5 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.53 (t, J = 10.4 Hz, 1H), 10.57 (s, 1H), 9.10 (t, J = 5.2 Hz, 1H), 8.06 (d, J = 9.1 Hz, 2H), 7.95 (s, 1H), 7.91 (d, J = 9.1 Hz, 2H), 7.84 (t, J = 5.4 Hz, 1H), 6.42 (s, 1H), 6.36 (s, 1H), 4.29 (dd, J = 5.6, 5.2 Hz, 1H), 4.13-4.10 (m, 1H), 3.63-3.25 (m, under the water signal), 3.17 (tt, J = 5.8, 5.8 Hz, 4H), 3.10-3.06 (m, 1H), 2.81 (dd, J = 12.4, 5.1 Hz, 1H), 2.58-2.49 (m, overlapped with the DMSO signal), 2.35-2.28 (m, 8H), 2.05 (t, J = 7.4 Hz, 2H), 1.93 (tt, J = 7.0 Hz, 2H), 1.64-1.40 (m, 4H), 1.34-1.23 (m, 2H), 0.94 (s, 6H); LC-MS: [M+H] + = 1380.38.

[0244] (4) Manufacturing of CPA-306 A mixture of the compound (CPA-305) (8.5 mg), NHS (1.4 mg), WSC·HCl (2.4 mg), and DMF (0.5 mL) was stirred at room temperature for 4 hours. NHS (1.4 mg) and WSC·HCl (2.4 mg) were added, and the mixture was stirred for 2 hours. The solvent was then evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the desired product (7.7 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.55-13.52 (m, 1H), 10.38 (s, 1H), 9.12-9.07 (m, 1H), 8.08-7.80 (m, 6H), 6.41 (s, 1H), 6.35 (s, 1H), 4.31-4.28 (m, 1H), 4.13-4.10 (m, 1H), 3.71 (t, J = 6.0 Hz, 1H), 3.63-3.25 (m, under the water signal), 3.17 (tt, J = 6.0, 6.0 Hz, 4H), 3.10-3.06 (m, 1H), 2.92 (t, J = 6.0 Hz, 2H), 2.83-2.79 (m, 4H), 2.58-2.44 (m, overlapped with the DMSO signal), 2.34-2.28 (m, 8H), 2.05 (t, J = 7.4 Hz, 2H), 1.97-1.90 (m, 2H), 1.64-1.40 (m, 4H), 1.32-1.23 (m, 2H), 0.94 (s, 6H); LC-MS: [M+2H] 2+ = 1478.07

[0245] Production Example 16: Production of compound (CPA-310) Compound (CPA-310) was produced according to the method shown in the following scheme. [ka]

[0246] (1) Manufacturing of CPA-307 Diethylene glycol bis(3-aminopropyl) ether (2.1 mL) was dissolved in DCM (200 mL), and allyl chloroformate (1.0 mL) was added dropwise under ice-cooling, followed by stirring for 4 hours. The mixture was warmed to room temperature and stirred for 14 hours, after which the solvent was evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (0.50 g). 1H NMR (400 MHz, DMSO-d6) δ 8.26-7.37 (brs, 2H), 7.19 (t, J = 5.4, 1H), 5.95-5.85 (m, 1H), 5.26 (ddt, J = 13.8, 1.6, 1.6 Hz, 1H), 5.17 (ddt, J = 6.2, 1.6, 1.6 Hz, 1H), 4.45 (d, J = 5.3 Hz, 2H), 3.54-3.36 (m, 12H), 3.03 (dt, J = 6.6, 6.6 Hz, 2H), 2.83 (t, J = 7.0 Hz, 2H), 1.78 (tt, J = 6.6, 6.6 Hz, 2H), 1.62 (tt, J = 7.0, 7.0 Hz, 2H); LC-MS: [M+H] + = 305.16.

[0247] (2) Manufacturing of CPA-308 To a DMF solution (20 mL) of the compound (CPA-307) (0.50 g) prepared above, Fmoc-Lys(Boc)-OH (0.77 g), WSC·HCl (0.38 g), and HOBt·HO (0.30 g) were added at room temperature and stirred for 15 hours. The solvent was then evaporated. A saturated aqueous solution of NaHCO3 was added, followed by extraction with AcOEt. The organic layer was washed with saturated brine and dried over Na2SO4. The solvent was evaporated, and the mixture was purified by column chromatography (silica gel, AcOEt / MeOH) to obtain the target product (0.80 g). 1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 7.6 Hz, 2H), 7.60 (d, J = 7.4 Hz, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.31 (td, J = 7.4, 1.0 Hz, 2H), 6.83-6.73 (brs, 1H), 5.92-5.84 (m, 1H), 5.72-5.62 (brs, 1H), 5.44-5.34 (brs, 1H), 5.26 (d, J = 17.4 Hz, 1H), 5.17 (d, J = 10.6 hz, 2H), 4.75-4.63 (brs, 1H), 4.52 (d, J = 5.3 Hz, 2H), 4.42 (d, J = 7.0 Hz, 2H), 4.21 (t, J = 6.9 Hz, 1H), 4.14-4.10 (m, 1H), 3.59-3.43 (m, 13H), 3.35-3.25 (m, 3H), 3.11-3.08 (brm, 2H), 1.85-1.73 (m, 4H), 1.66-1.60 (m, 1H), 1.50-1.35 (m, 13H); LC-MS: [M+H] + = 755.21.

[0248] (3) Manufacturing of CPA-309 To a solution of the compound (CPA-308) (0.80 g) and 1,3-dimethylbarbituric acid (0.33 g) prepared above in DCM (10 mL) was added Pd(PPh3)4 (0.12 g), and the mixture was stirred at room temperature for 2 hours, after which the solvent was evaporated. The residue was purified by column chromatography (silica gel, AcOEt / MeOH) to obtain the intermediate (0.41 g). Biotin-NHS (0.31 g), DIPEA (0.16 mL), and DMF (10 mL) were added to the intermediate (0.41 g), and the mixture was stirred at room temperature for 2 hours, after which the solvent was evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (0.15 g). 1H NMR (400 MHz, DMSO-d6) δ 7.89 (d, J = 7.5 Hz, 2H), 7.85 (t, J = 5.5 Hz, 1H), 7.74-7.72 (m, 3H), 7.44-7.40 (m, 3H), 7.33 (td, J = 7.4, 0.9 Hz, 2H), 6.76 (t, J = 5.3 Hz, 1H), 6.42 (s, 1H), 6.35 (s, 1H), 4.31-4.19 (m, 4H), 4.13-4.10 (m, 1H), 3.91-3.86 (m, 1H), 3.50-3.44 (m, 8H), 3.39-3.33 (m, 4H), 3.14-3.03 (m, 5H), 2.91-2.85 (m, 2H), 2.81 (dd, J = 12.4, 5.1 Hz, 1H), 2.57 (d, J = 12.4 Hz, 1H), 2.04 (t, J = 7.4 Hz, 2H), 1.64-1.43 (m, 10H), 1.36-1.21 (m, 15H); LC-MS: [M+H] + = 897.11.

[0249] (4) Manufacturing of CPA-310 A mixture of the compound (CPA-309) (0.15 g), piperidine (24 μL), and DMF (3 mL) was stirred at room temperature for 2 hours, and the solvent was evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (0.11 mg). 1H NMR (400 MHz, DMSO-d6) δ 7.81 (t, J = 5.7 Hz, 1H), 7.75 (t, J = 5.5 Hz, 1H), 6.75 (t, J = 5.5 Hz, 1H), 6.42 (s, 1H), 6.35 (s, 1H), 4.32-4.29 (m, 1H), 4.14-4.11 (m, 1H), 3.53-3.45 (m, 8H), 3.41-3.33 (m, 4H), 3.13-3.03 (m, 6H), 2.90-2.80 (m, 3H), 2.57 (d, J = 12.4 Hz, 1H), 2.04 (t, J = 7.4 Hz, 2H), 1.66-1.57 (m, 5H), 1.54-1.43 (m, 4H), 1.37-1.20 (m, 16H); LC-MS: [M+H] + = 675.21.

[0250] Production Example 17: Production of compound (CPA-311)

Chem.

[0251] Thionyl chloride (0.33 mL) was added dropwise to 6-aminocaproic acid (0.20 g) and allyl alcohol (1.0 mL), and the mixture was stirred at room temperature for 1 hour. The solvent was then evaporated. Succinic anhydride (0.18 g), DIPEA (0.80 mL), and DMF (5 mL) were added to the residue, and the mixture was stirred at room temperature for 20 hours. Succinic anhydride (0.15 g) and DIPEA (0.4 mL) were added, and the mixture was stirred at room temperature for 1 hour. The solvent was then evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the desired product (0.40 g). 1H NMR (400 MHz, CDCl3) δ 6.02 (s, 1H), 5.97-5.87 (m, 1H), 5.32 (ddt, J = 14.1, 1.4, 1.4 Hz, 1H), 5.24 (ddt, J = 10.4, 1.4, 1.4 Hz, 1H), 4.58 (dt, J = 5.8, 1.4 Hz, 2H), 3.27 (dt, J = 6.6, 6.6 Hz, 2H), 2.71-2.68 (m, 2H), 2.52-2.49 (m, 2H), 2.36 (t, J = 7.3 Hz, 2H), 1.65 (tt, J = 7.5, 7.5 Hz, 2H), 1.53 (tt, J = 7.3, 7.3 Hz, 2H), 1.40-1.32 (m, 2H); LC-MS: [M+H] + = 272.21.

[0252] Production Example 18: Production of compound (CPA-314) [ka]

[0253] (1) Manufacturing of CPA-312 Compound (CPA-310) (0.11 g) prepared in Preparation Example 16 and compound (CPA-311) (43 mg) prepared in Preparation Example 17 were dissolved in DMF (4 mL), and WSC·HCl (37 mg) and HOBt·HO (29 mg) were added. The mixture was stirred at room temperature for 14 hours, and the solvent was then evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (0.13 g). 1H NMR (400 MHz, DMSO-d6) δ 7.95 (d, J = 8.0 Hz, 1H), 7.86-7.82 (m, 2H), 7.74 (t, J = 5.5 Hz, 1H), 6.74 (t, J = 5.4 Hz, 1H), 6.42 (s, 1H), 6.35 (s, 1H), 5.96-5.86 (m, 1H), 5.28 (ddt, J = 17.3, 1.5, 1.5 Hz, 1H), 5.20 (ddt, J = 10.5, 1.5, 1.5 Hz, 1H), 4.54 (dt, J = 5.4, ​​1.4 Hz, 2H), 4.32-4.29 (m, 1H), 4.14-4.06 (m, 2H), 3.52-3.45 (m, 8H), 3.40-3.33 (m, 4H), 3.12-2.98 (m, 7H), 2.89-2.80 (m, 3H), 2.57 (d, J = 12.4 Hz, 1H), 2.37-2.25 (m, 6H), 2.04 (t, J = 7.4 Hz, 2H), 1.65-1.19 (m, 31H); LC-MS: [M+H] + = 928.30.

[0254] (2) Manufacturing of CPA-313 To a solution of the compound (CPA-312) (0.13 g) prepared above in DCM (3 mL) was added TFA (1.1 mL) dropwise, and the mixture was stirred at room temperature for 1 hour. The solvent was then evaporated. The residue was diluted with DMF (2 mL), and DIPEA (0.12 mL) and adipic anhydride (36 mg) were added, followed by stirring at room temperature for 2 hours. The stirring was stopped, and the mixture was allowed to stand for 4 days. The solvent was then evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (75 mg). 1H NMR (400 MHz, DMSO-d6) δ 8.02-7.95 (m, 1H), 7.89-7.84 (m, 2H), 7.78-7.75 (m, 2H), 6.43 (s, 1H), 6.36 (s, 1H), 5.96-5.86 (m, 1H), 5.28 (ddt, J = 17.2, 1.5, 1.5 Hz, 1H), 5.20 (ddt, J = 10.5, 1.5 1.5 Hz, 1H), 4.53 (dt, J = 5.5, 1.5, 2H), 4.32-4.29 (m, 1H), 4.14-4.06 (m, 2H), 3.52-3.33 (m, 12H), 3.12-2.96 (m, 9H), 2.82 (dd, J = 12.5, 5.2 Hz, 1H), 2.57 (d, J = 12.5 Hz, 1H), 2.38-2.27 (m, 6H), 2.21-2.16 (m, 2H), 2.06-2.02 (m, 4H), 1.65-1.21 (m, 26H); LC-MS: [M+H] + = 956.19.

[0255] (3) Manufacturing of CPA-314 The compound (CPA-313) (75 mg) prepared above was dissolved in DMF (2 mL), and dimedone (12 mg), WSC·HCl (16 mg), and DMAP (9.6 mg) were added. The mixture was stirred at room temperature for 14 hours, and the solvent was evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (31 mg). 1H NMR (400 MHz, DMSO-d6) δ 7.99 (d, J = 8.0 Hz, 1H), 7.89-7.82 (m, 2H), 7.74 (t, J = 10.6 Hz, 2H), 6.61 (d, J = 6.0 Hz, 1H), 6.42 (s, 1H), 6.35 (s, 1H), 5.96-5.86 (m, 1H), 5.28 (ddt, J = 17.2, 1.6, 1.6 Hz, 1H), 5.20 (ddt, J = 10.5, 1.5, 1.5 Hz, 1H), 4.53 (dt, J = 5.4, ​​1.4 Hz, 2H), 4.32-4.28 (m, 1H), 4.14-4.06 (m, 2H), 3.52-3.43 (m, 8H), 3.40-3.33 (m, 4H), 3.12-2.96 (m, 11H), 2.86-2.79 (m, 3H), 2.57 (d, J = 12.5 Hz, 1H), 2.36-2.30 (m, 8H), 2.06-1.96 (m, 4H), 1.65-1.21 (m, 26H), 0.97 (s, 6H); LC-MS: [M+2H] 2+ = 1079.50.

[0256] Production Example 19: Production of compound (CPA-318) [ka]

[0257] (1) Manufacturing of CPA-317 Compound (CPA-315) (0.11 g) and compound (CPA-316) (0.087 g) were dissolved in DMF (5 mL), and HATU (0.209 g) and DIPEA (0.160 mL) were added at room temperature. The mixture was stirred for 20 hours, and the solvent was evaporated. The residue was purified by column chromatography (silica gel, hexane / AcOEt) to obtain the target product (0.11 mg). 1H NMR (400 MHz, CDCl3) δ 8.22-8.19 (m, 2H), 7.74-7.64 (brs, 1H), 7.61-7.52 (m, 3H), 5.15-5.02 (brs, 1H), 3.80-3.71 (m, 4H), 3.69-3.64 (m,4H), 3.58 (t, J = 5.2 Hz, 2H), 3.36-3.33 (m, 2H), 1.43 (s, 9H); LC-MS: [M+H] + = 421.08.

[0258] (2) Manufacturing of CPA-318 The compound (CPA-317) (0.11 mg) prepared above was dissolved in DCM (2 mL), and TFA (0.97 mL) was added dropwise. After stirring at room temperature for 1 hour, the solvent was evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (89 mg). 1H NMR (400 MHz, DMSO-d6) δ 9.15 (t, J = 5.4 Hz, 1H), 8.15-8.12 (m, 2H), 7.74-7.64 (m, 6H), 3.61-3.58 (m, 8H), 3.54-3.49 (m, 2H), 2.97 (t, J = 5.2 Hz, 2H); LC-MS: [M+H] + = 321.06.

[0259] Production Example 20: Production of compound (CPA-321) [ka]

[0260] (1) Manufacturing of CPA-319 The compound (CPA-318) (12 mg) prepared in Preparation Example 19 and the compound (CPA-314) (30 mg) prepared in Preparation Example 18 were dissolved in DMF (1 mL), and the mixture with DIPEA (19 μL) was stirred at 60 °C for 2 hours. Purification by column chromatography (ODS, water / MeCN) gave the target product (30 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.35 (t, J = 4.5, 1H), 9.08 (t, J = 5.9 Hz, 1H), 8.13-8.10 (m, 2H), 7.96 (d, J = 8.0 Hz, 1H), 7.86-7.82 (m, 2H), 7.75-7.63 (m, 5H), 6.42 (s, 1H), 6.35 (s, 1H), 5.95-5.85 (m, 1H), 5.28 (ddt, J = 17.2, 1.5, 1.5 Hz, 1H), 5.20 (ddt, J = 10.4, 1.5, 1.5Hz, 1H), 4.53 (dt, J = 5.5, 1.5 Hz, 2H), 4.32-4.28 (m, 1H), 4.13-4.05 (m, 2H), 3.63-3.60 (m, 8H), 3.50-3.43 (m, 10H), 3.39-3.33 (m, 4H), 3.11-2.90 (m, 11H), 2.81 (dd, J = 12.4, 5.1 Hz, 1H), 2.57 (d, J = 12.4 Hz., 1H), 2.36-2.27 (m, 8H), 2.23 (s, 4H), 2.11-2.02 (m, 4H), 1.65-1.21 (m, 26H), 0.91 (s, 6H) ; LC-MS: [M+H] + = 1380.98.

[0261] (2) Manufacturing of CPA-320 A mixture of the compound (CPA-319) (29 mg), 1,3-dimethylbarbituric acid (6.6 mg), Pd(PPh3)4 (2.4 mg), and DCM (1 mL) was stirred at room temperature for 1 hour, and the solvent was evaporated. The residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (26 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.35 (t, J = 4.3 Hz, 1H), 9.09 (t, J = 5.5, 1H), 8.13-8.10 (m, 3H), 7.99-7.89 (brs, 1H), 7.85 (t, J = 5.5 Hz, 1H), 7.78-7.75 (m, 2H), 7.72-7.63 (m, 3H), 6.43 (s, 1H), 6.36 (s, 1H), 4.32-4.28 (m, 1H), 4.14-4.05 (m, 2H), 3.63-3.60 (m, 8H), 3.50-3.45 (m, 10H), 3.39-3.33 (m, 4H), 3.11-2.90 (m, 11H), 2.82 (dd, J = 12.4, 5.1 Hz, 1H), 2.57 (d, J = 12.4 Hz, 1H), 2.36-2.27 (m, 6H), 2.23 (s, 4H), 2.16-2.02 (m, 6H), 1.65-1.21 (m, 26H), 0.91 (s, 6H); LC-MS: [M+H] + = 1339.89.

[0262] (3) Manufacturing of CPA-321 A mixture of the compound (CPA-320) (25 mg), NHS (4.3 mg), WSC·HCl (7.2 mg), and DMF (0.4 mL) was stirred at room temperature for 3 hours. NHS (4.3 mg) and WSC·HCl (7.2 mg) were added, and the mixture was stirred at room temperature for an additional 3 hours. The product was purified by column chromatography (ODS, water / MeCN) to obtain the target compound (21 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.35 (t, J = 4.4 Hz, 1H), 9.08 (t, J = 5.6 Hz, 1H), 8.13-8.10 (m, 2H), 7.96 (d, J = 7.9 Hz, 1H), 7.87-7.83 (m, 2H), 7.75-7.63 (m, 5H), 6.42 (s, 1H), 6.35 (s, 1H), 4.32-4.28 (m, 1H), 4.14-4.05 (m, 2H), 3.63-3.60 (m, 8H), 3.50-3.43 (m, 10H), 3.39-3.33 (m, 4H), 3.11-2.90 (m, 11H), 2.84-2.79 (m, 5H), 2.65 (t, J = 7.4 Hz, 2H), 2.57 (d, J = 12.4 Hz, 1H), 2.38-2.28 (m, 6H), 2.23 (s, 4H), 2.09-2.02 (m, 4H), 1.65-1.21 (m, 26H), 0.91 (s, 6H); LC-MS: [M+H] + = 1437.12.

[0263] Production Example 21: Synthesis of CPF-242 [ka]

[0264] (1) Synthesis of CPF-241 To a mixture of CPI-104 (14 mg) and DMF (1 mL), CPI-006 (6.6 mg), WSC·HCl (6.8 mg), and HOBt·HO (5.4 mg) were added and stirred at room temperature for 3 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN). To a mixture of the purified product (8.9 mg) and DMF (1 mL), N-Boc-1,5-diaminopentane (12 μL), WSC·HCl (11 mg), and HOBt·HO (9.0 mg) were added and stirred at room temperature for 16 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the desired product (7.4 mg). 1 H NMR (400 MHz, DMSO-d6) δ 7.77 (t, J = 5.4 Hz, 1H), 7.72-7.65 (m, 3H), 7.57-7.50 (m, 3H), 7.47-7.43 (m, 2H), 7.20 (d, J = 2.3 Hz, 1H), 6.75 (t, J = 5.5 Hz, 1H), 4.55 (d, J = 10.5 Hz, 1H), 4.25-4.18 (m, 1H), 3.77 (d, J = 10.5 Hz, 1H), 3.70-3.63 (m, 1H), 2.98 (dt, J = 6.5, 6.5Hz, 2H), 2.90-2.80 (m, 4H), 1.86-1.77 (m, 4H), 1.58-1.31 (m, 19H), 1.24-1.18 (m, 2H); LC-MS: [M+H] + = 680.53.

[0265] (2) Synthesis of CPF-242 To a mixture of the compound (CPF-241) (6.6 mg) prepared above and DCM (1 mL) was added TFA (149 μL) and the mixture was stirred at room temperature for 2 hours. The solvent was evaporated, and to the residue were added DMF (1 mL), CPI-005 (6.0 mg), and DIPEA (17 μL), and the mixture was stirred at 60 °C for 3 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (7.9 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.46 (t, J = 4.9 Hz, 1H), 7.84-7.79 (m, 2H), 7.72-7.65 (m, 3H), 7.57-7.50 (m, 3H), 7.47-7.43 (m, 2H), 7.20 (d, J = 2.4 Hz, 1H), 6.41 (s, 1H), 6.35 (s, 1H), 4.55 (d, J = 10.5 Hz, 1H), 4.31-4.28 (m, 1H), 4.25-4.18 (m, 1H), 4.14-4.10 (m, 1H), 3.76 (d, J = 10.5 Hz, 1H), 3.70-3.58 (m, 3H), 3.52-3.48 (m, 14H), 3.42-2.99 (m, overlapped with water signal), 2.85-2.79 (m, 3H), 2.59-2.49 (m, overlapped with DMSO signal), 2.28 (s, 4H), 2.06 (t, J = 7.4 Hz, 2H), 1.87-1.77 (m, 4H), 1.65-1.23 (m, 18H), 0.94 (s, 6H); LC-MS: [M+H] + = 1175.70.

[0266] Production Example 22: Production of CPA-325 [ka]

[0267] (1) Manufacturing of CPA-322 The compound (CPA-316) (40 mg), N-Boc-1,3-diaminopropane (61 μL), and COMU (99 mg) were dissolved in DMF (1 mL), DIPEA (121 μL) was added, and the mixture was stirred at room temperature for 26 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (53 mg). 1H NMR (500 MHz, DMSO-d6) δ 9.15 (br, 1H), 8.13 (d, J = 7.9 Hz, 2H), 7.72-7.64 (m, 3H), 6.84 (br, 1H), 3.30 (br, overlapped with water signal), 2.99 (br, 2H), 1.67 (quin, J = 6.5 Hz, 2H), 1.38 (s, 9H); LC-MS: [M+Na] + = 369.41.

[0268] (2) Manufacturing of CPA-323 The compound (CPA-322) (51 mg) prepared above was dissolved in DCM (1 mL), and TFA (113 μL) was added under ice cooling. The mixture was stirred at room temperature for 1 hour. The solvent was evaporated, and 15-(Boc-amino)-4,7,10,13-tetraoxapentadecanoic acid (56 mg), COMU (76 mg), DIPEA (85 μL), and DMF (1 mL) were added to the residue, followed by stirring at room temperature for 12 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (76 mg). 1 H NMR (500 MHz, DMSO-d6) δ 9.15 (t, J = 5.7 Hz, 1H), 8.13 (d, J = 7.7 Hz, 2H), 7.89 (t, J = 5.4 Hz, 1H), 7.72-7.69 (m, 2H), 7.66 (t, J = 7.2 Hz, 1H), 6.74 (t, J = 5.2 Hz, 1H), 3.60 (t, J = 6.4 Hz, 2H), 3.49-3.46 (m, 12H), 3.37-3.36 (m, overlapped with water signal), 3.12 (q, J = 6.4 Hz, 2H), 3.05 (q, J = 5.9 Hz, 2H), 2.32 (t, J = 6.4 Hz, 2H), 1.68 (quin, J = 7.0 Hz, 2H), 1.36 (s, 9H); LC-MS: [M+H] += 594.60.

[0269] (3) Manufacturing of CPA-324 The compound (CPA-323) (67 mg) prepared above was dissolved in DCM (1 mL), and TFA (130 μL) was added, followed by stirring at room temperature for 2 hours. The solvent was evaporated to obtain the target product (68 mg). 1 H NMR (400 MHz, DMSO-d6) δ 9.16 (t, J = 5.8 Hz, 1H), 8.15-8.11 (m, 2H), 7.92 (t, J = 5.6 Hz, 1H), 7.75-7.64 (m, 6H), 3.62-3.48 (m, 16H), 3.34 (dt, J = 6.7, 6.7 Hz, 2H), 3.12 (dt, J = 6.7, 6.7 Hz, 2H), 3.01-2.94 (m, 2H), 2.33 (t, J = 6.4 Hz, 2H), 1.69 (tt, J = 6.7, 6.7Hz, 2H); LC-MS: [M+H]+ = 494.38.

[0270] (4) Manufacturing of CPA-325 This was produced in the same manner as in Production Example 20, except that CPA-318 was replaced with the compound (CPA-324) produced above. 1H NMR (400 MHz, DMSO-d6) δ 13.35 (s, 1H), 9.15 (t, J = 5.9 Hz, 1H), 8.14-8.11 (m, 2H), 7.97 (d, J = 8.4 Hz, 1H), 7.91-7.83 (m, 3H), 7.76-7.63 (m, 5H), 6.42 (s, 1H), 6.35 (s, 1H), 4.30 (dd, J = 7.6, 5.2 Hz, 1H), 4.14-4.05 (m, 2H), 3.61-3.34 (m, 32 H), 3.17-2.91 (m, 13H), 2.84-2.79 (m, 5H), 2.65 (t, J = 7.4 Hz, 2H), 2.57 (d, J = 12.4 Hz, 2H), 2.37-2.26 (m, 10H), 2.09-2.02 (m, 4H), 1.71-1.19 (m, 28H), 0.93 (s, 6H); LC-MS: [M+2H] 2+ = 1611.63.

[0271] Production Example 23: Production of CPA-326 [ka]

[0272] Production was carried out in the same manner as in Production Example 21, except that N-Boc-1,3-diaminopropane was changed to N-Boc-3,3'-((oxybis(ethane-2,1-diyl))bis(oxy))bis(propan-1-amine). 1H NMR (400 MHz, DMSO-d6) δ 13.35 (s, 1H), 9.14 (t, J = 5.8 Hz, 1H), 8.14-8.11 (m, 2H), 7.96 (d, J = 8.0 Hz, 1H), 7.85 (dd, J = 9.9, 5.4 Hz, 2H), 7.79 (t, J = 5.6 Hz, 1H), 7.87-7.63 (m, 5H), 6.42 (s, 1H), 6.35 (s, 1H), 4.30 (dd, J = 7.6, 5.2 Hz, 1H), 4.14-4.05 (m, 2H), 3.61-3.34 (m, 40H), 3.11-2.92 (m, 15H), 2.84-2.79 (m, 5H), 2.65 (t, J = 7.4 Hz, 2H), 2.57 (d, J = 12.4, 1H), 2.37-2.26 (m, 12H), 2.09-2.02 (m, 4H), 1.83-1.77 (m, 2H), 1.65-1.19 (m, 28H), 0.93 (s, 6H); LC-MS: [M+2H] 2+ = 1757.83.

[0273] Production Example 24: Production of CPA-332 [ka]

[0274] (1) Manufacturing of CPA-328 The compound (CPA-327) (60 mg) and Nα,Nε-di-Boc-L-lysine (87 mg) were dissolved in DMF (2 mL), and COMU (108 mg) and DIPEA (109 μL) were added. The mixture was stirred at room temperature for 1 hour. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (129 mg). 1H NMR (500 MHz, DMSO-d6) δ 8.13 (d, J = 7.4 Hz, 1H), 6.77-6.39 (m, 3H), 5.88 (ddt, J = 17.3, 10.5, 5.3 Hz, 1H), 5.30 (dd, J = 17.3, 1.6 Hz, 1H), 5.20 (dd, J = 10.5, 1.5 Hz, 1H), 4.59-4.52 (m, 2H), 4.22 (br, 1H), 3.91 (br, 1H), 2.92-2.82 (m, 4H), 1.72-1.66 (m, 1H), 1.64-1.52 (m, 2H), 1.49-1.42 (m, 1H), 1.37-1.24 (m, 35H); LC-MS: [M+H] + = 615.68.

[0275] (2) Manufacturing of CPA-329 The compound (CPA-328) (126 mg) synthesized above was dissolved in DCM (2 mL), TFA (474 ​​μL) was added, and the mixture was stirred at room temperature for 1 hour. The solvent was evaporated, and the compound (CPA-316) (128 mg), DMF (2 mL), COMU (288 mg), and DIPEA (235 μL) were added to the residue, and the mixture was stirred at room temperature for 1 hour. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (132 mg). 1H NMR (500 MHz, DMSO-d6) δ 9.18 (t, J = 6.0 Hz, 1H), 9.15 (t, J = 6.0 Hz, 1H), 8.92 (d, J = 8.0 Hz, 1H), 8.52 (d, J = 7.3 Hz, 1H), 8.12-8.09 (m, 6H), 7.70-7.62 (m, 9H), 5.87 (ddt, J = 17.3, 10.5, 5.3 Hz, 1H), 5.29 (dq, J = 17.2, 1.6 Hz, 1H), 5.17 (dq, J = 10.5, 1.5 Hz, 1H), 4.60 (q, J = 7.3 Hz, 1H), 4.56 (dq, J = 5.4, 1.6 Hz, 2H), 4.32-4.27 (m, 1H), 3.32-3.30 (m, overlapped with water signal), 1.86-1.77 (m, 3H), 1.73-1.66 (m, 1H), 1.64-1.55 (m, 4H), 1.49-1.36 (m, 4H); LC-MS: [M+H] + = 831.77.

[0276] (3) Manufacturing of CPA-330 The compound (CPA-329) (42 mg) prepared above was dissolved in DCM (3 mL), and Pd(PPh3)4 (5.8 mg) and N-methylaniline (27 μL) were added and stirred at room temperature for 1 hour. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (31 mg). 1H NMR (500 MHz, DMSO-d6) δ 12.60 (br, 1H), 9.18 (t, J = 5.8 Hz, 1H), 9.15 (t, J = 5.8 Hz, 1H), 8.92 (d, J = 8.0 Hz, 1H), 8.36 (d, J = 7.7 Hz, 1H), 8.12-8.07 (m, 5H), 7.80-7.77 (m, 1H), 7.71-7.62 (m, 9H), 4.60 (q, J = 7.5 Hz, 1H), 4.24-4.19 (m, 1H), 3.34-3.29 (m, overlapped with water signal), 1.87-1.76 (m, 3H), 1.70-1.54 (m, 5H), 1.49-1.35 (m, 4H); LC-MS: [M+H] + = 791.46.

[0277] (4) Manufacturing of CPA-331 Produced in the same manner as in Production Example 22. 1 H NMR (400 MHz, DMSO-d6) δ 9.18-9.14 (m, 2H), 8.99 (d, J = 8.0 Hz, 1H), 8.17-8.07 (m, 7H), 7.97 (t, J = 5.4 Hz, 1H), 7.71-7.62 (m, 12H), 4.56 (dt, J = 7.2, 7.2 Hz, 2H), 4.26 (dt, J = 7.2, 7.2 Hz, 2H), 3.59-3.53 (m, 6H), 3.42-3.16 (m, 8H), 3.00-2.96 (m, 2H), 1.86-1.80 (m, 2H), 1.70-1.28 (m, 10H); LC-MS: [M+H] + = 921.30.

[0278] (5) Manufacturing of CPA-332 Production was carried out in the same manner as in Production Example 21, except that CPA-318 was changed to CPA-331. 1H NMR (400 MHz, DMSO-d6) δ 13.34 (s, 1H), 9.17-9.12 (m, 2H), 8.98 (d, J = 8.4 Hz. 1H), 8.17 (d, J = 8.1 Hz, 1H), 8.12-8.06 (m, 6H), 8.06-7.96 (m, 2H), 7.87-7.83 (m, 2H), 7.76-7.74 (m, 2H), 7.70-7.53 (m, 9H), 6.42 (s, 1H), 6.36 (s, 1H), 4.57 (dt, J = 7.5, 7.5 Hz, 1H), 4.32-4.24 (m, 2H), 4.14-4.05 (m, 2H), 3.60-2.90 (m, 37H), 2.84-2.79 (m, 5H), 2.66-2.50 (m, 3H), 2.36-2.25 (m, 10H), 2.09-2.02 (m, 4H), 1.86-1.80 (m, 2H), 1.68-1.20 (m, 36H), 0.91 (s. 6H); LC-MS: [M+2H] 2+ = 2039.42.

[0279] Production Example 25: Production of CPA-403 [ka]

[0280] (1) Manufacturing of CPA-401 To a mixture of 4-benzoylbenzoic acid (50 mg), N-Boc-2,2'-(ethylenedioxy)diethylamine (55 mg), and DMF (3 mL), WSC·HCl (42 mg) and HOBt·HO (34 mg) were added and stirred at room temperature for 15 hours. The solvent was evaporated, and the residue was purified by column chromatography (silica gel, hexane / AcOEt) to give the desired product (61 mg). 1H NMR (400 MHz, DMSO-d6) δ 8.07-7.88 (brm, 2H), 7.86-7.79 (m, 4H), 7.62 (tt, J = 7.4, 1.2 Hz, 1H), 7.50 (t, J = 7.6 Hz, 2H), 6.99-6.88 (brs, 1H), 5.03-4.91 (brs, 1H), 3.71-3.56 (m, 10H), 3.36-3.23 (brm, 2H), 1.42 (s, 9H); LC-MS: [M+H] + = 457.45.

[0281] (2) Manufacturing of CPA-402 To a mixture of the compound (CBI-712) (61 mg) and DCM (2 mL), TFA (515 μL) was added and the mixture was stirred at room temperature for 14 hours. The solvent was evaporated to give the target product (63 mg). 1 H NMR (400 MHz, DMSO-d6) δ 8.73 (t, J = 5.6 Hz, 1H), 8.01-7.98 (m, 2H), 7.82-7.69 (m, 8H), 7.61-7.57 (m, 2H), 3.60-3.56 (m, 8H), 3.49-3.38 (m, 2H), 3.00-2.93 (m, 2H); LC-MS: [M+H] + = 357.36.

[0282] (3) Manufacturing of CPA-403 Production was carried out in the same manner as in Production Example 22, except that CPA-316 was changed to CPA-402. 1H NMR (400 MHz, DMSO-d6) δ 13.38-13.32 (brm, 1H), 8.72 (t, J = 5.6 Hz, 1H), 8.00-7.96 (m, 3H), 7.87-7.83 (m, 2H), 7.80-7.68 (m, 7H), 7.60-7.56 (m, 2H), 6.42 (s, 1H), 6.35 (s, 1H), 4.30 (dd, J = 7.6, 5.1 Hz, 1H), 4.14-4.05 (m, 3H), 3.62-3.36 (m, 22H), 3.11-2.91 (m, 11H), 2.84-2.79 (m, 5H), 2.65 (t, J = 7.2 Hz, 2H), 2.57 (d, J = 12.6 Hz, 1H), 2.37-2.24 (m, 10H), 1.68-1.19 (m, 26H), 0.91 (s, 6H); LC-MS: [M+2H] 2+ = 1474.68.

[0283] Production Example 26: Production of CPA-503

change

[0284] (1) Manufacture of CPA-502 The compound represented by the above formula (CPA-501) (40 mg) was dissolved in DCM (2 mL), added to TFA (258 μL), and stirred at room temperature for 1 hour. The solvent was left and the target compound (41 mg) was obtained. 1 H NMR (400 MHz, DMSO-d6) δ 7.93 (t, J = 5.4 Hz, 1H), 7.87-7.66 (brs, 3H), 3.60-3.52 (m, 6H), 3.41 (t, J = 6.0 Hz, 2H), 3.20 (dt, J = 5.9, 5.9 Hz, 2H), 2.97 (t, J = 5.2 Hz, 2H), 1.97 (m, 2H), 1.56 (m, 2H), 0.98 (s, 3H); LC-MS: [M+H]+ = 259.28.

[0285] (2) Manufacturing of CPA-503 Production was carried out in the same manner as in Production Example 22, except that CPA-316 was changed to CPA-502. 1 H NMR (400 MHz, DMSO-d6) δ 13.35 (s, 1H), 7.98-7.84 (m, 4H), 7.77-7.74 (m, 2H), 6.42 (s, 1H), 6.36 (s, 1H), 4.30 (dd, J = 7.6, 5.2 Hz, 1H), 4.14-4.06 (m, 2H), 3.61-3.16 (m, 22H), 3.12-2.92 (m, 11H), 2.84-2.80 (m, 5H), 2.65 (t, J = 7.3 Hz, 2H), 2.59-2.56 (m, 1H), 2.37-2.27 (m, 10H), 2.10-2.02 (m, 4H), 1.97 (t, J = 7.7 Hz), 1.65-1.23 (m, 28H), 0.97 (s, 3H), 0.94 (s, 6H); LC-MS: [M+2H] 2+ = 1376.31.

[0286] Production Example 27: Production of CPA-334 [ka]

[0287] To a mixture of the compound represented by the formula (CPA-333) and DCM (1 mL), TFA (80 μL) was added and stirred at room temperature for 1 hour. The solvent was evaporated, and the compound represented by the formula (CPA-321) (15 mg), DMF (1 mL), and DIPEA (5.5 μL) were added to the residue and stirred at room temperature for 8 hours. DIPEA (1.8 μL) was added, and the mixture was stirred at room temperature for 14 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (12 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.34 (t, J = 4.5 Hz, 1H), 9.96 (d, J = 0.7 Hz, 1H), 9.08 (t, J = 5.7 Hz, 1H), 8.13-8.10 (m, 2H), 8.04 (t, J = 7.5 Hz, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.87-7.82 (m, 3H), 7.78-7.63 (m, 6H), 6.42 (s, 1H), 6.35 (s, 1H), 4.30 (dd, J = 7.5, 5.3 Hz, 1H), 4.14-4.05 (m, 2H), 3.63-3.60 (m, 8H), 3.50-3.29 (m, overlapped with water signal), 3.11-2.89 (m, 11H), 2.83-2.79 (m, 1H), 2.59-2.23 (m, overlapped with DMSO signal), 2.09-2.02 (m, 4H), 1.65-1.24 (m, 26H), 0.91 (s, 6H); LC-MS: [M+H] + = 1528.03.

[0288] Manufacturing Example 28: Manufacturing of CPI-007 [ka]

[0289] To a mixture of adipic anhydride (100 mg) and DCM (5 mL), allyl alcohol (96 μL), DMAP (9.5 mg), and pyridine (170 μL) were added under ice cooling, and the mixture was stirred for 1 hour. The mixture was then warmed to room temperature and stirred for 15 hours. The reaction mixture was washed with 2 M aqueous HCl and saturated brine, and then dried over Na2SO4. The solvent was evaporated to give the desired product (101 mg). 1H NMR (400 MHz, CDCl3) δ 5.97-5.87 (m, 1H), 5.34-5.29 (m, 1H), 5.26-5.22 (m, 1H), 4.58 (ddt, J = 5.7, 1.3, 1.3 Hz, 2H), 2.42-2.34 (m, 4H), 1.75-1.64 (m, 4H); LC-MS: [M+H] + = 187.20.

[0290] Production Example 29: Production of CPA-338 [ka]

[0291] (1) Manufacturing of CPA-335 To a mixture of the compound (CPA-310) (139 mg) and DCM (5 mL), TEA (43 μL) and 2,2,2-trichloroethyl chloroformate (33 μL) were added and stirred under ice cooling for 1 hour. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (172 mg). 1 H NMR (400 MHz, DMSO-d6) δ 7.89 (t, J = 5.6 Hz, 1H), 7.78-7.73 (m, 2H), 6.75 (t, J = 5.4 Hz, 1H), 6.42 (s, 1H), 6.35 (s, 1H), 4.82 (d, J = 12.4 Hz, 1H), 4.75 (d, J = 12.4 Hz, 1H), 4.30 (dd, J = 7.6, 5.2 Hz, 1H), 4.14-4.12 (m, 1H), 3.93-3.87 (m, 1H), 3.52-3.54 (m, 8H), 3.40-3.34 (m, 4H), 3.12-3.04 (m, 5H), 2.88-2.80 (m, 3H), 2.57 (d, J = 12.4 Hz, 1H), 2.04 (t, J = 7.4 Hz, 2H), 1.65-1.41 (m, 10H), 1.37-1.21 (m, 15H); LC-MS: [M+H] + = 849.56.

[0292] (2) Manufacturing of CPA-336 To a mixture of the compound (CPA-335) (172 mg) prepared above and DCM (3 mL) was added TFA (468 μL) and stirred at room temperature for 1 hour. The solvent was evaporated, and the residue was mixed with DMF (3 mL), CBI-741 (41 mg), WSC·HCl (47 mg), HOBt·HO (37 mg), and DIPEA (141 μL) and stirred at room temperature for 13 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (134 mg). 1 H NMR (400 MHz, DMSO-d6) δ 7.89 (t, J = 5.6 Hz, 1H), 7.80-7.73 (m, 3H), 6.42 (s, 1H), 6.35 (s, 1H), 5.96-5.86 (m, 1H), 5.28 (ddt, J = 17.2, 1.5, 1.5 Hz, 1H), 5.20 (ddt, J = 10.5, 1.5, 1.5 Hz, 1H), 4.82 (d, J = 12.4 Hz, 1H), 4.75 (d, J = 12.4 Hz, 1H), 4.53 (dt, J = 5.4, ​​1.5 Hz, 2H), 4.30 (dd, J = 7.6, 5.2 Hz, 1H), 4.14-4.11 (m, 1H), 3.93-3.87 (m, 1H), 3.52-3.44 (m, 8H), 3.39-3.30 (m, 4H), 3.15-3.04 (m, 5H), 3.01-2.96 (m, 2H), 2.82 (dd, J = 12.4, 5.1 Hz, 1H), 2.57 (d, J = 12.4 Hz, 1H), 2.35-2.31 (m, 2H), 2.06-2.02 (m, 4H), 1.64-1.21 (m, 20H); LC-MS: [M+H] + = 917.63.

[0293] (3) Manufacturing of CPA-337 To a mixture of the compound CPA-336 (134 mg) prepared above and DCM (3 mL), Pd(PPh3)4 (17 mg) and 1,3-dimethylbarbituric acid (47 mg) were added and stirred at room temperature for 1 hour. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (96 mg). 1 H NMR (400 MHz, DMSO-d6) δ 7.91 (t, J = 5.5 Hz, 1H), 7.80-7.74 (m, 3H), 6.42 (s, 1H), 6.36 (s, 1H), 4.82 (d, J = 12.4 Hz, 1H), 4.75 (d, J = 12.4 Hz), 4.30 (dd, J = 7.6, 5.2 Hz, 1H), 4.14-4.11 (m, 1H), 3.93-3.87 (m, 1H), 3.52-3.35 (m, 12H), 3.12-3.04 (m, 5H), 3.01-2.96 (m, 2H), 2.82 (dd, J = 12.4, 5.1 Hz, 1H), 2.57 (d, J = 12.4 Hz, 1H), 2.18 (t, J = 6.8 Hz, 2H), 2.06-2.01 (m, 4H), 1.64-1.21 (m, 20H); LC-MS: [M+H] + = 877.54.

[0294] (4) Manufacturing of CPA-338 To a mixture of the compound (CPA-337) (96 mg) prepared above and DMF (2 mL), dimedone (31 mg), WSC·HCl (42 mg), and DMAP (27 mg) were added and stirred at 60 °C for 2 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN, containing 0.1% AcOH) to obtain the target product (69 mg). 1H NMR (400 MHz, DMSO-d6) δ 12.12-11.83 (brs, 1H), 7.90 (t, J = 5.5 Hz, 1H), 7.78-7.74 (m, 3H), 6.42 (s, 1H), 6.35 (s, 1H), 4.82 (d, J = 12.4 Hz, 1H), 4.75 (d, J = 12.4 Hz, 1H), 4.30 (dd, J = 7.6, 5.2 Hz, 1H), 4.14-4.11 (m, 1H), 3.93-3.87 (m, 1H), 3.52-3.30 (m, 12H), 3.12-3.04 (m, 5H), 3.01-2.96 (m, 2H), 2.93-2.90 (m, 2H), 2.82 (dd, J = 12.4, 5.1 Hz, 1H), 2.59-2.43 (m, 5H), 2.04 (t, J = 7.3 Hz, 2H), 1.64-1.21 (m, 20H), 0.99 (s, 6H); LC-MS: [M+H] + = 999.69.

[0295] Manufacturing Example 30: Manufacturing of CPI-009 [ka]

[0296] To a mixture of the compound (CPI-008) (80 mg), DMF (3 mL), and DIPEA (166 μL), TSTU (105 mg) was added and stirred at room temperature for 5 minutes. 6-Aminohexanoic acid (41 mg) was added and stirred at room temperature for 1 hour. The mixture was heated to 50 °C and stirred for 5 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (102 mg). 1H NMR (400 MHz, CD3OD) δ 8.53 (d, J = 2.1 Hz, 1H), 8.37 (t, J = 5.4 Hz, 1H), 7.99 (dd, J = 8.8, 2.3 Hz, 1H), 6.70 (d, J = 8.8 Hz, 1H), 3.39-3.32 (m, 2H), 2.31 (t, J = 7.3 Hz, 2H), 1.70-1.58 (m, 4H), 1.53-1.33 (m, 11H); LC-MS: [M+H] + = 367.30.

[0297] Production Example 31: Production of CPA-342 [ka]

[0298] (1) Manufacturing of CPA-339 DIPEA (25 μL) was added to a mixture of the compound (CPA-338) (29 mg), the compound (CPA-318) (13 mg) prepared in Preparation Example 19, and DMF (1 mL), and the mixture was stirred at 60° C. for 1 hour. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (31 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.36-13.34 (m, 1H), 9.06 (t, J = 5.8 Hz, 1H), 8.14-8.11 (m, 2H), 7.89 (t, J = 5.5 Hz, 1H), 7.78-7.63 (m, 6H), 6.42 (s, 1H), 6.35 (s, 1H), 4.82 (d, J = 12.3 Hz, 1H), 4.75 (d, J = 12.3 Hz, 1H), 4.32-4.29 (m, 1H), 4.14-4.11 (m, 1H), 3.93-3.87 (m, 1H), 3.63-3.60 (m, 10H), 3.52-3.30 (m, 14H), 3.11-3.04 (m, 5H), 3.00-2.96 (m, 2H), 2.93-2.90 (m, 2H), 2.82 (dd, J = 12.4, 5.1 Hz, 1H), 2.57 (d, J = 12.4 Hz, 1H), 2.23 (s, 4H), 2.09-2.02 (m, 4H), 1.63-1.21 (m, 20H), 0.91 (s, 6H); LC-MS: [M+H] + = 1301.83.

[0299] (2) Manufacturing of CPA-340 To a mixture of the compound (CPA-339) (31 mg) prepared above and 1,4-dioxane (1 mL), zinc (16 mg) and acetic acid (68 μL) were added and stirred at 60 °C for 2 hours. The reaction mixture was purified by column chromatography (ODS, water / MeCN, containing 0.1% AcOH) to obtain the target product (21 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.34 (t, J = 4.2 Hz, 1H), 9.09 (t, J = 5.7 Hz, 1H), 8.14-8.11 (m, 2H), 7.84 (t, J = 5.8 Hz, 1H), 7.77-7.63 (m, 5H), 6.42 (s, 1H), 6.36 (s, 1H), 4.32-4.29 (m, 1H), 4.14-4.11 (m, 1H), 3.63-3.04 (m, 32H), 3.01-2.96 (m, 2H), 2.94-2.90 (m, 2H), 2.82 (dd, J = 12.5, 5.1 Hz, 1H), 2.57 (d, J = 12.5 Hz, 1H), 2.24 (s, 4H), 2.09-2.02 (m, 4H), 1.65-1.21 (m, 20H), 0.91 (s, 6H); LC-MS: [M+H] + = 1127.92.

[0300] (3) Manufacturing of CPA-341 To a mixture of the compound (CPA-340) (10 mg), the compound (CPI-009) (4.7 mg) prepared in Preparation Example 28(1), and DMF (1 mL), WSC·HCl (3.4 mg), HOBt·HO (2.7 mg), and DIPEA (6.2 μL) were added and stirred at room temperature for 12 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (5.1 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.34 (t, J = 4.7 Hz, 1H), 9.09 (t, J = 5.7 Hz, 1H), 8.91 (s, 1H), 8.63 (s, 1H), 8.52 (d, J = 1.6 Hz, 1H), 8.24 (t, J = 5.5 Hz, 1H), 8.13-8.10 (m, 2H), 7.93 (dd, J = 8.9, 1.7 Hz, 1H), 7.86-7.84 (m, 2H), 7.77-7.63 (m, 5H), 6.43 (s, 1H), 6.36 (s, 1H), 4.32-4.29 (m, 1H), 4.17-4.11 (m, 2H), 3.62-2.89 (m, 35H), 2.81 (dd, J = 12.5, 5.1 Hz, 1H), 2.57 (d, J = 12.5 Hz, 1H), 2.23 (s, 4H), 2.13-2.02 (m, 6H), 1.67-1.24 (m, 35H), 0.90 (s, 6H); LC-MS: [M+H] + = 1476.01.

[0301] (4) Manufacturing of CPA-342 To a mixture of the compound (CPA-341) (4.7 mg) prepared above and MeOH (50 μL), 4M HCl in 1,4-dioxane was added and stirred at room temperature for 1 hour. MeOH (100 μL), acetone (100 μL), and DIPEA (70 μL) were added and stirred at room temperature for 1 hour. The reaction mixture was purified by column chromatography (ODS, water / MeCN) to obtain the desired product (2.9 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.34 (t, J = 4.6 Hz, 1H), 9.62 (s, 1H), 9.08 (t, J = 5.7 Hz, 1H), 8.57 (d, J = 1.8 Hz, 1H), 8.26 (t, J = 5.6 Hz, 1H), 8.14-8.11 (m, 2H), 7.99 (dd, J = 8.8, 2.3 Hz, 1H), 7.86-7.84 (m, 2H), 7.7607.63 (m, 5H), 7.04 (d, J = 8.8 Hz, 1H), 6.43 (s, 1H), 6.36 (s, 1H), 4.31-4.28 (m, 1H), 4.17-4.10 (m, 2H), 3.62-3.59 (m, 10H), 3.50-2.89 (m, 25H), 2.81 (dd, J = 12.6, 5.1 Hz, 1H), 2.57 (d, J = 12.6 Hz, 1H), 2.23 (s, 4H), 2.14-2.02 (m, 6H), 1.96 (s, 3H), 1.92 (s, 3H), 1.63-1.15 (m, 26H), 0.90 (s, 6H); LC-MS: [M+H] + = 1415.76.

[0302] Production Example 32: Production of CPA-344 [ka]

[0303] Production was carried out in the same manner as in Production Example 31, except that CPA-321 was changed to CPA-343. 11H NMR (400 MHz, DMSO-d6) δ 13.35 (s, 1H), 9.62 (s, 1H), 9.14 (t, J = 5.6 Hz, 1H), 8.57 (d, J = 1.9 Hz, 1H), 8.26 (t, J = 5.5 Hz, 1H), 8.14 - 8.12 (m, 2H), 7.99 (dd, J = 8.8, 2.4 Hz, 1H), 7.86 - 7.84 (m, 2H), 7.81 - 7.63 (m, 6H), 7.04 (d, J = 8.8 Hz, 1H), 6.43 (s, 1H), 6.36 (s, 1H), 4.32 - 4.28 (m, 1H), 4.17 - 4.10 (m, 2H), 3.60 - 2.91 (m, overlapped with water signal), 2.81 (dd, J = 12.4, 5.1 Hz, 1H), 2.61 - 2.45 (m, overlapped with DMSO signal), 2.29 - 2.26 (m, 6H), 2.14 - 2.02 (m, 8H), 1.96 (s, 3H), 1.93 (s, 3H), 1.80 (tt, J = 6.6, 6.6 Hz, 2H), 1.68 - 1.24 (m, 28H), 0.93 (s, 6H); LC-MS: [M+H] + = 1735.36.

[0304] Production Example 33: Production of CPI-016

Chem.

[0305] (1) Preparation of CPI-012 To a mixture of the compound (CPI-011) (97 mg) and DCM (2 mL), TFA (683 μL) was added and stirred at room temperature for 1 hour. The solvent was evaporated, and the residue was mixed with DMF (2 mL), N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-glutamic acid 5-tert-butyl hydrate (75 mg), HATU (81 mg), and DIPEA (155 μL) and stirred at room temperature for 1 hour. N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-glutamic acid 5-tert-butyl hydrate (75 mg) and HATU (68 mg) were added and stirred at room temperature for 1 hour. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (136 mg). 1 H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 7.4 Hz, 2H), 7.66-7.60 (m, 2H), 7.40 (dd, J = 7.4, 7.4 Hz, 2H), 7.32 (ddd, J = 7.4, 7.4, 1.0 Hz, 2H), 7.28-7.25 (m, 1H), 6.68 (s, 1H), 6.55 (s, 1H), 6.13-6.05 (brm, 1H), 4.89-4.78 (brm, 1H), 4.50-4.47 (m, 1H), 4.38-4.17 (m, 5H), 3.65-3.51 (m, 12H), 3.43-3.28 (m, 4H), 3.16-3.11 (m, 1H), 2.89 (dd, J = 12.8, 4.9 Hz, 1H), 2.68 (d, J = 12.8 Hz, 1H), 2.42-2.28 (m, 2H), 2.19-2.05 (m, 3H), 1.98-1.89 (m, 1H), 1.81-1.57 (m, 8H), 1.49-1.39 (m, 11H); LC-MS: [M+H] + = 854.39.

[0306] (2) Manufacturing of CPI-013 To a mixture of the compound (CPI-012) (136 mg) and DMF (2 mL), piperidine (24 μL) was added and stirred at room temperature for 5 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (87 mg). 1 H NMR (400 MHz, DMSO-d6) δ 7.83 (t, J = 5.7 Hz, 1H), 7.75 (t, J = 5.6 Hz, 1H), 6.42 (s, 1H), 6.35 (s, 1H), 4.32-4.29 (m, 1H), 4.14-4.11 (m, 1H), 3.53-3.45 (m, 8H), 3.41-3.33 (m, 6H), 3.13-3.04 (m, 6H), 2.82 (dd, J = 12.5, 5.1 Hz, 1H), 2.57 (d, J = 12.5 Hz, 1H), 2.24-2.20 (m, 2H), 2.04 (t, J = 7.4 Hz, 2H), 1.80-1.71 (m, 1H), 1.66-1.43 (m, 9H), 1.39 (s, 9H), 1.35-1.23 (m, 2H); LC-MS: [M+H] + = 632.38.

[0307] (3) Manufacturing of CPI-015 To a mixture of the compound (CPI-013) (87 mg), the compound (CPI-014) (22 mg), and DMF (2 mL), WSC·HCl (32 mg) and HOBt·HO (25 mg) were added and stirred at room temperature for 5 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (92 mg). 1H NMR (400 MHz, DMSO-d6) δ 7.80-7.71 (brm, 1H), 7.22 (t, J = 5.6 Hz, 1H), 6.85 (t, J = 5.3 Hz, 1H), 6.61 (s, 1H), 5.95-5.85 (m, 1H), 5.31 (dd, J = 15.8, 1.4 Hz, 1H), 5.22 (dd, J = 9.2, 1.4 Hz, 1H), 5.01-4.79 (brs, 1H), 4.57 (d, J = 5.7 Hz, 2H), 4.53-4.50 (m, 1H), 4.39-4.34 (m, 2H), 3.70-3.49 (m, 12H), 3.43-3.34 (m, 3H), 3.28-3.14 (m, 2H), 2.92 (dd, J = 12.8, 4.8 Hz, 1H), 2.72 (d, J = 12.8 Hz, 1H), 2.69-2.65 (m, 2H), 2.61-2.57 (m, 2H), 2.39-2.26 (m, 2H), 2.15-2.03 (m, 3H), 1.98-1.36 (m, 20H); LC-MS: [M+H] + = 772.45.

[0308] (4) Manufacturing of CPI-016 To a mixture of the compound (CPI-015) (92 mg) prepared above and DCM (1 mL), TFA (200 μL) was added and stirred at room temperature for 20 hours. The solvent was evaporated, and to the residue, DMF (2 mL), dimedone (33 mg), WSC·HCl (46 mg), and DMAP (29 mg) were added and stirred at 60 °C for 4 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN, containing 0.1% AcOH) to obtain the target product (45 mg). 1H NMR (400 MHz, DMSO-d6) δ 8.06 (d, J = 8.0 Hz, 1H), 7.84 (t, J = 5.4 Hz, 1H), 7.74 (t, J = 5.6 Hz, 1H), 6.42 (s, 1H), 6.35 (s, 1H), 5.94-5.84 (m, 1H), 5.29 (ddt, J = 17.2, 1.5, 1.5 Hz, 1H), 5.19 (ddt, J = 10.5, 1.5, 1.5 Hz, 2H), 4.52 (dt, J = 5.3, 1.5 Hz, 2H), 4.32-4.29 (m, 1H), 4.22-4.16 (m, 1H), 4.14-4.11 (m, 1H), 3.52-2.45 (m, 8H), 3.40-3.30 (m, 4H), 3.14-3.02 (m, 5H), 3.01-2.91 (brs, 2H), 2.82 (dd, J = 12.4, 5.1 Hz, 1H), 2.59-2.41 (m, 9H), 2.04 (t, J = 7.4 Hz, 2H), 1.95-1.87 (m, 1H), 1.80-1.71 (m, 1H), 1.65-1.57 (m, 5H), 1.54-1.41 (m, 3H), 1.35-1.24 (m, 2H), 0.99 (s, 6H); LC-MS: [M+H] + = 838.42.

[0309] Production Example 34: Production of CPA-346 [ka]

[0310] (1) Manufacturing of CPA-345 To a mixture of the compound (CPA-316) (8.8 mg) and DMF (1 mL), N-Boc-1,2-diaminoethane (7.3 μL), COMU (22 mg), and DIPEA (24 μL) were added and stirred at room temperature for 5 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (8.3 mg). 1 H NMR (400 MHz, CDCl3) δ 8.22-8.19 (m, 2H), 7.81-7.70 (brs, 1H), 7.61-7.52 (m, 3H), 4.99-4.87 (brs, 1H), 3.68 (dt, J = 5.7, 5.7 Hz, 2H), 3.45 (dt, J = 5.7, 5.7 Hz, 2H), 1.44 (s, 9H); LC-MS: [M+H] + = 333.16.

[0311] (2) Manufacturing of CPA-346 It was produced in the same manner as in Production Example 20 using CPA-345 and CPI-016. 1 H NMR (400 MHz, DMSO-d6) δ 13.31 (t, J = 5.9 Hz, 1H), 9.43 (t, J = 5.6 Hz, 1H), 8.15-8.12 (m, 2H), 7.85 (t, J = 5.6 Hz, 1H), 7.75-7.65 (m, 4H), 6.41 (s, 1H), 6.35 (s, 1H), 4.48 (dd, J = 10.8, 3.5 Hz, 1H), 4.32-4.28 (m, 1H), 4.14-4.10 (m, 1H), 3.70-3.67 (m, 2H), 3.58-3.29 (m, 14H), 3.12-2.89 (m, 7H), 2.84-2.66 (m, 5H), 2.59-2.49 (m, 5H), 2.26-2.19 (m, 5H), 2.04 (t, J = 7.4 Hz, 2H), 1.65-1.41 (m, 8H), 1.35-1.24 (m, 2H), 0.91 (s, 6H); LC-MS: [M+H] + = 1109.51.

[0312] Production Example 35: Production of CPA-347 [ka]

[0313] (1) Manufacturing of CPI-018HCL To a mixture of the compound represented by the formula above (CPI-017, isomer ratio 4:3) (63 mg) and THF (2 mL), triphenylphosphine (78 mg) and HO (0.40 mL) were added and stirred overnight at room temperature. 1 M aqueous HCl (0.10 mL) was added and stirred at room temperature for 1 hour. 1 M aqueous HCl (0.30 mL) was added and stirred at room temperature for 1 hour. The mixture was heated to 60 °C and stirred for 6 hours, then stirred at room temperature for 4 days. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the desired product (39 mg, isomer ratio 10:9). 1 H NMR (400 MHz, CD3OD) δ 8.20 (dd, J = 8.4, 0.8 Hz, 1H), 7.99 (d, J = 8.8 Hz, 0.9H), 7.69 (d, J = 2.6 Hz, 0.9H), 7.45-7.37 (m, 2 + 0.9H), 4.21-4.15 (m, 2 + 0.9 × 2H), 3.67 (s, 0.9 × 3H), 3.65 (s, 3H), 2.95 (t, J = 7.6 Hz, 2 + 0.9 × 2H), 1.94-1.85 (m, 2 + 0.9 × 2H), 1.76-1.66 (m, 2 + 0.9 × 2H), 1.65-1.45 (m, 4 + 0.9 × 4H); LC-MS: [M+H] + = 292.23.

[0314] (2) Manufacturing of CPI-019 To a mixture of the compound (CPI-018HCl) (39 mg), DMF (2 mL), and DIPEA (93 μL), succinic anhydride (13 mg) was added and stirred overnight at room temperature. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (32 mg, isomer ratio 2:1). 1H NMR (400 MHz, CD3OD) δ 8.19 (dd, J = 7.6, 2.0 Hz, 1H), 7.98 (d, J = 8.8 Hz, 0.5H), 7.69 (d, J = 2.6 Hz, 0.5H), 7.45-7.37 (m, 2 + 0.5H), 4.19-4.12 (m, 2 + 0.5 × 2H), 3.66 (s, 0.5 × 3H), 3.64 (s, 3H), 3.19 (t, J = 6.9 Hz, 2 + 0.5 × 2H), 2.60-2.55 (m, 2 + 0.5 × 2H), 2.45 (t, J = 6.8 Hz, 2 + 0.5 × 2H), 1.91-1.82 (m, 2 + 0.5 × 2H), 1.60-1.50 (m, 4 + 0.5 × 4H), 1.48-1.39 (m, 2 + 0.5 × 2H); LC-MS: [M+H] + = 392.33.

[0315] (3) Manufacture of CPA-347 Production example 18 is produced by using the same material as production example 20 (opposite sex ratio 2:1). 1 H NMR (400 MHz, DMSO-d6) δ 13.34 (t, J = 4.9 Hz, 1H), 9.10 (t, J = 5.5 Hz, 1H), 8.13-9.10 (m, 2.3H), 8.00 (d, J = 7.9 Hz, 1H), 7.89-7.63 (m, 8.3H), 7.52 (s, 0.7H), 7.36 (s, 0.7H), 6.43 (s, 1H), 6.36 (s, 1H), 4.31-4.28 (m, 1H), 4.13-4.06 (m, 4H), 3.62-2.89 (m, 36H), 2.81 (dd, J = 12.5, 5.2 Hz, 1H), 2.60-2.49 (m, 3H), 2.37-2.28 (m, 4H), 2.23 (s, 4H), 2.09-2.02 (m, 4H), 1.78-1.71 (m, 2H), 1.65-1.23 (m, 26H), 0.90 (s, 6H); LC-MS: [M+H] + = 1500.96.

[0316] Production Example 36: Production of CPA-353 [ka]

[0317] (1) Manufacturing of CPA-348 A mixture of the compound (CPA-338) (11 mg), N-Boc-2,2'-(ethylenedioxy)diethylamine (2.7 mg), and DMF (1 mL) was stirred at 60 °C for 2 hours. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (10 mg). 1 H NMR (400 MHz, DMSO-d6) δ 13.35 (s, 1H), 7.89 (t, J = 5.6 Hz, 1H), 7.78-7.74 (m, 3H), 6.77 (t, J = 5.3 Hz, 1H), 6.42 (s, 1H), 6.35 (s, 1H), 4.82 (d, J = 12.4 Hz, 1H), 4.75 (d, J = 12.4 Hz, 1H), 4.32-4.29 (m, 1H), 4.14-4.11 (m, 1H), 3.93-3.87 (m, 1H), 3.61-2.92 (m, 33H), 2.82 (dd, J = 12.4, 5.1 Hz, 1H), 2.59-2.49 (m, 1H), 2.27 (s, 4H), 2.10-2.02 (m, 4H), 1.73-1.21 (m, 29H), 0.94 (s, 6H); LC-MS: [M+H] + = 1229.65.

[0318] (2) Manufacturing of CPA-349 To a mixture of the compound (CPA-348) (10 mg) prepared above and 1,4-dioxane (1 mL), zinc (11 mg) and acetic acid (47 μL) were added and stirred at 60 °C for 30 minutes. Zinc (11 mg) was added and the mixture was stirred at 60 °C for 30 minutes. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN, containing 0.1% AcOH) to obtain the target product (8.6 mg). 1 H NMR (400 MHz, DMSO-d6) δ 13.35 (s, 1H), 7.98-7.92 (brs, 1H), 7.75 (t, J = 5.5 Hz, 2H), 6.77 (t, J = 5.4 Hz, 1H), 6.42 (s, 1H), 6.35 (s, 1H), 6.32-6.29 (m, 1H), 4.14-4.11 (m, 1H), 3.61-3.28 (m, 27H), 3.14-2.92 (m, 9H), 2.82 (dd, J = 12.4, 5.1 Hz, 1H), 2.59-2.49 (m, 1H), 2.27 (s, 4H), 2.10-2.02 (m, 4H), 1.64-1.23 (m, 29H), 0.94 (s, 6H); LC-MS: [M+H] + = 1055.66.

[0319] (3) Manufacturing of CPA-350 To a mixture of the compound (CPA-349) (8.6 mg), CPA-311 (2.2 mg), and DMF (1 mL), WSC·HCl (3.1 mg) and HOBt·HO (2.5 mg) were added and stirred at room temperature for 1 hour. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (2.3 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.34 (s, 1H), 7.96 (d, J = 7.9 Hz, 1H), 7.87-7.82 (m, 2H), 7.75-7.73 (m, 2H), 6.80-6.74 (brs, 1H), 6.42 (s, 1H), 6.35 (s, 1H), 5.96-5.86 (m, 1H), 5.32-5.26 (m, 1H), 5.22-5.19 (m, 1H), 4.54-4.53 (m, 2H), 4.32-4.28 (m, 1H), 4.14-4.04 (m, 2H), 3.61-3.16 (m, overlapped with water signal), 3.12-2.89 (m, 11H), 2.82 (dd, J = 12.4, 5.1 Hz, 1H), 2.61-2.49 (m, overlapped with DMSO signal), 2.33-2.22 (m, 10H), 2.10-2.02 (m, 4H), 1.85 (s, 2H), 1.65-1.21 (m, 33H), 0.94 (s, 6H); LC-MS: [M+H] + = 1309.01.

[0320] (4) Manufacturing of CPA-351 To a mixture of the compound (CPA-350) (2.2 mg) prepared above and DCM (0.50 mL) was added TFA (130 μL) and stirred at room temperature for 1 hour. The solvent was evaporated, and to the residue were added DMF (0.50 mL), 2-thiopheneglyoxylic acid (1.3 mg), COMU (3.6 mg), and DIPEA (15 μL), and the mixture was stirred at room temperature for 1 hour. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the desired product (1.4 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.36-13.33 (m, 1H), 8.88 (t, J = 5.5 Hz, 1H), 8.19-8.16 (m, 2H), 7.97 (d, J = 8.3 Hz, 1H), 7.87-7.83 (m, 2H), 7.77-7.73 (m, 2H), 7.29 (dd, J = 4.8, 4.0 Hz, 1H), 6.42 (s, 1H), 6.35 (s, 1H), 5.96-5.86 (m, 1H), 5.31-5.26 (m, 1H), 5.22-5.19 (m, 1H), 4.53 (dd, J = 5.4, ​​1.2 Hz, 2H), 4.32-4.28 (m, 1H), 4.14-4.05 (m, 2H), 3.61-2.91 (m, overlapped with water signal), 2.82 (dd, J = 12.4, 5.1 Hz, 1H), 2.61-2.49 (m, overlapped with DMSO signal), 2.34-2.25 (m, 10H), 2.09-2.02 (m, 4H), 1.63-1.19 (m, 26H), 0.92 (s, 6H); LC-MS: [M+H] + = 1346.89.

[0321] (5) Manufacturing of CPA-352 To a mixture of the compound (CPA-351) (1.4 mg) prepared above and DCM (0.50 mL), Pd(PPh3)4 (0.1 mg) and 1,3-dimethylbarbituric acid (0.3 mg) were added and stirred at room temperature for 1 hour. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (0.7 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.35-13.33 (m, 1H), 8.94-8.91 (m, 1H), 8.19-8.16 (m, 2H), 8.07-7.99 (m, 1H), 7.94-7.91 (m, 2H), 7.29 (dd, J = 4.8, 3.9 Hz, 1H), 6.47 (s, 1H), 6.37 (s, 1H), 4.31-4.28 (m, 1H), 4.14-4.04 (m, 2H), 3.62-2.94 (m, overlapped with water signal), 2.82 (dd, J = 12.4, 5.1 Hz, 1H), 2.61-2.49 (m, overlapped with DMSO signal), 2.34-2.25 (m, 10H), 2.11-2.03 (m, 4H), 1.94-1.88 (brm, 2H), 1.62-1.11 (m, 24H), 0.92 (s, 6H); LC-MS: [M+H] + = 1306.84.

[0322] (6) Manufacturing of CPA-353 To a mixture of the compound (CPA-352) (0.7 mg) prepared above and DMF (0.50 mL) were added N-hydroxysuccinimide (0.6 mg) and WSC·HCl (1.0 mg), and the mixture was stirred at room temperature for 16 hours. Further N-hydroxysuccinimide (0.6 mg) and WSC·HCl (1.0 mg) were added, and the mixture was stirred at room temperature for 1 hour. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (0.7 mg). 1H NMR (400 MHz, DMSO-d6) δ 13.37-13.33 (m, 1H), 8.94-8.87 (m, 1H), 8.19-8.16 (m, 2H), 7.98-7.96 (m, 1H), 7.87-7.83 (m, 2H), 7.76-7.73 (m, 2H), 7.29 (dd, J = 4.9, 3.9 Hz, 1H), 6.42 (s, 1H), 6.36 (s, 1H), 4.31-4.28 (m, 1H), 4.14-4.04 (m, 2H), 3.62-2.79 (m, overlapped with water signal), 2.61-2.25 (m, overlapped with DMSO signal), 2.09-2.02 (m, 4H), 1.63-1.15 (m, 26H), 0.92 (s, 6H); LC-MS: [M+2H] 2+ = 1404.71.

[0323] Production Example 37: Synthesis of CPI-018 [ka]

[0324] Synthesis of CPI-018 To a mixture of the compound (CPI-017) (20 mg), DMF (0.5 mL), and DIPEA (14 μL) was added succinic anhydride (2.4 mg) and stirred overnight at room temperature. AcOEt was added to the reaction solution, which was washed with water and saturated brine and then dried over Na2SO4. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the desired product (14 mg). 1H NMR (500 MHz, CD3OD) δ 4.30-4.23 (m, 1H), 3.53-3.38 (m, 8H), 3.31-3.25 (m, 2H), 3.20-3.13 (m, 4H), 2.54-2.40 (m, 4H), 2.30-2.24 (m, 2H), 2.20-2.10 (m, 1H), 1.89-1.80 (m, 1H), 1.69-1.27 (m, 66H); LC-MS: [M+H] + = 1073.20.

[0325] Production Example 38: Production of CPA-354 [ka]

[0326] Synthesis of CPA-353 To a mixture of the compound (CPI-018) (5.3 mg), the compound (CPA-340) (5.6 mg) prepared in Preparation Example 30 (2), and DMF (0.5 mL), HATU (2.3 mg) and DIPEA (2.6 μL) were added and stirred at room temperature for 1 hour. The solvent was evaporated, and the residue was purified by column chromatography (ODS, water / MeCN) to obtain the target product (5.8 mg). 1 H NMR (400 MHz, CD3OD) δ 8.20-8.15 (m, 2H), 7.69-7.59 (m, 3H), 4.52-4.47 (m, 1H), 4.33-4.19 (m, 3H), 3.78-3.38 (m, 34H), 3.29-3.10 (m, 11H), 3.00-2.89 (m, 3H), 2.70 (d, J = 12.7 Hz, 1H), 2.64-2.48 (m, 4H), 2.33-2.05 (m, 11H), 1.93-1.27 (m, 87H), 0.97 (s, 6H).

[0327] Synthesis of CPA-354 To a mixture of the compound (CPA-353) (5.8 mg) and DCM (1 mL), TFA (0.5 mL) was added and the mixture was stirred at room temperature for 9 hours. The solvent was evaporated to give the target product (4.5 mg). 1 H NMR (400 MHz, CD3OD) δ 8.19-8.15 (m, 2H), 7.69-7.59 (m, 3H), 4.52-4.46 (m, 1H), 4.34-4.20 (m, 3H), 3.80-3.46 (m, 34H), 3.29-3.10 (m, 11H), 3.00-2.89 (m, 3H), 2.74-2.68 (m, 1H), 2.67-2.48 (m, 4H), 2.33-2.09 (m, 11H), 1.93-1.26 (m, 33H), 0.97 (s, 6H); LC-MS: [M+H] 2+ = 923.48.

[0328] Production Example 39: Production of labeled antibody (CPA-321 labeled antibody) 20 μL of anti-CD71 antibody OKT9 (#16-0719-85, Thermo Fisher Scientific) or a mouse IgG1 isotype control antibody (#553447, BD Biosciences) was placed in a tube and diluted with NHS labeling buffer (25 mM HEPES-NaOH pH 8.2, 150 mM NaCl) to a final concentration of approximately 0.1 mg / ml. Approximately 1.5 μL of a DMSO solution (10 mg / ml in DMSO) of the compound (CPA-321) prepared in Preparation Example 20 was added and incubated at room temperature for 2 hours. Approximately 5 μL of a lysine solution (100 mM) was then added and incubated at room temperature for 20 minutes. The mixture was desalted using a desalting column (Zeba Spin, Thermo Fisher Scientific, 89882), and the solution containing the CPA-321-labeled antibody (CPA-321-labeled antibody solution) was recovered.

[0329] [Test example] The effects of the compounds of the present invention will be explained below by showing test results using representative compounds among the tetrafunctional compounds and tetrafunctional chemical probes of the present invention, although the present invention is not limited to these test examples.

[0330] Test Example 1: Isolation and detection of target molecules from living cells using compound (CPP-127) 1. Cell Culture and Methods for Treating Cultured Cells with the Compounds CHO-K1 cells stably expressing human dopamine receptor D2 (DRD2) were cultured on a 10 cm plate until confluent. After washing the cells with medium, the medium was replaced with medium containing DMSO or unmodified ligand and incubated for 30 minutes. The medium was then discarded and replaced with medium containing the compound (CPP-127) synthesized in Preparation Example 12, and the plate was incubated at 37°C for 30 minutes. The medium was discarded, the cells were washed with PBS, 1 ml of PBS was added to the cells on the plate, and the cells were irradiated with 302 nm UV light for 1 minute using a UV crosslinker. After UV irradiation, the cells were collected using a cell scraper, and the collected cell pellet was frozen and stored at -80°C.

[0331] 2. Membrane Fraction Protein Preparation and Purification Cryopreserved cells were suspended in 50 mM Tris homogenate buffer (pH 7.4, 2.5 mM EDTA, 5 mM MgCl2, 320 mM sucrose) and homogenized using an ultrasonic homogenizer. The homogenized suspension was centrifuged in a centrifuge (1,000 g, 15 minutes, 4°C). The supernatant was transferred to another tube and centrifuged (30,000 × g, 30 minutes, 4°C). After centrifugation, the supernatant was discarded, and the pellet was homogenized in 100 mM sodium carbonate buffer (Na2CO3) using an ultrasonic homogenizer and incubated on ice for 10 minutes. The suspension was then centrifuged in an ultracentrifuge (45,2000 × g, 30 minutes, 4°C), the supernatant was discarded, and the resulting pellet was rinsed with Milli-Q water. This was used as the membrane protein fraction.

[0332] 3. Elution of Membrane Proteins The membrane fraction proteins prepared by the above method were added to membrane lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% SDS, 0.5% SDC, 1% Triton-X-100, 8 M urea) and solubilized using an ultrasonicator. Streptavidin beads (#88816, Thermo Fisher Scientific) were added to the solubilized membrane protein solution and mixed for 60 minutes to adsorb the biotin-labeled proteins to the beads. After washing the beads with membrane lysis buffer, 40 μL of 2% N2H4 solution containing 0.05% SDS was added and the mixture was left to stand at room temperature for 30 minutes. The beads were removed by magnetic separation, and the solution was recovered. The recovered solution was neutralized with 20% TFA solution and then concentrated using a centrifugal evaporator. The concentrated solution was reduced and alkylated with dithiothreitol and iodoacetamide, and then digested with trypsin solution overnight at 37°C.

[0333] 4. LC-MS Analysis and Target Protein Identification The resulting tryptic digest was desalted and purified, and protein analysis was performed by LC-MS. LC-MS was performed using an LC-MS system combining a nano-HPLC system (Ultimate 3000, Thermo Scientific) and a mass spectrometer (Q Exactive, Thermo Scientific). The nano-HPLC system used a column (stationary phase) consisting of a trap column (C18 Acclaim PepMap, Thermo Scientific) and a tip column (75 μm id, 12 cm long, 3 μm C18 particles, Nikkyo Technos). Reverse-phase chromatography was performed using a 0.1% formic acid-acetonitrile mobile phase. The flow rate was 300 nL / min, and peptides were eluted with a linear gradient increasing the acetonitrile concentration from 4% to 35% over 48 min. Mass spectrometry was performed in positive ion mode with nanoelectrospray ionization. Full-scan MS spectra were obtained from m / z 380 to 1600, and product ion spectra were collected using data-dependent acquisition mode. The data obtained by LC-MS were analyzed using the Proteome Discovere 2.2 (PD 2.2, Thermo Fisher Scientific) analysis software. Protein identification was performed using the Sequest search engine in PD2.2, searching against a database downloaded from Uniprot. False discovery rates were calculated and filtering was performed to reduce the false-positive protein identification rate to less than 1%. To further select reliable candidate proteins, proteins with a PSM score of 5 or higher were identified. The number of unique peptides identified was further refined. Proteins were quantified using the LFQ quantitative method in PD2.2, and specific proteins were identified by quantitative comparison with the unmodified ligand-treated group. Analysis was performed in two independent experiments, and the results are expressed as a ratio to the unmodified ligand-treated group.

[0334] 5.Results The results are shown in Figure 1. As shown in Figure 1, when the tetrafunctional chemical probe of the present invention (compound (CPP-127)) was used, dopamine receptor D2 was significantly suppressed in the unmodified ligand-treated group, confirming that dopamine receptor D2 (DRD2) was specifically recovered.

[0335] Experimental Example 2: Isolation and Detection of Target Molecules from Live Tissues Using Compounds (CPF-224, CPF-202, CPF-242) DBA / 2CrSlc mice were anesthetized with isoflurane inhalation and then decapitated, and the brains were rapidly removed. The removed brains were immersed in 0°C artificial cerebrospinal fluid (ACSF) for 60 seconds while continuously aerating with a mixed gas (95% O2, 5% CO2). The olfactory bulbs and cerebellum were removed with a scalpel, and the brains were fixed to the stage of a vibrating blade microtome (Leica, VT1200) using instant glue. Brain slices (0.3 mm thick) containing the striatum and hippocampus were prepared in 0°C ACSF while continuously aerating with a mixed gas. The slices were placed in a dish containing artificial cerebrospinal fluid, and unmodified ligand or DMSO was added. The slices were then left at room temperature for 5 minutes. Next, the compound (CPF-224) prepared in Preparation Example 10 or the compound (CPF-202) prepared in Preparation Example 8 was added and allowed to stand at room temperature for 30 minutes. The solution was removed, and the slices were irradiated with 302 nm UV light for 1 minute (CPF-224 and CPF-202) or 365 nm UV light for 15 minutes (CPF242). The slices were washed with PBS and collected, and the supernatant was removed by centrifugation (3000 × g, 4 °C, 5 minutes). The resulting pellet was analyzed using the same method as in Test Example 1. Analysis was performed in two independent experiments and can be expressed as a ratio to the unmodified ligand-treated group.

[0336] The results using compound (CPF-224) are shown in Figure 2, the results using compound (CPF-202) in Figure 3, and the results using compound (CPF-242) in Figure 4. As shown in these figures, when the tetrafunctional chemical probe of the present invention was used, AMPA-type glutamate receptors (Gria1, Gria2, Gria3, Gria4) and GABA receptor subunits (Gabra1, Gabra2, Gabra5, Gabrg2) contained in brain tissue in the unmodified ligand-treated group were significantly suppressed by CPF-224, CPF-202, and CPF242, respectively, confirming that each membrane protein was specifically recovered.

[0337] Test Example 3: Capture of cell surface antigens using labeled antibodies Antigen molecules were isolated from THP-1 cells using the CPA-321-labeled anti-CD71 antibody or CPA-321-labeled anti-mouse IgG1 control antibody prepared in Preparation Example 21. Specifically, THP-1 cells were harvested and washed with SB buffer (PBS buffer containing 2% FCS). After washing, the cells were suspended in SB buffer containing 0.1% Fc block reagent (Invitrogen, 16-9161-73) and incubated at 4°C for 15 minutes. The supernatant was removed by centrifugation (600 × g, 3 minutes, 4°C), and an antibody solution (prepared in Preparation Example 21, 20 μg, with SB buffer to make 1 mL) was added and incubated at 4°C for 20 minutes. The antibody solution was removed by centrifugation, and the cells were washed with SB buffer and then suspended in 0.5 mL of SB buffer. The cell suspension was irradiated with 302 nm UV light for 1 minute, and the SB buffer was removed by centrifugation. The obtained cells were analyzed by the method described in Test Example 1. The analysis was performed in two independent experiments, and the results were expressed as the ratio of the amount of protein obtained with the anti-CD71 antibody to that obtained with the mouse IgG1 isotype control antibody (anti-mouse IgG antibody) [Figure 5].

[0338] As shown in Figure 5, it was confirmed that TFRC (CD71) was significantly obtained in two analyses.

[0339] Test Examples 4 to 13: Capture of cell surface antigens using labeled antibodies (1) The labeled antibodies listed in Table 1 were used to probe antigen molecules from the cells listed in Table 1. Specifically, the cells listed in Table 1 were washed with SB buffer (PBS buffer containing 2% FCS), suspended in SB buffer containing 0.1% Fc block reagent (Invitrogen, 16-9161-73), and incubated at 4°C for 15 minutes. The supernatant was removed by centrifugation (600 × g, 3 minutes, 4°C), and a solution containing the labeled antibody prepared according to the labeled antibody preparation instructions was added and incubated at 4°C for 20 minutes. The antibody solution was removed by centrifugation, and the cells were washed with SB buffer and then suspended in 0.5 ml of SB buffer. The cell suspension was irradiated with ultraviolet light at the wavelengths listed in the table for the times indicated, and the SB buffer was removed by centrifugation. The collected cells were analyzed using the method described in Test Example 1. Two independent experiments were performed, and the amounts of the target membrane proteins listed in the table were compared (Figures 6 to 8).

[0340] Production of labeled antibodies (Test Examples 4 to 12) The antibodies listed in Table 1 were placed in tubes and diluted with NHS labeling buffer (25 mM HEPES-NaOH pH 8.2, 150 mM NaCl) to a final concentration of approximately 0.05 mg / ml. Approximately 0.9 μL of a DMSO solution (10 mg / ml in DMSO) of the probes listed in Table 1 was added to each tube, and the mixture was incubated at room temperature for 2 hours. Next, approximately 5 μL of a lysine solution (100 mM) was added, and the mixture was incubated at room temperature for 20 minutes. The mixture was then stored at 4°C until use.

[0341] Preparation of labeled ligand (Test Example 13) 12.5 μg of His-Tag EGF was placed in a tube and diluted with NHS labeling buffer to a final concentration of approximately 0.25 mg / ml. 48 μL of this solution was mixed with 2 μL of 5 mM CPA-354 to prepare the labeled ligand solution. This solution was stored at 4°C until use.

[0342] [Table 2]

[0343] Test Example 14: Capture of cell surface antigens using labeled antibodies (2) Antigen molecules were probed in A431 cells using labeled OKT9 prepared as described in the table below. First, the cells were washed with SB buffer (PBS buffer containing 2% FCS), and then SB buffer containing 0.1% Fc block reagent (Invitrogen, 16-9161-73) was added and incubated at 4°C for 15 minutes. The supernatant was removed, and 250 μL of ligand solution diluted 10-fold with SB buffer was added and incubated at 4°C for 20 minutes. The ligand solution was removed, and the cells were washed with PBS and then irradiated with 302 nm UV light for 1.5 minutes. Proteins were extracted from the cells using lysis buffer containing 1% NP-40 and analyzed by the method described in Test Example 1. The unprobed OKT9 antibody was used as a negative control (No Probe), and the amount of CD71 detected by each probe ligand was compared (Figure 9).

[0344] Preparation of labeled antibodies (1) 10 μg of anti-CD71 antibody OKT9 (Thermo Fisher Scientific, Cat # 16-0719-85) was placed in a tube and diluted with NHS labeling buffer (25 mM HEPES-NaOH pH 8.2, 150 mM NaCl) to a final concentration of approximately 0.1 mg / ml. 0.9 μL of CPA-321 solution (10 mg / ml in DMSO) was added and incubated at room temperature for 2 hours. Approximately 5 μL of lysine solution (100 mM) was then added and incubated at room temperature for 20 minutes. The mixture was then stored at 4°C until use. (2) 10 μg of the anti-CD71 antibody OKT9 was placed in a tube, and 5 μL of 30 mM sodium periodate (in phosphate buffer, pH 6.5) was added, followed by phosphate buffer, pH 6.5, to bring the total volume to 100 μL. After 15 minutes of incubation at room temperature, the solvent was exchanged with PBS using a ZabaSpin column (Thermo Scientific). To 100 μL of the resulting antibody solution, 1 μL of 500 mM 5-MA aqueous solution was added, followed by 1 μL of 10 mg / ml CPA-342 or CPA-344 DMSO solution. The mixture was then incubated at room temperature for 1.5 hours in the dark. After the incubation, the solvent was exchanged with PBS using a ZabaSpin column, and excess reagent was removed.

[0345] [Table 3]

[0346] Test Example 17: Capture of cell surface antigens using labeled antibodies (secondary antibody method) Antigen molecules were probed in THP-1 cells using a labeled secondary antibody prepared by the CBP-198-labeled secondary antibody manufacturing method. Specifically, THP-1 cells were washed with SB buffer (PBS buffer containing 2% FCS) and suspended in SB buffer containing 0.1% Fc block reagent (Invitrogen, 16-9161-73), followed by incubation at 4°C for 15 minutes. The supernatant was removed by centrifugation (600 × g, 3 minutes, 4°C). A solution of anti-CD71 antibody DF1513 diluted to 5 μg / mL in SB buffer was added to the cells, and the incubation was continued for 20 minutes at 4°C. The antibody solution was removed by centrifugation, and the cells were washed with SB buffer. Next, the cells were suspended in a labeled secondary antibody prepared by the CPA-503-labeled secondary antibody manufacturing method (prepared by adding SB buffer to 1 mL) and incubated for 20 minutes at 4°C. The antibody solution was removed by centrifugation, and the cells were washed with SB buffer and then suspended in 0.5 ml of SB buffer. The cells were irradiated with 365 nm UV light for 15 minutes, and the SB buffer was removed by centrifugation. The collected cells were analyzed by the method described in Test Example 1. The test was performed independently twice, and the amount of CD71 detected was compared. As a comparative example, the amount of CD71 detected was calculated in the same manner as in Test Example 11, except that the anti-CD71 antibody OKT9 was replaced with the anti-CD71 antibody DF1513 (FIG. 10).

[0347] Preparation of CPA-503-labeled secondary antibody Goat anti-mouse IgG (5 μg) was placed in a tube and diluted with NHS labeling buffer (25 mM HEPES-NaOH pH 8.2, 150 mM NaCl) to a final concentration of approximately 0.05 mg / ml as shown in the table. Approximately 0.9 μL of a DMSO solution of CPA-503 (10 mg / ml in DMSO) was added to each tube, and the mixture was incubated at room temperature for 2 hours. Approximately 5 μL of a 100 mM lysine solution was then added, and the mixture was incubated at room temperature for 20 minutes. The mixture was then stored at 4°C until use.

[0348] By using the secondary antibody method, it is possible to detect membrane proteins that could not be detected by the primary antibody method. [Sequence List Free Text]

[0349] SEQ ID NOs: 1 and 2 are both amino acid sequences of peptides which are one embodiment of the compound constituting the cleavable site (E) of the tetrafunctional compound or tetrafunctional chemical probe of the present invention.

Claims

1. A tetrafunctional compound comprising a ligand binding site (A), a reactive site (D), a cleavable site (E), and a biotin site (B) linked via a spacer, The tetrafunctional compound is represented by the following formula (I) or the following formula (II): 【Chemistry 1】 (wherein A, D, E, and B represent the ligand binding site (A), the reactive site (D), the cleavable site (E), and the biotin site (B), respectively; S １ , S ２ , S ３ , S ４ , S ５ , S ６ and S ７ are each independently a spacer.) the ligand binding site (A) is an N-hydroxysuccinimide ester group; The reactive site (D) is a group represented by formula (2) [in which n in formula (2) is 1] or a group represented by formula (2'-1) when the tetrafunctional compound adopts the structure of formula (I), and is a group represented by formula (2) [in which n in formula (2) is 0], a group represented by formula (2'-2), or a group having a benzophenone structure (moiety) when the tetrafunctional compound adopts the structure of formula (II), 【Chemistry 2】 (In the formula, * means a bond to an adjacent group.) The cleavable site (E) is a group having a structure of Dde represented by the following formula (3): 【Transformation 3】 (In the formula, R １ , R ２ , R ３ , R ４ , R ５ and R ６ Each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. * represents a bond to the adjacent group. The biotin moiety (B) is a group represented by the following formula (1): 【Chemistry 4】 (wherein * means a bond to an adjacent group); The spacer is a linear or branched alkylene group having 1 to 20 carbon atoms, or a linear or branched alkylene group having 3 to 20 carbon atoms, in which non-adjacent —CH ２ The groups are independently —O—, —NR ａ - [R ａ means a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. The same applies hereinafter.], —CO—NR ａ -, -NR ａ -CO- is a group substituted with at least one bridging group selected from the group consisting of Or a tetrafunctional compound represented by any one of the following formulas (III) to (V): 【Transformation 5】

2. The tetrafunctional compound according to claim 1, which is a compound represented by the following formula (i), (ii), or (iii): 【Transformation 6】 (In the formula, p represents 2.)

3. A tetrafunctional chemical probe in which a ligand is bonded to the ligand binding site (A) of the tetrafunctional compound according to claim 1 or 2 by replacing a free functional group in the ligand binding site (A), or a tetrafunctional chemical probe represented by any one of the following formulas (VI) to (IX): 【Transformation 7】

4. 4. The tetrafunctional chemical probe according to claim 3, wherein the ligand is any one selected from the group consisting of proteins, peptides, lipids, sugars, and low molecular weight compounds having affinity for membrane proteins derived from living cells or living tissues.

5. A method for detecting a target protein in a cell or tissue using the tetrafunctional chemical probe according to claim 3 or 4, comprising: (1) reacting a tetrafunctional chemical probe with cells or tissues to bind a ligand in the tetrafunctional chemical probe to a target protein; (2) forming a covalent bond between the reactive site (D) in the tetrafunctional chemical probe and the target protein; (3) purifying a fraction containing the target protein bound to the tetrafunctional chemical probe; (4) binding the biotin moiety in the tetrafunctional chemical probe with avidin to form a tetrafunctional chemical probe-avidin complex; (5) cleaving the formed tetrafunctional chemical probe-avidin complex at the cleavable site in the tetrafunctional chemical probe; and (6) detecting fractions containing the target protein A method for detecting a target protein, comprising the steps of:

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