Quadruple-functional chemical probe and method for identifying target membrane proteins from living cells or living tissues using the same

Abstract

A tetrafunctional compound, which is formed by connecting a ligand binding site (A) or a ligand, a reactive site (D), a potential cleavable site (E) and a biotin site (B) through a spacer as needed, wherein: the ligand binding site (A) is an activated 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-carboxytetrazolyl; the potential cleavable site (E) is a group having a structure of a compound such as 1-(4,4-dimethyl-2,6-dioxocyclohexyl-1-ylidene)ethyl; and the spacer is a group obtained by substitution with a cross-linking group, such as a straight or branched alkylene group having one or more carbon atoms.

Description

Technical Field

[0001] The present invention relates to a probe capable of unbiased and comprehensive identification of target proteins of specific ligands directly from living cells or tissues reflecting phenotypes using chemical proteomics, and also relates to an identification method using the probe. Specifically, the present invention relates to a probe capable of detecting various target membrane proteins, including ion channels, GPCRs, or receptors forming multiple transmembrane segments or multimers, from living cells or brain slices as living tissue, and also relates to a method for identifying target membrane proteins using the probe. Background Art

[0002] In the field of chemical biology research, organic chemistry and molecular biology methods have been used to detect and quantify small amounts of biomolecules, such as proteins or physiologically active substances, as well as to understand the functions and reactions of these biomolecules. As part of these efforts, compounds that act specifically on biomolecules (i.e., chemical probes) are being developed with the goal of revealing the functions and reactions of biomolecules.

[0003] In this research field, chemical proteomics is one of the methods that uses organic chemistry methods to elucidate the structure and function of proteins.

[0004] Chemical proteomics is a method that allows for the unbiased and comprehensive identification of target proteins directly from living cells or tissues that reflect their phenotypes. It is believed to have a significant impact on drug development, for example, in the identification of unknown target proteins for drug candidates discovered in phenotypic screening or in the identification of antigens for antibodies with unknown antigens.

[0005] There are known conventional techniques for detecting known membrane proteins: a method for labeling living cells using a reactive group having photoaffinity (NPL 1), a method for identifying membrane proteins from the cell membrane fraction of living cells by proteomics (NPL 2), a method for detecting target membrane proteins from genetically engineered cells using chemical probes (NPLs 3 and 4), a method for detecting target membrane proteins from cultured cells or primary cultured cells using chemical probes (NPL 5), a method for detecting target proteins within cells or on the membrane from cultured cells using chemical probes (NPL 6), a method for performing fluorescent imaging of target membrane proteins on brain slices using chemical probes (NPL 7), and a method for identifying target membrane proteins using proteins such as growth factors (e.g., EGF and FGFR antibodies) (NPL 8).

[0006] Such target protein identification uses a probe with a part (ligand component) that specifically binds to the target protein. For example, a method for identifying a target protein using a compound that is bound to biotin at one end and to a ligand with affinity for the target protein at the other end is known (e.g., NPL 1 and 9). This probe is used to identify the target protein as follows: the target protein is bound to the probe via the ligand, a complex of biotin and avidin is then formed via biotin, and the target protein is isolated and purified using this complex as an indicator. However, membrane proteins with low expression levels are not easy to identify.

[0007] Specifically, NPL 1 discloses a method for detecting a target protein with affinity for a ligand using a probe containing a tetrazolium positioned near the ligand. This method involves irradiating the target protein with UV light after the ligand is bound to the target protein to form a covalent bond between the tetrazolium and the protein. Consequently, the protein bound to the ligand is not released during purification, for example.

[0008] NPL 9 discloses a probe having a cleavable moiety incorporated at a suitable position between a ligand and biotin to remove nonspecific proteins, including avidin, under mild conditions.

[0009] As a probe for identifying target glycoproteins present in living cells or biological fluids, PTL 1 discloses a trifunctional cross-linking reagent having: (i) a ligand-reactive group for coupling to a ligand of interest having at least one binding site on a target glycoprotein receptor, (ii) a hydrazone group for capturing oxidized receptor-glycoproteins, and (iii) an affinity group selected from azide and alkyne for detection, separation and purification of the captured glycoprotein.

[0010] However, none of the aforementioned methods are considered to be highly reproducible and practical techniques for identifying target membrane proteins from living cells or tissues that accurately reflect their phenotypes. This is because many proteins on the cell membrane are expressed at low levels, and the reactive groups widely used in known chemical proteomics techniques are inefficient at capturing target membrane proteins; this makes it difficult to specifically and sensitively detect the true target membrane proteins. Furthermore, nonspecific proteins generated by heat treatment of avidin beads or protein digestion on the beads are mixed in the purified sample. For these reasons, there are currently no reports of cases where target membrane proteins have been identified from living tissues using chemical proteomics.

[0011] Citation List

[0012] Patent Literature

[0013] PTL 1: WO2017 / 081069A

[0014] Non-patent literature

[0015] NPL 1: J.Am.Chem.Soc.,138,14609-14615(2016)

[0016] NPL 2: CellChemicalBiology 2017,24,3-8

[0017] NPL 3: ChemBioChem 2017,18,1639-1649

[0018] NPL 4: Mol.Pharmacol.2019,95,196-209

[0019] NPL 5: J.Am.Chem.Soc.2018,140,6067-6075

[0020] NPL 6: J.Am.Chem.Soc.2018,140,4259-4268

[0021] NPL 7: Nature Communications,8,14850(2017)

[0022] NPL 8: Nature Communications,9,1519(2018)

[0023] NPL 9: Chem Commun,49,5366(2013) Summary of the Invention

[0024] Technical issues

[0025] An object of the present invention is to provide a novel compound that can be used as a probe to identify 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 to identify membrane proteins expressed at very low levels. Another object of the present invention is to provide a method for using the compound as a probe to identify a target protein, preferably a target membrane protein, present in living cells or living tissues.

[0026] Technical Solutions

[0027] The present invention designs a tetrafunctional compound having four functionalities as a means to achieve the above objectives: the four functionalities specifically include 1) a ligand-binding component for coupling a ligand with affinity (binding properties) for a target protein, 2) a reactive component for capturing the target protein, 3) a cleavable component, which is a cleavage site for selectively eluting the target protein captured by avidin beads, and 4) a biotin tag for enriching or purifying the captured target protein. The present inventors have discovered that by binding a small molecule compound or antibody with affinity (binding properties) 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 as living tissue.

[0028] The present invention has been completed through further studies based on these findings, and includes the following subject matters.

[0029] Item 1. A tetrafunctional compound comprising

[0030] Ligand-binding component (A),

[0031] Reactive component (D),

[0032] a cleavable moiety (E), and

[0033] a biotin tag (B), wherein the components (A), (D), (E) and (B) are optionally connected via a spacer,

[0034] in

[0035] The ligand-binding component (A) is at least one activated functional group selected from amine-reactive groups, hydroxyl-reactive groups, thiol-reactive groups, aldehyde-reactive or ketone-reactive groups, alkyl halide-reactive or aryl halide-reactive groups, alkyl sulfonate-reactive or aryl sulfonate-reactive groups, amide-reactive groups, sulfonamide-reactive groups, aryl-reactive groups, diol-reactive groups and carboxyl-reactive groups, or at least one reactive functional group selected from -COOH, -NH2, -OH, -SH, -CH=CH-, -(C=O)-CH=CH-, alkyl halide, vinyl halide, aryl halide, alkyl sulfonate, vinyl sulfonate, aryl sulfonate, alkynyl, azide, epoxy and click tag,

[0036] The reactive component (D) is a group having a structure of at least one compound selected from 2-aryl-5-carbonyltetrazolyl, phenyl azide, diazirine, α-ketoamide, 4-hydroxybenzene, phthalic acid hydrazide and benzophenone,

[0037] The cleavable moiety (E) is a group having the structure of at least one compound selected from the group consisting of 1-(4,4-dimethyl-2,6-dioxocyclohexan-1-ylidene)ethyl, levulinate, vicinal diol, diazobenzene, diarylhydrazone, dialkoxydiphenylsilane, disulfide, a peptide containing the sequence ENLYFQG (SEQ ID NO: 1) and a peptide containing the sequence ENLYFQS (SEQ ID NO: 2),

[0038] The biotin tag (B) is a group represented by the following formula (1):

[0039]

[0040] The symbol * indicates a bond to an adjacent group;

[0041] The spacer is a linear or branched alkylene group having one or more carbon atoms; or a linear or branched alkylene group having 3 or more carbon atoms, wherein non-adjacent -CH2- groups are independently replaced by at least one cross-linking group selected from the group consisting of: -O-, -OCH2-, -CH2O-, -CO-, -NR a -(where R a Represents a hydrogen atom or C 1-6 alkyl, which also applies hereinafter), -CO-NR a -,-NR a -CO-, a group represented by the following formula (1-1) and a group represented by the following formula (1-2)

[0042]

[0043] Wherein m represents 0 or 1, and n represents 1 or 2.

[0044] Item 2. The tetrafunctional compound according to Item 1, wherein the reactive component (D) is a group represented by the following formula (2) or (2′),

[0045]

[0046] wherein the symbol * represents a bond to an adjacent group, and n represents 0 or 1,

[0047] and / or

[0048] The cleavable moiety (E) is a group represented by the following formula (3):

[0049]

[0050] wherein R1, R2, R3, R4, R5 and R6 each independently represent a hydrogen atom or a C 1-6 alkyl, and the symbol * represents a bond to an adjacent group.

[0051] Item 3. The tetrafunctional compound according to Item 1 or 2, which is represented by the following formula (I) or formula (II):

[0052] A-S1-D-S2-E-S3-B (1)

[0053]

[0054] wherein A, B, C, D and E represent the corresponding components in Item 1, and S1, S2, S3, S4, S5, S6 and S7 each independently represent a spacer.

[0055] Item 4. The tetrafunctional compound according to any one of items 1 to 3, wherein the ligand-binding moiety (A) is an N-hydroxysuccinimide ester group.

[0056] Item 5. The tetrafunctional compound according to Item 4, which is represented by the following formula (i), (ii) or (iii):

[0057]

[0058] Where p represents 2.

[0059] Item 6. A tetrafunctional chemical probe comprising:

[0060] The tetrafunctional compound according to any one of items 1 to 5, and

[0061] A ligand that binds to the ligand-binding component (A) of the tetrafunctional compound.

[0062] Item 7. The tetrafunctional chemical probe according to Item 6, wherein the ligand is a member selected from proteins, peptides, lipids, carbohydrates and small molecule compounds, each of which has affinity for membrane proteins derived from living cells or living tissues.

[0063] Item 8. The tetrafunctional chemical probe according to Item 6 or 7, which is represented by formula (iv):

[0064]

[0065] wherein S1 represents a spacer, and p represents 2.

[0066] 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, the method comprising:

[0067] (1) reacting cells or tissues with the tetrafunctional chemical probe so that the ligand in the tetrafunctional chemical probe binds to the target protein,

[0068] (2) forming a covalent bond between the reactive component (D) in the tetrafunctional chemical probe and the target protein,

[0069] (3) purifying the fraction containing the target protein bound to the tetrafunctional chemical probe,

[0070] (4) binding the biotin tag in the tetrafunctional chemical probe to avidin to form a conjugate of the tetrafunctional chemical probe and the avidin,

[0071] (5) cleaving the conjugate of the tetrafunctional chemical probe and the avidin formed at the cleavable component in the tetrafunctional chemical probe, and

[0072] (6) Detecting the fraction containing the target protein.

[0073] Beneficial effects

[0074] The tetrafunctional compound according to the present invention includes a ligand-binding component (A), a reactive component (D), a cleavable component (E) and a biotin tag (B) optionally connected by a spacer, and by binding a ligand having affinity (binding properties) for the target protein to be detected or identified to the ligand-binding component, the tetrafunctional compound can be used as a tetrafunctional chemical probe.

[0075] The tetrafunctional chemical probe according to the present invention has a reactive component that exhibits high reactivity and high selectivity and a cleavable component that cleaves under mild conditions, both of which are derived from the structure of the tetrafunctional compound. Due to this structure, the tetrafunctional chemical probe according to the present invention particularly exhibits excellent membrane protein collection, detection sensitivity, and repeatability. Therefore, the use of the tetrafunctional chemical probe according to the present invention can identify target membrane proteins in living cells with higher sensitivity than the prior art. In addition, the tetrafunctional chemical probe according to the present invention can also be used to identify target membrane proteins in living tissues, which are more sophisticated and complex physiological environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 Shown are the results of Experimental Example 1, in which the target molecule was isolated from living cells using a compound (CPP-127).

[0077] Figure 2 Shown are the results of Experimental Example 2, in which the target molecule was isolated from living tissue using a compound (CPF-224).

[0078] Figure 3Shown are the results of Experimental Example 2, in which the target molecule was isolated from living tissue using a compound (CPF-202).

[0079] Figure 4 Shown are the results of Experimental Example 2, in which the target molecule was isolated from living tissue using a compound (CPF-242).

[0080] Figure 5 The results of Experimental Example 3 are shown, in which the cell surface antigen was captured using a labeled antibody (CPA-321-labeled anti-CD71 antibody).

[0081] Figure 6 is a graph showing the results of detecting the amount of CD71 as an antigen from THP-1 cells using anti-CD71 antibodies labeled with compounds having different linker lengths.

[0082] Figure 7 : is a graph showing the results of detecting the amount of CD71 as an antigen from A431 cells using anti-CD71 antibodies labeled with probes having different cross-linking groups.

[0083] Figure 8 It is a graph showing the results of detecting the amount of EGF receptor as an antigen from MDA-MB-231 cells using His-tagged EGF labeled with various probes.

[0084] Figure 9 Graphs showing the results of detecting the amount of CD71 as an antigen from A431 cells using anti-CD71 antibodies labeled with different methods.

[0085] Figure 10 : is a graph showing the results of detecting the amount of CD71 as an antigen from THP-1 cells using the anti-CD71 antibody DF1513 labeled by the secondary antibody method. DETAILED DESCRIPTION

[0086] (I) Explanation of terms used in this specification

[0087] Unless otherwise indicated, the terms and phrases used in this specification are used with the meanings described below.

[0088] The term "tetrafunctional" refers to the presence of four functional groups. As stated above, these four functional groups include one that constitutes the ligand-binding moiety or a ligand moiety formed at the site of the ligand-binding moiety, one that constitutes the reactive moiety, one that constitutes the cleavable moiety, and finally, a biotin residue that can bind to avidin. Biotin refers to (5-[(3aS,4S,6aR)-2-oxahexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoic acid), a water-soluble vitamin classified as a B vitamin.

[0089] In the present specification, the term "alkylene" refers to a divalent free radical represented by the formula -(CHR)n-, wherein R represents a hydrogen atom or any substituent derived from a hydrocarbon. The alkylene group preferably has about 1 to 30 carbon atoms (n = 1 to 30) or preferably about 1 to 10 carbon atoms.

[0090] In this specification, the term "alkyl" refers to a group having 1 to 6 carbon atoms (C 1-6 ) is a straight or branched short chain alkyl group. Specific examples of such alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl and 3-methylpentyl. The alkyl group is preferably a straight or branched short chain alkyl group having 1 to 3 carbon atoms. "C 1-6 "Alkyl" also includes short chain alkyl groups in which 1 to 3 hydrogen atoms are replaced by deuterium atoms.

[0091] In this specification, the terms "protein" and "peptide" have the same meaning and refer to amino acid polymers of any length (usually, peptides are referred to as "protein fragments"). Such polymers may have straight, branched, or cyclic chains. The amino acids may be natural or non-natural amino acids or mutated amino acids.

[0092] In the present invention, the protein can be a naturally occurring protein or a synthetic protein. The protein can also have a sequence prepared by carrying out synthetic engineering on a naturally occurring protein. The protein can be an intracellular protein, a cell surface protein (i.e., a protein that is bound to the cell surface) or a protein in solution (i.e., a protein that is secreted into the culture medium). The protein can also be a glycoprotein or a membrane protein. In the present invention, the protein of interest can be any drug or commercially relevant protein with useful biological or chemical activity, such as a receptor, an antibody, an enzyme, a hormone, a regulator, an antigen, and a conjugate. The protein used in the method according to the present invention listed below is merely an example, and the protein of interest in the present invention is not limited to these proteins. Those skilled in the art will appreciate that any protein can be the target protein of the probe and method of the present invention.

[0093] In this specification, the phrase "target protein" refers to a protein that can bind to one or more specific types of ligands. These target proteins are preferably proteins present in biological fluids, cells or tissues derived from mammals, including humans and non-human animals, although not limited thereto. These target proteins are more preferably proteins expressed and present in living cells or living tissues. Therefore, if the target protein is, for example, a membrane protein expressed on the surface of a cell, the protein is bound to the cell membrane of the living cell and may have at least one amino acid exposed to the extracellular space, so that one or more ligands can bind to the protein.

[0094] The target protein in the present invention is preferably a membrane protein, although not particularly limited thereto. A membrane protein refers to a protein that can interact directly or indirectly with a lipid membrane, particularly a lipid bilayer membrane, wherein at least one of the amino acid molecules constituting the membrane protein is present in the extracellular environment. Examples include G protein-coupled receptors, seven-transmembrane receptors, receptor tyrosine kinases, immunoglobulin superfamily and related proteins, scavenger receptors, other similar receptors, transporters, ion channels, solution transporters, active transporters, co-transporters, enzymes, and other similar proteins.

[0095] 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, lysophospholipid 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 receptor, anaphylatoxin chemoattractant receptor, gastrin-releasing peptide receptor, orexin receptor, histamine receptor, serotonin receptor, interleukin receptor, leucine-rich repeat-containing G protein-coupled receptor, leukotriene receptor, adrenocorticotropic hormone receptor, melanocortin receptor, melanin-concentrating hormone receptor, melatonin receptor, neuromodulatory peptide receptor, neuropeptide receptor, neurotensin receptor, opioid receptor, orphan receptor, oxoglutarate receptor, oxytocin receptor, P2Y purinergic receptor, prostaglandin receptor, rhodopsin, relaxin receptor, somatostatin receptor, succinate receptor, substance P receptor, substance K receptor, coagulation ane receptors, urotensin receptors, calcitonin receptors, taste receptors, metabotropic glutamate receptors, and olfactory receptors.

[0096] 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, enterotoxin receptors, and their precursors.

[0097] Examples of the immunoglobulin superfamily and related proteins include immunoglobulin receptors, killer cell immunoglobulin-like receptors, leukocyte immunoglobulin-like receptors, axon guidance factor receptors, T-cell receptors, various cytokine receptors, various fragments and superfamilies of T-cell surface glycoprotein CDs and their precursors. Other examples of receptors include adiponectin receptor, progesterone receptor, contactin-related protein, delrin, integrins and their precursors, axonal proteins, neuropilins, Notch receptors, plexin and its 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.

[0098] Examples of transport proteins include aquaporins, chloride channels, bestrophins, ryanodine receptors, voltage-gated potassium channels, cyclic nucleotide-gated channels, calcium-activated potassium channels, transient receptor potential channels (TRP channels), voltage-gated sodium channels, potential voltage-gated calcium channels, other voltage-gated channels, serotonin receptors, acetylcholine receptors, γ-monovalent butyrate receptors, glycine receptors, ionotropic glutamate receptors, and P2X purinergic receptors. Examples of solution transport proteins include various SLC family proteins. Examples of active transport proteins include ATP-degrading enzymes and ABC transporters that transport ions. In addition, examples of enzymes include NADH-ubiquinone oxidoreductase, cytochrome c, flavin-containing monooxygenases, cytochrome P450, other oxidoreductases; acyltransferases, glucosyltransferases, sulfotransferases, other transferases; ligases, tyrosine phosphatases, phosphodiesterases, glycosidases, serine peptide internal hydrolases, metalloendopeptidases, nucleoside diphosphate phosphatases, and other hydrolases.

[0099] In this specification, phrase " sample " or " biological sample " refers to any living cell or solid or fluid sample (biological sample) obtained from organism.They include for example tissue culture, bioreactor, human or non-human animal tissue, plant (including fruit and vegetables), unicellular microorganism (such as bacterium and yeast) and multicellular organism.Described biological sample can also be the biological fluid obtained from for example blood, plasma, serum, urine, bile, semen, cerebrospinal fluid, aqueous humor or vitreous humor, any body secretion, transudate or exudate (such as pus or the fluid from any other infection or inflammation site), or the fluid from joint (such as normal joint or the joint affected by disease such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis).Described biological sample can also be the sample obtained from any organ or tissue, including biopsy or autopsy specimen.Described cell also includes primary cell and cultured cell.

[0100] The biological sample is preferably derived from living cells or living tissues of humans or non-human animals. The living cells that can be used in the present invention are preferably cells in which proteins that can be detected using ligands, preferably membrane proteins, are expressed, although not limited thereto. Examples include, but are not limited to, CHO cells, MDCK cells, 3T3-L1, 293 cells, MCF7 cells, A431 cells, 3T3 cells, CV-1 cells, HeLa cells, L cells, BHK21 cells, HL-60 cells, U937 cells, HaK cells, Jurkat cells, THP-1 cells, other cell lines obtained by transformation, blood cells isolated from living organisms, and cell lines derived from in vitro culture of primary tissues or primary transplants. In addition to cells cultured in two-dimensional culture, examples also include cells cultured in three-dimensional culture systems such as organoid culture. The biological sample can also be a cell in which a gene encoding a specific protein is introduced using a vector as a host organism. Without constituting any limitation, for example, CHO-K1 cells stably expressing dopamine D2 receptors can be used.

[0101] The tissue that can be used in the present invention may be any tissue in which proteins, preferably membrane proteins, that can be detected by using ligands are expressed, and is not particularly limited. Examples include, but are not limited to, tissue slices and organ slice cultures of brain, liver, kidney, etc.

[0102] In this specification, phrase " living cell " refers to the cell that is established as cell line derived from mammal (human or non-human animal), or the cell that has been cultured and not denatured that is established as cell line derived from non-mammalian source.Described living cell also includes the cell (such as blood cell, the cell of organ and primary culture cell) separated from any organ or tissue under non-denaturing condition.In addition to the cell cultivated in two-dimensional culture, example also includes the cell cultivated in three-dimensional culture system such as organoid culture.In this specification, phrase " living tissue " refers to the tissue slice or tissue culture sample (such as tissue slice and organ slice culture of brain, liver, kidney etc.) separated from any organ or tissue under non-denaturing condition from mammal (human or non-human animal).

[0103] In the present invention, the term "ligand" refers to a compound that has affinity for a target protein and can therefore bind to the target protein. The ligand is preferably any compound that is bound to a cell membrane and can interact or bind to a target membrane protein expressed in and present on the cell surface, although not limited thereto. Examples of ligands include, but are not limited to, peptides, polypeptides, proteins containing 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 and lectins), transmitters, autologous active substances, growth factors (e.g., PDGF, VEGF, EGF, TGFa, TBFβ, GM-CSF, G-CSF, M-CSF, FGF, IGF, bombesin, thrombopoietin, erythropoietin, oncostatin and endothelin 1), interleukins (e.g., interleukins 1 to 15), lymphokines, cell signaling molecules, cytokines (e.g., including tumor necrosis factors (tumor necrosis factor α and β) and interferons (e.g., interferon α, β and γ)), prosthetic groups, coenzymes, cofactors, regulators, natural or synthetic organic molecules that can specifically bind to receptors, fragments, analogs and other derivatives of these ligands that maintain the same binding properties.

[0104] 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. The antibody is preferably a monoclonal antibody and may be, for example, a therapeutic antibody such as trastuzumab and bevacizumab. The antibody may also be a humanized antibody.

[0105] Either the first antibody method or the second antibody method can be used. The first antibody method uses an antibody (first antibody) with affinity for the target protein as a ligand. The second antibody method uses a second antibody that is integrated with the ligand-binding component of the probe, and the first antibody (ligand) and the second antibody (ligand-binding component) form a conjugate. The affinity of the antibody for the target protein that cannot be sensitively detected by the first antibody method may be sensitively detected by the second antibody method.

[0106] The ligand can also be a manipulated affinity binder, such as an ankyrin repeat binder, particularly a His-tag binder, an affinity binder generated by phage display, an oligonucleotide, or a peptide aptamer. The ligand can also be a protein, such as a protein receptor, or a cell surface protein, such as a domain of a cell surface protein receptor. In addition, the ligand can be a microorganism or a virus.

[0107] In the present invention, the ligand interacts with its target protein through a binding site. The binding site is a specific peptide fragment of the target protein, such as a specific amino acid sequence or a fragment of the three-dimensional structure of the target protein, and is referred to as a "binding site". For a ligand that binds to a binding site of a target protein (cell surface or secreted target protein), the term "interaction" includes temporary 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., the dissociation equilibrium constant, Kd. The typical binding affinity of a ligand for its target protein can be at least 10 -5 M, preferably 10 -6 M or higher, such as about 10 -7 M to about 10 -12 M.

[0108] As described above, the ligand can be any compound that has an affinity for the target protein; however, the ligand is preferably a compound that is used as an active ingredient in a pharmaceutical product for treating a disease in humans or non-human animals (a pharmaceutical compound). The pharmaceutical compound can be a small molecule compound, a medium molecular weight compound, a natural product, or a protein including an antibody. The pharmaceutical compound can be modified so as to bind to the ligand-binding component of the tetrafunctional compound according to the present invention, as long as the modification does not affect the affinity for the target protein. In the tetrafunctional chemical probe according to the present invention, the ligand can be bound to the reactive component via a spacer.

[0109] In the present specification, alkylation, hydrolysis, amination, esterification, amidation, etherification, nucleophilic substitution, addition, oxidation and reduction refer to methods known per se. These methods are described, for example, in Jikken Kagaku Koza (5th edition, The Chemical Society of Japan, Maruzen Publishing), Organic Functional Group Preparations (2nd edition, Academic Press, Inc., published in 1989), Comprehensive Organic Transformations (VCH Publishers Inc., published in 1989), and Greene's Protective Groups in Organic Synthesis (PGM Wuts and TW Greene, 4th edition, 2006).

[0110] In this specification, there is no particular limitation on the “palladium compound”. Examples include tetravalent palladium catalysts such as sodium hexachloropalladium (IV) tetrahydrate and potassium hexachloropalladium (IV); divalent palladium catalysts such as [1,1'-bis(diphenylphosphino)ferrocene]palladium (II) dichloride dichloromethane adduct (Pd(dppf)Cl2·CH2Cl2), (2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium (II) methanesulfonate (XPhos Pd G3), palladium (II) chloride, palladium (II) bromide, palladium (II) acetate, palladium (II) acetylacetonate, dichlorobis (benzonitrile) palladium (II), dichlorobis (acetonitrile) palladium (II), dichlorobis (triphenylphosphine) palladium (II), dichlorotetraaminopalladium (II), dichloro (cycloocta-1,5-diene) palladium (II), palladium (II) trifluoroacetate and 1,1'-bis (diphenylphosphino) ferrocene dichloropalladium (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 can be used alone or in combination of two or more.

[0111] In the present specification, examples of carbodiimides 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 methyl iodide, N-tert-butyl-N'-ethylcarbodiimide, N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate, N,N'-di-tert-butylcarbodiimide and N,N'-di-p-tolylcarbodiimide.

[0112] In the present specification, examples of the protecting group include tert-butoxycarbonyl (BOC), 9-fluorenylmethoxycarbonyl (Fmoc), 2,2,2-trichloroethoxycarbonyl (Troc), benzyloxycarbonyl (Z), allyloxycarbonyl (Alloc), trifluoroacetyl, phthaloyl, p-toluenesulfonyl (Ts) and 2-nitrobenzenesulfonyl (Ns).

[0113] In this specification, the term "solvent" refers to a solvent that is inactive for the reaction. Examples include water, ethers (e.g., dioxane, tetrahydrofuran, diethyl ether, 1,2-dimethoxyethane, diethylene glycol dimethyl ether, and ethylene glycol dimethyl ether), halogenated hydrocarbons (e.g., dichloromethane, chloroform, 1,2-dichloroethane, and carbon tetrachloride), aromatic hydrocarbons (e.g., benzene, toluene, and xylene), short-chain alcohols (e.g., methanol, ethanol, and isopropanol), polar solvents (e.g., N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), hexamethylphosphoric triamide, and acetonitrile). These solvents can be used alone or in combination of two or more.

[0114] In the present specification, amines include trialkylamines (eg, trimethylamine, triethylamine, N,N-diisopropylethylamine) and dialkylamines (eg, diethylamine and diisopropylamine).

[0115] In the present specification, examples of the base include inorganic bases and organic bases. Inorganic bases include alkali metal hydroxides (e.g., lithium hydroxide, sodium hydroxide, and potassium hydroxide), alkaline earth metal hydroxides (e.g., magnesium hydroxide, calcium hydroxide, and barium hydroxide), alkali metal carbonates (e.g., sodium carbonate, potassium carbonate, and cesium carbonate), alkaline earth metal carbonates (e.g., magnesium carbonate, calcium carbonate, and barium carbonate), alkali metal bicarbonates (e.g., sodium bicarbonate and potassium bicarbonate), alkali metal phosphates (e.g., sodium phosphate, potassium phosphate, and cesium phosphate), alkaline earth metal phosphates (e.g., magnesium phosphate and calcium phosphate), alkali metal alcoholates (e.g., sodium methoxide, sodium ethoxide, potassium tert-butoxide), and alkali metal hydrides (e.g., sodium hydride and potassium hydride). Organic bases include trialkylamines (e.g., trimethylamine, triethylamine (TEA), N,N-diisopropylethylamine (DIPEA)), dialkylamines (e.g., diethylamine and 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 used in combination.

[0116] In the present specification, examples of the condensing agent include 1-hydroxybenzotriazole (HOBt), 3-hydroxy-3,4-dihydro-1,2,3-benzotriazine-4-one (HOOBt), N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC), 2-(1H-7-azabenzotriazol-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-(pyrrolidinyl)-phosphonium hexafluorophosphate (PyBop), (3,4-dihydro-1,2,3-benzotriazin-4-one-3-oxy)diethyl phosphate (DEPB), t), 3,4-dihydro-1,2,3-benzotriazine-4-one-3-oxytris-(pyrrolidinyl)-phosphonium hexafluorophosphate (PDOP), 2-(benzotriazol-1-yloxy)-1,3-dimethyl-2-pyrrolidin-1-yl-1,3,2-diazaphosphonium 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-dimethylmethylimine hexachloroantimonate (BOMI), N,N'-bis(2-oxo-3- oxazolidinyl)phosphine chloride (BOP-Cl), 1-(1H-benzotriazol-1-yloxy)phenylmethylenepyrrolidinium hexachloro-antimonate (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-ethyl Pyridinium hexachloroantimonate (FEPH), 1-(1-pyrrolidinyl-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene)pyrrolidinium hexafluorophosphate N-oxide (HAPyU), 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)uronium hexafluorophosphate (HBPyU), (1H- 7-Azabenzotriazol-1-yloxy)tris(pyrrolidinyl)phosphonium hexafluorophosphate (PyAOP), bromo-tripyrrolidinylphosphonium hexafluorophosphate (PyBrOp), chloro-tripyrrolidinylphosphonium hexafluorophosphate (PyClOP), 1,1,3,3-bis(tetramethylene)chlorouronium hexafluorophosphate (PyClU), tetramethylfluoro-amidinium hexafluorophosphate (TFFH), triphosgene, triazine-based reagents (cyanuric chloride, cyanuric fluoride, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl) chlorophenyl), diphenyl chlorophosphate, diphenylphosphoryl azide (DPPA), N-[1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino(morpholino)]uronium hexafluorophosphate (COMU), and N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU).

[0117] In the present specification, the deprotecting agent may be any agent capable of removing a protecting group. Examples include bases such as piperidine and lithium diisopropylamide, acids such as zinc and TFA, nucleophiles in the presence of a zero-valent palladium catalyst, aqueous alkaline solutions, methylamine, hydrazine, single-electron reducing agents, and thiols.

[0118] (II) Tetrafunctional Compounds and Tetrafunctional Chemical Probes

[0119] The tetrafunctional compound according to the present invention is a compound comprising a ligand-binding component (A), a reactive component (D), a cleavable component (E), and a biotin tag (B), optionally connected by a spacer. Tetrafunctional chemical probes useful in the detection, identification, or purification of a target protein can be prepared by combining a ligand having an affinity (binding property) for the target protein with the ligand-binding component of the compound. Specifically, the tetrafunctional chemical probe is a compound comprising a ligand component, a reactive component (D), a cleavable component (E), and a biotin tag (B), optionally connected by a spacer.

[0120] The following describes the four functional components of the tetrafunctional compound and tetrafunctional chemical probe according to the present invention and the spacer connecting the four components.

[0121] Ligand-binding component

[0122] The ligand-binding moiety (A) of the tetrafunctional compound according to the present invention is the site at which the ligand binds to the tetrafunctional compound (coupling site). Thus, the ligand-binding moiety (A) can be a reactive functional group or an activated functional group that reacts with a reactive counterpart group present on the ligand (these groups are collectively referred to as "ligand-reactive groups").

[0123] The activated functional group refers to a reactive functional group that is activated by standard chemical techniques to obtain a corresponding activated functional group. In a specific embodiment, the activated functional group is selected from an amine reactive group, a hydroxyl reactive group, a thiol reactive group, an aldehyde reactive group or a ketone reactive group, an alkyl halide reactive group or an aryl halide reactive group, an alkyl sulfonate reactive group or an aryl sulfonate reactive group, an amide reactive group, a sulfonamide reactive group, an aryl reactive group, a diol reactive group, and a carboxyl reactive group.

[0124] The amine-reactive group refers to an activated functional group that reacts with a primary or secondary amine. Typical amine-reactive groups include aryl-activated or alkyl-activated carboxylates -COOR, such as N-hydroxysuccinimide esters or derivatives thereof (e.g., sulfo-N-hydroxysuccinimide esters) and phenol esters or derivatives thereof (e.g., R represents phenol, p-nitrophenol, or tetrafluorophenol). Other amine-reactive groups include acid chlorides (-COCl), aryl or alkyl imide esters (-C(NH)OMe), alkyl or aryl isocyanates (-NCO), isothiocyanates (-NCS), aldehydes, particularly 2-pyridinecarboxyaldehyde, carbonyl groups, epoxy groups, α,β-unsaturated carbonyl groups, alkyl or aryl halides, and alkyl or aryl sulfonates.

[0125] The hydroxyl-reactive group refers to an activated functional group that reacts with a hydroxyl group. Typical examples of hydroxyl-reactive groups include alkyl or aryl isocyanates -NCO, aryl-activated or alkyl-activated carboxylates -COOR, epoxy groups, α,β-unsaturated carbonyl groups, alkyl or aryl halides, and alkyl or aryl sulfonates.

[0126] The thiol-reactive group refers to an activated functional group that reacts with thiols. Typical examples of thiol-reactive groups include maleimide, α-haloamide (-NH-CO-CH2-Hal), epoxy, α,β-unsaturated carbonyl, alkyl or aryl halide, and alkyl or aryl sulfonate.

[0127] The aldehyde-reactive or ketone-reactive group refers to an activated functional group that reacts with an aldehyde or ketone. Examples of typical aldehyde-reactive or ketone-reactive groups include aryl or alkylamine, aryl or alkylhydrazine (-NHNH ), aryl or alkylhydrazide (-CO-NHNH ), alkyl or arylhydroxylamine (-ONH ) and alkyl or arylmagnesium halide.

[0128] The alkyl halide reactive or aryl halide reactive group refers to an activated reactive group that reacts with an alkyl halide or an aryl halide. Typical alkyl halide activated or aryl halide activated reactive groups include amines, alcohols, sulfhydryls, amides, sulfonamides, carboxylic acids, alkenes, alkynes, boric acid, borate esters, and alkyl tins.

[0129] The alkylsulfonate reactive or arylsulfonate reactive group refers to an activated reactive group that reacts with an alkylsulfonate or arylsulfonate. Typical alkylsulfonate or arylsulfonate activated reactive groups include amines, alcohols, sulfhydryls, amides, sulfonamides, carboxylic acids, alkenes, alkynes, boric acid, borate esters, and alkyl tins.

[0130] The amide reactive group refers to an activated reactive group that reacts with unsubstituted or substituted amides. Typical amide reactive groups include epoxy groups, α,β-unsaturated carbonyl groups, alkyl or aryl halides, and alkyl or aryl sulfonates.

[0131] The sulfonamide reactive group refers to an activated reactive group that reacts with unsubstituted or substituted sulfonamides. Typical sulfonamide reactive groups include epoxy groups, α,β-unsaturated carbonyl groups, alkyl or aryl halides, and alkyl or aryl sulfonates.

[0132] The aryl-reactive group refers to an activated functional group that reacts with an aryl group. Typical examples of the aryl-reactive group include 4-hydroxybenzene and phthalhydrazide.

[0133] The diol-reactive group refers to an activated functional group that reacts with a diol group. Typical examples of the diol-reactive group include boric acid.

[0134] The carboxyl-reactive group refers to an activated functional group that reacts with a carboxyl group. Typical examples of carboxyl-reactive groups include halogen, alkyl or aryl sulfonate, hydroxyl, epoxy, mercapto, amino, isocyanate, and carbodiimide.

[0135] Unless otherwise indicated, the reactive functional group refers to an unprotected free functional group. Specifically, the reactive functional group can be selected from -COOH, -NH2, -OH, -SH, -CH=CH-, -(C=O)-CH=CH-, alkyl, vinyl or aryl halide, alkyl, vinyl or aryl sulfonate, alkynyl, azide, epoxy and click tag.

[0136] Examples of activating reagents for activating reactive functional groups include, but are not limited to, 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-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (H ATU), 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-(pyrrolidinyl)-phosphonium hexafluorophosphate (PyBop), (3,4-dihydro-1,2,3-benzotriazin-4-one-3-oxy)diethyl phosphate (DEPBt), 3,4-dihydro-1,2,3-benzotriazine-4-one-3-oxytris-(pyrrolidinyl)-phosphonium hexafluorophosphate (PDOP), 2-(benzotriazol-1-yloxy)-1,3-dimethyl-2-pyrrolidin-1-yl-1,3,2-diazaphosphonium hexafluorophosphate (BOMP), 5-(1H-7-azabenzotriazol-1-yloxy)-3,4-dihydro-1-methyl 2H-pyrrolium hexachloroantimonate (AOMP), (1H-7-azabenzotriazol-1-yloxy)tris(dimethyl)phosphonium Amino)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-dimethylmethylimine hexachloroantimonate (BOMI), N,N'-bis(2-oxo-3- oxazolidinyl)phosphine chloride (BOP-Cl), 1-(1H-benzotriazol-1-yloxy)phenylmethylenepyrrolidinium hexachloro-antimonate (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 -triazolo[4,5-b]pyridin-1-ylmethylene)pyrrolidinium hexafluorophosphate N-oxide (HAPyU), 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)uronium hexafluorophosphate (HBPyU), (1H-7-azabenzotriazol-1-yloxy)tris(pyrrolidinyl)phosphonium hexafluorophosphate (PyAOP), bromo-tripyrrolidinylphosphonium hexafluorophosphate (PyBrOp), chloro-tripyrrolidinylphosphonium Hexafluorophosphate (PyClOP), 1,1,3,3-bis(tetramethylene)chlorouronium hexafluorophosphate (PyClU), tetramethylfluoro-amidinium 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) chlorophosphate, diphenyl chlorophosphate, diphenylphosphoryl azide (DPPA), N-[1-(cyano-2-ethoxy)-1,3,5-dimethylthiazol-2-yl]-4-methylmorpholinium chloride (DMT-MM), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT)]

[00145] The present invention relates to a novel nanostructured carbonyl group comprising: a nanostructured carbonyl group, ...

[0137] Those skilled in the art will readily understand which ligand reactive group should be selected for coupling to the chosen ligand and will be able to appropriately select a ligand reactive group from those listed above for use.

[0138] In a preferred embodiment, the ligand-binding moiety (A) is an activated functional group selected from amine-reactive groups, hydroxyl-reactive groups, thiol-reactive groups, aldehyde-reactive or ketone-reactive groups, alkyl halide-reactive or aryl halide-reactive groups, alkyl sulfonate-reactive or aryl sulfonate-reactive groups, amide-reactive groups, sulfonamide-reactive groups, aryl-reactive groups, diol-reactive groups, and carboxyl-reactive groups. Particularly preferably, the ligand-binding moiety (A) is an aryl-activated or alkyl-activated carboxylate -COOR, such as N-hydroxysuccinimide ester represented by the following formula:

[0139]

[0140] The symbol * indicates the binding site with the adjacent group.

[0141] In another preferred embodiment, the ligand-binding moiety (A) is a reactive functional group selected from -COOH, -NH2, -OH, -SH, -CH=CH-, -(C=O)-CH=CH-, alkyl, vinyl, or aryl halides, alkyl, vinyl, or aryl sulfonates, alkynyl, azide, epoxy, and click tags. The click tag is a functional group for bioorthogonal reactions. Examples include diphenylphosphinylphenyl, 1,2,4,5-tetrazin-3-yl, trans-cyclooctenyl, norbornenyl, cyclopropenyl, dihydroxyboronylvinyl, dihydroxyboronylaryl, and halogenated aryl.

[0142] Reactive component: D

[0143] The reactive component (D) as another functional component of the tetrafunctional compound according to the present invention is a group that can form a covalent bond with the target protein. This group can be any group with such an effect, and is preferably a group that forms a covalent bond under simple reaction conditions. Examples of such groups include groups having a structure (component) of at least one compound selected from 2-aryl-5-carbonyl tetrazole (ACT), phenyl azide, diazines, α-ketoamides, 4-hydroxybenzene, phthalohydrazide and benzophenone. ACT can mainly covalently bind to the carboxyl group of the protein, and phenyl azide can mainly covalently bind to the amino group of the protein. 4-hydroxybenzene and phthalohydrazide can mainly covalently bind to the 4-hydroxyphenyl group of the protein. Diazines, α-ketoamides and benzophenone can non-specifically form covalent bonds with proteins.

[0144] A preferred group is a group having a structure of ACT represented by the following formula (2):

[0145]

[0146] Wherein the symbol * represents the binding site to the adjacent group; n represents 0 or 1; n being 0 means that no group is coordinated to the benzene ring (i.e., ACT is located at the terminal end of the tetrafunctional compound according to the present invention). When n is 1, the bond to the adjacent group may be at the ortho, meta, or para position relative to the tetrazole group coordinated to the benzene ring, and is preferably at the para position.

[0147] In the above embodiment, the simple reaction is, for example, irradiation with UV light. The wavelength and irradiation time of the irradiated UV light are not particularly limited, as long as the reactive moiety of the tetrafunctional compound according to the present invention can covalently bind to the target protein. The wavelength is preferably 254 to 365 nm, more preferably 302 nm. The irradiation time is preferably 1 second to 10 minutes, more preferably 30 seconds to 5 minutes, and even more preferably 1 minute.

[0148] Another preferred group is a group having a structure of diazirine represented by the following formula (2'):

[0149]

[0150] The symbol * indicates the binding site with the adjacent group.

[0151] In the above embodiment, the simple reaction is, for example, irradiation with UV light. The wavelength and irradiation time of the irradiated UV light are not particularly limited, as long as the reactive moiety of the tetrafunctional compound according to the present invention can covalently bind to the target protein. The wavelength is preferably 254 to 380 nm, more preferably 365 nm. The irradiation time is preferably 5 to 30 minutes, more preferably 10 to 20 minutes, and even more preferably 15 minutes.

[0152] Cuttable component: E

[0153] The cleavable component (E) as another functional component of the tetrafunctional compound according to the present invention is a group that can be cleaved under specific conditions. This group can be any group having such an effect, and is preferably a group that can be cleaved under mild conditions. Examples of such groups include groups having a structure (component) of at least one compound selected from 1-(4,4-dimethyl-2,6-dioxocyclohexane-1-ylidene)ethyl (Dde), levulinate, vicinal diol, diazobenzene, diarylhydrazone, dialkoxydiphenylsilane, disulfide, a peptide containing the sequence ENLYFQG (SEQ ID NO: 1) and a peptide containing the sequence ENLYFQS (SEQ ID NO: 2) or a disulfide group. The group preferably has the structure of Dde represented by the following formula (3):

[0154]

[0155] wherein R1, R2, R3, R4, R5 and R6 each independently represent a hydrogen atom or a C 1-6 Alkyl; in a preferred embodiment, R1 and R2 are alkyl, and R3, R4, R5 and R6 are hydrogen atoms; the alkyl is preferably C 1-3 an alkyl group, more preferably a methyl group; and the symbol * represents a bond to an adjacent group.

[0156] In the above embodiment, the mild conditions refer to conditions under which the protein captured by the treatment is not degraded and the contaminating proteins non-specifically bound to the avidin beads are not eluted. Specifically, the mild conditions refer to conditions under which the cleavable component (E) can be chemically or biochemically cleaved; for example, 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 or a dilute aqueous dithiothreitol solution. More preferably, the mild conditions refer to treatment with a dilute aqueous hydrazine solution. When used herein, the term "dilute" refers to a concentration of 5% by mass or less, preferably 3% by mass or less, but is not limited thereto.

[0157] Biotin tag: B

[0158] The biotin tag (E), which is another functional component of the tetrafunctional compound according to the present invention, is a group having a biotin structure represented by the following formula (1):

[0159]

[0160] The symbol * represents a bond to an adjacent group.

[0161] The biotin tag is a tag that can specifically bind to a solid support (such as magnetic particles, beads, plates, filters, membranes, and chromatography resins) bound to avidin or streptavidin. The biotin tag effectively plays a role in detecting, identifying, or purifying target proteins.

[0162] spacer

[0163] The four functional components are connected to each other by independent spacers, thereby forming a tetrafunctional compound according to the present invention. The spacer connecting the four functional components can be a straight chain or branched alkylene group having one or more carbon atoms, or a straight chain or branched alkylene group having three or more carbon atoms, in which non-adjacent -CH2- groups in the alkylene chain are independently selected from -O-, -OCH2-, -CH2O-, -CO-, -NRa -(where R a Represents a hydrogen atom or C 1-6 Alkyl, the same applies below), -CO-NR a -、-NR a The cross-linking group may replace the -CH2- group in the alkylene chain or the -CH2- group at the end of the alkylene chain. a Preferred is a hydrogen atom.

[0164]

[0165] Wherein m represents 0 or 1, and n represents 1 or 2.

[0166] Specific examples of the group represented by formula (1-1) or the group represented by formula (1-2) include the following formula (1-3) to formula (1-16).

[0167]

[0168] The type and length of the spacer can be selected so as to minimize steric crowding and not impair the effects of the four functional groups. In this regard, there is no limitation on the number of carbon atoms in the alkylene group (alkylene chain length); for example, the number of carbon atoms in the alkylene group is preferably 1 to 20, more preferably 2 to 10.

[0169] The tetrafunctional compound according to the present invention includes a linear compound represented by the following formula (I) and a branched compound represented by the following formula (II).

[0170] A-S1-D-S2-E-S3-B (I)

[0171]

[0172] (II-1) Linear tetrafunctional compounds and tetrafunctional chemical probes

[0173] In formula (I), the ligand-binding moiety represented by A, the reactive moiety represented by D, the cleavable moiety represented by E, and the biotin tag represented by B are as defined above. In formula (I), the moieties represented by S1, S2, and S3 each independently represent a spacer group.

[0174] The spacer group represented by S1 may be, for example, a group represented by the following formula (5).

[0175]

[0176] wherein a represents an integer from 0 to 10; and b, c, d, e, f, g and h each independently represent an integer selected from 0 to 6.

[0177] In one embodiment, S1 is a straight chain alkylene group having one or more carbon atoms. This corresponds to an embodiment in which g, c, and h in formula (5) are 0 and a is an integer of 1 or greater. Although a can be an integer of 1 or greater, a is preferably an integer of 1 to 30, more preferably an integer of 1 to 10.

[0178] In another embodiment, S1 is a linear or branched alkylene group having 3 or more carbon atoms, wherein non-adjacent -CH2- groups in the alkylene chain are independently selected from -O-, -OCH2-, -CH2O-, -CO-, -NR a -(where R a Represents a hydrogen atom or C 1-6 Alkyl, the same applies below), -CO-NR a -and-NR a In the formula (5), at least one crosslinking group of -CO- is replaced, preferably by at least one crosslinking group selected from -O-, -CO- and -NH-CO-. For example, the number of carbon atoms may be 3 to 40, more preferably 10 to 35, but is not limited thereto. Specifically, examples include the case where g is 0, a, b, d and e are 2, c and f are 4, and h is 1 in the formula (5), and the case where g, c and f are 0, a is 3, d is 4, and h is 1 in the formula (5).

[0179] The spacer groups represented by S2 and S3 may be, for example, independently groups represented by the following formula (6).

[0180]

[0181] wherein a, b, c, d, e, f, g, and h each independently represent an integer selected from 0 to 6; i represents 0 or 1; however, a, c, h, and i are not 0 at the same time.

[0182] In one embodiment, S2 is a straight chain alkylene having one or more carbon atoms, wherein non-adjacent -CH2- groups in the alkylene chain are independently selected from -O-, -OCH2-, -CH2O-, -CO-, -NR a -(where R a Represents a hydrogen atom or C 1-6 Alkyl, the same applies below), -CO-NR a -and-NR aAt least one crosslinking group of -CO- is replaced, preferably by at least one crosslinking group selected from -O-, -NH- and -NH-CO-. For example, the number of carbon atoms may be 1 to 20, more preferably 1 to 5, but is not limited thereto. Specifically, examples include the case where a and c are 0, d is 3, f and g are 0, and i is 1 in formula (6).

[0183] In one embodiment, S3 is a straight chain alkylene group having 3 or more carbon atoms, wherein non-adjacent -CH2- groups in the alkylene chain are independently selected from -O-, -OCH2-, -CH2O-, -CO-, -NR a -(where R a Represents a hydrogen atom or C 1-6 Alkyl, the same applies below), -CO-NR a -and-NR a At least one crosslinking group of -CO- is replaced, preferably by at least one crosslinking group selected from -O- and -NH-. For example, the number of carbon atoms may be 3 to 20, more preferably 5 to 15, but is not limited thereto. Specifically, examples include the case where a and b are 2, c is 4, and h and i are 0 in formula (6).

[0184] There is no limitation on the molecular weight of the linear tetrafunctional compound (i.e., tetrafunctional chemical probe) having a ligand binding component and a ligand to which it binds, as long as the linear tetrafunctional compound can suitably coordinate a protein with a high affinity for the ligand; the molecular weight is generally 500 to 3000, preferably 800 to 2000, and more preferably 1000 to 1500. Linear tetrafunctional compounds having molecular weights within these ranges can inhibit membrane permeability. According to the literature discussing the cell membrane permeability of various ligands (Methods Mol Biol. 2015; 1266: 29-53), from the viewpoint of inhibiting membrane permeability, preferably, the number of hydrogen bond donors is 6 or more, the number of hydrogen bond acceptors is 15 or more, or the polar surface area is or greater, more preferably The tetrafunctional compounds and tetrafunctional chemical probes according to the present invention described hereinafter have 6 to 9 hydrogen bond donors, 15 to 28 hydrogen bond acceptors and or greater polar surface area. In recent years, it has also been believed that membrane permeability depends on molecular size rather than molecular weight. There are examples of cyclic peptides that have actually been measured in the case of medium molecules. It is known that membrane permeability is higher in molecules with a size of or larger, and weakens when the molecular size is The tetrafunctional compound and tetrafunctional chemical probe according to the present invention described below have or larger molecular size. According to the literature mentioned above, when the number of rotatable bonds is 10 or less, membrane permeability is good. More specifically, from the viewpoint of inhibiting membrane permeability, it is believed that the number of rotatable bonds is preferably 10 or greater, more preferably 30 or greater. The tetrafunctional compound and tetrafunctional chemical probe according to the present invention have 33 to 60 rotatable bonds. The tetrafunctional chemical probe according to the present invention thus designed exhibits suppressed cell membrane permeability and does not allow membrane penetration; therefore, the tetrafunctional chemical probe can be effectively coordinated to membrane proteins.

[0185] The following describes a method for producing a linear tetrafunctional compound whose ligand-binding moiety is an N-hydroxysuccinimide ester group (NHS ester group), using compound (CPA-306) (see Production Example 15) as an example. Compounds whose ligand-binding moiety is an NHS ester group can be used to prepare antibody probes capable of recognizing and binding to specific antigens serving as target proteins by conjugating antibodies to the ligand-binding moiety. Producing its ligand Method for preparing linear tetrafunctional compounds whose synthetic component is an NHS ester group (Example) 1]

[0187] 2]

[0189] 3]

[0191] 4]

[0193] 5]

[0195] 6]

[0197] 7]

[0199] 8]

[0201]

[0202] wherein q independently represents an integer from 0 to 6.

[0203] first step

[0204] This step can be carried out by stirring PEG-NH2 and allyl alcohol in the presence of thionyl chloride without a solvent or in a solvent inert to the reaction at a temperature ranging from 0°C to a temperature at which heating is performed under reflux for usually 0.1 hour to 5 days.

[0205] Step 2

[0206] This step can be carried out by stirring the compound obtained in the first step and 2-aryl-5-carboxytetrazole (ATC) in the presence of N-[1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino(morpholine)]uronium hexafluorophosphate (COMU) and an amine in a solvent inert to the reaction at a temperature within a range of 0°C to a temperature reaching heating reflux for usually 0.1 hour to 5 days.

[0207] Step 3: De-protect

[0208] This step can be carried out by stirring the compound obtained in the second step in the presence of morpholine and a palladium compound in a solvent inert to the reaction at a temperature ranging from 0°C to a temperature up to heating reflux for usually 0.1 hour to 5 days.

[0209] Step 4: Sidechain extension

[0210] 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 solvent inert to the reaction at a temperature ranging from 0°C to a temperature up to heating reflux for usually 0.1 hour to 5 days.

[0211] Step 5: Protect

[0212] This step can be carried out by stirring the compound obtained in the fourth step and a deprotecting agent in a solvent inert to the reaction at a temperature ranging from 0°C to a temperature up to heating under reflux for usually 0.1 hour to 5 days.

[0213] Step 6

[0214] 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 at a temperature ranging from 0°C to a temperature up to heating reflux for usually 0.1 hour to 5 days.

[0215] Step 7: De-protect

[0216] 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 solvent inert to the reaction at a temperature ranging from 0°C to heating under reflux for usually 0.1 hour to 5 days.

[0217] Step 8

[0218] 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 solvent inert to the reaction at a temperature ranging from 0°C to a temperature at reflux for usually 0.1 hour to 5 days.

[0219] The desired antibody can be attached to the ligand-binding moiety (NHS ester group) of the thus prepared linear tetrafunctional compound according to known methods, thereby producing the linear tetrafunctional chemical probe according to the present invention. An example of a method for conjugating the desired antibody to the NHS ester group is described below, although the method is not limited to this example.

[0220] Method for labeling antibodies with tetrafunctional compounds

[0221] The concentration of a desired antibody with affinity for the target protein is adjusted using a commonly used NHS labeling buffer, and the tetrafunctional compound according to the present invention is added thereto, followed by reaction at a temperature of 0 to 37°C for approximately 5 to 60 minutes. Subsequently, a lysine solution is added, and the mixture is reacted at 0 to 37°C for approximately 5 to 60 minutes. Thus, a linear tetrafunctional chemical probe can be obtained, comprising the tetrafunctional compound according to the present invention and having the desired antibody (labeled antibody) bound to its ligand-binding moiety (NHS ester group).

[0222] Among the linear tetrafunctional compounds, compounds whose ligand-binding moieties are ligand-reactive groups other than NHS ester groups can be produced such that the desired ligand is directly bound to the ligand-binding moiety (i.e., tetrafunctional chemical probes). Examples of methods for producing these compounds are described below.

[0223] Method for producing linear tetrafunctional chemical probes (Example)

[0224] first step

[0225]

[0226] wherein r represents an integer from 1 to 6; and R1 and R2 are as defined above.

[0227] 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 solvent inert to the reaction at a temperature within a range from 0°C to heating under reflux for usually 0.1 hour to 5 days.

[0228] Steps 2 and 3

[0229]

[0230] Here, m represents an integer from 1 to 6.

[0231] Step 2

[0232] 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 solvent inert to the reaction at a temperature ranging from 0°C to a temperature reaching heating reflux, usually for 0.1 hour to 5 days.

[0233] Step 3

[0234] This step can be carried out by stirring compound 1-5 in the presence of a base in a solvent inert to the reaction at a temperature ranging from 0°C to heating under reflux for usually 0.1 hour to 5 days.

[0235] Step 4

[0236]

[0237] wherein s represents an integer from 1 to 10; and m is as defined above.

[0238] 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 solvent inert to the reaction at a temperature ranging from 0°C to heating under reflux for usually 0.1 hour to 5 days.

[0239] Step 5

[0240]

[0241] wherein m, r, s, R1 and R2 are as defined above.

[0242] This step can be carried out as follows: Compound 1-8 obtained in the fourth step is stirred in the presence of a weak acid in a solvent inert to the reaction at a temperature in the range of 0°C to reflux, usually for 0.1 hour to 5 days, to perform a concentration operation, and the concentrated product is stirred in the presence of compound 1-2 obtained in the first step and an amine in a solvent inert to the reaction at a temperature in the range of 0°C to reflux, usually for 0.1 hour to 5 days. Weak acids used in this reaction include formic acid, acetic acid, trifluoroacetic acid, citric acid, and oxalic acid. Amines used in this reaction include diisopropylethylamine and triethylamine.

[0243] The resulting compounds 1-9 contain the tetrafunctional compounds according to the present invention and the desired ligand bound to their ligand-binding components, and are therefore useful as tetrafunctional chemical probes. Examples of ligands useful herein include, but are not limited to, those described above except for antibodies.

[0244] (II-2) Branched tetrafunctional compounds and tetrafunctional chemical probes

[0245] In the above formula (II), the ligand-binding moiety represented by A, the reactive moiety represented by D, the cleavable moiety represented by E, and the biotin tag represented by B are as defined above. In formula (II), the sites represented by S4, S5, S6, and S7 independently represent spacer groups.

[0246] The spacer group represented by S4 is, for example, a group represented by the following formula (7):

[0247]

[0248] wherein a, b, c, d, e, f, g, h, and i independently represent an integer selected from 0 to 6; however, a, c, and i are not 0 at the same time, and d, f, and g are not 0 at the same time.

[0249] In one embodiment, S4 is a straight chain alkylene group having 3 or more carbon atoms, in which non-adjacent -CH2- groups in the alkylene chain are independently selected from -O-, -OCH2-, -CH2O-, -CO-, -NR a -(where R a Represents a hydrogen atom or C 1-6 Alkyl, the same applies below), -CO-NR a -and-NR a At least one crosslinking group of -CO- is replaced, preferably by at least one crosslinking group selected from -O- and -NH-. For example, the number of carbon atoms may be 1 to 20, preferably 1 to 15, more preferably 3 to 10, but is not limited thereto. Specifically, examples include the case where a is 2, b is 2, c is 2, h is 0, and i is 0 in the above formula.

[0250] The spacer group represented by S5 is, for example, a group represented by the following formulae (8) to (10).

[0251]

[0252] wherein w, x, y and z each independently represent an integer selected from 0 to 6; however, x and y are not 0 at the same time.

[0253] In one embodiment, S5 is a linear alkylene group (with a substituent) having 3 or more carbon atoms, wherein non-adjacent -CH2- groups in the alkylene chain are independently selected from -O-, -OCH2-, -CH2O-, -CO-, -NR a -(where R a Represents a hydrogen atom or C 1-6 Alkyl, the same applies below), -CO-NR a -and-NR a At least one crosslinking group of -CO- is replaced, preferably by at least one crosslinking group selected from -CO-, -NH-, -CO-NH- and -NH-CO-. For example, the number of carbon atoms may be 3 to 20, preferably 3 to 15, more preferably 5 to 15, but is not limited thereto. Specifically, examples include the case where x and y in formula (8) are 4.

[0254] The spacer group represented by S6 is, for example, a group represented by the following formula.

[0255]

[0256] wherein a, b, c and d each independently represent an integer selected from 0 to 6; however, a and c are not 0 at the same time.

[0257] In one embodiment, S6 is a straight chain alkylene group having 3 or more carbon atoms, wherein non-adjacent -CH2- groups in the alkylene chain are independently selected from -O-, -OCH2-, -CH2O-, -CO-, -NR a -(where R a Represents a hydrogen atom or C 1-6 Alkyl, the same applies below), -CO-NR a -and-NR a At least one crosslinking group of -CO- is replaced, preferably by at least one crosslinking group selected from -O- and -NH-. For example, the number of carbon atoms may be 3 to 20, preferably 5 to 15, but is not limited thereto. Specifically, examples include the case where a is 3, b is 2, c is 3, and d is 1 in formula (11).

[0258] The spacer group represented by S7 is, for example, a group represented by the following formula.

[0259]

[0260] wherein a, b, c, d, e, f, g, and h each independently represent an integer selected from 0 to 6; however, a and c are not 0 at the same time, and a, c, g, and h are not 0 at the same time.

[0261] In one embodiment, S7 is a straight chain alkylene group having 3 or more carbon atoms, wherein non-adjacent -CH2- groups in the alkylene chain are independently selected from -O-, -OCH2-, -CH2O-, -CO-, -NR a -(where R a Represents a hydrogen atom or C 1-6 Alkyl, the same applies below), -CO-NR a -and-NR a At least one crosslinking group of -CO- is replaced, preferably by at least one crosslinking group selected from -O-, -CO- and -NH-CO-. For example, the number of carbon atoms may be 3 to 40, preferably 3 to 30, more preferably 3 to 20, but is not limited thereto. Specifically, examples include the case where g is 0, a is 5, c is 0, d is 2, f is 0, and h is 1 in the above formula.

[0262] In another embodiment, the spacer represented by S7 is, for example, a group represented by the following formula.

[0263]

[0264] wherein a, b, c, d, e, f, and g each independently represent an integer selected from 0 to 6; however, a and c are not 0 at the same time, and a, c, and h are not 0 at the same time.

[0265] In another embodiment, S7 is a straight chain alkylene group having 3 or more carbon atoms, wherein non-adjacent -CH2- groups in the alkylene chain are independently selected from -O-, -OCH2-, -CH2O-, -CO-, -NR a -(where R a Represents a hydrogen atom or C 1-6 Alkyl, the same applies below), -CO-NR a -、-NR a-CO-, a group represented by the following formula (1-1) and a group represented by the following formula (1-2) are replaced by at least one cross-linking group, preferably by at least one cross-linking group selected from -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). For example, the number of carbon atoms may be 3 to 40, preferably 3 to 30, more preferably 3 to 20, but is not limited thereto. Specifically, examples include those wherein g is 0, a is 5, c is 0, d is 2, f is 0 and h is 1 in formula (13).

[0266]

[0267] Wherein m represents 0 or 1, and n represents 1 or 2.

[0268]

[0269] There is no limitation on the molecular weight of the branched tetrafunctional compound (i.e., tetrafunctional chemical probe) having a ligand binding component with a ligand bound thereto, as long as the branched tetrafunctional compound can suitably coordinate a protein with a high affinity for the ligand; the molecular weight is generally 500 to 3000, preferably 800 to 2000, and more preferably 1000 to 1500. Branched tetrafunctional compounds having molecular weights within these ranges can inhibit membrane permeability. According to the literature discussing the cell membrane permeability of various ligands (Methods Mol Biol. 2015; 1266: 29-53), from the viewpoint of inhibiting membrane permeability, preferably, the number of hydrogen bond donors is 6 or more, the number of hydrogen bond acceptors is 15 or more, or the polar surface area is or greater, more preferably The tetrafunctional compounds and tetrafunctional chemical probes according to the present invention described hereinafter have 6 to 9 hydrogen bond donors, 15 to 28 hydrogen bond acceptors and or greater polar surface area. In recent years, it has also been believed that membrane permeability depends on molecular size rather than molecular weight. There are examples of cyclic peptides that have actually been measured in the case of medium molecules. It is known that membrane permeability is higher in molecules with a size of or larger, and weakens when the molecular size is The tetrafunctional compound and tetrafunctional chemical probe according to the present invention described below have or larger molecular size. According to the literature mentioned above, when the number of rotatable bonds is 10 or less, membrane permeability is good. More specifically, from the viewpoint of inhibiting membrane permeability, it is believed that the number of rotatable bonds is preferably 10 or greater, more preferably 30 or greater. The tetrafunctional compound and tetrafunctional chemical probe according to the present invention have 33 to 60 rotatable bonds. The tetrafunctional chemical probe according to the present invention thus designed exhibits suppressed cell membrane permeability and does not allow membrane penetration; therefore, the tetrafunctional chemical probe can be effectively coordinated to membrane proteins.

[0270] The following describes a method for producing a branched tetrafunctional compound whose ligand-binding component is an N-hydroxysuccinimide ester group (NHS ester group), using compound (CPA-306) (see Production Example 15) as an example. Compounds whose ligand-binding component is an NHS ester group can be used to prepare antibody probes capable of recognizing and binding to specific antigens serving as target proteins by binding an antibody to the ligand-binding component. Producing its ligand Method for preparing branched tetrafunctional compounds whose synthetic component is an NHS ester group (Example)

[0271] The reaction process is broadly divided into five parts to facilitate the following explanation.

[0272] Reaction Scheme I

[0273]

[0274] wherein t represents an integer from 0 to 6, and v represents an integer from 1 to 5.

[0275] first step

[0276] This step can be carried out by stirring the diamine and the acid chloride in a solvent inert to the reaction at a temperature ranging from 0°C to a temperature at which the mixture is heated to reflux for usually 0.1 hour to 5 days.

[0277] Step 2

[0278] This step can be carried out by stirring the diamine to which the protecting group is attached in the first step and the linker having a branched amine in the presence of carbodiimide (WSC) and 1-hydroxybenzotriazole (HOBt) in a solvent inert to the reaction at a temperature within a range of 0° C. to a temperature up to heating reflux for usually 0.1 hour to 5 days.

[0279] Step 3

[0280] 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 solvent inert to the reaction at a temperature ranging from 0°C to heating under reflux for usually 0.1 hour to 5 days.

[0281] Step 4

[0282] 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 solvent inert to the reaction at a temperature ranging from 0°C to a temperature up to heating reflux for usually 0.1 hour to 5 days.

[0283] Step 5

[0284] This step can be carried out by stirring the compound obtained in the third step in the presence of piperidine in a solvent inert to the reaction at a temperature ranging from 0°C to a temperature up to heating reflux for usually 0.1 hour to 5 days.

[0285] Reaction Scheme II

[0286]

[0287] Here, w represents an integer from 0 to 6.

[0288] first step

[0289] This step can be carried out by stirring 1-aminoalkylcarboxylic acid and allyl alcohol in the presence of thionyl chloride in a solvent inert to the reaction at a temperature ranging from 0°C to a temperature at which heating is performed under reflux for usually 0.1 hour to 5 days.

[0290] Step 2

[0291] This step can be carried out by stirring the 1-aminoalkylcarboxylic acid obtained in the first step and an alkyl dicarboxylic anhydride in the presence of an amine in a solvent inert to the reaction at a temperature ranging from 0°C to a temperature at which heating to reflux is performed for usually 0.1 hour to 5 days. Examples of the alkyl dicarboxylic anhydride used in the reaction include, but are not limited to, succinic anhydride, glutaric anhydride, and adipic anhydride.

[0292] Reaction Scheme III

[0293]

[0294] wherein c and d each represent an integer from 0 to 6.

[0295] first step

[0296] This step can be carried out by stirring a diamine protected with BOC and a tetrazole-containing carboxylic acid in the presence of an amine and HATU in a reaction solvent inert to the reaction at a temperature ranging from 0°C to heating under reflux for usually 0.1 hour to 5 days.

[0297] Step 2

[0298] This step can be carried out by stirring the compound obtained in the first step and a deprotecting agent in a solvent inert to the reaction at a temperature ranging from 0°C to a temperature up to heating under reflux for usually 0.1 hour to 5 days.

[0299] Step 3

[0300] This step and the fourth step are optional steps for further extending the length of the linker. This step can be carried out by stirring the compound obtained in the second step and an aminocarboxylic acid whose amino group is protected by BOC in the presence of an amine and COMU in a solvent inert to the reaction at a temperature ranging from 0°C to reflux, usually for 0.1 hour to 5 days.

[0301] Step 4

[0302] This step can be carried out by stirring the compound obtained in the third step and a deprotecting agent in a solvent inert to the reaction at a temperature ranging from 0°C to a temperature up to heating under reflux for usually 0.1 hour to 5 days.

[0303] Reaction Scheme IV

[0304]

[0305] wherein t and u each independently represent an integer from 0 to 6, and v, w, R1 and R2 are as defined above.

[0306] first step

[0307] This step can be carried out by stirring the compounds obtained in Reaction Schemes II and III in the presence of water-soluble carbodiimide (WSC) and 1-hydroxybenzotriazole (HOBt) in a solvent inert to the reaction at a temperature within a range of 0° C. to a temperature up to heating reflux for usually 0.1 hour to 5 days.

[0308] Step 2

[0309] This step can be carried out by stirring the compound obtained in the first step and a deprotecting agent in a solvent inert to the reaction at a temperature ranging from 0°C to a temperature up to heating under reflux for usually 0.1 hour to 5 days.

[0310] Step 3

[0311] This step can be carried out by stirring the compound obtained in the second step and an alkyl dicarboxylic anhydride in the presence of an amine in a solvent inert to the reaction at a temperature ranging from 0°C to a temperature up to heating reflux for usually 0.1 hour to 5 days.

[0312] Step 4

[0313] 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 solvent inert to the reaction at a temperature within a range of 0° C. to a temperature up to heating reflux for usually 0.1 hour to 5 days.

[0314] Reaction Scheme V

[0315]

[0316] wherein c, u, v, w, t, R1 and R2 are as defined above.

[0317] first step

[0318] This step can be carried out by stirring the compound obtained in Reaction Schemes III and IV in the presence of an amine in a solvent inert to the reaction at a temperature ranging from 0°C to a temperature up to heating reflux for usually 0.1 hour to 5 days.

[0319] Step 2

[0320] 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 solvent inert to the reaction at a temperature ranging from 0°C to a temperature up to heating under reflux for usually 0.1 hour to 5 days.

[0321] Step 3

[0322] 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 solvent inert to the reaction at a temperature ranging from 0°C to a temperature at reflux for usually 0.1 hour to 5 days.

[0323] Step 4

[0324] This step is an optional step for converting the ligand-binding moiety. This step 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 solvent inert to the reaction at a temperature ranging from 0°C to reflux, usually for 0.1 hour to 5 days.

[0325] A desired antibody can be attached to the ligand-binding moiety (NHS ester group) of the thus prepared branched tetrafunctional compound according to a known method, thereby producing a branched tetrafunctional chemical probe according to the present invention. An example of a method for binding a desired antibody to the NHS ester group is a method of labeling an antibody with the above-mentioned tetrafunctional compound, although the method is not limited to this example.

[0326] In each reaction of the above reaction scheme, the product can be used as it is as a reaction solution or as a crude product in subsequent reactions. However, the product can also be separated from the reaction mixture according to conventional methods and easily purified by typical separation methods. Examples of typical separation methods include recrystallization, distillation and chromatography.

[0327] The starting material compound, intermediate compound and target compound (tetrafunctional compound, tetrafunctional chemical probe according to the present invention) in each step include geometric isomers, stereoisomers, optical isomers and tautomers. These isomers can be separated by typical optical resolution methods and can also be produced from suitable optically active starting material compounds.

[0328] The tetrafunctional compounds and tetrafunctional chemical probes according to the present invention are not limited to the synthetic methods shown in the above reaction schemes and can be produced by methods similar to those methods or other methods. Unless otherwise specified, the starting material compounds used in the production can be commercially available compounds or compounds produced by methods known per se or methods similar to such known methods.

[0329] The starting material compound and target compound in each step can be used in the form of a suitable salt. Depending on the type of substituent, these salts include but are not limited to acid addition salts and salts with alkali. Examples of acids forming such acid addition salts include inorganic acids (e.g., hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, and phosphoric acid) and organic acids (e.g., methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, citric acid, tartaric acid, maleic acid, fumaric acid, malic acid, and lactic acid). Examples of alkalis forming salts include inorganic bases (e.g., sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate) and organic bases (e.g., methylamine, diethylamine, trimethylamine, triethylamine, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, tris(hydroxymethyl)methylamine, dicyclohexylamine, N,N'-diphenylmethylethylenediamine, guanidine, pyridine, picoline, and choline) and ammonium salts. Salts can also be formed with, for example, amino acids such as lysine, arginine, aspartic acid, or glutamic acid.

[0330] (II) Method for detecting target protein

[0331] The target protein in the sample can be detected using the tetrafunctional chemical probe according to the present invention. The target protein can be detected using the tetrafunctional chemical probe according to the present invention by a method comprising the following steps, although the method is not limited thereto.

[0332] (1) A step of reacting the tetrafunctional chemical probe with cells or tissues in a sample to allow the ligand of the tetrafunctional chemical probe to bind to the target protein.

[0333] (2) A step of forming a covalent bond between the reactive component (D) of the tetrafunctional chemical probe and the target protein.

[0334] (3) A step of purifying the fraction containing the target protein bound to the tetrafunctional chemical probe.

[0335] (4) A step of binding the biotin tag of the tetrafunctional chemical probe to avidin to form a conjugate of the tetrafunctional chemical probe and the avidin.

[0336] (5) A step of cleaving the formed conjugate of the tetrafunctional chemical probe and the avidin at the cleavable component of the tetrafunctional chemical probe.

[0337] (6) A step of detecting or identifying the target protein.

[0338] Each step is described below.

[0339] (1) Step of binding ligand to protein

[0340] Although not limited, this step can be performed as follows. Cells expressing the target protein are cultured at 37° C. in a 5% CO 2 atmosphere. After confirming that the cells have become confluent, the tetrafunctional chemical probe according to the present invention adjusted to a desired concentration with DMSO is added thereto, and the cells are allowed to stand at 37° C. in a 5% CO 2 atmosphere for approximately 30 to 60 minutes.

[0341] (2) Step of forming a covalent bond between the reactive component (D) of the tetrafunctional chemical probe and the target protein

[0342] Although not limited, this step can be performed by irradiating using a UV crosslinking agent at a wavelength of 254 to 365 nm, preferably 302 nm, for 1 second to 10 minutes, preferably 30 seconds to 5 minutes, more preferably 1 minute.

[0343] (3) Step of purifying the fraction containing the target protein bound to the tetrafunctional chemical probe

[0344] Although not limited to, this step can be performed, for example, by the following method. First, a tris homogenization buffer is added to the cells, and the mixture is homogenized and then centrifuged. The precipitate is separated and a sodium carbonate buffer is added thereto, followed by homogenization and incubation. The precipitate is separated by centrifugation to purify and collect a fraction (e.g., a membrane fraction) containing the target protein bound to the tetrafunctional chemical probe.

[0345] (4) combining the biotin tag of the tetrafunctional chemical probe with avidin to form a tetrafunctional chemical probe and Avidin conjugate steps

[0346] Although not limited to, this step can be performed as follows. For example, RIPA buffer containing 8M urea is added to the collected fractions, and the fractions are dissolved in the buffer using an ultrasonic disintegrator. The supernatant is separated by centrifugation and then streptavidin magnetic particles are added thereto. Thus, the biotin tag in the chemical probe binds to the avidin, thereby forming a conjugate of the tetrafunctional chemical probe and the avidin. In addition, the conjugate of the tetrafunctional chemical probe and the avidin can be purified and collected by performing magnetic separation according to a conventional method and discarding the supernatant.

[0347] (5) The tetrafunctional chemical probe is cleaved at the cleavable component of the tetrafunctional chemical probe to form a tetrafunctional chemical probe and an affinity Steps for the conjugate of Hesulin

[0348] Although not limiting, this step can be performed as follows: SDS (sodium dodecyl sulfate) and an aqueous hydrazine solution are added to the conjugate of the tetrafunctional chemical probe and avidin collected in the previous step (magnetic particle conjugate), and the mixture is allowed to stand at room temperature (1 to 30°C) for 30 minutes. This reaction allows the cleavable component in the tetrafunctional chemical probe to be cleaved, thereby separating the conjugate of the tetrafunctional chemical probe and avidin into a target protein binding domain and a biotin-avidin binding domain. The reaction solution is magnetically separated to obtain a supernatant; thereby, the target protein binding domain fragments can be collected.

[0349] (6) Steps for detecting or identifying target proteins

[0350] In detecting or identifying the target protein, the binding domain fragment of the target protein collected above can be optionally purified and hydrolyzed. Although not limited, specifically, for example, dithiothreitol is added to the supernatant obtained, and the mixture is stirred at 70°C for 15 minutes. Iodoacetamide is subsequently added thereto, and the mixture is allowed to stand at room temperature for 30 minutes. SP3 beads are added to the solution thus obtained, followed by MeCN, and then incubated for 18 minutes. Magnetic separation is performed after the incubation is complete to discard the supernatant. For peptide degradation, for example, a trypsin solution is added to the collected residue, and the mixture is incubated at 37°C for 12 to 16 hours. MeCN is added, and the mixture is incubated for 18 minutes, and then magnetic separation is performed to discard the supernatant. A TFA aqueous solution is added to the residue and the mixture is incubated for 3 minutes. Magnetic separation is performed to obtain a supernatant.

[0351] The peptides thus prepared were desalted and resuspended in a suitable buffer solution for analysis using a high-quality mass spectrometer. The mass spectrometer was operated in data-dependent acquisition mode, wherein the device automatically switches from MS to MS / MS mode for ion signals exceeding a predetermined threshold, in order to generate collision-induced dissociation (CID) or high-energy collision dissociation (HCD) spectra of the peptides. All MS / MS spectra were searched against a standard protein database.

[0352] The peptides thus obtained are analyzed. Any method available in the art for analyzing such compounds can be used, and a preferred method is mass spectrometry. Methods for performing mass spectrometry 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); and Aebersold and Goodlett, Chem. Rev. 101: 269-295 (2001)). For high-resolution polypeptide fragment separation, liquid chromatography ESI-MS / MS or automatic LC-MS / MS using capillary reverse phase chromatography can be used 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 dissociation (HCD) involving dynamic exclusion is used as the mass spectrometry of choice (Goodlett et al., Anal. Chem. 72: 1112-1118 (2000)). For such analysis, the mass spectrometer is typically operated in data-dependent acquisition mode, wherein the device automatically initiates switching from MS to MS / MS mode for ion signals exceeding a predetermined threshold to generate a collision-induced dissociation (CID) spectrum or a high-energy collision dissociation (HCD) spectrum of the peptide.

[0353] For protein identification, all MS / MS spectra were searched against standard protein databases using standard algorithms (eg, SEQUEST, Mascot, X!tandem, and MS Aanda) and typical filtering was performed to reduce false positive protein identifications to less than 1%.

[0354] In one embodiment, the membrane protein concentration in the sample can be quantitatively compared with the control sample. This allows the specific enrichment of the target membrane protein receptor to be detected. For this unlabeled mass spectrometry, the reversed-phase chromatography performed immediately before mass spectrometry can be displayed as an MS characteristic diagram, which plots the characteristics of retention time relative to mass / charge ratio. As detected by a mass spectrometer, the peptides in this diagram appear with a clear isotope pattern within a predetermined time and appear according to the ion current intensity determined by their amount in the sample. Once the peptide has been fragmented and identified by MS / MS analysis, this information can be assigned to the specific peptide features on the MS diagram and can be combined with the 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 ProteomeDiscoverer (Thermo Fischer Scientific). The MS characteristic diagrams of different samples (e.g., sample and control) can be superimposed and compared to determine the ratio of the amount of the peptide. The ratio of the randomly labeled peptide derived from membrane proteins should be about 1. The membrane protein peptides that were specifically captured based on the ligand had a higher ratio than the control sample.

[0355] In another embodiment, 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; e.g., Nilsson et al., Nat Methods (2010) Vol. 7(9), pp. 681-5), mass spectrometry data-independent acquisition (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), and 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).

[0356] MS analysis can be replaced by other analytical methods. These other methods are included as part of the present invention.

[0357] The use of chemical probes according to the present invention allows the determination of the site of target protein binding to a ligand by identifying a peptide sequence to which a portion of the probe structure has been added using mass spectrometry. This enables identification of the binding site of the target protein to the ligand, thereby making identification of the target protein more accurate. Furthermore, this allows the design of probes with enhanced ability to detect target proteins.

[0358] The disclosures of all patent and non-patent literature cited in this specification are incorporated herein by reference in their entirety.

[0359] Example

[0360] The present invention is described in further detail below with reference to Test Examples and Production Examples; however, these examples do not limit the present invention, and modifications may be made without departing from the scope of the present invention.

[0361] In this specification, the following abbreviations may be used.

[0362] Table 1

[0363]

[0364]

[0365] In the following examples, "room temperature" generally refers to about 10 to 35° C. Unless otherwise specified, the ratios indicated for mixed solvents refer to volume ratios. Unless otherwise specified, % refers to mass %.

[0366] Proton NMR ( 1 H NMR) spectra were measured by Fourier transform NMR (using either a Bruker AVANCE III 400 (400 MHz) or a Bruker AVANCE III HD (500 MHz). Spectral splitting patterns measured using tetramethylsilane as a standard were designated as singlet (s), doublet (d), triplet (t), quartet (q), multiple or more overlapping signals (m), and broad signals (br). The solvent is shown in parentheses.

[0367] Mass spectra were measured using a Waters ACQUITY H-Class coupled to an SQD mass spectrometer using electrospray ionization (ESI). High-resolution mass spectrometry (HRMS) was performed using a Waters Xevo G2-XS QTof mass spectrometer using electrospray ionization (ESI). [M+H] + Refers to monoisotopic molecular weight.

[0368] For silica gel column chromatography in the examples, Hi-Flash column (Yamazen Corporation) or SNAP Ultra HP-Sphere 25 μm (Biotage) was used. For ODS column chromatography, octadecyl C18 (Yamazen Corporation) or SNAP Ultra C18 (Biotage) was used.

[0369] In the experimental example, anti-CD71 antibody OKT9 (catalog number 16-0719-85) produced by Thermo Fisher Scientific Inc., anti-CD71 antibody DF1513 (ab212863) produced by Abcam, His-tagged EGF (47061000) produced by Oriental Yeast Co., Ltd., and goat anti-mouse IgG antibody (catalog number 1030-01) produced by Southern Biotech were used.

[0370] Production Example 1: Production of Compound (CPI-003)

[0371] The compound (CPI-003) was produced according to the method shown in the following scheme.

[0372]

[0373] (1) Production of compound (CPI-002)

[0374] N-(tert-Butoxycarbonyl)-4-aminobutyric acid (0.24 g), the compound shown in the above scheme (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 give the desired product (CPI-002) (0.45 g).

[0375] 1H NMR (500MHz, DMSO-d6) δ10.31(s,1H),8.08(d,J=9.0Hz,2H),7.89(d,J=9.0Hz,2H),6.86(t,J=5.5Hz,1H),4.4 7(q,J=7.0Hz,2H),2.98(q,J=6.5Hz,2H),2.36(t,J=7.5Hz,2H),1.71(m,J=7.2Hz,2H),1.38(t,J=7.1Hz,12H);

[0376] HRMS (ESI) is C19 H 27 The calculated value of N6O5 is 419.2043[M+H] + , the measured value is 419.2054.

[0377] (2) Production of compound (CPI-003)

[0378] Under ice cooling, a solution of the above-synthesized CPI-002 (0.45 g) in MeOH (15 ml) and a 0.2 M LiOH aqueous solution (5.9 ml) were added, followed by stirring for 30 minutes. AcOH (0.30 μL) was added to the reaction mixture, and the solvent was removed. The residue was purified by silica gel chromatography (ODS, water / MeCN, containing 0.1% AcOH) to give the desired product (0.45 g).

[0379] 1H NMR(500MHz,DMSO-d6)δ10.30(s,1H),8.06(d,J=9.1Hz,2H),7.89(d,J=9.1Hz,2H),6.86(t,J=5.5Hz ,1H),2.98(q,J=6.6Hz,2H),2.36(t,J=7.4Hz,2H),1.71(m,J=7.2Hz,2H),1.38(s,9H); HRMS(ESI) is C 17 H 23 The calculated value of N6O5 is 391.1730[M+H] + , the measured value is 391.1724.

[0380] Production Example 2: Production of Compound (CPI-005)

[0381] Compound (CPI-005) was produced according to the method shown in the following scheme.

[0382]

[0383] A mixture of dimedone (63 mg), the compound shown above (CPI-004) (0.20 g), WSC hydrochloride (82 mg) and DMAP (50 mg) in DMF (3 ml) was stirred at room temperature for 14 hours, and then the reaction mixture was concentrated. The residue was purified by silica gel chromatography (ODS, water / MeCN, containing 0.1% AcOH) to give the desired product (0.14 g).

[0384] 1H NMR(500MHz,DMSO-d6)δ7.82(t,J=5.6Hz,1H),6.41(br,1H),6.35(br,1H),4.30(dd,J=7.9,5.0Hz,1H),4.14- 4.11(m,1H),3.68(t,J=6.4Hz,2H),3.52-3.49(m,12H),3.39(t,J=5.9Hz,2H),3.23(t,J=6.4Hz,2H),3.18(q, J=5.7Hz,2H),3.11-3.07(m,1H),2.82(dd,J=12.5,5.1Hz,1H),2.63(s,2H),2.57(d,J=12.5Hz,1H),2.33(s,2 H), 2.06 (t, J = 7.5Hz, 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) is C 29 H 48 The calculated value of N3O9S is 614.3111 [M+H] + , the measured value is 614.3112.

[0385] Production Example 3: Production of Compound (CPI-104)

[0386] Compound (CPI-104) was produced according to the method shown in the following scheme.

[0387]

[0388] (1) Production of compound (CPI-102)

[0389] Use microwave reactor by the mixture of compound (CPI-101) (0.21g) and 1-bromo-3-chloropropane (5.0ml) in NMP (5ml) as shown above to react 6 hours at 120 ℃.Add water to reaction mixture, then extract with AcOEt.By organic layer saturated brine washing, then Na2SO4 upper drying, and distill off solvent.Residue is purified by column chromatography (silica gel, hexane / AcOEt) to give intermediate (0.18g).A suspension of described intermediate (0.18g), phthalimide (0.20g), potassium carbonate (0.19g) and sodium iodide (14mg) in DMF (9ml) is stirred 10 hours at 70 ℃.After distilling off solvent, water is added thereto, then extract with AcOEt.By organic layer saturated brine washing, then Na2SO4 upper drying and distill off solvent. The residue is purified by column chromatography (silica gel, hexane / AcOEt) to provide compound (0.24g). The compound (0.24g) obtained is dissolved in DCM (9ml), and chloroacetyl chloride (0.15ml) and triethylamine (0.25ml) are added thereto, then stirred at room temperature for 2 hours. Water is added to the reaction mixture, then extracted with DCM. The organic layer is washed with saturated brine, then on Na2SO4 and solvent is distilled off. The residue is purified by column chromatography (silica gel, hexane / AcOEt) to provide required product (0.18g).

[0390] 1H NMR (400MHz, CDCl3) δ7.83-7.69(m,6H),7.63-7.59(m,2H),7.55(d,J=8.4Hz,1H),7.49-7.45(m,3H),4.15-4.05(m ,1H),3.99(d,J=13.0Hz,1H),3.87(d,J=13.0Hz,1H),3.64(t,J=7.5Hz,2H),3.02-2.95(m,1H),1.82-1.75(m,2H);

[0391] LC-MS: [M+H] + =495.04.

[0392] (2) Production of compound (CPI-103)

[0393] The compound (CPI-102) (0.18g) synthesized as mentioned above is dissolved among the EtOH (5ml).Add hexamethylenetetramine (0.10g) and AcONH to it (56mg), and mixture was heated under reflux for 9 hours.Further add hexamethylenetetramine (51mg) and AcONH to it (28mg), and mixture was heated under reflux for 9 hours.Add water to reaction mixture, then use AcOEt extraction.With organic layer saturated brine washing, then Na SO Upper drying and distilling off solvent.Residue is carried out to purification (silica gel, hexane / AcOEt) by column chromatography, to provide required product (0.14g).

[0394] 1H NMR (400MHz, CDCl3) δ7.83-7.79(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),4.8 0(d,J=10.6Hz,1H),4.38(dt,J=14.4,7.9Hz,1H),3.74(d,J=10.6Hz,1H),3.71-3.52(m,3H),1.93(tt,J=7.2,7.2Hz,2H);

[0395] LC-MS: [M+2H] 2+ =458.08.

[0396] (3) Production of compound (CPI-104)

[0397] To a solution (0.5 ml) of the compound (CPI-103) (10 mg) synthesized as described above in MeOH, methylamine and 40% MeOH solution (22 μ L) were added, followed by stirring at room temperature for 2 hours. Methylamine and 40% MeOH solution (22 μ L) were further added thereto, followed by stirring 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, then dried over Na SO and the solvent was distilled off. The residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (1.6 mg).

[0398] 1H NMR (400MHz, CD3OD) δ7.66(d,J=1.4Hz,2H),7.58-7.45(m,6H),7.24(t,J=1.4Hz,1H),4.64(d,J=10.6Hz,1H),4 .53-4.43(m,1H),3.86(d,J=10.7Hz,1H),3.82-3.76(m,1H),2.46-2.43(m,2H),1.73-1.53(m,2H); LC-MS: [M+H] + =328.17.

[0399] Production Example 4: Production of Compound (CPP-112)

[0400] Compound (CPP-112) was produced according to the method shown in the following scheme.

[0401]

[0402] The mixture of the compound (CPP-111) (0.44g) shown in the above-mentioned route, hydroxylamine hydrochloride (0.18g), pyridine (1.0ml) and EtOH (20ml) was heated under reflux for 2 hours. The solvent was distilled off, and the residue was acidified with 2M hydrochloric acid, then extracted with DCM. The organic layer was washed with saturated brine, then dried over Na2SO4. The solvent was distilled off to provide a colorless oily crude product (0.51g). Under ice-cooling, thionyl chloride (0.31ml) was added to a solution of DMAP (0.26g) in DCM (15ml), and the mixture was stirred for 30 minutes. Under ice-cooling, a solution (5ml) and DMAP (0.39g) of the crude product (0.51g) obtained in the previous step in DCM was added thereto, the mixture was warming to room temperature and stirred for 3 days. Water was added to the reaction mixture, then extracted with DCM. The organic layer was washed with saturated brine, then dried over Na2SO4 and the solvent was distilled off. Purification by column chromatography (silica gel, hexane / AcOEt) gave the desired product (0.30 g).

[0403] 1H NMR (400MHz, CDCl3) δ7.94-7.89(m,1H),7.87(s,1H),7.42-7.37(m,2H),3.92(s ,3H),2.84(t,J=7.3Hz,2H),2.34(t,J=7.3Hz,2H),2.02(tt,J=7.3,7.3Hz,2H);

[0404] LC-MS: [M+H] + =204.02.

[0405] Production Example 5: Production of Compound (CPP-117)

[0406] Compound (CPP-117) was produced according to the method shown in the following scheme.

[0407]

[0408] (1) Production of CPP-114

[0409] To a mixture of the compound (CPP-113) (100 mg) and THF (3 ml) shown in the above route was added 1.6 M n-butyl lithium and n-hexane solution (0.53 ml) at -78 ° C, and the mixture was stirred for 1 hour. A solution (2 ml) of the compound (CPP-112) (84 mg) produced in Production Example 4 in THF was added thereto, and the mixture was stirred at -78 ° C for 5 hours. The mixture was warmed to room temperature over a 3-hour period 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, then dried on Na2SO4 and the solvent was distilled off. The residue was purified by column chromatography (silica gel, hexane / AcOEt) to give the desired product (48 mg).

[0410] 1H NMR(400MHz, CDCl3)δ7.66-7.36(m,6H),7.31-7.13(brm,1H),3.14-2.92(brm,3H),2 .90-2.77(brs,2H),2.34(t,J=6.9Hz,2H),2.00(tt,J=6.9,6.9Hz,2H),1.26(s,9H);

[0411] LC-MS: [M+Na] + =434.94.

[0412] (2) Production of CPP-115

[0413] To the solution (2ml) of compound (CPP-114) (48mg) produced as described above in DCM, TFA (0.45ml) was added. The mixture was stirred at room temperature for 2 hours, and the solvent was distilled off. The residue was diluted with DCM (2ml). TEA (49 μL) and chloroacetyl chloride (28 μL) were added thereto, and 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, then dried over Na2SO4 and the solvent was distilled off. The residue was purified by column chromatography (silica gel, hexane / AcOEt). A mixture of purified product (38mg), hexamethylenetetramine (33mg), AcONH4 (18mg) and EtOH (2ml) was heated under reflux for 6 hours. The solvent was distilled off, and purified by column chromatography (silica gel, hexane / AcOEt), to provide the desired product (28mg).

[0414] 1H NMR (400MHz, CDCl3) δ7.53 (dd, J=8.8, 2.5Hz, 1H), 7.50 (s, 1H), 7.41-7.30 (m, 4H), 7.27 (d, J=2.5Hz, 1H), 4.84 (d, J=10.8Hz,1H),3.77(d,J=10.8Hz,1H),3.40(s,3H),2.88-2.77(m,2H),2.36(t,J=7.0Hz,2H),2.05-1.97(m,2H);

[0415] LC-MS: [M+H] + =352.08.

[0416] (3) Production of CPP-116

[0417] A mixture of the compound (CPP-115) (28 mg) produced as described above, 30% hydrogen peroxide solution (0.16 ml), KCO (11 mg), and DMSO (1 ml) was stirred at room temperature for 2 hours. A sodium thiosulfate aqueous solution was added, and the mixture was stirred, then extracted with AcOEt. The organic layer was washed with saturated brine, then dried over NaSO, and the solvent was distilled off. The residue was purified by column chromatography (silica gel, AcOEt / MeOH) to give the desired product (28 mg).

[0418] 1H NMR (400MHz, CDCl3) δ7.52 (dd, J=8.8, 2.5Hz, 1H), 7.50-7.46 (brm, 1H), 7.37-7.28 (m, 5H), 5.60-5.18 (brm, 2H), 4.82 (d,J=10.8Hz,1H),3.77(d,J=10.8Hz,1H),3.40(s,3H),2.79-2.65(m,2H),2.23(t,J=7.5Hz,2H),2.08-1.91(m,2H);

[0419] LC-MS: [M+H] + =370.00.

[0420] (4) Production of CPP-117

[0421] A mixture of compound (CPP-116) (28 mg), lead tetraacetate (40 mg) and t-BuOH (0.72 ml) produced as described above was stirred at 80 ° C for 3 hours. Lead tetraacetate (40 mg) was further added thereto, and the mixture was stirred at 80 ° C for 3 hours. Water / AcOEt was then added thereto, and the mixture was filtered through diatomaceous earth. The filtrate was extracted with AcOEt. The organic layer was washed with saturated brine, then dried on Na SO and the solvent was distilled off. The residue was purified by column chromatography (silica gel, hexane / AcOEt) to give the desired product (19 mg).

[0422] 1H NMR (400MHz, CDCl3) δ7.53-7.49(m,2H),7.33-7.28(m,5H),4.83(d,J=10.8Hz,1H),4.62-4.47(brs,1H),3.77(d, J=10.8Hz,1H),3.40(s,3H),3.22-3.10(brm,2H),2.68(t,J=7.6Hz,2H),1.82(tt,J=7.6,7.6Hz,2H),1.44(s,9H);

[0423] LC-MS: [M+H] + =442.11.

[0424] Production Example 6: Production of Compound (CPP-124)

[0425] Compound (CPP-124) was produced according to the method shown in the following scheme.

[0426]

[0427] (1) Production of compound (CPP-122)

[0428] A mixture of the compound (CPP-121) (0.51 g) shown in the above route, 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, which was then concentrated. AcOEt was added to the residue. The mixture was washed with water, then dried over Na2SO4 and the solvent was distilled off. The residue was purified by column chromatography (silica gel, AcOEt / MeOH) to give the desired product (0.45 g).

[0429] 1H NMR (500MHz, CDCl3) δ7.61(d,J=2.7Hz,1H),7.55(d,J=8.1Hz,1H),7.42(d,J=5.5Hz,1H),7.38(d,J=5.5Hz, 1H),7.27(t,J=7.8Hz,1H),7.09(d,J=8.3Hz,1H),6.99(dd,J=8.3,2.7Hz,1H),6.90(d,J=7.6Hz,1H),4.11(t ,J=6.3Hz,2H),3.70(t,J=6.8Hz,2H),3.63(t,J=6.4Hz,2H),3.59(t,J=6.7Hz,2H),3.20(br,4H),2.94(t,J =6.6Hz,2H),2.73(br,4H),2.64(t,J=7.3Hz,2H),2.15(m,J=6.7Hz,2H),2.04(m,J=6.5Hz,2H); HRMS(ESI) is C 27 H 33 The calculated value of N3O2S is 498.1982[M+H] + , the measured value is 498.1979.

[0430] (2) Production of compound (CPP-123)

[0431] A suspension of compound (CPP-122) (0.45 g), phthalimide (0.16 mg), K CO (0.15 g) and NaI (41 mg) produced as described above in DMF (10 ml) was stirred at 70° C. for 17 hours. The reaction mixture was concentrated, and water was added to the residue, which was then extracted with EtOAc. The organic layer was dried over Na SO and the solvent was distilled off. The residue was purified by column chromatography (silica gel, AcOEt / MeOH) to give the desired product (0.43 g).

[0432] 1H NMR (500MHz, CDCl3) δ7.84(d,J=3.0Hz,1H)7.83(d,J=3.0Hz,1H),7.60(d,J=2.7Hz,1H),7.54(d,J=8.1Hz,1H),7 .41(dd,J=5.5,0.6Hz,1H),7.38(d,J=5.5Hz,1H),7.27(t,J=7.8Hz,1H),7.09(d,J=8.3Hz,1H),6.98(dd,J=8.3, 2.8Hz,1H),6.90(dd,J=7.7,0.6Hz,1H),4.10(t,J=6.4Hz,2H),3.77(t,J=7.3Hz,2H),3.66(t,J=7.1Hz,2H),3.5 7(t,J=6.7Hz,2H),3.20(br,4H),2.96(t,J=6.6Hz,2H),2.75(br,4H),2.65(t,J=7.4Hz,2H),2.07-2.01(m,4H);

[0433] HRMS (ESI) is C 35 H 37 The calculated value of N4O4S is 609.2535[M+H] + , the measured value is 609.2538.

[0434] (3) Production of compound (CPP-124)

[0435] The mixture of compound (CPP-123) (0.43g) produced as described above, hydrazine monohydrate (0.069ml) and EtOH (12ml) was heated under reflux for 8 hours. The reaction mixture was concentrated, and the 1M NaOH aqueous solution (50ml) was added to the residue, which was then extracted with AcOEt. The organic layer was washed with saturated brine, then dried over Na2SO4 and the solvent was distilled off. The residue was purified by column chromatography (silica gel, DCM / MeOH). The purified product was dissolved in MeOH (10ml). 2M HCl aqueous solution (2.1ml) was added dropwise thereto, and the mixture was stirred at room temperature for 15 hours. The solvent was then distilled off. AcOEt was added to the residue, then stirred. The solid formed was collected by filtration to give the desired product (0.35g).

[0436] 1H NMR (500MHz, CDCl3) δ11.4(br,1H),7.98(br,3H),7.78(d,J=5.5Hz,1H),7.71(d,J=8.1Hz,1H),7.50(d, J=5.5Hz,1H),7.43(d,J=2.7Hz,1H),7.33(t,J=7.8Hz,1H),7.26(d,J=8.4Hz,1H),7.10(dd,J=8.3,2.7Hz , 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 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);

[0437] HRMS (ESI) is C 27 H 35 The calculated value of N4O2S is 479.2481[M+H] + , the measured value is 479.2482.

[0438] Production Example 7: Production of Compound (CPP-125)

[0439] Compound (CPP-125) was produced according to the method shown in the following scheme.

[0440]

[0441] A mixture of the compound (CPP-124) (20 mg) produced in Production Example 6, Boc-5-amino-n-pentanoic 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 give the desired product (15 mg).

[0442] 1H NMR (500MH, DMSO-d6) δ7.78(t,J=5.5Hz,1H),7.69(d,J=5.6Hz,1H),7.61(d,J=7.9Hz,1H),7.40(d,J=5.6Hz,1H),7.39(d,J=2.7Hz,1H) ,7.27(t,J=7.8Hz,1H),7.20(d,J=8.4Hz,1H),7.05(dd,J=8.3,2.6Hz,1H),6.90(d,J=7.7Hz,1H),6.77(t,J=5.4Hz,1H),4.06(t,J=6.3H z, 2H), 3.50 (t, J = 7.0 Hz, under water signal), 3.46 (t, J = 6.6 Hz, under 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) is C 37 H 52 The calculated value of N5O5S is 678.3689[M+H] + , the measured value is 678.3693.

[0443] Production Example 8: Production of Compound (CPF-202)

[0444] The compound (CPF-202) was produced according to the method shown in the following scheme.

[0445]

[0446] (1) Production of CPF-201

[0447] A mixture of the compound produced in Production Example 3 (CPI-104) (9.0 mg), the compound produced in Production Example 1 (CPI-003) (11 mg), COMU (13 mg), DIPEA (11 μL) and DMF (1 ml) was stirred at room temperature for 2 hours. The solvent was distilled off, and purification was performed by column chromatography (ODS, water / MeCN, containing 0.1% AcOH) to give the desired product (10 mg).

[0448] 1H NMR (400MHz, CDCl3) δ9.57(s,1H),8.13(dt,J=9.1,2.4Hz,2H),7.87(d,J=8.9Hz,2H),7.64- 7.59(m,3H),7.54-7.47(m,2H),7.45-7.39(m,3H),7.30(d,J=2.4Hz),4.88-4.83(m,2H),4.5 4-4.47(m,1H),3.81-3.71(m,2H),3.64-3.56(m,1H),3.29(dt,J=6.1,6.1Hz,2H),3.11-3.0 3(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.

[0449] (2) Production of CPF-202

[0450] A mixture of the compound (CPF-201) (10 mg) produced as described above, TFA (0.11 ml) and DCM (1 ml) was stirred at room temperature for 2 hours, and then the solvent was distilled off. A mixture of the residue, the compound (CPI-005) (10 mg) produced in Production Example 2, DIPEA (10 μL) and DMF (1 ml) was stirred at 60° C. for 1 hour, and then the solvent was distilled off. The residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (11 mg).

[0451] 1H NMR (400MHz, CDCl3) δ13.69-13.66(m,1H),9.41(s,1H),8.09(dt,J=9.0,2.4Hz,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=2.4Hz,1H),6.44-6.39(m,1H),5.61-5.58(m,1H),4.85(d,J=10.5Hz,1H),4.68(d,J=7.4Hz,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=12.8,4.0Hz,1H ),2.57(t,J=7.1Hz,2H),2.34(s,4H),2.14(dd,J=7.1,7.1Hz,4H),1.99-1.91(m,1H),1.77-1.52(m,6H),1.47-1.41(m,2H),1.00(s,6H);

[0452] LC-MS: [M+H] + =1195.3.

[0453] Production Example 9: Production of Compound (CPF-212)

[0454]

[0455] (1) Production of CPF-211

[0456] To the solution of the compound (CPP-117) (19 mg) produced in Production Example 5 in DCM (1 ml) was dropwise added TFA (0.17 ml). The mixture was stirred at room temperature for 2 hours, and then the solvent was distilled off. The residue was purified by column chromatography (ODS, water / MeCN) to give an 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). COMU (18 mg) and DIPEA (0.23 ml) were added thereto, and the mixture was stirred at room temperature for 3 hours. The solvent was then distilled off. The residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (18 mg).

[0457] 1H NMR (400MHz, CDCl3) δ9.62 (s, 1H), 8.14 (dt, J = 9.1, 2.4Hz, 2H), 7.90-7.87 (m, 2H), 7.53-7.50 (m,2H),7.38-7.33(m,3H),7.30(dd,J=5.6,3.1Hz,2H),7.26-7.22(m,1H),4.84-4.82(m,2H), 3.77(d,J=10.8Hz,1H),3.60(dt,J=6.8,6.8Hz,2H),3.40(s,3H),3.29(dt,J=6.1,6.1Hz,2H) ,2.79(t,J=7.7Hz,2H),2.44-2.41(m,2H),2.07-2.00(m,2H),1.92-1.86(m,2H),1.50(s,9H);

[0458] LC-MS: [M+H] + =714.08.

[0459] (2) Production of CPF-212

[0460] To the solution of compound (CPF-211) (18mg) produced as described above in DCM (1ml) TFA (97 μ L) is added dropwise. The mixture is stirred at room temperature for 1.5 hours, and then the solvent is distilled off. To the solution of the compound (CPI-005) (22mg) produced in Production Example 2 in DMF (1ml) DIPEA (18 μ L) is added, and the mixture is stirred at 60 ℃ for 3 hours. DIPEA (18 μ L) is further added thereto, and the mixture is continued to be stirred at 60 ℃ for 3 hours. The solvent is then distilled off. The residue is purified by column chromatography (ODS, water / MeCN), to provide the required product (13 mg).

[0461] 1H NMR (400MHz, DMSO-d6) δ13.53(t,J=5.1Hz,1H),10.37(s,1H),9.19(t,J=5.7Hz,1H),8 .06(dt,J=9.1,2.3Hz,2H),7.89(dt,J=9.1,2.3Hz,2H),7.81(t,J=5.6Hz,1H),7.70(d d,J=8.9,2.5Hz,1H),7.60(d,J=8.9Hz,1H),7.49(s,1H),7.42-7.36(m,2H),7.30(dt, J=7.4,1.6Hz,1H),7.22(d,J=2.5Hz,1H),6.40(s,1H),6.35(s,1H),4.57(d,J=10.7Hz ,1H),4.31-4.28(m,1H),4.13-4.10(m,1H),3.78(d,J=10.7Hz,1H),3.63-3.58(m,3H),3.47-3.46(m,9H),3.38-3.05(m,under water signal),2.80(dd,J=12.4,5.1Hz,1H),2.71-2.66(m , 3H), 2.58-2.49 (m, overlapped with 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.

[0462] Production Example 10: Production of Compound (CPF-224)

[0463] The compound (CPF-224) was produced according to the method shown in the following scheme.

[0464]

[0465] (1) Production of compound (CPF-222)

[0466] The compound (CPI-003) (20 mg) produced in Production Example 1 and the compound (CPF-221) (23 mg) shown in the above route were dissolved in DMF (2 ml). COMU (24 mg) and DIPEA (0.020 ml) were added thereto, and the mixture was stirred for 1 hour. The solvent was then distilled off. The residue was purified by silica gel chromatography (ODS, water / MeCN, containing 0.1% AcOH) to give the desired product (33 mg).

[0467] 1H NMR (400MHz, DMSO-d6) δ12.46(br,1H),10.30(s,1H),9.43(t,J=6.1Hz,1H),8.06(d,J=9. 2Hz,2H),7.89(d,J=9.1Hz,2H),7.60(s,1H),7.52(br,1H),6.88-6.83(m,3H),6.28(dd,J =2.9,1.7Hz,1H),5.00(s,2H),4.41(d,J=6.0Hz,2H),4.13(q,J=7.1Hz,2H),2.98(q,J=6. 6Hz,2H),2.36(t,J=7.3Hz,2H),1.71(m,J=7.3Hz,2H),1.38(s,9H),1.17(t,J=7.1Hz,3H);

[0468] LC-MS: [M+H] + =783.31.

[0469] (2) Production of compound (CPF-223)

[0470] To the solution of compound (CPF-222) (33mg) synthesized as described above in MeOH (2ml) was added the 1M LiOH aqueous solution (0.13ml) and water (0.36ml), and the mixture was stirred for 21 hours. The reaction mixture was neutralized with the 0.1% AcOH aqueous solution, and the solution obtained was concentrated. Residue was purified by silica gel chromatography (ODS, water / MeCN, containing 0.1% AcOH), to provide required product (30mg).

[0471] 1H NMR (400MHz, DMSO-d6) δ12.46 (s, 1H), 10.30 (s, 1H), 9.43 (t, J = 5.9Hz, 1H), 8. 06(d,J=9.1Hz,2H),7.88(d,J=9.1Hz,2H),7.61(s,1H),7.47(br,1H),6.90-6. 83(m,3H),6.28(dd,J=2.9,1.7Hz,1H),4.92(s,2H),4.41(d,J=5.9Hz,2H),2. 98(q,J=6.5Hz,2H),2.36(t,J=7.4Hz,2H),1.71(m,J=7.2Hz,2H),1.38(s,9H);

[0472] LC-MS: [M+H] + =754.99.

[0473] (3) Production of compound (CPF-224)

[0474] The compound (CPF-223) (29mg) synthesized as described above is dissolved in DCM (3ml). TFA (0.29ml) is added dropwise thereto, then stirred at room temperature for 2 hours. The solution obtained is then concentrated. DIPEA (27 μ L), the compound (CBI-455) (39mg) produced in Production Example 2 and DMF (2ml) are added to the residue, then stirred at 60 ℃ for 5 hours. The reaction mixture is then concentrated. The residue is purified by silica gel chromatography (ODS, water / MeCN, containing 0.1% TFA), to provide required product (17mg).

[0475] 1H NMR (400MHz, DMSO-d6) δ13.53(t,J=5.5Hz,1H),12.47(s,1H),10.38(s,1H),9.44(t,J=6.2Hz,1H),8.07(d, J=9.1Hz,2H),7.89(d,J=9.1Hz,2H),7.82(t,J=5.7Hz,1H),7.61(s,1H),7.49(br,1H),6.90(br,1H),6.84(t , J=2.4Hz,1H),6.41(br,1H),6.35(br,1H),6.28(dd,J=2.8,1.7Hz,1H),4.95(s,2H),4.41(d,J=5.9Hz,2H),4.30(dd,J=7.8,4.4Hz,1H),4.12(dd,J=7.6,4.4Hz,1H),3.61(t,J=6.3Hz,4H),3.49-3.46(m,under 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 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);

[0476] HRMS (ESI) is C 57 H 71 N 13 O 14 The calculated value of F3S is 1250.4916 [M+H] + , the measured value is 1250.4919.

[0477] Production Example 11: Production of Compound (CPF-232)

[0478] The compound (CPF-232) was produced according to the method shown in the following scheme.

[0479]

[0480] (1) Production of compound (CPF-231)

[0481] The compound (CPP-124) (40 mg) produced in Production Example 6 and the compound (CPI-003) (26 mg) produced in Production Example 1 were diluted with DMF (2 ml). HATU (26 mg) and DIPEA (0.036 ml) were added thereto, followed by stirring 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 give the desired product (50 mg).

[0482] 1H NMR (500MHz, DMSO-d6) δ11.96(br,1H),10.30(s,1H),9.16(t,J=5.8Hz,1H),8.06(d,J=9.1Hz,2H),7.89(d,J=9.1Hz,2H),7.74(d,J=5.1Hz,1 H),7.67(d,J=7.7Hz,1H),7.46(d,J=4.1Hz,1H),7.43(d,J=2.3Hz,1H),7.30(t,J=7.8Hz,1H),7.23(d,J=8.4Hz,1H),7.07(dd,J=8.3,2.7Hz, 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 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.

[0483] (2) Production of compound (CPF-232)

[0484] The compound (CPF-231) (19 mg) produced as described above was dissolved in DCM (2 ml). TFA (0.16 ml) was added dropwise thereto, then stirred at room temperature for 1 hour. The reaction mixture was then concentrated. The compound (CPI-005) (15 mg), DIPEA (17 μ L) and DMF (2 ml) produced in Production Example 2 were added to the residue, then 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 the desired product (8.7 mg).

[0485] 1H NMR (500MHz, DMSO-d6) δ13.53(t,J=5.3Hz,1H),10.38(s,1H),9.51(br,1H),9.17(t,J=5.9Hz,1H),8. 06(d,J=9.1Hz,2H),7.89(d,J=9.1Hz,2H),7.81(t,J=5.5Hz,1H),7.78(d,J=5.5Hz,1H),7.72(d,J=8. 1Hz,1H),7.51(d,J=5.6Hz,1H),7.45(d,J=2.7Hz,1H),7.33(t,J=7.9Hz,1H),7.25(d,J=8.5Hz,1H),7 .08(dd,J=8.3,2.7Hz,1H),6.99(d,J=7.7Hz,1H),6.41(br,1H),6.36(br,1H),4.29(dd,J=7.8,4.9Hz, 1H), 4.14-4.10 (m, 3H), 3.70 (br, 2H), 3.62-3.55 (m, under water signal), 3.41-3.34 (m, under 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), 2.5 7(d,J=12.4Hz,1H),2.28(s,4H),2.22-2.17(m,2H),2.05(t,J=7.4Hz,2H),1.93(m,J=7.1Hz,2H),1.8 6(m,J=6.8Hz,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) is C 68 H 92 N 13 O 12 The calculated value of S2 is 1346.6429 [M+H]+ , the measured value is 1346.6445.

[0486] Production Example 12: Production of Compound (CPP-127)

[0487] Compound (CPP-127) was produced according to the method shown in the following scheme.

[0488]

[0489] (1) Production of compound (CPP-126)

[0490] The compound (CPP-125) (15 mg) produced in Production Example 7 was dissolved in DCM (3 ml). TFA (0.15 μ L) was added dropwise thereto, and then stirred at room temperature for 2 hours. The reaction mixture was then concentrated. The residue was diluted with the compound (CPI-003) (9.1 mg) and DMF (2 ml) produced in Production Example 1. COMU (9.35 mg) and DIPEA (15 μ L) were added thereto, and then 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 give the desired product (16 mg).

[0491] 1H NMR (500MHz, DMSO-d6) δ11.94(br,1H),10.29(s,1H),9.14(t,J=5.9Hz,1H),8.04(d,J=9.1Hz,2H),7.87(d,J=9.1Hz,2H),7.81(t,J=5.5Hz,1H), 7.69(br,1H),7.61(br,1H),7.41-7.38(m,2H),7.28(t,J=7.6Hz,1H),7.19(d,J=8.6Hz,1H),7.04(dd,J=8.5,2.7Hz,1H),6.90(br,1H),6.86(t, J=5.4Hz,1H),4.05(br,2H),3.50(t,J=6.5Hz,2H),3.46(t,J=7.1Hz,2H),3.09-3.04(m,6H),2.98(q,J=6.5Hz,2H),2.87(t,J=6.7Hz,2H),2.64( br,4H),2.36(t,J=7.4Hz,2H),2.10(br,2H),1.94(br,2H),1.91(s,3H),1.71(m,J=7.2Hz,2H),1.67(m,J=7.0Hz,2H),1.55(br,4H),1.37(s,9H);

[0492] HRMS (ESI) is C 49 H 64 N 11 The calculated value of O7S is 950.4711[M+H] + , the measured value is 950.4713.

[0493] (2) Production of compound (CPP-127)

[0494] The compound (CPP-126) (14 mg) produced as described above was dissolved in DCM (5 ml). TFA (0.23 ml) was added dropwise thereto, and then stirred at room temperature for 12 hours. The reaction mixture was then concentrated. The compound (CPI-005) (9.2 mg), DIPEA (12 μL) and DMF (2 ml) produced in Production Example 2 were added to the residue, and then 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 the desired product (13 mg).

[0495] 1H NMR (500MHz, DMSO-d6) δ13.53(t,J=5.4Hz,1H),10.38(s,1H),9.63(br,1H),9.15(t,J=5.9Hz,1H),8.05(d,J=9 .1Hz,2H),7.88(d,J=9.1Hz,2H),7.84-7.81(m,2H),7.78(d,J=5.6Hz,1H),7.72(d,J=8.1Hz,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 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=5.9Hz,2H),3.32(br,2H),3.27 (t,J=6.1Hz,2H),3.17(q,J=5.8Hz,2H),3.11-3.04(m,5H),2.88(t,J=6.4Hz,2H),2.81(dd,J=12.4,5.1Hz,1H), 2.57 (d, J = 12.3 Hz, 1H), 2.48 (d, overlapped with 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) is C 73 H 101 N 14 O 13 The calculated value of S2 is 1445.7114[M+H] + , the measured value is 1445.7126.

[0496] Production Example 13: Production of Compound (CPP-133)

[0497] Compound (CPP-133) was produced according to the method shown in the following scheme.

[0498]

[0499] (1) Production of CPP-132

[0500] Compound (CPP-131) (15 mg) and the compound (CPI-003) (16 mg) produced in Production Example 1 were dissolved in a DMF solution (1 ml), and COMU (19 mg) and DIPEA (15 μL) were added thereto. The mixture was stirred at room temperature for 20 hours, and then the solvent was distilled off. The residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (22 mg).

[0501] 1H NMR (400MHz, CDCl3) δ9.30(s,1H),9.11(s,1H),8.10(dt,J=9.1,2.4Hz,2H),7.85(d,J=9.1Hz,2H),7.31-7.25( m,2H),7.05(d,J=2.4Hz,1H),7.01(td,J=7.5,0.9Hz,1H),6.86(dd,J=7.7,0.7Hz,1H),6.82(dd,J=8.3,2.4Hz,1 H),6.65(d,J=8.3Hz,1H),5.21(s,1H),4.87-4.78(brm,1H),3.70(dt,J=5.6,5.6Hz,2H),3.66-3.50(brs,4H), 3.28(dt,J=6.0,6.0Hz,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.

[0502] (2) Production of CPP-133

[0503] The compound (CPP-132) (22 mg) produced as described above was diluted with DCM (1 ml). TFA (0.11 ml) was added dropwise thereto, and the mixture was stirred at room temperature for 20 hours. The solvent was then distilled off. The compound (CPI-005) (18 mg), DIPEA (21 μL) and DMF (1.5 ml) produced in Production Example 2 were added to the residue, and the mixture was stirred at 60 ° C for 3 hours. DIPEA (26 μL) was further added thereto, and the mixture was continued to be stirred at 60 ° C for 1 hour. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN, containing 0.1% AcOH) to give the desired product (16 mg).

[0504] 1H NMR (400MHz, CDCl3) δ13.73-13.66(brm,1H),9.28-9.16(brm,2H),8.06(dt,J=9.1,2.4Hz,2H),7.86(d,J=9.1Hz,2H),7.31-7.26(m,2H),7.04(d,J =2.4Hz,1H),7.00(td,J=7.5,0.9Hz,1H),6.87(d,J=7.9Hz,1H),6.81(dd, J=8.3,2.4Hz,1H),6.68(d,J=8.3Hz,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=11.4Hz,2H),3.72-3.48(m,23H),3.38-3.28(m,4H),3.16-3.12(m,1H ),2.92(dd,J=12.9,5.0Hz,1H),2.72-2.54(m,8H),2.35(s,4H),2.19-2.0 9(m,4H),1.90-1.82(m,2H),1.73-1.41(m,6H),1.01(s,6H); LC-MS: [M+H] + =1237.14.

[0505] Production Example 14: Production of Compound (CPA-302)

[0506] Compound (CPA-302) was produced according to the method shown in the following scheme.

[0507]

[0508] (1) Production of CPA-301

[0509] To a mixture of 3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propionic acid (22 mg) and allyl alcohol (78 μL) was added thionyl chloride (0.17 ml), followed by stirring at room temperature for 1 hour. After adding water thereto, the mixture was concentrated under reduced pressure. The residue and the compound (CPI-003) (30 mg) produced in Production Example 1 were dissolved in DMF (2 ml), and DIPEA (40 μL) and COMU (36 mg) were added thereto. The mixture was stirred at room temperature for 3 hours, and the solvent was then distilled off. The residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (37 mg).

[0510] 1H NMR (400MHz, CDCl3) δ9.60(s,1H),8.14(dt,J=9.0,2.5Hz,2H),7.88(d,J=9.0Hz,2H),7.71(t ,J=5.4Hz,1H),5.95-5.86(m,1H),5.31(ddt,J=17.2,1.4,1.4Hz,1H),5.22(ddt,J=10.4,1.4 ,1.4Hz,1H),4.86-4.82(m,1H),4.59(dt,J=5.6,1.4Hz,2H),3.78-3.60(m,18H),3.32-3.27( m,2H),2.63(t,J=6.5Hz,2H),2.44-2.41(m,2H),1.92-1.86(m,2H),1.50(s,9H); LC-MS: [M+H] + =678.27.

[0511] (2) Production of CPA-302

[0512] A mixture of compound (CPA-301) (36 mg), morpholine (9.3 μ L), Pd(PPh ) (3.1 mg) and THF (1 ml) produced as described above was stirred at room temperature for 6 hours. Morpholine (4.6 μ L) and Pd(PPh ) (3.1 mg) were further added thereto, and then stirred at room temperature for 1 hour. The solvent was then distilled off. The residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (27 mg).

[0513] 1H NMR (400MHz, DMSO-d6) δ10.54(s,1H),9.09(t,J=5.7Hz,1H),8.05(dt,J=9.2,2.4Hz,2H),7.91(dt,J=9.2,2.3Hz,2H),6.91(t,J=5.2Hz,1H),3.60-3. 42(m,18H),2.98(dt,J=6.4,6.4Hz,2H),2.66(t,J=6.4Hz,1H),2.37(t,J=7 .4Hz,2H),2.29(t,J=6.4Hz,2H),1.71(tt,J=6.4,6.4Hz,2H),1.37(s,9H);

[0514] LC-MS: [M+H] + =638.23.

[0515] Production Example 15: Production of Compound (CPA-306)

[0516] Compound (CPA-306) was produced according to the method shown in the following scheme.

[0517]

[0518] (1) Production of CPA-303

[0519] 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. After adding water thereto, the mixture was concentrated under reduced pressure. The residue and the compound (CPA-302) (22 mg) produced in Production Example 14 were dissolved in DMF (1 ml), and COMU (16 mg) and DIPEA (18 μL) were added thereto. The mixture was stirred at room temperature for 5 hours, and the solvent was then distilled off. The residue was purified by column chromatography (ODS, water / MeCN) to give the desired compound (17 mg).

[0520] 1H NMR(400MHz, CDCl3)δ9.63(s,1H),8.13(dt,J=9.0,2.3Hz,2H),7.89(d,J=9.0Hz,2H),7.78(t,J=5.3Hz,1H), 6.72(s,1H),5.96-5.86(m,1H),5.31(ddt,J=17.2,1.4,1.4Hz,1H),5.23(ddt,J=10.4,1.4,1.4Hz,1H),4.89( s,1H),4.59(dt,J=5.7,1.4Hz,2H),3.78-3.60(m,32H),3.54(t,J=5.3Hz,2H),3.43(dt,J=5.3,5.3Hz,2H),3 .28(dt,J=5.9Hz,2H),2.62(t,J=6.5Hz,2H),2.48-2.42(m,4H),1.93-1.87(m,2H),1.49(s,9H); LC-MS: [M+H] + =925.27.

[0521] (2) Production of CPA-304

[0522] The compound (CPA-303) (17 mg) produced as described above was dissolved in DCM (1 ml). TFA (0.14 ml) was added dropwise thereto and the compound was stirred at room temperature for 2 hours. The solvent was then distilled off. DIPEA (13 μL) was added to the residue and a solution (1 ml) of the compound (CPI-005) (17 mg) produced in Production Example 2 in DMF, and the mixture was stirred at 60° C. for 4 hours. The solvent was then distilled off. The residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (22 mg).

[0523] 1H NMR (400MHz, CDCl3) δ13.68(t,J=5.0Hz,1H),9.50(s,1H),8.10(d,J=9.0Hz,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=17.2,1.4,1.4Hz,1H),5.23(ddt,J=10.4,1.4,1.4Hz,1H),5.11-5.06(m,1H),4. 59(dt,J=5.6,1.4Hz,2H),4.54-4.51(m,1H),4.34-4.31(m,1H),3.78-3.50(m,50H), 3.43(dt,J=5.4,5.4Hz,2H),3.38(dt,J=5.3,5.3Hz,2H),3.33(dt,J=5.5,5.5Hz,2H ),3.17-3.12(m,1H),2.92(dd,J=12.8,4.9Hz,1H),2.74(d,J=12.8Hz,1H),2.63(t,J =6.5Hz,2H),2.58(t,J=7.1Hz,2H),2.47(t,J=6.1Hz,2H),2.34(s,4H),2.18(t,J=7 .2Hz,2H),2.12(t,J=6.9Hz,2H),1.82-1.60(m,6H),1.47-1.40(m,2H),1.01(s,6H);

[0524] LC-MS: [M+H] + =1420.69.

[0525] (3) Production of CPA-305

[0526] A mixture of compound (CPA-304) (10 mg) produced as described above, 1,3-dimethylbarbituric acid (2.2 mg), Pd(PPh 3 ) 4 (0.81 mg) and AcOEt / DCM (1:1, 0.5 ml) was stirred at room temperature for 1 hour, and then the solvent was distilled off. The residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (9.5 mg).

[0527] 1H NMR (400MHz, DMSO-d6) δ13.53(t,J=10.4Hz,1H),10.57(s,1H),9.10(t,J=5.2Hz,1H),8.06(d,J=9.1Hz,2H),7.95(s,1H) ,7.91(d,J=9.1Hz,2H),7.84(t,J=5.4Hz,1H),6.42(s,1H),6.36(s,1H),4.29(dd,J=5.6,5.2Hz,1H),4.13-4.10(m,1H),3 .63-3.25 (m, under 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 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);

[0528] LC-MS: [M+H] + =1380.38.

[0529] (4) Production of CPA-306

[0530] A mixture of the compound (CPA-305) (8.5 mg) produced as described above, 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 further added, and the mixture was stirred for 2 hours. The solvent was then distilled off. The residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (7.7 mg).

[0531] 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 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 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);

[0532] LC-MS: [M+2H] 2+ =1478.07.

[0533] Production Example 16: Production of Compound (CPA-310)

[0534] Compound (CPA-310) was produced according to the method shown in the following scheme.

[0535]

[0536] (1) Production of CPA-307

[0537] Diethylene glycol bis (3-aminopropyl) ether (2.1ml) is dissolved among the DCM (200ml), and under ice-cooling, dropwise add allyl chloroformate (1.0ml), then stirred 4 hours.Described mixture is warming up to room temperature and stirred 14 hours, then distills off solvent.Residue is carried out to purifying (ODS, water / MeCN) by column chromatography, to provide required product (0.50g).

[0538] 1H NMR(400MHz,DMSO-d6)δ8.26-7.37(brs,2H),7.19(t,J=5.4,1H),5.95-5.85 (m,1H),5.26(ddt,J=13.8,1.6,1.6Hz,1H),5.17(ddt,J=6.2,1.6,1.6Hz,1H ),4.45(d,J=5.3Hz,2H),3.54-3.36(m,12H),3.03(dt,J=6.6,6.6Hz,2H),2. 83(t,J=7.0Hz,2H),1.78(tt,J=6.6,6.6Hz,2H),1.62(tt,J=7.0,7.0Hz,2H);

[0539] LC-MS: [M+H] + =305.16.

[0540] (2) Production of CPA-308

[0541] To a solution (20 ml) of the compound (CPA-307) produced as described above (0.50 g) in DMF were added Fmoc-Lys(Boc)-OH (0.77 g), WSC·HCl (0.38 g), and HOBt·H O (0.30 g) at room temperature, and the mixture was stirred for 15 hours. The solvent was then distilled off. A saturated aqueous NaHCO solution was added, followed by extraction with AcOEt. The organic layer was washed with saturated brine and then dried over Na SO The solvent was distilled off, and purification was performed by column chromatography (silica gel, AcOEt / MeOH) to give the desired product (0.80 g).

[0542] 1H NMR (400MHz, CDCl3) δ7.76(d,J=7.6Hz,2H),7.60(d,J=7.4Hz,2H),7.40(t,J=7.5Hz,2H),7.31(td,J=7.4,1.0Hz,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=17.4Hz,1H),5.17(d,J=10 .6hz,2H),4.75-4.63(brs,1H),4.52(d,J=5.3Hz,2H),4.42(d,J=7.0Hz,2H),4.21(t,J=6.9Hz,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);

[0543] LC-MS: [M+H] + =755.21.

[0544] (3) Production of CPA-309

[0545] To a solution of compound (CPA-308) (0.80 g) and 1,3-dimethylbarbituric acid (0.33 g) produced as described above in DCM (10 ml) was added Pd(PPh 3 ) 4 (0.12 g), and the mixture was stirred at room temperature for 2 hours. The solvent was then distilled off. The residue was purified by column chromatography (silica gel, AcOEt / MeOH) to give an 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. The solvent was then distilled off. The residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (0.15 g).

[0546] 1H NMR(400MHz, DMSO-d6)δ7.89(d,J=7.5Hz,2H),7.85(t,J=5.5Hz,1H),7.74-7.72(m,3H),7.44-7.40(m,3H),7.33 (td,J=7.4,0.9Hz,2H),6.76(t,J=5.3Hz,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, 5.1Hz, 1H), 2.57 (d, J = 12.4Hz, 1H), 2.04 (t, J = 7.4Hz, 2H), 1.64-1.43 (m, 10H), 1.36-1.21 (m, 15H); LC-MS: [M+H] + =897.11.

[0547] (4) Production of CPA-310

[0548] A mixture of compound (CPA-309) (0.15 g) produced as described above, piperidine (24 μL) and DMF (3 ml) was stirred at room temperature for 2 hours, and then the solvent was distilled off. The residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (0.11 mg).

[0549] 1H NMR (400MHz, DMSO-d6) δ7.81(t,J=5.7Hz,1H),7.75(t,J=5.5Hz,1H),6.75(t,J=5.5Hz, 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=12.4Hz,1H),2.04( t,J=7.4Hz,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.

[0550] Production Example 17: Production of Compound (CPA-311)

[0551]

[0552] To 6-aminocaproic acid (0.20g) and allyl alcohol (1.0ml), thionyl chloride (0.33ml) is dropwise added, and the mixture is stirred at room temperature 1 hour.Then solvent is distilled off.Succinic anhydride (0.18g), DIPEA (0.80ml) and DMF (5ml) are added to residue, and the mixture is stirred at room temperature 20 hours.Succinic anhydride (0.15g) and DIPEA (0.4ml) are further added to it, and the mixture is stirred at room temperature 1 hour.Then solvent is distilled off.Residue is carried out to purification (ODS, water / MeCN) by column chromatography, to provide required product (0.40g).

[0553] 1H NMR (400MHz, CDCl3) δ6.02 (s, 1H), 5.97-5.87 (m, 1H), 5.32 (ddt, J = 14.1, 1.4, 1. 4Hz,1H),5.24(ddt,J=10.4,1.4,1.4Hz,1H),4.58(dt,J=5.8,1.4Hz,2H),3.27( dt,J=6.6,6.6Hz,2H),2.71-2.68(m,2H),2.52-2.49(m,2H),2.36(t,J=7.3Hz,2 H),1.65(tt,J=7.5,7.5Hz,2H),1.53(tt,J=7.3,7.3Hz,2H),1.40-1.32(m,2H);

[0554] LC-MS: [M+H] + =272.21.

[0555] Production Example 18: Production of Compound (CPA-314)

[0556]

[0557] (1) Production of CPA-312

[0558] The compound (CPA-310) (0.11 g) produced in Production Example 16 and the compound (CPA-311) (43 mg) produced in Production Example 17 were dissolved in DMF (4 ml). WSC·HCl (37 mg) and HOBt·H2O (29 mg) were added, and the mixture was stirred at room temperature for 14 hours. The solvent was then distilled off. The residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (0.13 g).

[0559] 1H NMR (400MHz, DMSO-d6) δ7.95(d,J=8.0Hz,1H),7.86-7.82(m,2H),7.74(t,J=5.5Hz,1H),6.74(t,J=5.4Hz,1H),6.42( s,1H),6.35(s,1H),5.96-5.86(m,1H),5.28(ddt,J=17.3,1.5,1.5Hz,1H),5.20(ddt,J=10.5,1.5,1.5Hz,1H),4.54(d t,J=5.4,1.4Hz,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=12.4Hz,1H),2.37-2.25(m,6H),2.04(t,J=7.4Hz,2H),1.65-1.19(m,31H); LC-MS: [M+H] + =928.30.

[0560] (2) Production of CPA-313

[0561] To a solution of compound (CPA-312) (0.13 g) produced as described above in DCM (3 ml) was dropwise added TFA (1.1 ml), and the mixture was stirred at room temperature for 1 hour. The solvent was then distilled off. The residue was diluted with DMF (2 ml). DIPEA (0.12 ml) and adipic anhydride (36 mg) were then added thereto, and the mixture was stirred at room temperature for 2 hours. Stirring was stopped, and the mixture was allowed to stand for 4 days. The solvent was then distilled off. The residue was purified by column chromatography (ODS, water / MeCN), to give the desired product (75 mg).

[0562] 1H NMR(400MHz,DMSO-d6)δ8.02-7.95(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=17.2,1.5,1.5Hz,1H),5.20(ddt,J=10.5,1.5 1.5Hz,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 .5,5.2Hz,1H),2.57(d,J=12.5Hz,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.

[0563] (3) Production of CPA-314

[0564] Compound (CPA-313) (75 mg) produced as described above was dissolved in DMF (2 ml). Dimethicone (12 mg), WSC·HCl (16 mg), and DMAP (9.6 mg) were added, and the mixture was stirred at room temperature for 14 hours. The solvent was then distilled off. The residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (31 mg).

[0565] 1H NMR (400MHz, DMSO-d6) δ7.99(d,J=8.0Hz,1H),7.89-7.82(m,2H),7.74(t,J=10.6Hz,2H),6.61(d,J=6.0Hz,1H),6.42 (s,1H),6.35(s,1H),5.96-5.86(m,1H),5.28(ddt,J=17.2,1.6,1.6Hz,1H),5.20(ddt,J=10.5,1.5,1.5Hz,1H),4.53 (dt,J=5.4,1.4Hz,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,11 H),2.86-2.79(m,3H),2.57(d,J=12.5Hz,1H),2.36-2.30(m,8H),2.06-1.96(m,4H),1.65-1.21(m,26H),0.97(s,6H);

[0566] LC-MS: [M+2H] 2+ =1079.50.

[0567] Production Example 19: Production of Compound (CPA-318)

[0568]

[0569] (1) Production of CPA-317

[0570] The compound (CPA-315) (0.11 g) shown in the above route and the compound (CPA-316) (0.087 g) shown in the above route were dissolved in DMF (5 ml). HATU (0.209 g) and DIPEA (0.160 ml) were added thereto at room temperature, and the mixture was stirred for 20 hours. The solvent was then distilled off. The residue was purified by column chromatography (silica gel, hexane / AcOEt) to give the desired product (0.11 mg).

[0571] 1H NMR (400MHz, CDCl3) δ8.22-8.19(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=5.2Hz,2H),3.36-3.33(m,2H),1.43(s,9H); LC-MS: [M+H] + =421.08.

[0572] (2) Production of CPA-318

[0573] The compound (CPA-317) (0.11 mg) produced as described above was dissolved in DCM (2 ml). TFA (0.97 ml) was added dropwise and the mixture was stirred at room temperature for 1 hour. The solvent was then distilled off. The residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (89 mg).

[0574] 1H NMR(400MHz,DMSO-d6)δ9.15(t,J=5.4Hz,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=5.2Hz,2H); LC-MS: [M+H] + =321.06.

[0575] Production Example 20: Production of Compound (CPA-321)

[0576]

[0577] (1) Production of CPA-319

[0578] The compound (CPA-318) (12 mg) produced in Production Example 19 and the compound (CPA-314) (30 mg) produced in Production Example 18 were dissolved in DMF (1 ml). DIPEA (19 μL) was added thereto, and the mixture was stirred at 60° C. for 2 hours. Purification was performed by column chromatography (ODS, water / MeCN) to give the desired product (30 mg).

[0579] 1H NMR (400MHz, DMSO-d6) δ13.35(t,J=4.5,1H),9.08(t,J=5.9Hz,1H),8.13-8.10(m,2H),7.96(d,J=8.0Hz,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=17.2,1.5,1.5Hz,1H),5.20(ddt,J=10.4,1.5,1.5Hz,1H),4.53(dt, J=5.5,1.5Hz,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=12.4,5.1Hz,1H),2.57(d,J=12.4Hz.,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);

[0580] LC-MS: [M+H] + =1380.98.

[0581] (2) Production of CPA-320

[0582] A mixture of compound (CPA-319) (29 mg) produced as described above, 1,3-dimethylbarbituric acid (6.6 mg), Pd(PPh 3 ) 4 (2.4 mg) and DCM (1 ml) was stirred at room temperature for 1 hour, and then the solvent was distilled off. The residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (26 mg).

[0583] 1H NMR (400MHz, DMSO-d6) δ13.35(t,J=4.3Hz,1H),9.09(t,J=5.5,1H),8.13-8.10(m,3H),7.99-7.89(brs,1H),7.85(t ,J=5.5Hz,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=12.4,5.1Hz,1H),2.57 (d,J=12.4Hz,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.

[0584] (3) Production of CPA-321

[0585] A mixture of the compound (CPA-320) (25 mg) produced as described above, 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 further added, and the mixture was stirred at room temperature for another 3 hours. Purification by column chromatography (ODS, water / MeCN) gave the desired product (21 mg).

[0586] 1H NMR (400MHz, DMSO-d6) δ13.35(t,J=4.4Hz,1H),9.08(t,J=5.6Hz,1H),8.13-8.10(m,2H),7.96(d,J=7.9Hz,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=7.4Hz,2H),2.57(d ,J=12.4Hz,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.

[0587] Production Example 21: Synthesis of CPF-242

[0588]

[0589] (1) Synthesis of CPF-241

[0590] To a mixture of CPI-104 (14 mg) and DMF (1 ml) were added CPI-006 (6.6 mg), WSC HCl (6.8 mg) and HOBt HO (5.4 mg), and the mixture was stirred at room temperature for 3 hours. The solvent was distilled off, 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) were added N-Boc-1,5-diaminopentane (12 μL), WSC HCl (11 mg) and HOBt HO (9.0 mg), and the mixture was stirred at room temperature for 16 hours. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (7.4 mg).

[0591] 1H NMR(400MHz,DMSO-d6)δ7.77(t,J=5.4Hz,1H),7.72-7.65(m,3H),7.57-7.50(m,3H), 7.47-7.43(m,2H),7.20(d,J=2.3Hz,1H),6.75(t,J=5.5Hz,1H),4.55(d,J=10.5Hz,1H ),4.25-4.18(m,1H),3.77(d,J=10.5Hz,1H),3.70-3.63(m,1H),2.98(dt,J=6.5,6.5 Hz,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);

[0592] LC-MS: [M+H] + =680.53.

[0593] (2) Synthesis of CPF-242

[0594] To a mixture of compound (CPF-241) (6.6 mg) produced as described above and DCM (1 ml) was added TFA (149 μL), and the mixture was stirred at room temperature for 2 hours. The solvent was distilled off. DMF (1 ml), CPI-005 (6.0 mg) and DIPEA (17 μL) were added to the residue, and the mixture was stirred at 60° C. for 3 hours. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (7.9 mg).

[0595] 1H NMR(400MHz, DMSO-d6)δ13.46(t,J=4.9Hz,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=2.4Hz,1H),6.41(s,1H),6.35(s,1H),4.55(d,J=10.5Hz,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);

[0596] LC-MS: [M+H] + =1175.70.

[0597] Production Example 22: Production of CPA-325

[0598]

[0599] (1) Production of CPA-322

[0600] The compound (CPA-316) (40 mg) shown in the above route, 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 distilled off and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (53 mg).

[0601] 1 H 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);

[0602] LC-MS: [M+Na] + =369.41.

[0603] (2) Production of CPA-323

[0604] The compound (CPA-322) (51 mg) produced as described above was dissolved in DCM (1 ml). TFA (113 μL) was added thereto under ice cooling, and the mixture was stirred at room temperature for 1 hour. The solvent was distilled off. 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, and the mixture was stirred at room temperature for 12 hours. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (76 mg).

[0605] 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);

[0606] LC-MS: [M+H] + =594.60.

[0607] (3) Production of CPA-324

[0608] The compound (CPA-323) (67 mg) produced as described above was dissolved in DCM (1 ml). TFA (130 μL) was added thereto, and the mixture was stirred at room temperature for 2 hours. The solvent was distilled off to give the desired product (68 mg).

[0609] 1H NMR(400MHz, DMSO-d6)δ9.16(t,J=5.8Hz,1H),8.15-8.11(m,2H),7.92(t,J=5.6Hz,1H),7.75-7.64(m,6H),3.62-3.48(m,16H), 3.34(dt,J=6.7,6.7Hz,2H),3.12(dt,J=6.7,6.7Hz,2H),3.01-2.94(m,2H),2.33(t,J=6.4Hz,2H),1.69(tt,J=6.7,6.7Hz,2H);

[0610] LC-MS: [M+H] + =494.38.

[0611] (4) Production of CPA-325

[0612] The desired product was produced in the same manner as in Production Example 20, except that the compound produced as described above (CPA-324) was used instead of CPA-318.

[0613] 1 H NMR (400MHz, DMSO-d6) δ13.35(s,1H),9.15(t,J=5.9Hz,1H),8.14-8.11(m,2H),7.97(d,J=8.4Hz, 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=7.6,5.2Hz,1H),4 .14-4.05(m,2H),3.61-3.34(m,32H),3.17-2.91(m,13H),2.84-2.79(m,5H),2.65(t,J=7.4Hz,2H ),2.57(d,J=12.4Hz,2H),2.37-2.26(m,10H),2.09-2.02(m,4H),1.71-1.19(m,28H),0.93(s,6H);

[0614] LC-MS: [M+2H] 2+ =1611.63.

[0615] Production Example 23: Production of CPA-326

[0616]

[0617] The desired product was produced in the same manner as in Production Example 21, except that N-Boc-3,3′-((oxybis(ethane-2,1-diyl))bis(oxy))bis(propan-1-amine) was used instead of N-Boc-1,3-diaminopropane.

[0618] 1 H NMR (400MHz, DMSO-d6) δ13.35(s,1H),9.14(t,J=5.8Hz,1H),8.14-8.11(m,2H),7.96(d,J=8.0Hz,1H),7.85(d d,J=9.9,5.4Hz,2H),7.79(t,J=5.6Hz,1H),7.87-7.63(m,5H),6.42(s,1H),6.35(s,1H),4.30(dd,J=7.6,5.2H z,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=7.4Hz,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);

[0619] LC-MS: [M+2H] 2+ =1757.83.

[0620] Production Example 24: Production of CPA-332

[0621]

[0622] (1) Production of CPA-328

[0623] The compound (CPA-327) (60 mg) shown in the above route and Nα, Nε-di-Boc-L-lysine (87 mg) were dissolved in DMF (2 ml). COMU (108 mg) and DIPEA (109 μL) were added thereto, and the mixture was stirred at room temperature for 1 hour. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (129 mg).

[0624] 1H NMR(500MHz,DMSO-d6)δ8.13(d,J=7.4Hz,1H),6.77-6.39(m,3H),5.88(ddt, J=17.3,10.5,5.3Hz,1H),5.30(dd,J=17.3,1.6Hz,1H),5.20(dd,J=10.5,1. 5Hz,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);

[0625] LC-MS: [M+H] + =615.68.

[0626] (2) Production of CPA-329

[0627] The compound (CPA-328) (126 mg) synthesized as described above was dissolved in DCM (2 ml). TFA (474 μL) was added thereto, and the mixture was stirred at room temperature for 1 hour. The solvent was distilled off. To the residue were added the compound (CPA-316) (128 mg) shown in the above route, DMF (2 ml), COMU (288 mg) and DIPEA (235 μL), and the mixture was stirred at room temperature for 1 hour. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (132 mg).

[0628] 1 H NMR (500MHz, DMSO-d6) δ9.18(t,J=6.0Hz,1H),9.15(t,J=6.0Hz,1H),8.92(d,J=8.0Hz,1H),8.52(d,J=7. 3Hz,1H),8.12-8.09(m,6H),7.70-7.62(m,9H),5.87(ddt,J=17.3,10.5,5.3Hz,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);

[0629] LC-MS: [M+H] + =831.77.

[0630] (3) Production of CPA-330

[0631] The compound (CPA-329) (42 mg) produced as described above was dissolved in DCM (3 ml). Pd(PPh ) (5.8 mg) and N-methylaniline (27 μL) were added thereto, and the mixture was stirred at room temperature for 1 hour. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (31 mg).

[0632] 1 H 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);

[0633] LC-MS: [M+H] + =791.46.

[0634] (4) Production of CPA-331

[0635] The desired product was produced in a similar manner to that in Production Example 22.

[0636] 1 H NMR (400MHz, DMSO-d6) δ9.18-9.14(m,2H),8.99(d,J=8.0Hz,1H),8.17-8.07(m,7H),7.97(t,J=5.4Hz,1H),7.71-7.62(m,12H),4.56(dt,J=7 .2,7.2Hz,2H),4.26(dt,J=7.2,7.2Hz,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);

[0637] LC-MS: [M+H]+ =921.30.

[0638] (5) Production of CPA-332

[0639] The desired product was produced in the same manner as in Production Example 21, except that CPA-331 was used instead of CPA-318.

[0640] 1 H NMR (400MHz, DMSO-d6) δ13.34(s,1H),9.17-9.12(m,2H),8.98(d,J=8.4Hz.1H),8.17(d,J=8.1Hz,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,1 H),4.57(dt,J=7.5,7.5Hz,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);

[0641] LC-MS: [M+2H] 2+ =2039.42.

[0642] Production Example 25: Production of CPA-403

[0643]

[0644] (1) Production of CPA-401

[0645] To a mixture of 4-benzoylbenzoic acid (50 mg), N-Boc-2,2'-(ethylenedioxy)diethylamine (55 mg) and DMF (3 ml) were added WSC·HCl (42 mg) and HOBt·H2O (34 mg), and the mixture was stirred at room temperature for 15 hours. The solvent was distilled off, and the residue was purified by column chromatography (silica gel, hexane / AcOEt) to give the desired product (61 mg).

[0646] 1H NMR (400MHz, DMSO-d6) δ8.07-7.88(brm,2H),7.86-7.79(m,4H),7.62(tt,J=7.4,1.2Hz,1H),7.50(t,J=7. 6Hz,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);

[0647] LC-MS: [M+H] + =457.45.

[0648] (2) Production of CPA-402

[0649] To a mixture of the compound (CBI-712) produced as described above (61 mg) and DCM (2 ml) was added TFA (515 μL), and the mixture was stirred at room temperature for 14 hours. The solvent was distilled off to give the desired product (63 mg).

[0650] 1 H NMR(400MHz, DMSO-d6)δ8.73(t,J=5.6Hz,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);

[0651] LC-MS: [M+H] + =357.36.

[0652] (3) Production of CPA-403

[0653] The desired product was produced in the same manner as in Production Example 22, except that CPA-402 was used instead of CPA-316.

[0654] 1H NMR (400MHz, DMSO-d6) δ13.38-13.32(brm,1H),8.72(t,J=5.6Hz,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, 5.1Hz,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=7.2Hz,2H),2.57(d,J=12.6Hz,1H),2.37-2.24(m,10H),1.68-1.19(m,26H),0.91(s,6H);

[0655] LC-MS: [M+2H] 2+ =1474.68.

[0656] Production Example 26: Production of CPA-503

[0657]

[0658] (1) Production of CPA-502

[0659] The compound (CPA-501) (40 mg) shown in the above route was dissolved in DCM (2 ml). TFA (258 μL) was added thereto, and the mixture was stirred at room temperature for 1 hour. The solvent was distilled off to give the desired product (41 mg).

[0660] 1 H NMR (400MHz, DMSO-d6) δ7.93(t,J=5.4Hz,1H),7.87-7.66(brs,3H),3.60-3.52(m,6H),3.41(t,J=6. 0Hz,2H),3.20(dt,J=5.9,5.9Hz,2H),2.97(t,J=5.2Hz,2H),1.97(m,2H),1.56(m,2H),0.98(s,3H);

[0661] LC-MS: [M+H] + =259.28.

[0662] (2) Production of CPA-503

[0663] The desired product was produced in the same manner as in Production Example 22, except that CPA-502 was used instead of CPA-316.

[0664] 1 H NMR(400MHz,DMSO-d6)δ13.35(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=7.6,5.2Hz,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=7.3Hz,2H),2.59-2.56(m,1H),2.37-2.27(m,1 0H),2.10-2.02(m,4H),1.97(t,J=7.7Hz),1.65-1.23(m,28H),0.97(s,3H),0.94(s,6H);

[0665] LC-MS: [M+2H] 2+ =1376.31.

[0666] Production Example 27: Production of CPA-334

[0667]

[0668] To the mixture of the compound (CPA-333) shown in the above route and DCM (1ml) was added TFA (80 μL), and the mixture was stirred at room temperature for 1 hour. The solvent was distilled off. The compound (CPA-321) (15 mg), DMF (1ml) and DIPEA (5.5 μL) shown in the above route were added to the residue, and the mixture was stirred at room temperature for 8 hours. DIPEA (1.8 μL) was further added thereto, and the mixture was stirred at room temperature for 14 hours. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (12 mg).

[0669] 1H NMR (400MHz, DMSO-d6) δ13.34(t,J=4.5Hz,1H),9.96(d,J=0.7Hz,1H),9.08(t,J=5.7Hz,1H),8.13-8.10(m, 2H),8.04(t,J=7.5Hz,1H),7.96(d,J=8.0Hz,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);

[0670] LC-MS: [M+H] + =1528.03.

[0671] Production Example 28: Production of CPI-007

[0672]

[0673] Under ice-cooling, add allyl alcohol (96 μ L), DMAP (9.5 mg) and pyridine (170 μ L) to the mixture of adipic anhydride (100 mg) and DCM (5 ml), and stir the mixture under ice-cooling for 1 hour. The mixture is warming up to room temperature and stirred for 15 hours. By reaction mixture 2M HCl aqueous solution and saturated brine washing, then Na SO Upper drying. Distill off solvent, to provide required product (101 mg).

[0674] 1 H NMR (400MHz, CDCl3) δ5.97-5.87(m,1H),5.34-5.29(m,1H),5.26-5.22(m,1 H),4.58(ddt,J=5.7,1.3,1.3Hz,2H),2.42-2.34(m,4H),1.75-1.64(m,4H);

[0675] LC-MS: [M+H] + =187.20.

[0676] Production Example 29: Production of CPA-338

[0677]

[0678] (1) Production of CPA-335

[0679] To a mixture of compound (CPA-310) (139 mg) shown in the above route and DCM (5 ml) was added TEA (43 μL) and 2,2,2-trichloroethyl chloroformate (33 μL), and the mixture was stirred under ice cooling for 1 hour. The solvent was distilled off and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (172 mg).

[0680] 1 H NMR (400MHz, DMSO-d6) δ7.89(t,J=5.6Hz,1H),7.78-7.73(m,2H),6.75(t,J=5.4Hz,1H),6.42(s,1 H),6.35(s,1H),4.82(d,J=12.4Hz,1H),4.75(d,J=12.4Hz,1H),4.30(dd,J=7.6,5.2Hz,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=12.4Hz,1H),2.04(t,J=7.4Hz,2H),1.65-1.41(m,10H),1.37-1.21(m,15H);

[0681] LC-MS: [M+H] + =849.56.

[0682] (2) Production of CPA-336

[0683] To a mixture of compound (CPA-335) (172 mg) produced as described above and DCM (3 ml) was added TFA (468 μL), followed by stirring at room temperature for 1 hour. The solvent was distilled off. To the residue were added DMF (3 ml), CBI-741 (41 mg), WSC·HCl (47 mg), HOBt·H 2 O (37 mg) and DIPEA (141 μL), and the mixture was stirred at room temperature for 13 hours. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (134 mg).

[0684] 1H NMR (400MHz, DMSO-d6) δ7.89(t,J=5.6Hz,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. 2,1.5,1.5Hz,1H),5.20(ddt,J=10.5,1.5,1.5Hz,1H),4.82(d,J=12.4Hz,1H),4.75(d,J=12.4Hz,1H),4.53(dt,J=5.4,1.5Hz,2H ),4.30(dd,J=7.6,5.2Hz,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=12.4,5.1Hz,1H),2.57(d,J=12.4Hz,1H),2.35-2.31(m,2H),2.06-2.02(m,4H),1.64-1.21(m,20H);

[0685] LC-MS: [M+H] + =917.63.

[0686] (3) Production of CPA-337

[0687] To a mixture of compound (CPA-336) (134 mg) produced as described above and DCM (3 ml) were added Pd(PPh 3 ) 4 (17 mg) and 1,3-dimethylbarbituric acid (47 mg), followed by stirring at room temperature for 1 hour. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (96 mg).

[0688] 1H NMR (400MHz, DMSO-d6) δ7.91 (t, J = 5.5Hz, 1H), 7.80-7.74 (m, 3H), 6.42 (s, 1H), 6.36 (s, 1H), 4. 82(d,J=12.4Hz,1H),4.75(d,J=12.4Hz),4.30(dd,J=7.6,5.2Hz,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=12.4Hz,1H),2.18(t,J=6.8Hz,2H),2.06-2.01(m,4H),1.64-1.21(m,20H);

[0689] LC-MS: [M+H] + =877.54.

[0690] (4) Production of CPA-338

[0691] To a mixture of compound (CPA-337) (96 mg) produced as described above and DMF (2 ml) were added dimedone (31 mg), WSC·HCl (42 mg) and DMAP (27 mg), followed by stirring at 60° C. for 2 hours. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN, containing 0.1% AcOH) to give the desired product (69 mg).

[0692] 1 H NMR(400MHz,DMSO-d6)δ12.12-11.83(brs,1H),7.90(t,J=5.5Hz,1H),7.78-7.74(m,3H),6.42(s,1H), 6.35(s,1H),4.82(d,J=12.4Hz,1H),4.75(d,J=12.4Hz,1H),4.30(dd,J=7.6,5.2Hz,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=12.4,5.1Hz,1H),2.59-2.43(m,5H),2.04(t,J=7.3Hz,2H),1.64-1.21(m,20H),0.99(s,6H);

[0693] LC-MS: [M+H] + =999.69.

[0694] Production Example 30: Production of CPI-009

[0695]

[0696] To the mixture of compound (CPI-008) (80 mg) shown in the above route, DMF (3 ml) and DIPEA (166 μ L) was added TSTU (105 mg), then stirred at room temperature for 5 minutes. 6-aminocaproic acid (41 mg) was added thereto, and the mixture was stirred at room temperature for 1 hour. The mixture was warmed to 50° C. and stirred for 5 hours. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (102 mg).

[0697] 1 H NMR (400MHz, CD3OD) δ8.53(d,J=2.1Hz,1H),8.37(t,J=5.4Hz,1H),7.99(dd,J=8.8,2.3Hz,1H),6.7 0(d,J=8.8Hz,1H),3.39-3.32(m,2H),2.31(t,J=7.3Hz,2H),1.70-1.58(m,4H),1.53-1.33(m,11H);

[0698] LC-MS: [M+H] + =367.30.

[0699] Production Example 31: Production of CPA-342

[0700]

[0701] (1) Production of CPA-339

[0702] To a mixture of the compound (CPA-338) (29 mg) shown in the above scheme, the compound (CPA-318) (13 mg) produced in Production Example 19, and DMF (1 ml), DIPEA (25 μL) was added, followed by stirring at 60° C. for 1 hour. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (31 mg).

[0703] 1H NMR (400MHz, DMSO-d6) δ13.36-13.34(m,1H),9.06(t,J=5.8Hz,1H),8.14-8.11(m,2H),7.89(t,J=5.5Hz,1H),7.78-7.6 3(m,6H),6.42(s,1H),6.35(s,1H),4.82(d,J=12.3Hz,1H),4.75(d,J=12.3Hz,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=12.4,5.1Hz,1H),2.57(d,J=12.4Hz,1H),2.23(s,4H),2.09-2.02(m,4H),1.63-1.21(m,20H),0.91(s,6H);

[0704] LC-MS: [M+H] + =1301.83.

[0705] (2) Production of CPA-340

[0706] To a mixture of compound (CPA-339) (31 mg) produced as described above and 1,4-dioxane (1 ml) were added zinc (16 mg) and acetic acid (68 μL), followed by stirring at 60° C. for 2 hours. The reaction mixture was purified by column chromatography (ODS, water / MeCN containing 0.1% AcOH) to give the desired product (21 mg).

[0707] 1H NMR (400MHz, DMSO-d6) δ13.34(t,J=4.2Hz,1H),9.09(t,J=5.7Hz,1H),8.14-8.11(m,2H),7.8 4(t,J=5.8Hz,1H),7.77-7.63(m,5H),6.42(s,1H),6.36(s,1H),4.32-4.29(m,1H),4.14-4.1 1(m,1H),3.63-3.04(m,32H),3.01-2.96(m,2H),2.94-2.90(m,2H),2.82(dd,J=12.5,5.1Hz, 1H),2.57(d,J=12.5Hz,1H),2.24(s,4H),2.09-2.02(m,4H),1.65-1.21(m,20H),0.91(s,6H);

[0708] LC-MS: [M+H] + =1127.92.

[0709] (3) Production of CPA-341

[0710] To a mixture of the compound (CPA-340) (10 mg) produced as described above, the compound (CPI-009) (4.7 mg) produced in Production Example 28 (1), and DMF (1 ml) were added WSC·HCl (3.4 mg), HOBt·H2O (2.7 mg), and DIPEA (6.2 μL), followed by stirring at room temperature for 12 hours. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (5.1 mg).

[0711] 1H NMR (400MHz, DMSO-d6) δ13.34(t,J=4.7Hz,1H),9.09(t,J=5.7Hz,1H),8.91(s,1H),8.63(s,1H),8.52(d,J= 1.6Hz,1H),8.24(t,J=5.5Hz,1H),8.13-8.10(m,2H),7.93(dd,J=8.9,1.7Hz,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 =12.5,5.1Hz,1H),2.57(d,J=12.5Hz,1H),2.23(s,4H),2.13-2.02(m,6H),1.67-1.24(m,35H),0.90(s,6H);

[0712] LC-MS: [M+H] + =1476.01.

[0713] (4) Production of CPA-342

[0714] To a mixture of compound (CPA-341) (4.7 mg) produced as described above and MeOH (50 μL) was added a 4M HCl solution in 1,4-dioxane, followed by stirring at room temperature for 1 hour. MeOH (100 μL), acetone (100 μL), and DIPEA (70 μL) were added, followed by stirring at room temperature for 1 hour. The reaction mixture was purified by column chromatography (ODS, water / MeCN) to give the desired product (2.9 mg).

[0715] 1H NMR (400MHz, DMSO-d6) δ13.34(t,J=4.6Hz,1H),9.62(s,1H),9.08(t,J=5.7Hz,1H),8.57(d,J=1.8Hz,1H),8.26(t,J=5.6H z,1H),8.14-8.11(m,2H),7.99(dd,J=8.8,2.3Hz,1H),7.86-7.84(m,2H),7.7607.63(m,5H),7.04(d,J=8.8Hz,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=12.6,5.1H z,1H),2.57(d,J=12.6Hz,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);

[0716] LC-MS: [M+H] + =1415.76.

[0717] Production Example 32: Production of CPA-344

[0718]

[0719] The desired product was produced in the same manner as in Production Example 31, except that CPA-343 was used instead of CPA-321.

[0720] 1H NMR (400MHz, DMSO-d6) δ13.35(s,1H),9.62(s,1H),9.14(t,J=5.6Hz,1H),8.57(d,J=1.9Hz,1H),8.26(t,J=5.5Hz,1H),8.14 -8.12(m,2H),7.99(dd,J=8.8,2.4Hz,1H),7.86-7.84(m,2H),7.81-7.63(m,6H),7.04(d,J=8.8Hz,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);

[0721] LC-MS: [M+H] + =1735.36.

[0722] Production Example 33: Production of CPI-016

[0723]

[0724] (1) Production of CPI-012

[0725] To the mixture of compound (CPI-011) (97 mg) and DCM (2 ml) shown in the above route, TFA (683 μ L) is added, and the mixture is stirred at room temperature for 1 hour. The solvent is then distilled off. DMF (2 ml), N-[(9H-fluorenes-9-ylmethoxy) carbonyl]-L-glutamic acid 5-tert-butyl ester hydrate (75 mg), HATU (81 mg) and DIPEA (155 μ L) are added to the residue, and the mixture is stirred at room temperature for 1 hour. N-[(9H-fluorenes-9-ylmethoxy) carbonyl]-L-glutamic acid 5-tert-butyl ester hydrate (75 mg) and HATU (68 mg) are further added thereto, and the mixture is stirred at room temperature for 1 hour. The solvent is distilled off, and the residue is purified by column chromatography (ODS, water / MeCN), to give the desired product (136 mg).

[0726] 1H NMR (400MHz, CDCl3) δ7.76 (d, J=7.4Hz, 2H), 7.66-7.60 (m, 2H), 7.40 (dd, J=7.4, 7.4Hz, 2H), 7.32 (ddd, J=7.4, 7.4, 1.0Hz,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,1 H),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=12.8,4.9Hz,1H),2.68 (d,J=12.8Hz,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);

[0727] LC-MS: [M+H] + =854.39.

[0728] (2) Production of CPI-013

[0729] To a mixture of compound (CPI-012) (136 mg) produced as described above and DMF (2 ml) was added piperidine (24 μL), followed by stirring at room temperature for 5 hours. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (87 mg).

[0730] 1 H NMR (400MHz, DMSO-d6) δ7.83(t,J=5.7Hz,1H),7.75(t,J=5.6Hz,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=12.5,5.1Hz,1H),2.57(d,J=12.5Hz,1H),2.24-2.20(m,2H),2.0 4(t,J=7.4Hz,2H),1.80-1.71(m,1H),1.66-1.43(m,9H),1.39(s,9H),1.35-1.23(m,2H);

[0731] LC-MS: [M+H]+ =632.38.

[0732] (3) Production of CPI-015

[0733] To a mixture of the compound (CPI-013) (87 mg) produced as described above, the compound (CPI-014) (22 mg) shown in the above route, and DMF (2 ml) were added WSC·HCl (32 mg) and HOBt·H 2 O (25 mg), followed by stirring at room temperature for 5 hours. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (92 mg).

[0734] 1 H NMR (400MHz, DMSO-d6) δ7.80-7.71(brm,1H),7.22(t,J=5.6Hz,1H),6.85(t,J=5.3Hz,1H),6.61(s,1H),5.95-5.85(m, 1H),5.31(dd,J=15.8,1.4Hz,1H),5.22(dd,J=9.2,1.4Hz,1H),5.01-4.79(brs,1H),4.57(d,J=5.7Hz,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=12.8,4.8Hz,1H),2. 72(d,J=12.8Hz,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);

[0735] LC-MS: [M+H] + =772.45.

[0736] (4) Production of CPI-016

[0737] To a mixture of the compound (CPI-015) (92 mg) produced as described above and DCM (1 ml) was added TFA (200 μL), followed by stirring at room temperature for 20 hours. The solvent was distilled off, and DMF (2 ml), dimedone (33 mg), WSC·HCl (46 mg), and DMAP (29 mg) were added to the residue, followed by stirring at 60° C. for 4 hours. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN, containing 0.1% AcOH) to give the desired product (45 mg).

[0738] 1 H NMR (400MHz, DMSO-d6) δ8.06(d,J=8.0Hz,1H),7.84(t,J=5.4Hz,1H),7.74(t,J=5.6Hz,1H),6.42(s,1H),6.35(s,1H),5.94-5.84(m,1H),5 .29(ddt,J=17.2,1.5,1.5Hz,1H),5.19(ddt,J=10.5,1.5,1.5Hz,2H),4.52(dt,J=5.3,1.5Hz,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=12.4,5.1Hz,1H),2.59-2.41( m,9H),2.04(t,J=7.4Hz,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);

[0739] LC-MS: [M+H] + =838.42.

[0740] Production Example 34: Production of CPA-346

[0741]

[0742] (1) Production of CPA-345

[0743] To a mixture of the compound (CPA-316) (8.8 mg) shown in the above scheme and DMF (1 ml) was added N-Boc-1,2-diaminoethane (7.3 μL), COMU (22 mg) and DIPEA (24 μL), followed by stirring at room temperature for 5 hours. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (8.3 mg).

[0744] 1H NMR(400MHz, CDCl3)δ8.22-8.19(m,2H),7.81-7.70(brs,1H),7.61-7.52(m,3H),4.99 -4.87(brs,1H),3.68(dt,J=5.7,5.7Hz,2H),3.45(dt,J=5.7,5.7Hz,2H),1.44(s,9H);

[0745] LC-MS: [M+H] + =333.16.

[0746] (2) Production of CPA-346

[0747] The desired product was produced in a similar manner to Production Example 20 using CPA-345 and CPI-016.

[0748] 1 H NMR (400MHz, DMSO-d6) δ13.31(t,J=5.9Hz,1H),9.43(t,J=5.6Hz,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=10.8,3.5Hz,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=7.4Hz,2H),1.65-1.41(m,8H),1.35-1.24(m,2H),0.91(s,6H);

[0749] LC-MS: [M+H] + =1109.51.

[0750] Production Example 35: Production of CPA-347

[0751]

[0752] (1) Production of CPI-018HCL

[0753] To the mixture of the compound (CPI-017, isomer ratio is 4: 3) (63mg) and THF (2ml) shown in the above route, triphenylphosphine (78mg) and H o (0.40ml) were added, and then stirred at room temperature overnight. 1M HCl aqueous solution (0.10ml) was added thereto, and then stirred at room temperature for 1 hour. 1M HCl aqueous solution (0.30ml) was further added thereto, and then stirred at room temperature for 1 hour. The mixture was warmed to 60°C and stirred for 6 hours, and then stirred at room temperature for 4 days. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN), to give the desired product (39mg, isomer ratio is 10: 9).

[0754] 1 H NMR (400MHz, CD3OD) δ8.20(dd,J=8.4,0.8Hz,1H),7.99(d,J=8.8Hz,0.9H),7.69(d,J=2.6Hz,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=7.6Hz,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);

[0755] LC-MS: [M+H] + =292.23.

[0756] (2) Production of CPI-019

[0757] To a mixture of compound (CPI-018HCL) (39 mg) produced as described above, DMF (2 ml) and DIPEA (93 μL) was added succinic anhydride (13 mg), followed by stirring at room temperature overnight. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (32 mg, 2:1 isomer ratio).

[0758] 1H NMR (400MHz, CD3OD) δ8.19 (dd, J=7.6, 2.0Hz, 1H), 7.98 (d, J=8.8Hz, 0.5H), 7.69 (d, J= 2.6Hz,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=6.9Hz,2+0.5×2H),2.60-2.55(m,2+0.5×2H),2.45(t,J=6.8Hz,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);

[0759] LC-MS: [M+H] + =392.33.

[0760] (3) Production of CPA-347

[0761] The desired product was produced in a similar manner to Production Example 18 and Production Example 20 (isomer ratio was 2:1).

[0762] 1 H NMR (400MHz, DMSO-d6) δ13.34(t,J=4.9Hz,1H),9.10(t,J=5.5Hz,1H),8.13-9.10(m,2.3H),8.00(d, J=7.9Hz,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=12.5,5.2Hz,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);

[0763] LC-MS: [M+H] + =1500.96.

[0764] Production Example 36: Production of CPA-353

[0765]

[0766] (1) Production of CPA-348

[0767] A mixture of the compound (CPA-338) shown in the above scheme (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 distilled off and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (10 mg).

[0768] 1 H NMR (400MHz, DMSO-d6) δ13.35(s,1H),7.89(t,J=5.6Hz,1H),7.78-7.74(m,3H),6.77(t,J=5 .3Hz,1H),6.42(s,1H),6.35(s,1H),4.82(d,J=12.4Hz,1H),4.75(d,J=12.4Hz,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);

[0769] LC-MS: [M+H] + =1229.65.

[0770] (2) Production of CPA-349

[0771] To a mixture of the compound (CPA-348) (10 mg) produced as described above and 1,4-dioxane (1 ml) were added zinc (11 mg) and acetic acid (47 μL), followed by stirring at 60° C. for 30 minutes. Zinc (11 mg) was further added thereto, followed by stirring at 60° C. for 30 minutes. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN, containing 0.1% AcOH) to give the desired product (8.6 mg).

[0772] 1H NMR(400MHz,DMSO-d6)δ13.35(s,1H),7.98-7.92(brs,1H),7.75(t,J=5.5Hz,2H ),6.77(t,J=5.4Hz,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=12.4,5.1Hz,1H),2.5 9-2.49(m,1H),2.27(s,4H),2.10-2.02(m,4H),1.64-1.23(m,29H),0.94(s,6H);

[0773] LC-MS: [M+H] + =1055.66.

[0774] (3) Production of CPA-350

[0775] To a mixture of compound (CPA-349) (8.6 mg) produced as described above, CPA-311 (2.2 mg) and DMF (1 ml) were added WSC·HCl (3.1 mg) and HOBt·H 2 O (2.5 mg), followed by stirring at room temperature for 1 hour. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (2.3 mg).

[0776] 1 H NMR (400MHz, DMSO-d6) δ13.34(s,1H),7.96(d,J=7.9Hz,1H),7.87-7.82(m,2H),7.75-7.73(m,2H),6.80-6.74(b rs,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);

[0777] LC-MS: [M+H]+ =1309.01.

[0778] (4) Production of CPA-351

[0779] To a mixture of the compound (CPA-350) (2.2 mg) produced as described above and DCM (0.50 ml) was added TFA (130 μL), followed by stirring at room temperature for 1 hour. The solvent was distilled off, and DMF (0.50 ml), 2-thiophene glyoxylic acid (1.3 mg), COMU (3.6 mg) and DIPEA (15 μL) were added to the residue, followed by stirring at room temperature for 1 hour. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (1.4 mg).

[0780] 1 H NMR (400MHz, DMSO-d6) δ13.36-13.33(m,1H),8.88(t,J=5.5Hz,1H),8.19-8.16(m,2H),7.97(d,J=8.3Hz,1H),7.87-7.8 3(m,2H),7.77-7.73(m,2H),7.29(dd,J=4.8,4.0Hz,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=5.4,1.2Hz,2H),4.32-4.28(m,1H),4.14-4.05(m,2H),3.61-2.91(m,overlapping with water signal),2.82(dd,J=12.4,5.1Hz,1H),2.61-2.49(m,overlapping 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);

[0781] LC-MS: [M+H] + =1346.89.

[0782] (5) Production of CPA-352

[0783] To a mixture of compound (CPA-351) (1.4 mg) produced as described above and DCM (0.50 ml) was added Pd(PPh 3 ) 4 (0.1 mg) and 1,3-dimethylbarbituric acid (0.3 mg), followed by stirring at room temperature for 1 hour. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (0.7 mg).

[0784] 1 H NMR (400MHz, DMSO-d6) δ13.35-13.33(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=4.8,3.9Hz,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);

[0785] LC-MS: [M+H] + =1306.84.

[0786] (6) Production of CPA-353

[0787] To a mixture of the compound (CPA-352) (0.7 mg) produced as described above and DMF (0.50 ml) were added N-hydroxysuccinimide (0.6 mg) and WSC·HCl (1.0 mg), followed by stirring at room temperature for 16 hours. N-hydroxysuccinimide (0.6 mg) and WSC·HCl (1.0 mg) were further added thereto, followed by stirring at room temperature for 1 hour. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (0.7 mg).

[0788] 1 H NMR(400MHz,DMSO-d6)δ13.37-13.33(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=4.9,3.9Hz,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,overlapping with water signal),2.61-2.25(m,overlapping with DMSO signal),2.09-2.02(m,4H),1.63-1.15(m,26H),0.92(s,6H);

[0789] LC-MS: [M+2H] 2+ =1404.71.

[0790] Production Example 37: Synthesis of CPI-018

[0791]

[0792] Synthesis of CPI-018

[0793] To a mixture of the compound (CPI-017) (20 mg) shown in the above route, DMF (0.5 ml) and DIPEA (14 μL) was added succinic anhydride (2.4 mg), followed by stirring at room temperature overnight. AcOEt was added to the reaction solution, and the solution was washed with water and saturated brine and dried over Na2SO4. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (14 mg).

[0794] 1 H NMR(500MHz,CD3OD)δ4.30-4.23(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);

[0795] LC-MS: [M+H] + =1073.20.

[0796] Production Example 38: Production of CPA-354

[0797]

[0798] Synthesis of CPA-353

[0799] To a mixture of the compound (CPI-018) (5.3 mg) produced as described above, the compound (CPA-340) (5.6 mg) produced in Production Example 30 (2) and DMF (0.5 ml) were added HATU (2.3 mg) and DIPEA (2.6 μL), followed by stirring at room temperature for 1 hour. The solvent was distilled off, and the residue was purified by column chromatography (ODS, water / MeCN) to give the desired product (5.8 mg).

[0800] 1H NMR (400MHz, CD3OD) δ8.20-8.15(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=12.7Hz,1H),2.64-2.48(m,4H),2.33-2.05(m,11H),1.93-1.27(m,87H),0.97(s,6H).

[0801] Synthesis of CPA-354

[0802] To a mixture of the compound (CPA-353) (5.8 mg) produced as described above and DCM (1 ml) was added TFA (0.5 ml), followed by stirring at room temperature for 9 hours. The solvent was distilled off to give the desired product (4.5 mg).

[0803] 1 H NMR (400MHz, CD3OD) δ8.19-8.15(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);

[0804] LC-MS: [M+H] 2+ =923.48.

[0805] Production Example 39: Production of labeled antibody (CPA-321 labeled antibody)

[0806] The anti-CD71 antibody OKT9 (#16-0719-85, Thermo Fisher Scientific) or a mouse IgG1 isotype control antibody (#553447, BD Biosciences) as a control antibody was placed in a 20-μL tube and diluted with NHS labeling buffer (25mM HEPES-NaOH pH 8.2, 150mM NaCl) to a final concentration of about 0.1 mg / ml. About 1.5 μL of a solution of the compound (CPA-321) produced in Production Example 20 in DMSO (10 mg / ml in DMSO) was added thereto, and the mixture was reacted at room temperature for 2 hours. About 5 μL of lysine solution (100mM) was then added thereto, and the mixture was reacted at room temperature for 20 minutes. The resulting mixture was desalted by applying it to a desalting column (Zeba Spin, Thermo Scientific, 89882), and a solution containing the CPA-321-labeled antibody (CPA-321-labeled antibody solution) was collected.

[0807] Test example

[0808] The effects of the compounds of the present invention are described below by showing the test results using the tetrafunctional compounds of the present invention and representative compounds among the tetrafunctional chemical probes. However, the present invention is not limited to these test examples.

[0809] Experimental Example 1: Isolation and detection of target molecules from living cells using compound (CPP-127)

[0810] 1. Cell culture and methods of treating cultured cells with this compound

[0811] CHO-K1 cells stably expressing human dopamine receptor D2 (DRD2) were cultured on 10-cm culture plates until they became confluent.

[0812] The cells were washed with culture medium, and the culture medium was replaced with a culture medium containing DMSO or an unmodified ligand, and then reacted for 30 minutes. Subsequently, the culture medium was discarded and replaced with a culture medium containing the compound (CPP-127) synthesized in Production Example 12, and the cells were allowed to stand at 37°C for 30 minutes. The culture medium was discarded, and the cells were washed with PBS. PBS (1 ml) was added to the cells on the culture plate, and the cells were then irradiated with 302 nm UV light using a UV crosslinker for 1 minute. Cells were collected using a cell scraper after UV irradiation, and the collected cell mass was cryopreserved at -80°C.

[0813] 2. Preparation and Purification of Membrane Fraction Proteins

[0814] The cryopreserved cells were suspended in 50 mM Tris homogenization buffer (pH 7.4, 2.5 mM EDTA, 5 mM MgCl 2 , 320 mM sucrose) and homogenized using an ultrasonic instrument. The homogenized suspension was centrifuged using a centrifuge (1,000 g, 15 minutes, 4° C.). The supernatant obtained after centrifugation was transferred to another tube and centrifuged (30,000 × g, 30 minutes, 4° C.). After centrifugation, the supernatant was discarded, and 100 mM sodium carbonate buffer (Na 2 CO 3 ) was added to the precipitate, which was then homogenized using an ultrasonic instrument and incubated on ice for 10 minutes. The suspension was centrifuged by ultracentrifugation (45, 2000 × g, 30 minutes, 4° C.), the supernatant was discarded, and the resulting precipitate was rinsed with Milli-Q water and used as a membrane fraction protein.

[0815] 3. Membrane Protein Elution

[0816] Membrane lysis buffer (50mM Tris-HCl (pH 7.5), 150mM NaCl, 0.1% SDS, 0.5% SDC, 1% Triton-X-100, 8M urea) was added to the membrane fraction protein prepared by the above method, and the membrane fraction protein was dissolved using a sonicator. Streptavidin beads (#88816, Thermo Fisher Scientific) were added to the dissolved membrane protein solution, and the biotin-labeled protein was adsorbed onto the beads by stirring and mixing for 60 minutes. After the beads were washed with membrane lysis buffer, 40 μL of 2% N2H4 solution containing 0.05% SDS was added, and the beads were allowed to stand at room temperature for 30 minutes. The beads were removed by magnetic separation and the solution was collected. The collected solution was neutralized by adding 20% TFA solution and then concentrated using a centrifugal evaporator. Dithiothreitol and iodoacetamide were added to the concentrated solution for reduction and alkylation, and trypsin solution was added, and then digested at 37°C overnight.

[0817] 4. LC-MS Analysis and Identification of Target Proteins

[0818] The obtained tryptic digest was desalted and purified, and protein analysis was performed by LC-MS. LC-MS was performed using a combined LC-MS system utilizing a nano-HPLC device (Ultimate 3000, Thermo Scientific) and a mass spectrometer (Q Exactive, Thermo Scientific). In the nano-HPLC device, a column (stationary phase) consisting of a capture column (C18Acclaim PepMap, Thermo Scientific) and a chip column (75 μm i.d., 12 cm long, 3 μm C18 particles, NikkyoTechnos, Co., Ltd.) was used, and a 0.1% formic acid-acetonitrile system was used as the mobile phase for gradient analysis by reversed-phase chromatography. The flow rate was 300 nl / min, and the peptides were eluted with a linear gradient of acetonitrile concentration from 4% to 35% over a period of 48 minutes. Mass spectrometry was performed in positive ion mode by nano-electrospray ionization. Full scan MS spectra are obtained in the m / z range of 380 to 1600, and product ion spectra are measured in data dependent acquisition mode. The data obtained by LC-MS are analyzed by Proteome Discoverer 2.2 analysis software (PD 2.2, Thermo Fisher Scientific). For protein identification, the search engine Sequest provided in PD 2.2 is used to search in the database downloaded from UniProt. False discovery rate is used to calculate. After filtering to make the false positive protein identification rate less than 1%, the protein with a PSM value greater than or equal to 5 is considered as the protein identified, so as to select more reliable candidate proteins. In addition, the number of unique peptides identified is used to narrow the data range. In quantitative analysis, protein is quantitatively analyzed by the quantitative method LFQ provided in PD 2.2, and quantitative comparative analysis is carried out with the unmodified ligand treatment group to distinguish the proteins collected specifically. Analysis is carried out in two independent experiments, and the result can be expressed as the ratio relative to the unmodified ligand treatment group.

[0819] 5. Results

[0820] Figure 1 The results are shown. Figure 1 As shown in the results, when the tetrafunctional chemical probe of the present invention (compound (CPP-127)) was used, the detection of dopamine receptor D2 was significantly inhibited in the unmodified ligand-treated group, confirming that dopamine receptor D2 (DRD2) was specifically collected. Experimental Example 2: Isolation and Detection of Target Molecules from Living Tissue Using Compounds (CPF-224, CPF-202, and CPF-242)

[0821] By isoflurane inhalation, DBA / 2CrSlc mice are anesthetized, then decapitated and brain is taken out rapidly.The brain taken out is soaked 60 seconds at 0 ℃ in artificial cerebrospinal fluid (ACSF), while constantly blasting gas mixture (95% O , 5% CO ). Remove olfactory bulb and cerebellum with a scalpel, and use viscose glue to fix brain to the sample carrier of a vibrating blade microtome (Leica, VT1200). At 0 ℃, constantly blast into the ACSF of described mixed gas and prepare the brain slice (thickness is the thin slice of 0.3mm) containing striatum and hippocampus. Section is placed in the culture dish containing artificial cerebrospinal fluid. Add unmodified part or DMSO, and culture dish was placed 5 minutes in room temperature. Subsequently add the compound (CPF-224) produced in Production Example 10 or the compound (CPF-202) produced in Production Example 8, and culture dish was placed 30 minutes in room temperature. The solution was removed and the sections were irradiated with UV light (in the case of CPF-224 and CPF-202, irradiated with 302nm UV light for 1 minute, and in the case of CPF242, irradiated with 365nm UV light for 15 minutes). The sections were washed with PBS and collected, and the supernatant was removed by centrifugation (3000 × g, 4°C, 5 minutes). The resulting sediment was analyzed by the same method as in Experimental Example 1. The analysis was performed in two independent experiments, and the results can be expressed as a ratio relative to the unmodified ligand-treated group.

[0822] Figure 2 Results obtained using compound (CPF-224) are shown. Figure 3 Results obtained using compound (CPF-202) are shown. Figure 4 The results obtained using the compound (CPF-242) are shown. As shown in these figures, when the tetrafunctional chemical probes of the present invention, CPF-224, CPF-202, and CPF242, were used, the detection of AMPA-type glutamate receptors (Gria1, Gria2, Gria3, Gria4) and GABA receptor subunits (Gabra1, Gabra2, Gabra5, Gabrg2) in brain tissue was significantly inhibited in the unmodified ligand-treated group, confirming that the membrane proteins were specifically collected.

[0823] Experimental Example 3: Capturing cell surface antigens using labeled antibodies

[0824] Antigen molecules were obtained from THP-1 cells using an anti-CD71 antibody labeled with CPA-321 produced in Production Example 21 or an anti-mouse IgG1 control antibody labeled with CPA-321 produced in Production Example 21. Specifically, THP-1 cells were collected and washed with SB buffer (PBS buffer containing 2% FCS). The washed cells were suspended in SB buffer containing 0.1% Fc blocking reagent (Invitrogen, 16-9161-73) and reacted at 4°C for 15 minutes. The supernatant was removed by centrifugation (600 × g, 3 minutes, 4°C), and an antibody solution was added (obtained by adding SB buffer to a solution (20 μg) of the antibody labeled with CPA-321 produced in Production Example 21 so that the total volume was 1 ml), and then reacted at 4°C for 20 minutes. The antibody solution was removed by centrifugation, the cells were washed with SB buffer, and then suspended in 0.5 ml SB buffer. The cell suspension was irradiated with a 302nm UV tube for 1 minute, and the SB buffer was removed by centrifugation. The obtained cells were analyzed by the method described in Experimental Example 1. The analysis was performed in two independent experiments, and the results were expressed as the ratio of the amount of protein obtained using the anti-CD71 antibody to the amount of protein obtained in the case of a mouse IgG1 isotype control antibody (anti-mouse IgG antibody) ( Figure 5 ).

[0825] like Figure 5 As shown in , two analytical tests confirmed the significant acquisition of TFRC (CD71). Experimental Examples 4 to 13: Capturing Cell Surface Antigens Using Labeled Antibodies (1)

[0826] The investigation of the antigen molecules from the cells shown in Table 2 was carried out using the labeled antibodies shown in Table 2. Specifically, the cells shown in Table 2 were washed with SB buffer (PBS buffer containing 2% FCS), suspended in SB buffer containing 0.1% Fc blocking reagent (Invitrogen, 16-9161-73), and reacted at 4°C for 15 minutes. Each supernatant was removed individually by centrifugation (600×g, 3 minutes, 4°C), and each solution containing the labeled antibody produced according to the labeled antibody production method described below was added individually, and then reacted at 4°C for 20 minutes. Each antibody solution was removed individually by centrifugation, and the cells were washed with SB buffer and then suspended in 0.5ml SB buffer. Each cell suspension was irradiated individually with ultraviolet light of the wavelength shown in the table for the time period shown in the table, and the SB buffer was removed by centrifugation. The collected cells were analyzed by the method shown in Experimental Example 1. The analysis was carried out in two independent experiments, and the detection amounts of the membrane proteins to be analyzed shown in the table were compared ( Figures 6 to 8 ).

[0827] Production of labeled antibodies (Test Examples 4 to 12)

[0828] The antibodies shown in Table 2 are placed separately in independent tubes and diluted to a final concentration of about 0.05 mg / ml with NHS labeling buffer (25 mM HEPES-NaOH pH 8.2, 150 mM NaCl). A solution of the probe shown in each of Table 2 of about 0.9 μL in DMSO (10 mg / ml in DMSO) is added thereto separately, followed by reaction at room temperature for 2 hours. Subsequently, about 5 μL lysine solution (100 mM) is added thereto, followed by reaction at room temperature for 20 minutes. The resulting solution is stored at 4°C until use.

[0829] Production of labeled ligand (Test Example 13)

[0830] 12.5 μg of His-tagged EGF was placed in a tube and diluted with NHS labeling buffer to a final concentration of approximately 0.25 mg / ml. To 48 μL of this solution, 2 μL of 5 mM CPA-354 was added to prepare a labeled ligand solution. This solution was stored at 4°C until use.

[0831] Table 2

[0832]

[0833] Experimental Example 14: Capturing cell surface antigens using labeled antibodies (2)

[0834] The investigation of antigen molecules from A431 cells was carried out using labeled OKT9 produced by the method shown in Table 3. First, the cells were washed with SB buffer (PBS buffer containing 2% FCS). SB buffer containing 0.1% Fc blocking reagent (Invitrogen, 16-9161-73) was then added, and the cells were reacted at 4°C for 15 minutes. The supernatant was removed, and 250 μL of ligand solution diluted 10 times with SB buffer was added, and then reacted at 4°C for 20 minutes. The ligand solution was removed, the cells were washed with PBS, and then irradiated with 302nm UV light for 1.5 minutes. Proteins were extracted from the cells using a lysis buffer containing 1% NP-40 and analyzed by the method shown in Experimental Example 1. The amount of CD71 detected when using each ligand formed in the probe was compared with the amount detected when using the OKT9 antibody not formed in the probe as a negative control (no probe) ( Figure 9 ).

[0835] Production of labeled antibodies

[0836] (1) 10 μg of anti-CD71 antibody OKT9 (Thermo Fisher Scientific, catalog number 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 about 0.1 mg / ml. 0.9 μL of CPA-321 solution (10 mg / ml in DMSO) was added thereto, followed by reaction at room temperature for 2 hours. Subsequently, about 5 μL of lysine solution (100 mM) was added thereto, followed by reaction at room temperature for 20 minutes. The resulting solution was stored at 4°C until use.

[0837] (2) 10 μg of 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. Phosphate buffer (pH 6.5) was then added to bring the total volume to 100 μL. After reacting at room temperature for 15 minutes, the solvent was replaced with PBS using a Zeba Spin column (Thermo Scientific). 1 μL of a 500 mM 5-MA aqueous solution was added to 100 μL of the resulting antibody solution. 1 μL of a 10 mg / ml CPA-342 or CPA-344 solution in DMSO was subsequently added and the mixture was reacted at room temperature for 1.5 hours in the dark. After the reaction, the solvent was replaced with PBS using a Zeba Spin column and excess reagents were removed.

[0838] Table 3

[0839] Test example probe Methods for producing labeled antibodies Test Example 14 CPA-321 (1) Test Example 15 CPA-342 (2) Test Example 16 CPA-344 (2)

[0840] Experimental Example 17: Capturing cell surface antigens using labeled antibodies (secondary antibody method)

[0841] The investigation of antigen molecules from THP-1 cells is carried out using the labeled second antibody produced by the production method of the second antibody for CBP-198 labeling. Specifically, THP-1 cells are washed with SB buffer (PBS buffer containing 2% FCS) and suspended in SB buffer containing 0.1% Fc blocking reagent (Invitrogen, 16-9161-73), and the cells are reacted at 4 ° C for 15 minutes. The supernatant is removed by centrifugation (600 × g, 3 minutes, 4 ° C). An anti-CD71 antibody DF1513 solution diluted to 5 μg / mL with SB buffer is added to the cells, and the cells are reacted at 4 ° C for 20 minutes. The antibody solution is removed by centrifugation, and the cells are washed with SB buffer. Subsequently, the cells are suspended in a solution (obtained by adding SB buffer to a total volume of 1 ml to the labeled second antibody produced by the production method for the second antibody for CPA-503 labeling) and reacted at 4 ° C for 20 minutes. The antibody solution is removed by centrifugation, and the cells are washed with SB buffer and then suspended in 0.5 ml 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 Experimental Example 1. The analysis was performed in two independent experiments, and the amount of CD71 detected was compared.

[0842] 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 DF1513 was used instead of the anti-CD71 antibody OKT9 ( Figure 10 ).

[0843] Production of CPA-503 labeled secondary antibodies

[0844] Goat anti-mouse IgG antibody (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. About 0.9 μL of a DMSO solution of CPA-503 (10 mg / ml in DMSO) was added, followed by reaction at room temperature for 2 hours. Subsequently, about 5 μL of a lysine solution (100 mM) was added, followed by reaction at room temperature for 20 minutes. The resulting solution was stored at 4°C until use.

[0845] By using the secondary antibody method, membrane proteins that could not be detected by the primary antibody method can be detected.

[0846] Sequence Listing Free Text

[0847] SEQ ID NO: 1 and 2 are each an amino acid sequence of a peptide, which is an embodiment of a compound that forms the cleavable moiety (E) of the tetrafunctional compound or tetrafunctional chemical probe of the present invention. Sequence Listing <110> Otsuka Pharmaceutical Co., Ltd. <120> Tetrafunctional Chemical Probe and Method for Identifying Target Membrane Proteins Derived from Living Cells or Tissues Using the Probe <130> P20 - 079WO <150> JP 2019 - 107511 <151> 2019 - 06 - 07 <160> 2 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <230> Peptide Designed for the Cleavable Moiety of a Tetrafunctional Compound or Tetrafunctional Chemical Probe <400> 1 Glu Asn Leu Tyr Phe Gln Gly 5 <210> 2<( <211> 7 <212> PRT <213> Artificial Sequence <220> <230> Peptide Designed for the Cleavable Moiety of a Tetrafunctional Compound or Tetrafunctional Chemical Probe <400> 2 Glu Asn Leu Tyr Phe Gln Ser 5 It should be noted that there seems to be a small formatting issue in the original text where "(0002172)" is written with a parenthesis which might be a mistake. I've translated it as is but it might need to be corrected in the original source for better clarity.

Claims

1. A tetrafunctional compound represented by the following formula (i), (ii), (iii), CPA-325, CPA-326, CPA-346, CPA-332, CPA-403, CPA-354, CPA-342, or CPA-344: Where p represents 2, 2. A tetrafunctional chemical probe represented by the following formula: CPP-127, CPF-224, CPF-202, or CPF-242:

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