Human transferrin receptor-binding peptide

Abstract

To provide a peptide or the like capable of passing through a blood-brain barrier (BBB) by binding to a human transferrin receptor (hTfR).SOLUTION: A peptide having the amino acid sequence set forth in SEQ ID NO: 1 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys); or an amino acid sequence having a substitution, deletion, addition, and / or insertion of 1 or more and 10 or less amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] This invention relates to peptides that can bind to the human transferrin receptor (hTfR). This invention relates to peptides that can pass through the blood-brain barrier (BBB), peptides that are tropic to muscle tissue, and peptides that have cell-penetrating properties. Furthermore, this invention relates to methods for delivering any substance into the brain or muscle tissue using these peptides. [Background technology]

[0002] Capillaries supply blood to most brain tissues, except for a few areas including the circumventricular organs (e.g., the pineal gland, pituitary gland, and area postrema), unlike capillaries in other tissues such as muscle. The endothelial cells that make up the capillaries are tightly connected by strong intercellular junctions. This prevents passive transport of substances from blood to the brain, and, although there are exceptions, only highly lipid-soluble substances or substances with small molecular weights (less than 200-500 daltons) and electrically neutral at near physiological pH are able to pass through capillaries into the brain. This mechanism, which restricts the exchange of substances between blood and brain tissue fluid via the capillary endothelium in the brain, is called the blood-brain barrier (BBB). The BBB also restricts the exchange of substances between the blood and tissue fluid in the central nervous system, including the brain and spinal cord, and not just the brain. The existence of the blood-brain barrier allows the majority of cells in the central nervous system to maintain their biochemical homeostasis without being affected by fluctuations in the concentrations of hormones, lymphokines, and other substances in the blood.

[0003] As a method for delivering a polymeric substance to the brain through the blood-brain barrier, various methods have been reported in which the polymeric substance is modified so that it has affinity for the transferrin receptor, a membrane protein present on the endothelial cells of intracerebral capillaries (Patent Documents 1 to 3). For example, Patent Document 1 describes a blood-brain barrier shuttle that has affinity for the transferrin receptor and can bind to the receptor. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2015-528452 [Patent Document 2] Japanese Patent Application Laid-Open No. H06-228199 [Patent Document 3] WO2016 / 208695 [Patent Document 4] WO2019 / 151539 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the invention described herein is to provide novel peptides that bind to the human transferrin receptor (hTfR). Another object of the present invention is to provide peptides that can pass through the blood-brain barrier (BBB), peptides that are tropic to muscle tissue and can be efficiently transported into muscle tissue, and peptides that have cell-penetrating properties. Another object of the present invention is to provide various uses of the above-mentioned novel peptides. [Means for solving the problem]

[0006] Certain inventions described herein relate to peptides that bind to the transferrin receptor. This peptide has the amino acid sequence set forth in SEQ ID NO: 1 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys); or an amino acid sequence having substitutions, deletions, additions and / or insertions of 1 to 10 amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1. [Effects of the Invention]

[0007] According to the invention described in this specification, as demonstrated by the examples, it is possible to provide peptides that bind to the human transferrin receptor (hTfR), peptides that can pass through the blood-brain barrier (BBB), peptides that are directed toward muscle tissue, peptides that have cell-penetrating properties, and the like. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 is a photograph, instead of a drawing, showing the results of measuring the fluorescence intensity in each tissue. [Figure 1-2] Figure 1-2 is a photograph, instead of a drawing, showing the results of measuring the fluorescence intensity in each tissue. [Figure 1-3] Figures 1-3 are photographs, instead of drawings, showing the results of measuring the fluorescence intensity in each tissue. [Figure 2] FIG. 2 is a photograph, instead of a drawing, showing the results of measuring fluorescence intensity in an enlarged brain. [Figure 3] FIG. 3 is a photograph, instead of a drawing, showing the results of a mouse brain localization test (single dose). [Figure 4] FIG. 4 is a photograph, instead of a drawing, showing the results of a mouse brain localization test (multi-dose). [Figure 5] FIG. 5 is a fluorescence micrograph in place of a drawing showing migration into human breast cancer cells. DETAILED DESCRIPTION OF THE INVENTION

[0009] The following describes embodiments of the present invention with reference to the drawings. The present invention is not limited to the embodiments described below, and also includes appropriate modifications of the embodiments below within the scope obvious to those skilled in the art.

[0010] Certain inventions described herein relate to peptides that bind to the transferrin receptor and are capable of crossing the blood-brain barrier.

[0011] Transferrin receptor Transferrin receptor refers to a receptor that binds to transferrin, a protein contained in plasma that binds to iron ions, and incorporates it into cells. Transferrin receptors are expressed on various cells, including reticulocytes, placental trophoblast cells, and lymphocytes, and their expression has been suggested to be particularly prominent in tumor cells. Furthermore, because transferrin receptors have the property of triggering cellular endocytosis upon stimulation by binding with iron ions in plasma, research is underway to use antibodies that bind to transferrin receptors as DDS to allow substances to pass through the BBB. In this specification, unless otherwise specified, human transferrin receptors will be referred to as human TfR, hTfR, or simply TfR.

[0012] Transferrin receptor-binding peptides Binding to the transferrin receptor (also referred to as having binding activity or affinity) means specifically binding to the transferrin receptor. Affinity, represented by the equilibrium constant (KD) for the dissociation of the transferrin receptor and the binding peptide, is a measure of the binding strength between the transferrin receptor and the antigen-binding site on the binding peptide: the smaller the value of KD, the stronger the binding strength between the transferrin receptor and the binding peptide (alternatively, affinity can be expressed as an affinity constant (KA), which is 1 / KD). As will be apparent to those skilled in the art (e.g., based on the further disclosure herein), affinity can be determined in a manner known per se depending on the particular antigen of interest. Avidity is a measure of the strength of binding between the transferrin receptor and the binding peptide. Avidity is related to both the affinity between the transferrin receptor and its binding site on the binding peptide and the number of relevant binding sites present on the binding molecule.

[0013] The specific binding of the binding peptide to the transferrin receptor can be determined in any suitable manner known per se, including, for example, the surface plasmon resonance (SPR) assay described herein, Scatchard analysis, and / or competitive binding assays such as radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays, as well as different variants thereof known per se in the art. Preferably, the affinity of the peptides of the invention for the transferrin receptor is a KD of less than 100 nM, preferably less than 50 nM.

[0014] Able to pass the blood-brain barrier (BBB) Being able to pass through the BBB means, for example, that a substance can pass through the BBB into the brain, and that the substance or its metabolites can be detected at any site in the brain at a certain time after administration, or that knowledge can be obtained that allows one to infer that the substance has had an effect in the brain.

[0015] Brain-related diseases Brain-related diseases are diseases caused by some kind of abnormality in the brain, such as central nervous system (CNS) diseases. Examples of brain-related diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, prion diseases, Huntington's disease, lysosomal storage diseases, central nervous system disorders, central nervous system tumors including brain tumors, cerebral ischemia, diseases accompanied by brain damage, traumatic central nervous system disorders, viral and bacterial central nervous system diseases, and mental disorders such as schizophrenia and depression.

[0016] Muscle tissue-directed The muscle tissue may be any of cardiac muscle, skeletal muscle, and smooth muscle. Particularly preferred muscle tissue is cardiac muscle or skeletal muscle. "Muscle tissue-directed" means having the property of specifically and efficiently transferring to muscle tissue.

[0017] Neuromuscular disorders Neuromuscular diseases refer to diseases that cause movement disorders such as muscle weakness due to lesions in the nerves or muscles of the brain, spinal cord, or peripheral nerves. Examples of neuromuscular diseases include, but are not limited to, spinocerebellar degeneration, amyotrophic lateral sclerosis, myasthenia gravis, muscular dystrophy, polymyositis, hereditary myopathy, neuromuscular disease, muscular atrophy, drug-induced myopathy, acute heart failure, chronic heart failure, myocardial infarction, chronic fatigue syndrome, mitochondrial disease, mitochondrial respiratory chain complex disorder, and Guillain-Barré syndrome.

[0018] Cell-penetrating peptides Peptides with cell-permeability are known, for example, as described in Japanese Patent No. 6478632 and Japanese Patent No. 6708770 (Peptides with Cell-Permeability). Furthermore, as shown in the examples, the peptides of the present invention bind to transferrin receptors and are taken up into cells. Therefore, by using the peptides of the present invention or their complexes, it becomes possible to deliver a target active ingredient into cells, for example, to deliver a nucleic acid drug into cells.

[0019] peptide It refers to a structure in which multiple amino acids are consecutively linked, and includes polypeptides and proteins. In this application, amino acids include not only naturally occurring amino acids (natural amino acids) that are incorporated into peptide chains after mRNA translation in cells, but also non-naturally occurring amino acids (unnatural amino acids) that can form part of a peptide chain through peptide bonds. Amino acids may be artificially synthesized or may exist in nature. In addition, in this application, peptides in which a cyclic portion is formed by cyclization after synthesis (also called cyclic peptides), peptides obtained by further chemically modifying such peptides, complexes of peptides and substances bound to peptides, and complexes in which peptides and substances are bound via a linker are also included in the peptides and complexes of peptides and substances of the present invention. As used herein, a cyclic peptide refers to a peptide in which two amino acids separated by one or more amino acid residues in the amino acid sequence are bonded to each other, resulting in a cyclic structure in whole or in part. The type of bond between the two amino acids is not particularly limited, and examples of cyclic peptides include an amide bond between the carboxyl group of one amino acid and the amino group of the other, a thioether bond between the carboxyl group of one amino acid and the thiol group of the other, a thiol bond between the thiol group of one amino acid and the thiol group of the other, a cyclic structure formed by lactam ring formation or macrocyclization, and a lasso peptide-like structure. However, when the two amino acids are bonded by an amide bond, the amide bond is not limited to one formed by bonding the carboxyl group of one amino acid and the amino group of the other amino acid, as long as the amide bond is formed as a result of a synthetic reaction. The same applies to other bond types. That is, in the present application, the cyclic peptide may be one that partially forms a cyclic structure, and may also have a linear portion.

[0020] In this specification, some amino acids may be modified to cyclize the peptide. Such partially modified amino acids are also included. For example, a chloroacetyl group may be added to the amino acid at the N-terminus, which is then bonded to a cysteine ​​residue in the peptide to cyclize it. Various (natural / unnatural) amino acids to which a chloroacetyl group has been added are also included in the amino acids of this application.

[0021] Unnatural amino acids are compounds other than natural amino acids that have the properties of amino acids. Examples include, but are not limited to, β-amino acids, γ-amino acids, L-amino acids; D-amino acids (also called D-amino acids); chemically modified amino acids such as amino acid variants and amino acid derivatives; and amino acids that do not become components of proteins in vivo, such as norleucine, β-alanine, and ornithine. Examples include N-methyl amino acids, N-ethyl amino acids, D-amino acids, histidine-like amino acids, amino acids with structures such as extra methylene or aromatic rings in the side chain, and amino acid derivatives with a structure in which the carboxylic acid functional group in the side chain is replaced with a sulfonic acid group. Examples of unnatural amino acids and their abbreviations used herein are listed below. The CAS reference number or supplier name is shown in parentheses, and newly synthesized amino acids are indicated by synthesis example numbers. The CAS numbers refer to the unnatural amino acid alone or to a compound bound to a protecting group. However, non-standard amino acids are not limited to these. For example, non-standard amino acids include those in which one or more hydrogen atoms in the molecule are substituted with alkyl groups. When a hydrogen atom is substituted with an alkyl group, the alkyl group is preferably a methyl group or an ethyl group, and more preferably a methyl group. In this specification, amino acids preceded by Me or N-Me- refer to N-methylamino acids unless otherwise specified. For example, N-methylated amino acids of alanine (Ala or A) are represented as MeAla, N-MeAla, MeA, or N-MeA. Furthermore, amino acids represented by a single-letter amino acid designation preceded by d refer to D-amino acids. For example, D-amino acids of alanine (Ala or A) are represented as da. Amino acids without a CAS number or supplier can be purchased as general reagents. The following amino acids can be used in peptide synthesis by protecting the alpha amino group with Fmoc by known methods.

[0022] Yph (S)-2-amino-3-(4-phenoxyphenyl)propanoic acid (CAS number: 180414-93-1) W7OMe (S)-2-amino-3-(7-methoxy-1H-indol-3-yl)propanoic acid (CAS number: 2416720-26-6) W7N (S)-2-Amino-3-(1H-pyrrolo[2,3-β]pyridin-3-yl)propanoic acid (CAS number: 737007-45-3) W7F (S)-2-amino-3-(7-fluoro-1H-indol-3-yl)propanoic acid (CAS number: 1956434-65-3) W6N (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propanoic acid (Kishida Chemical Co., Ltd.) W6F (S)-2-amino-3-(6-fluoro-1H-indol-3-yl)propanoic acid (CAS number: 908847-01-8) W5OMe 5-Methoxy-L-tryptophan (CAS number: 460751-69-3) W5F (S)-2-amino-3-(5-fluoro-1H-indol-3-yl)propanoic acid (CAS number: 908846-88-8) W4OMe 4-Methoxy-L-tryptophan (CAS number: 1205553-56-5) W4N (S)-2-Amino-3-(1H-pyrrolo[3,2-β]pyridin-3-yl)propanoic acid (CAS number: 149818-23-5) W4F (S)-2-((((9H-Fluoren-9-yl)-methoxy)carbonyl)amino)-3-(1-(tert-butoxycarbonyl)-4-fluoro-1H-indol-3-yl)propanoic acid (CAS number: 2244532-65-6) W4C (S)-2-amino-3-(4-chloro-1H-indol-3-yl)propanoic acid (CAS number: 2244532-68-9) W2N (S)-2-Amino-3-(1H-indol-3-yl)propanoic acid (CAS number: 2305185-20-8) W1iPr 1-Isopropyl-L-tryptophan (CAS number: 1496563-42-8) W1Et7Cl (S)-2-amino-3-(7-chloro-1-ethyl-1H-indol-3-yl)propanoic acid (Synthesis Example 2-1) W1Et 1-Ethyl-L-tryptophan (CAS number: 168471-23-6) Tbg (S)-2-amino-3,3-dimethylbutanoic acid (CAS number: 33105-81-6) pHPeG N-(4-hydroxyphenethyl)glycine (CAS number: 258332-56-8) PeG N-(2-phenylethyl)-glycine (CAS number: 540483-58-7) Nva L-Norvaline (CAS number: 6600-40-4) Nle N-α-chloroacetyl-L-norleucine (CAS number: 688-12-0) Nal2 β-(2-naphthyl)L-alanine (CAS number: 58438-03-2) Nal1 β-(1-naphthyl)L-alanine (CAS number: 2353616-32-5) MeoBph N-α-methyl-2-phenyl-L-phenylalanine MeNal2 N-α-methyl-β-(2-naphthyl)-L-alanine (CAS number: 179385-30-9) MeNal1 N-α-methyl-β-(1-naphthyl)-L-alanine (CAS number: 1380327-68-3) MemBph N-α-methyl-3-phenyl-L-phenylalanine Hph L-Homophenylalanine (CAS number: 943-73-7) Hly (S)-2,7-diaminoheptanoic acid (CAS number: 498-56-6) F4OMe (S)-2-amino-3-(4-methoxyphenyl)propanoic acid (CAS number: 7635-29-2) F4G (4-Guanidine)-L-phenylalanine (CAS number: 59574-11-7) F4F 4-Fluoro-L-phenylalanine (CAS number: 1132-68-9) F4C N-α-chloroacetyl-4-chloro-L-phenylalanine (CAS number: 14173-39-8) F3OMe (S)-2-amino-3-(3-methoxyphenyl)propanoic acid (CAS number: 98813-19-5) F3F 3-Fluoro-L-phenylalanine (CAS number: 19883-77-3) F3C N-α-chloroacetyl-3-chloro-L-phenylalanine (CAS number: 80126-51-8) FOMe (S)-2-amino-3-(2-methoxyphenyl)propanoic acid (CAS number: 206060-41-5) F2C (S)-2-amino-3-(2-chlorophenyl)propanoic acid (CAS number: 198560-41-7) MeF4OMe (s)-3-(4-methoxyphenyl)-2-(methylamino)propanoic acid (CAS number: 1260595-45-6) MeF4F N-α-methyl-4-fluoro-L-phenylalanine (CAS number: 1979176-87-8) MeF3F N-α-methyl-3-fluoro-L-phenylalanine (CAS number: 1820567-10-9) MeF3C N-α-methyl-3-chlorofluoro-L-phenylalanine (CAS number: 1446478-28-9) MeBph N-α-methyl-4-phenyl-L-phenylalanine Me4Py N-α-methyl-4-pyridyl-L-alanine Me3Py N-α-methyl-3-pyridyl-L-alanine dr D-Arginine dp D-proline dc D-cysteine dk D-lysine Dap L-α,β-diaminopropionic acid (CAS number: 515-94-6) Dab (S)-4-amino-2-(2-chloroacetamido)butanoic acid (CAS number: 25691-37-6) Cit 2-amino-5-ureidopentanoic acid (CAS number: 627-77-0) Cha β-Cyclohexyl-L-alanine (CAS number: 4441-50-3) CeG N-(2-carboxyethyl)-glycine (CAS number: 174799-89-4) Cbg (S)-2-amino-2-cyclobutylacetic acid (CAS number: 1391630-31-1) Cba Cyclobutylalanine (CAS number: 478183-62-9) aMeY α-Methyl-L-tyrosine (CAS number: 658-48-0) aMeW α-methyl-tryptophan (CAS number: 153-91-3) aMeK α-methyl-lysine (CAS number: 111717-28-3) aMeC α-methyl-L-cysteine ​​(CAS number: 441317-73-3) Aib α-methylalanine (CAS number: 62-57-7) Ahp / Alahp (S)-2-aminoheptanoic acid (CAS number: 1115-90-8) Abu L-α-aminobutanoic acid (CAS number: 1492-24-6) A4paa (S)-2-amino-3-(1-(carboxymethyl)piperazin-4-yl)propanoic acid (Kishida Chemical Co., Ltd.) 5Ind (S)-2-amino-3-(1H-indol-5-yl)propanoic acid (CAS number: 1655518-66-3) 4Py2NH2 (S)-2-amino-3-(2-aminopyridin-4-yl)propanoic acid (Kishida Chemical Co., Ltd.) 4Py 4-Pyridyl-L-alanine (CAS number: 1956-21-4) 3Py6NH2 2-amino-3-(6-aminopyridin-3-yl)propanoic acid (Synthesis Example 2-3) 3Py 3-Pyridyl-L-alanine (CAS number: 17470-24-5) W1aa 1-(carboxymethyl)-L-tryptophan (CAS number: 773823-50-0) KCOpipzMe N6-(4-methylpiperazine-1-carbonyl)-L-lysine (Kishida Chemical Co., Ltd.) W1mCON 1-(2-amino-2-oxoethyl)-L-tryptophan (Synthesis Example 2-5) W1EtOH 1-(2-hydroxyethyl)-L-tryptophan (Synthesis Example 2-9) 3Py6OMe (S)-2-amino-3-(6-methoxypyridin-3-yl)propanoic acid (CAS number: 1270317-99-1) Epyrl2RCOO 2-((5-((R)-2-((allyloxy)carbonyl)pyrrolidin-1-yl)-5-oxopentanoic acid Dpyrl2RCOO 2-((4-((R)-2-((allyloxy)carbonyl)pyrrolidin-1-yl)-4-oxobutanoic acid (Examples 9-16) MeF3COO 3-Carboxy-N-methyl-phenylalanine (CAS number: 1499826-56-0) 3Imp 2-amino-3-(imidazo[1,2-a]pyridin-3-yl)propanoic acid (CAS number: 2276942-95-9) KaAc N6-glycyl-L-lysine (Synthesis Example 2-8) A1Me4pip 4-Amino-1-methylpiperazine-4-carboxylic acid (CAS number: 15580-66-2) Har N6-Carbamimidoyl-L-lysine (CAS number: 156-86-5) Acpr (S)-2-amino-3-cyclopropylpropanoic acid (CAS number: 1492156-90-7) Atb (S)-2-amino-4,4-dimethylpentanoic acid (CAS number: 1934633-35-8) MeF35dC (S)-3-(3,5-dichlorophenyl)-2-(methylamino)propanoic acid (CAS number: 1542508-65-5) Adod 12-aminododecanoic acid Hly L-homolysine W5C 5-chloro-L-tryptophan F3COO L-3-carboxyphenylalanine F3CON L-3-carbamoylphenylalanine Hgl L-2-aminoadipic acid Ndm N,N-dimethyl-L-asparagine KN3 or LysN3 6-azido-L-norleucine KAc N6-acetyl-L-lysine Dorn D-Ornithine Nle L-norleucine F3H 3-hydroxy-L-phenylalanine Yae O-(2-aminomethyl)-L-tyrosine F4aao O-(2-carboxymethyl)-L-tyrosine F4OEt O-Ethyl-L-tyrosine F34dOMe 3,4-dimethoxy-L-phenylalanine aLT L-Allothreonine alI L-Alloisoleucine MeK N-methyl-L-lysine Tbg (S)-2-amino-3,3-dimethylbutyric acid Nva L-Norvaline Abu (S)-(+)-2-aminobutyric acid da D-alanine Bph 4-phenyl-L-phenylalanine de D-glutamic acid MeA N-methyl-L-alanine PEG4c or PEG3 1-amino-3,6,9,12-tetraoxapentadecan-15-oic acid MeR N-methyl-L-arginine MeW N-methyl-L-tryptophan PEG8c 1-amino-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-oic acid PEG12c or PEG11 or PEG12 1-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonateriacontan-39-oic acid Furthermore, newly synthesized amino acids are useful because they may be able to add new functions to various peptides when producing various peptide derivatives.

[0023] The peptide of the present invention is a peptide having the amino acid sequence set forth in SEQ ID NO: 1 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys); or an amino acid sequence having substitutions, deletions, additions and / or insertions of 1 to 10 amino acid residues in the amino acid sequence set forth in SEQ ID NO: 1.

[0024] About the peptide sequence The number of substituted, deleted, added, and / or inserted amino acids may be between 1 and 10, with the lower limit being 1. The upper limit is 10, 9, 8, 7, 6, 5, 4, 3, or 2, with the minimum being 1. Such amino acid substitutions are preferably conservative amino acid substitutions.

[0025] Conservative amino acid substitutions A "conservative amino acid substitution" refers to a substitution with a functionally equivalent or similar amino acid. Conservative amino acid substitutions in a peptide result in a static change in the amino acid sequence of the peptide. For example, one or more amino acids with similar polarity act functionally equivalently, resulting in a static change in the amino acid sequence of the peptide. In general, substitutions within a group can be considered conservative in structure and function. However, as those skilled in the art will recognize, the role played by a particular amino acid residue can be determined by its context in the three-dimensional structure of the molecule containing that amino acid. For example, cysteine ​​residues can adopt an oxidized (disulfide) form that is less polar than its reduced (thiol) form. The long aliphatic portion of the arginine side chain can constitute a structurally and functionally important feature. Also, side chains containing aromatic rings (tryptophan, tyrosine, phenylalanine) can contribute to ion-aromatic or cation-pi interactions. In such cases, substitution of amino acids with these side chains with amino acids belonging to acidic or nonpolar groups can be structurally and functionally conservative. Residues such as proline, glycine, and cysteine ​​(disulfide form) can have direct effects on the main-chain conformation and often cannot be substituted without structural distortion.

[0026] Conservative amino acid substitutions include specific substitutions based on side chain similarity (L. Lehninger, Biochemistry, 2nd edition, pp73-75, Worth Publisher, New York (1975)) and typical substitutions, as shown below.

[0027] Preferred examples of such peptides are those of the following group: (I) substitution of the first alanine residue of SEQ ID NO: 1 with an aliphatic amino acid or a methylated aliphatic amino acid; (II) substitution of the second valine residue of SEQ ID NO: 1 with a basic amino acid residue or a methylated basic amino acid residue; (III) substitution of the third phenylalanine residue of SEQ ID NO: 1 with an aromatic amino acid residue, a methylated aromatic amino acid residue, an amino acid residue having an added aromatic ring, or an amino acid residue having an added fused ring; (IV) substitution of the fourth valine residue of SEQ ID NO: 1 with a methylated valine residue; (V) substitution of the fifth tryptophan residue in SEQ ID NO: 1 with an aromatic amino acid residue, a methyltryptophan residue, a methylated aromatic amino acid residue, an amino acid residue having an added aromatic ring, or an amino acid residue having an added fused ring; (VI) substitution of the asparagine residue at position 6 of SEQ ID NO: 1 with a neutral amino acid or a methylated neutral amino acid; (VII) substitution of the tyrosine residues at positions 7 and 8 of SEQ ID NO: 1 with an aromatic amino acid residue, a methylated aromatic amino acid residue, an amino acid residue having an added aromatic ring, or an amino acid residue having an added fused ring; (VIII) substitution of the isoleucine residue at position 9 of SEQ ID NO: 1 with an aliphatic amino acid residue, a methylated aliphatic amino acid residue, or an amino acid residue having a branched chain structure; (IX) Substitution of the 10th isoleucine residue of SEQ ID NO: 1 with any amino acid; and (X) substitution of the serine residue at position 11 of SEQ ID NO: 1 with a neutral amino acid residue, The amino acid sequence includes an amino acid sequence having one or more substitutions selected from:

[0028] (1) Nonpolar amino acid group: alanine (hereinafter referred to as "Ala" or simply "A"), valine (hereinafter referred to as "Val" or simply "V"), leucine (hereinafter referred to as "Leu" or simply "L"), isoleucine (hereinafter referred to as "Ile" or simply "I"), proline (hereinafter referred to as "Pro" or simply "P"), phenylalanine (hereinafter referred to as "Phe" or simply "F"), tryptophan (hereinafter referred to as "Trp" or simply "W"), methionine (hereinafter referred to as "Met" or simply "M")

[0029] (2) Uncharged polar amino acid group: glycine (hereinafter referred to as "Gly" or simply "G"), serine (hereinafter referred to as "Ser" or simply "S"), threonine (hereinafter referred to as "Thr" or simply "T"), cysteine ​​(hereinafter referred to as "Cys" or simply "C"), tyrosine (hereinafter referred to as "Tyr" or simply "Y"), asparagine (hereinafter referred to as "Asn" or simply "N"), glutamine (hereinafter referred to as "Gln" or simply "Q")

[0030] (3) Acidic amino acid group: aspartic acid (hereinafter referred to as "Asp" or simply "D"), glutamic acid (hereinafter referred to as "Glu" or simply "E")

[0031] (4) Basic amino acid group: lysine (hereinafter referred to as "Lys" or simply "K"), arginine (hereinafter referred to as "Arg" or simply "R"), histidine (hereinafter referred to as "His" or simply "H") Furthermore, naturally occurring amino acids can be divided into the following groups based on the properties of their common side chains:

[0032] (1) Hydrophobic amino acid group: Norleucine, Met, Ala, Val, Leu, Ile (2) Neutral hydrophilic amino acid group: Cys, Ser, Thr, Asn, Gln (3) Acidic amino acid group: Asp, Glu (4) Basic amino acid group: His, Lys, Arg (5) Group of amino acids that influence the direction of the main chain: Gly, Pro (6) Aromatic amino acid group: Trp, Tyr, Phe Each group also includes unnatural amino acids such as N-methylated amino acids.

[0033] A preferred example of this peptide is a peptide having the amino acid sequence set forth in SEQ ID NO: 2 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Arg-Arg-Tyr-MeTyr-Cys).

[0034] A preferred example of this peptide is a peptide having the amino acid sequence set forth in SEQ ID NO: 2, which is selected from the following groups: (I) substitution of the first alanine residue of SEQ ID NO: 2 with an aliphatic amino acid or a methylated aliphatic amino acid; (II) substitution of the second valine residue of SEQ ID NO: 2 with a basic amino acid residue or a methylated basic amino acid residue; (III) substitution of the third phenylalanine residue of SEQ ID NO: 2 with an aromatic amino acid residue, a methylated aromatic amino acid residue, an amino acid residue having an added aromatic ring, or an amino acid residue having an added fused ring; (IV) substitution of the fourth valine residue of SEQ ID NO: 2 with a methylated valine residue; (V) substitution of the fifth tryptophan residue in SEQ ID NO: 2 with an aromatic amino acid residue, a methylated aromatic amino acid residue, an amino acid residue having an added aromatic ring, or an amino acid residue having an added fused ring; (VI) substitution of the asparagine residue at position 6 of SEQ ID NO: 2 with a neutral amino acid or a methylated neutral amino acid; (VII) substitution of the tyrosine residues at positions 7 and 8 of SEQ ID NO: 2 with an aromatic amino acid residue, a methylated aromatic amino acid residue, an amino acid residue having an added aromatic ring, or an amino acid residue having an added fused ring; (VIII) substitution of the 9th isoleucine residue of SEQ ID NO: 2 with an aliphatic amino acid residue, a methylated aliphatic amino acid residue, or an amino acid residue having a branched chain structure; (IX) substitution of the isoleucine residue at position 10 of SEQ ID NO: 2 with any amino acid; (XI) substitution of the arginine residues at positions 11 and 12 of SEQ ID NO: 2 with basic amino acid residues; (XII) substitution of the tyrosine residue at position 13 of SEQ ID NO: 2 with a hydrophilic amino acid residue; (XIII) substitution of the methyltyrosine residue at position 14 of SEQ ID NO: 2 with a tyrosine residue, an aromatic amino acid residue, or a methylated aromatic amino acid residue; and (XIV) substitution of the cysteine ​​residue at position 15 of SEQ ID NO: 2 with a methylated cysteine ​​residue, The amino acid sequence includes an amino acid sequence having one or more substitutions selected from:

[0035] Another preferred example of this peptide is a peptide having a length of 10 to 17, wherein peptide A comprises the amino acid sequence set forth as positions 1 to 10 of SEQ ID NO: 18 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys): Peptide A, or Peptide A is a peptide having an amino acid sequence with substitution, deletion, and / or insertion of 1 to 6 amino acid residues.

[0036] Preferred examples of peptides in this specification are peptides that can bind to the human transferrin receptor (hTfR), similar to the peptides described above. Also preferred examples are peptides that can cross the blood-brain barrier (BBB), peptides that are tropic to muscle tissue, or peptides that have cell-penetrating properties.

[0037] Peptide A may be a peptide having an amino acid sequence in which any of the amino acid residues at positions 2, 3, 5, 8, and 10 of SEQ ID NO: 18 is substituted. "An amino acid residue has been substituted" means that a specific amino acid residue has been substituted with another amino acid residue that may be modified.

[0038] In peptide A, the second amino acid residue of SEQ ID NO: 18 is an optionally modified valine (Val) or an optionally modified glutamic acid (Glu); The third amino acid residue of SEQ ID NO: 18 is an optionally modified phenylalanine (Phe), the fifth amino acid residue of SEQ ID NO: 18 is an optionally modified tryptophan (Trp); the 8th amino acid residue of SEQ ID NO: 18 is an optionally modified tyrosine (Tyr); The peptide may be one in which the 10th amino acid residue of SEQ ID NO: 18 is an optionally modified isoleucine (Ile) or an optionally modified valine (Val).

[0039] "May be modified" means that known amino acid modifications or alterations may be made. Examples of modifications include N-methylation, amino acid modifications with the abbreviations described below, modification (conversion) to the D-type, and conversion to a known derivative of the amino acid.

[0040] The peptide preferably has a peptide length of 11 or more and 13 or less.

[0041] More preferably, this peptide has the following amino acid sequence: The second amino acid residue of SEQ ID NO: 18 is Val or Glu, the third amino acid residue of SEQ ID NO: 18 is Phe or MeF3C; The fifth amino acid residue of SEQ ID NO: 18 is Trp or MeTrp, the 8th amino acid residue of SEQ ID NO: 18 is Tyr or F4OMe; The peptide may be one in which the 10th amino acid residue of SEQ ID NO: 18 is Ile or Val.

[0042] Furthermore, this peptide has the following amino acid sequence at the N-terminus of peptide A. The 11th amino acid residue of SEQ ID NO: 18 is Ser or His, The peptide may be one in which the 12th amino acid residue of SEQ ID NO: 18 is Cys or Hgl.

[0043] Another preferred example of this peptide is a peptide having a length of 10 to 19, comprising the amino acid sequence set forth as positions 1 to 10 of SEQ ID NO: 15 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Val-Pro-Arg-Asp-Cys): Peptide B, or Peptide B is a peptide having an amino acid sequence with substitution, deletion, and / or insertion of 1 to 5 amino acid residues.

[0044] Peptide B may be a peptide having an amino acid sequence in which any of the amino acid residues at positions 2, 3, 5, 8, and 10 of SEQ ID NO: 15 is substituted.

[0045] In peptide B, the second amino acid residue of SEQ ID NO: 15 is an optionally modified valine (Val) or an optionally modified glutamic acid (Glu); The third amino acid residue of SEQ ID NO: 15 is optionally modified phenylalanine (Phe) or tryptophan (Trp), the fifth amino acid residue of SEQ ID NO: 15 is an optionally modified tryptophan (Trp); the 8th amino acid residue of SEQ ID NO: 15 is an optionally modified tyrosine (Tyr); The peptide may be one in which the 10th amino acid residue of SEQ ID NO: 15 is an optionally modified isoleucine (Ile) or an optionally modified valine (Val).

[0046] These peptides may have a peptide length of 13 or more and 15 or less.

[0047] More preferably, this peptide has the following amino acid sequence: The second amino acid residue of SEQ ID NO: 15 is Val or Glu, the third amino acid residue of SEQ ID NO: 15 is Phe, Trp, or MeF3C; The fifth amino acid residue of SEQ ID NO: 15 is Trp or MeTrp, the 8th amino acid residue of SEQ ID NO: 15 is Tyr, Phe, or F4OMe; The peptide may be one in which the 10th amino acid residue of SEQ ID NO: 15 is Ile or Val.

[0048] Furthermore, this peptide has the following amino acid sequence at the N-terminus of peptide B. The 11th amino acid residue of SEQ ID NO: 15 is Phe; the 12th amino acid residue of SEQ ID NO: 15 is Arg; the 13th amino acid residue of SEQ ID NO: 15 is Glu, Asn, Asp, His, Gln, or MeTrp; The peptide may be one in which the 14th amino acid residue of SEQ ID NO: 15 is Cys.

[0049] Another preferred example of this peptide is a peptide having a length of 11 to 19, comprising the amino acid sequence set forth as positions 1 to 10 of SEQ ID NO: 214 (MeA-Val-MeF3C-Val-MeW-Asn-Tyr-F4OMe-Ile-Ile-Arg-Arg-Phe-MeY-Cys): Peptide C, or Peptide C is a peptide having an amino acid sequence with substitution, deletion, and / or insertion of 1 to 5 amino acid residues.

[0050] Peptide C may be a peptide having an amino acid sequence in which any of the 1st, 3rd, 5th, and 8th amino acid residues of SEQ ID NO: 214 is substituted.

[0051] In peptide C, the first amino acid residue of SEQ ID NO: 214 is an optionally modified alanine (Ala) or an optionally modified glutamic acid (Glu); The third amino acid residue of SEQ ID NO: 214 is an optionally modified phenylalanine (Phe), The fifth amino acid residue of SEQ ID NO: 214 is an optionally modified tryptophan (Trp), The peptide may be one in which the 8th amino acid residue of SEQ ID NO: 214 is an optionally modified phenylalanine (Phe).

[0052] These peptides preferably have a peptide length of 15 or more and 18 or less.

[0053] More preferably, this peptide has the following amino acid sequence: The first amino acid residue of SEQ ID NO: 214 is Ala, Aib, Abu, Glu, Gly, Ser, Phe, Pro, or MeA, and particularly preferably Ala or MeA; the third amino acid residue of SEQ ID NO: 214 is Phe, F3C, F2C, F2OMe, F4C, Cha, MeF, MeF35dC, MeF4F, MeF4Ome, MeNal1, Me3Py, Me4Py, Me3OMe, MeF3COO, MeF3F, Glu, Epyr2RC00, Dpyr2RC00 or MeF3C, particularly preferably Phe, MeF or MeF3C; the fifth amino acid residue of SEQ ID NO: 214 is Trp, MeW, aMeW, dp, F3C, F3F, F3OMe, F4C, F4F, Hph, MemBph, MeNal1, MeNal2, MeoBph, W4OMe, W1Et, W1Et7Cl, W1iPr, Yph, W1Pr, W5C, W5F, W1aa, W1EtOH, W4OMe, W1mCON or W6F, particularly preferably Trp or MeW; The peptide may be one in which the 8th amino acid residue of SEQ ID NO: 214 is Phe, Tyr, Typ, Ahp, MeY, F4OMe, 3Imp, 4Py, 3Py, 3Py6OMe, F3C, F3CON, F4C, F4aao, F4F, F4OEt, MeF34dOMe, Yae, Lys, Orn, or Nall, and particularly preferably Tyr or F4OMe.

[0054] Furthermore, this peptide has the following amino acid sequence at the N-terminus of peptide C. The 11th amino acid residue of SEQ ID NO: 214 is Arg, Ala, Asp, Gly, Glu, Lys, MeK, MeR, Dap, Dap, Abu, Aib, Hly, dorn, aMeK, A1Me4pip, KCOpipzMe, F4G, Nle, Nva or Orn, and particularly preferably Lys or Arg; The 12th amino acid residue of SEQ ID NO: 214 is Lys, Arg, dr, Tyr, F4G, Orn, Hly, da, Cit, Dap or Dab, and particularly preferably Lys, Arg or dr; The 13th amino acid residue of SEQ ID NO: 214 is Ala, Phe, Asn, Tyr, or pHPeG, and particularly preferably Phe or Tyr; The 14th amino acid residue of SEQ ID NO: 214 is MeTyr, Tyr, Phe, Ala, aMeY, Glu, Gly, Arg, Val, MeoBphMeBph, MeF, MemBph, MeNal1, MeNal2, MeoBph, MeW or pHPeG, particularly preferably Phe or MeW; The peptide may be one in which the 15th amino acid residue of SEQ ID NO: 214 is Cys or Hgl.

[0055] Another preferred example of this peptide is a peptide having a length of 11 or more and 19 or less, comprising the amino acid sequence set forth as positions 1 to 10 of SEQ ID NO: 219 (Ala-Glu-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Arg-Arg-Tyr-MeY-Cys): Peptide D, or Peptide D is a peptide having an amino acid sequence with substitution, deletion, and / or insertion of 1 to 5 amino acid residues.

[0056] Peptide D may be a peptide having an amino acid sequence in which any of the 2nd, 3rd, 5th, 8th, and 10th amino acid residues of SEQ ID NO: 219 is substituted.

[0057] In peptide D, the second amino acid residue of SEQ ID NO: 219 is an optionally modified valine (Val), an optionally modified glutamic acid (Glu), an optionally modified arginine (Arg), an optionally modified lysine (Lys), an optionally modified aspartic acid (Asp), or an optionally modified phenylalanine (Phe), The third amino acid residue of SEQ ID NO: 219 is an optionally modified phenylalanine (Phe), The fifth amino acid residue of SEQ ID NO: 219 is an optionally modified tryptophan (Trp), the 8th amino acid residue of SEQ ID NO: 219 is an optionally modified tyrosine (Tyr); The peptide may be one in which the 10th amino acid residue of SEQ ID NO: 219 is an optionally modified isoleucine (Ile), an optionally modified glutamic acid (Glu), or an optionally modified lysine (Lys).

[0058] These peptides preferably have a peptide length of 15 or more and 18 or less.

[0059] More preferably, this peptide has the following amino acid sequence: The second amino acid residue of SEQ ID NO: 219 is Val, Glu, Ala, Arg, Lys, Asp, Phe, Dap, Har, Abu, Nva, AcPr, AtbAhp, or Hgl, and particularly preferably Gln or Val; The third amino acid residue in SEQ ID NO: 219 is Phe, F3C, F2C, F2OMe, F4C, Cha, MeF, MeF35dC, MeF4F, MeF4Ome, MeNal1, Me3Py, Me4Py, Me3OMe, MeF3COO, MeF3F, Glu, Epyrl2RCOO, Dpyrl2RCOO or MeF3C, particularly preferably Phe, MeF or MeF3C; the fifth amino acid residue of SEQ ID NO: 219 is Trp, MeW, aMeW, dp, F3C, F3F, F3OMe, F4C, F4F, Hph, MemBph, MeNal1, MeNal2, MeoBph, W4OMe, W1Et, W1Et7Cl, W1iPr, Yph, W1Pr, W5C, W5F, W1aa, W1EtOH, W4OMe, W1mCON or W6F, particularly preferably Trp or MeW; The 8th amino acid residue of SEQ ID NO: 219 is Phe, Tyr, Typ, Ahp, MeY, F4OMe, 3Imp, 4Py, 3Py, 3Py6OMe, F3C, F3CON, F4C, F4aao, F4F, F4OEt, MeF34dOMe, Yae, Lys, Orn, or Nall, and particularly preferably Tyr or F4OMe; The peptide may also include one in which the 10th amino acid residue of SEQ ID NO: 219 is Ala, Abu, Acpr, Ahp, Aib, all, all, Atb, Dab, Dap, dawn, Gln, Hly, Ile, Lys, KCOpipzMe, Leu, Nle, Nva, Pro, Arg, Ser, Thr, Tbg, Val, or Tyr, and is particularly preferably Ile or all.

[0060] Furthermore, this peptide has the following amino acid sequence at the N-terminus of peptide C. The 11th amino acid residue of SEQ ID NO: 219 is Arg, Ala, Asp, Gly, Glu, Lys, MeK, MeR, Dap, Dap, Abu, Aib, Hly, dorn, aMeK, A1Me4pip, KCOpipzMe, F4G, Nle, Nva or Orn, and particularly preferably Lys or Arg; The 12th amino acid residue of SEQ ID NO: 219 is Lys, Arg, dr, Tyr, F4G, Orn, Hly, da, Cit, Dap or Dab, and particularly preferably Lys, Arg or dr; the 13th amino acid residue of SEQ ID NO: 219 is Ala, Phe, Asn, Tyr, or pHPeG, and particularly preferably Phe or Tyr; The 14th amino acid residue of SEQ ID NO: 219 is MeTyr, Tyr, Phe, Ala, aMeY, Glu, Gly, Arg, Val, MeoBphMeBph, MeF, MemBph, MeNal1, MeNal2, MeoBph, MeW or pHPeG, particularly preferably Phe or MeW; The peptide may be one in which the 15th amino acid residue of SEQ ID NO: 219 is Cys or Hgl.

[0061] Another preferred example of this peptide is a peptide having a length of 15 to 18, comprising the amino acid sequence set forth as positions 1 to 15 of SEQ ID NO: 296 (Ala-Val-MeF-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Arg-Arg-Tyr-MeY-Cys): Peptide E, or Peptide E is a peptide having an amino acid sequence with substitution, deletion, and / or insertion of 1 to 5 amino acid residues.

[0062] Peptide E may be a peptide having an amino acid sequence in which any of the amino acid residues at positions 3, 5, 7, 8, 11, 12, and 13 of SEQ ID NO: 296 is substituted.

[0063] In peptide E, The third amino acid residue of SEQ ID NO: 296 is an optionally modified phenylalanine (Phe), The fifth amino acid residue of SEQ ID NO: 296 is an optionally modified tryptophan (Trp), the seventh amino acid residue of SEQ ID NO: 296 is an optionally modified tyrosine (Tyr); the 8th amino acid residue of SEQ ID NO: 296 is an optionally modified tyrosine (Tyr); The 11th amino acid residue of SEQ ID NO: 296 is an optionally modified arginine (Arg) or an optionally modified alanine (Ala), the 12th amino acid residue of SEQ ID NO: 296 is an optionally modified arginine (Arg) or an optionally modified lysine (Lys); The peptide may be one in which the 13th amino acid residue of SEQ ID NO: 296 is either an optionally modified tyrosine (Tyr) or an optionally modified phenylalanine (Phe).

[0064] In addition, in peptide E, The third amino acid residue in SEQ ID NO: 296 is Phe, F3C, F2C, F2OMe, F4C, Cha, MeF, MeF35dC, MeF4F, MeF4Ome, MeNal1, Me3Py, Me4Py, Me3OMe, MeF3COO, MeF3F, Glu, Epyrl2RCOO, Dpyrl2RCOO or MeF3C, particularly preferably Phe, MeF or MeF3C; the fifth amino acid residue of SEQ ID NO: 296 is Trp, MeW, aMeW, dp, F3C, F3F, F3OMe, F4C, F4F, Hph, MemBph, MeNal1, MeNal2, MeoBph, W4OMe, W1Et, W1Et7Cl, W1iPr, Yph, W1Pr, W5C, W5F, W1aa, W1EtOH, W4OMe, W1mCON or W6F, particularly preferably Trp or MeW; the seventh amino acid residue of SEQ ID NO: 296 is Tyr, PyOMe, Ala, Ahp, Phe, FH, FC, Na1, Arg, or Trp, and particularly preferably Tyr; The 8th amino acid residue of SEQ ID NO: 296 is Phe, Tyr, Typ, Ahp, MeY, F4OMe, 3Imp, 4Py, 3Py, 3Py6OMe, F3C, F3CON, F4C, F4aao, F4F, F4OEt, MeF34dOMe, Yae, Lys, Orn, or Nall, and particularly preferably Tyr or F4OMe; The 11th amino acid residue of SEQ ID NO: 296 is Arg, Ala, Asp, Gly, Glu, Lys, MeK, MeR, Dap, Dap, Abu, Aib, Hly, dorn, aMeK, A1Me4pip, KCOpipzMe, F4G, Nle, Nva or Orn, and particularly preferably Lys or Arg; The 12th amino acid residue of SEQ ID NO: 296 is Lys, Arg, dr, Tyr, F4G, Orn, Hly, da, Cit, Dap or Dab, and particularly preferably Lys, Arg or dr; The peptide may be one in which the 13th amino acid residue of SEQ ID NO: 296 is Ala, Phe, Asn, Tyr, or pHPeG, and particularly preferably Phe or Tyr.

[0065] Furthermore, this peptide has the following amino acid sequence at the N-terminus of peptide C. The 11th amino acid residue of SEQ ID NO: 296 is Arg, Ala, Asp, Gly, Glu, Lys, MeK, MeR, Dap, Dap, Abu, Aib, Hly, dorn, aMeK, A1Me4pip, KCOpipzMe, F4G, Nle, Nva or Orn, and particularly preferably Lys or Arg; The 12th amino acid residue of SEQ ID NO: 296 is Lys, Arg, dr, Tyr, F4G, Orn, Hly, da, Cit, Dap or Dab, and particularly preferably Lys, Arg or dr; The 13th amino acid residue of SEQ ID NO: 296 is Ala, Phe, Asn, Tyr, or pHPeG, and particularly preferably Phe or Tyr; The 14th amino acid residue of SEQ ID NO: 214 is MeTyr, Tyr, Phe, Ala, aMeY, Glu, Gly, Arg, Val, MeoBphMeBph, MeF, MemBph, MeNal1, MeNal2, MeoBph, MeW or pHPeG, particularly preferably Phe or MeW; The peptide may be one in which the 15th amino acid residue of SEQ ID NO: 296 is Cys or Hgl.

[0066] Furthermore, in peptide E, The third amino acid residue of SEQ ID NO: 296 is phenylalanine (Phe), methylated phenylalanine (MeF), or N-α-methyl-N-α-chloroacetyl-3-chloro-L-phenylalanine (MeF3C), The fifth amino acid residue of SEQ ID NO: 296 is tryptophan (Trp) or methylated tryptophan (MeW), The seventh amino acid residue of SEQ ID NO: 296 is tyrosine (Tyr), The 8th amino acid residue of SEQ ID NO: 296 is tyrosine (Tyr) or (S)-2-amino-3-(4-methoxyphenyl)propanoic acid (F4OMe), The 11th amino acid residue of SEQ ID NO: 296 is arginine (Arg) or lysine (Lys), The 12th amino acid residue of SEQ ID NO: 296 is arginine (Arg) or D-arginine (dr), The peptide may be one in which the 13th amino acid residue of SEQ ID NO: 296 is tyrosine (Tyr) or phenylalanine (Phe).

[0067] Preferred examples of this peptide include a peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 3 to 200, or a peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 3 to 200 in which the N-terminus is chloroacetyl-Ala.

[0068] A preferred example of this peptide is any of the peptides described above, which is a cyclic peptide.

[0069] A preferred example of this peptide is a peptide that contains the 1st to 10th amino acid sequence of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 552 or a complex of the amino acid sequence and a linker, and in which the amino acid sequence portion has a cyclic structure.

[0070] Particularly preferred examples of this peptide are peptides consisting of the 1st to 15th amino acids in the amino acid sequence set forth in any one of SEQ ID NOs: 2, 9, 21 to 148, 159 to 200, 213 to 448, and 450 to 552, or a complex of the amino acid sequence and a linker, in which the amino acid sequence portion has a cyclic structure.

[0071] About cyclic peptides It refers to a peptide in which two amino acids are bonded together, and the whole or part of the peptide is cyclic. In this application, it also includes peptides in which the amino acids in the peptide form a cross-linked structure, peptides in which a cyclic structure is formed by lactam ring formation or macrocyclization reaction, and peptides with a lasso peptide-like structure. In other words, in this application, a cyclic peptide is a peptide in which a part of the peptide forms a cyclic structure, and it may also have a linear portion. Peptides generally have poor metabolic stability in vivo, and their large size makes them difficult to penetrate cell membranes. To address these issues, peptide cyclization has been used. Cyclization of peptides improves protease resistance and metabolic stability, and also restricts conformational changes, increasing their rigidity, suggesting improved membrane permeability and affinity for target proteins.

[0072] cyclization method Peptide cyclization can be carried out according to known methods. For example, but not limited to, by designing a peptide to contain two or more cysteine ​​residues, a cyclic structure can be formed by disulfide bonds after translation. Alternatively, cyclization can be achieved by synthesizing a peptide with a chloroacetyl group at the N-terminus and placing a cysteine ​​residue in the peptide using genetic code reprogramming technology, according to the method of Goto et al. (Y. Goto, et al. Acss Chem. Biol. 3 120-129 (2008)). This allows spontaneous nucleophilic attack of the mercapto group on the chloroacetyl group after translation, resulting in cyclization of the peptide through a thioether bond. Cyclization can also be achieved by placing other combinations of amino acids in the peptide that bond to form a ring using genetic code reprogramming technology. Cyclization can also be achieved by synthesizing a peptide with a cycloamide at the N-terminus and placing an Hgl residue in the peptide. As described above, any known cyclization method can be used without particular limitations.

[0073] A preferred example of this peptide is any of the above-mentioned peptides, which consists of 15 amino acid residues.

[0074] Peptide length The number of amide bonds (number of amino acids, length) in the peptide or peptide portion is not particularly limited, but the total number of amino acid residues (excluding amino acids in cases where the substance bound to the peptide or the linker connecting the substance to the peptide contains amino acids) is preferably 20 or less. Preferably, the number of amino acids is 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more, and more preferably, the number of amino acids is 19 or less, 18 or less, 17 or less, 16 or less, or 15 or less.

[0075] An invention described in this specification relates to a conjugate (complex). This complex comprises any of the peptides described above, a linker bound to the peptide, and a substance bound to the linker. It is preferable that at least the substance is a complex that can pass through the blood-brain barrier. The complex as a whole may be able to pass through the blood-brain barrier. It is preferable that the complex has tropism toward muscle tissue. It is preferable that the complex is at least capable of transporting the substance to muscle tissue. It is preferable that the complex has cell permeability. It is preferable that the complex is at least capable of transporting the substance to cells.

[0076] Examples of linkers include those having an amino acid length of 1 to 15, and containing one or more glycines (Gly) or serines (Ser). A preferred example of this linker is an optionally modified cysteine ​​(Cys) or optionally modified lysine (Lys) at the N-terminus.

[0077] Another example of a linker is one having a length of 1 to 5 amino acids, and containing either or both of D-glutamic acid (de) and methylated glycine (MeG). A preferred example of this linker is a conjugate in which the N-terminus is an optionally modified cysteine ​​(Cys) or an optionally modified lysine (Lys).

[0078] Another example of a linker is a PEG linker that includes polyethylene glycol (PEG) or a derivative of polyethylene glycol. The derivative of polyethylene glycol includes all of those known as PEG linkers. Preferably, the PEG linker further comprises glycine (Gly), serine (Ser), glutamic acid (Glu), arginine (Arg), or lysine (Lys). A preferred example of this linker is a conjugate in which the N-terminus is an optionally modified cysteine ​​(Cys) or an optionally modified lysine (Lys).

[0079] Another example of a linker is a linker having a sequence shown by any of SEQ ID NOs: 201, 553-644.

[0080] A preferred example of this conjugate is one in which the linker is Polyethylene glycol (PEG), G linker, which is a peptide linker consisting of Gly or MeG; GS linker, which is a peptide linker consisting of Gly or MeG and Ser; Or, it is a linker having an amino acid sequence shown in any one of SEQ ID NOs: 201, 553 to 644.

[0081] As used herein, a linker (also referred to as a crosslinker) refers to an intermolecular link between a peptide that binds to a transferrin receptor and a substance to be delivered to the brain, and may be any linker known or described herein. In certain embodiments, the linker is, for example, a chemical linker, a fatty acid linker, or a peptide linker (polypeptide linker). It may also be a complex of, for example, a chemical linker and a peptide linker. For example, it may be a linker structure having both PEG and an amino acid residue or a peptide moiety, such as those shown in SEQ ID NO: 616 or Sequence Listing 627. The linker may be, for example, one that dissociates or separates depending on the environment or conditions, or one that maintains a stable structure.

[0082] Chemical Linkers: In some embodiments, the linker may be a chemical linker. Examples of chemical linkers include, but are not limited to, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, and / or substituted or unsubstituted heteroarylene. Peptides and linkers can also be conjugated via sulfhydryl groups, amino groups (amines), and / or carbohydrates or any suitable reactive group. Homobifunctional and heterobifunctional cross-linkers (conjugation agents) are available from many commercial sources. Cross-linkers may contain flexible arms, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms. Examples of cross-linkers include BSS3 (bis(sulfosuccinimidyl)suberate), NHSS / EDC (N-hydroxysuccinimide and N-ethyl-(dimethylaminopropyl)carbodiimide), sulfo-EMCSS (Ne-maleimidocaproic acid) hydrazide, and SSATA (N-succinimidyl-SS-acetylthioacetate).

[0083] A preferred example of a chemical linker is a PEG (Polyethyleneglycol) linker. For example, the PEG linker may be a PEG linker consisting of 1 to 24 ethylene glycol units.

[0084] Fatty acid linker: The linker may be a fatty acid linker that includes a divalent chemical moiety derived from a fatty acid. For example, the fatty acid linker may be a linker having 12-aminododecanoic acid.

[0085] Peptide Linker: A peptide linker comprises at least one amino acid (e.g., a peptide of at least 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 40, or 50 amino acids). In certain embodiments, the linker is a single amino acid (e.g., any naturally occurring amino acid, such as Cys). In other embodiments, glycine-rich peptides are used, such as peptides having the sequence [Gly-Gly-Gly-Gly-Ser]n, where n is 1, 2, 3, 4, 5, or 6, as described in U.S. Pat. No. 7,271,149. In other embodiments, serine-rich peptide linkers are used, such as those described in U.S. Pat. No. 5,525,491. Serine-rich peptide linkers include those of the formula [XXXX-Gly]y, where up to two of X are Thr, the remaining X are Ser, and y is 1 to 5 (e.g., Ser-Ser-Ser-Ser-Gly, where y is 2 or greater). In some cases, the linker is a single amino acid (eg, any amino acid such as Gly or Ala).

[0086] An invention described in this specification relates to a preventive or therapeutic agent for brain-related diseases. This preventive or therapeutic agent for brain-related diseases contains the above-mentioned complex, and the above-mentioned substance is an active ingredient.

[0087] The substance is a substance to be delivered to the brain. The substance may be any substance desired by a person skilled in the art, as long as it is a substance desired to be delivered to the brain. However, since passage through the BBB is by binding to transferrin receptors and by mechanisms such as endocytosis and transcytosis, substances that are too large to be transported by these mechanisms are not preferred. Examples of such substances include, but are not limited to, the following: Compounds: Not only low-molecular-weight compounds and medium-molecular-weight compounds, but also any compounds that can be introduced by the cytosis mechanism of cells are acceptable, including known low-molecular-weight drugs. Peptide: It may be a peptide that binds to a target in the body and exerts some effect, for example, it may be a cyclic peptide. RI: Any compound that can be labeled with a radioisotope, such as a small or medium molecule compound or antibody labeled with a radioisotope. For example, compounds used in PET scans can be used. Protein: Any protein that has a useful function in the body, such as an antibody or enzyme, for example, an enzyme used in enzyme replacement therapy. Nucleic acid: Anything that contains a base sequence, such as DNA or RNA. Examples include nucleic acid drugs. DDS: May be a DDS molecule such as a liposome or micelle. The DDS molecule may further contain a compound such as a pharmaceutical agent. and may be a composite of any of the above.

[0088] An invention described in this specification relates to a method for producing a preventive or therapeutic agent for brain-related diseases, which method comprises the step of obtaining the above-mentioned complex.

[0089] A preferred example of this method is one in which the linker is Polyethylene glycol (PEG), G linker, GS linker, Alternatively, it is a linker having an amino acid sequence shown in any one of SEQ ID NOs: 201, 553 to 644.

[0090] One invention described in this specification relates to a diagnostic agent for brain-related diseases, which comprises the above-mentioned complex.

[0091] An invention described in this specification is a preventive or therapeutic agent for neuromuscular diseases, comprising the above-mentioned complex. In this case, the substance is an active ingredient in the preventive or therapeutic agent for neuromuscular diseases. An invention described in this specification comprises the above-mentioned complex. An invention described in this specification relates to a diagnostic agent for neuromuscular diseases, comprising the above-mentioned complex.

[0092] The peptides of the present invention can be produced by known methods for producing peptides, such as chemical synthesis methods including liquid phase methods, solid phase methods, and hybrid methods combining liquid phase and solid phase methods; and genetic recombination methods.

[0093] In the solid-phase method, for example, the hydroxyl group of a hydroxyl-containing resin is esterified with the carboxyl group of a first amino acid (usually the C-terminal amino acid of the target peptide) whose α-amino group is protected with a protecting group. Known dehydration condensation agents such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIPCDI) can be used as esterification catalysts. Next, the protecting group on the α-amino group of the first amino acid is removed, and a second amino acid in which all functional groups except the carboxy group in the main chain are protected is added, the carboxy group is activated, and the first and second amino acids are linked. Furthermore, the α-amino group of the second amino acid is deprotected, and a third amino acid in which all functional groups except the carboxy group in the main chain are protected is added, the carboxy group is activated, and the second and third amino acids are linked. This process is repeated until a peptide of the desired length is synthesized, and then all functional groups are deprotected.

[0094] Resins for solid-phase synthesis include Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGA resin (Merck), HMPA-PEGA resin (Merck), etc. These resins can be used after washing with a solvent (dimethylformamide (DMF), 2-propanol, methylene chloride, etc.). Examples of the protecting group for the α-amino group include a benzyloxycarbonyl (Cbz or Z) group, a tert-butoxycarbonyl (Boc) group, a fluorenylmethoxycarbonyl (Fmoc) group, a benzyl group, an allyl group, and an allyloxycarbonyl (Alloc) group. The Cbz group can be deprotected by hydrofluoric acid, hydrogenation, etc., the Boc group can be deprotected by trifluoroacetic acid (TFA), and the Fmoc group can be deprotected by treatment with piperidine. The α-carboxy group can be protected using methyl ester, ethyl ester, benzyl ester, tert-butyl ester, cyclohexyl ester, etc. Other functional groups of amino acids include the hydroxyl groups of serine and threonine, which can be protected with benzyl or tert-butyl groups, the hydroxyl group of tyrosine with 2-bromobenzyloxycarbonyl or tert-butyl groups, the amino group of the lysine side chain, and the carboxyl groups of glutamic and aspartic acids, which can be protected as well as the α-amino and α-carboxyl groups.

[0095] Activation of the carboxy group can be carried out using a condensation agent, such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), or 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU).

[0096] Cleavage of the peptide chain from the resin can be carried out by treating with an acid such as TFA or hydrogen fluoride (HF).

[0097] The production of peptides by genetic recombination (translation synthesis) can be carried out using nucleic acids encoding the peptides of the present invention. The nucleic acids encoding the peptides of the present invention may be DNA or RNA. Nucleic acids encoding the peptides of the present invention can be prepared by known methods or methods equivalent thereto. For example, they can be synthesized using an automatic synthesizer. Restriction enzyme recognition sites may be added to insert the resulting DNA into a vector, or a base sequence encoding an amino acid sequence for excising the resulting peptide chain with an enzyme or the like may be incorporated. As mentioned above, when the peptide of the present invention is fused with a membrane-permeable peptide or the like, the nucleic acid also includes a nucleic acid encoding the membrane-permeable peptide. To prevent degradation by proteases derived from the host, a chimeric protein expression method can be used in which the peptide of interest is expressed as a chimeric peptide with another peptide. In this case, the nucleic acid used is a nucleic acid encoding the peptide of interest and a peptide that binds to it.

[0098] Next, an expression vector is prepared using a nucleic acid encoding the peptide of the present invention. The nucleic acid can be inserted downstream of the promoter of the expression vector either directly or after digestion with a restriction enzyme or the addition of a linker. Examples of vectors include E. coli-derived plasmids (pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pUC118, pBluescript II, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, pC1912, pTP4, pE194, pC194, etc.), yeast-derived plasmids (pSH19, pSH15, YEp, YRp, YIp, YAC, etc.), bacteriophages (e phage, M13 phage, etc.), viruses (retrovirus, vaccinia virus, adenovirus, adeno-associated virus (AAV), cauliflower mosaic virus, tobacco mosaic virus, baculovirus, etc.), and cosmids.

[0099] The promoter can be selected appropriately depending on the type of host. When the host is an animal cell, for example, a promoter derived from SV40 (simian virus 40) or a promoter derived from CMV (cytomegalovirus) can be used. When the host is Escherichia coli, a trp promoter, T7 promoter, lac promoter, etc. can be used. Expression vectors can also incorporate nucleic acids encoding DNA replication origins (ori), selection markers (antibiotic resistance, auxotrophy, etc.), enhancers, splicing signals, poly(A) addition signals, tags (FLAG, HA, GST, GFP, etc.), etc.

[0100] Next, suitable host cells are transformed with the expression vector. The host can be selected appropriately in relation to the vector, and for example, Escherichia coli, Bacillus subtilis, Bacillus bacteria, yeast, insects or insect cells, animal cells, etc. can be used. Examples of animal cells that can be used include HEK293T cells, CHO cells, COS cells, myeloma cells, HeLa cells, and Vero cells. Transformation can be carried out according to known methods such as lipofection, calcium phosphate method, electroporation, microinjection, and particle gun method, depending on the type of host. The target peptide is expressed by culturing the transformant according to standard methods.

[0101] To purify peptides from the culture of a transformant, the cultured cells are harvested, suspended in an appropriate buffer, disrupted by ultrasonication or freeze-thawing, and then centrifuged or filtered to obtain a crude extract. If the peptide is secreted into the culture medium, the supernatant is collected. Purification from crude extracts or culture supernatants can also be carried out by known methods or methods equivalent thereto (e.g., salting out, dialysis, ultrafiltration, gel filtration, SDS-PAGE, ion exchange chromatography, affinity chromatography, reverse-phase high performance liquid chromatography, etc.). The obtained peptide may be converted from a free form to a salt, or from a salt to a free form, by a known method or a method similar thereto.

[0102] The translation synthesis system may be a cell-free translation system. The cell-free translation system contains, for example, ribosomal proteins, aminoacyl-tRNA synthetases (ARSs), ribosomal RNAs, amino acids, rRNAs, GTP, ATP, translation initiation factors (IFs), elongation factors (EFs), release factors (RFs), and ribosome recycling factors (RRFs), as well as other factors necessary for translation. Escherichia coli extract or wheat germ extract may be added to increase expression efficiency. Additionally, rabbit erythrocyte extract or insect cell extract may also be added. By continuously supplying energy to a system containing these via dialysis, it is possible to produce protein in amounts ranging from several hundred μg to several mg / mL. A system containing RNA polymerase may also be used to simultaneously perform transcription from genetic DNA. Commercially available cell-free translation systems include E. coli-based systems such as Roche Diagnostics' RTS-100®, Gene Frontier's PURESYSTEM, and New England Biolabs' PURExpress In Vitro Protein Synthesis Kit, as well as wheat germ extract-based systems such as those from Zoigene and Cell Free Sciences. Using a cell-free translation system, expression products can be obtained in a highly pure form without purification.

[0103] In a cell-free translation system, instead of the aminoacyl-tRNA synthesized by a natural aminoacyl-tRNA synthetase, an artificial aminoacyl-tRNA in which a desired amino acid or hydroxy acid is linked (acylated) to a tRNA may be used. Such an aminoacyl-tRNA can be synthesized using an artificial ribozyme. Such ribozymes include flexizyme (H. Murakami, H. Saito, and H. Suga, (2003), Chemistry & Biology, Vol. 10, 655-662; H. Murakami, D. Kourouklis, and H. Suga, (2003), Chemistry & Biology, Vol. 10, 1077-1084; Murakami, A. Ohta, H. Ashigai, H. Suga (2006) Nature Methods 3, 357-359 “The flexizyme system: a highly flexible tRNA aminoacylation tool for the synthesis of nonnatural peptides”;N. Niwa, Y. Yamagishi, H. Murakami, H. Suga (2009) Bioorganic & Medicinal Chemistry Letters 19, 3892-3894 “A Examples of such compounds include "flexizyme that selectively charges amino acids activated by a water-friendly leaving group"; and WO2007 / 066627. Flexizyme is also known as the original flexizyme (Fx), and modified forms such as dinitrobenzyl flexizyme (dFx), enhanced flexizyme (eFx), and aminoflexizyme (aFx).

[0104] By using tRNA produced by Flexizyme and linked to a desired amino acid or hydroxy acid, a desired codon can be translated in association with the desired amino acid or hydroxy acid. A non-standard amino acid may also be used as the desired amino acid. For example, the unnatural amino acid required for the above-mentioned cyclization can also be introduced into the linked peptide using this method.

[0105] The macrocyclic peptides and analogs of the present invention can be chemically synthesized using a variety of methods commonly used in the art, including stepwise solid-phase synthesis, semisynthesis of peptide fragments via conformationally assisted religation, and chemical ligation. The peptides and analogs described herein are synthesized using various solid-phase techniques, such as those described in KJ Jensen, PT Shelton, and SL Pedersen, Peptide Synthesis and Applications, 2nd Edition, Springer, 2013. A preferred strategy is based on the combination of the Fmoc group, which temporarily protects the α-amino group and allows selective base removal, with a protecting group that temporarily protects side chain functional groups and is stable under Fmoc deprotection conditions. Such general peptide side chain selection is known from the aforementioned Peptide Synthesis and Applications, 2nd edition, and GB Fields, RL Noble, Solid Phase Peptide Synthesis Utilizing 9-Fluorenylmethoxycarbonyl Amino Acids, Int. J. Peptide Protein Res. 35, 1990, 161-214, among others. Preferred peptide side chain protecting groups include the Boc group and Mtt group for amino groups such as lysine, the tert-butyl group for carboxyl groups of glutamic acid and aspartic acid, and the Trt and Mmt groups for thiol groups of cysteine.

[0106] The peptides and their analogs described in this invention can be synthesized in a stepwise manner on the solid-phase resin described above. The α-amino protecting groups of the C-terminal amino acid used and all amino acids and peptides used in the synthesis must be selectively removed during the synthesis process. Preferably, the solid-phase resin described above is used, and the synthesis begins by converting the C-terminal carboxyl group of a peptide whose N-terminus is appropriately protected by Fmoc or the C-terminal carboxyl group of an amino acid protected with Fmoc into an activated ester with an appropriate reagent, followed by addition to the amino group on the solid-phase resin. Subsequent peptide chain elongation can be achieved by sequentially repeating the removal of the N-terminal protecting group (Fmoc group) and the condensation of the protected amino acid derivative according to the amino acid sequence of the desired peptide. Furthermore, this allows the desired peptide to be released at the final stage. For example, the release can be achieved using a TFA solution containing water, silyl hydride, and thiol as scavengers in TFA, as described in Teixeira, WE Benckhuijsen, PE de Koning, ARPM Valentijn, and JW Drijfhout, Protein Pept. Lett., 2002, 9, 379-385. A typical example is TFA / Water / TIS / DODT (volume ratio 92.5:2.5:2.5:2.5).

[0107] Synthesis of the peptide analogs described herein can be carried out using a single or multi-channel peptide synthesizer, such as a CEM Liberty Blue synthesizer or a Biotage Syro I synthesizer.

[0108] Activation of the carboxy group can be carried out using a condensation agent, such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), or 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU).

[0109] Another embodiment disclosed in this specification relates to a pharmaceutical. This pharmaceutical contains the above-mentioned peptide, or a pharmaceutically acceptable salt or solvate thereof (for simplicity, these will hereinafter also be referred to simply as peptide). This pharmaceutical preferably contains an effective amount of the above-mentioned peptide as an active ingredient.

[0110] In this specification, the administration form of the pharmaceutical composition is not particularly limited, and may be oral or parenteral. Examples of parenteral administration include injection administration such as intramuscular injection, intravenous injection, and subcutaneous injection, transdermal administration, and transmucosal administration (nasal, oral, ocular, pulmonary, vaginal, and rectal) administration.

[0111] The above pharmaceutical compositions can be modified in various ways, taking into account the tendency of polypeptides to be easily metabolized and excreted. For example, polyethylene glycol (PEG) or sugar chains can be added to the polypeptide to increase its blood circulation time and reduce its antigenicity. Furthermore, the polypeptide can be encapsulated in biodegradable polymer compounds such as polylactic acid glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, nanospheres, etc., which are used as sustained-release bases. For transdermal administration, a weak electric current can be applied to the skin surface to penetrate the stratum corneum (iontophoresis).

[0112] The above pharmaceutical compositions may use the active ingredient as is, or may be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc. Dosage forms include, for example, liquids (e.g., injections), dispersions, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, lozenges, inhalants, ointments, eye drops, nasal drops, ear drops, and poultices. Formulation can be carried out in a conventional manner using, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavorings, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, etc. as appropriate. Examples of ingredients used in formulations include, but are not limited to, purified water, saline, phosphate buffer, dextrose, glycerol, ethanol and other pharmaceutically acceptable organic solvents, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, anhydrous silicic acid, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, tragacanth, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, human serum albumin, etc. In view of the poor transmucosal absorption of peptides, the pharmaceutical composition may contain an absorption enhancer that improves the absorption of poorly absorbed drugs. Examples of such absorption enhancers include surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponin; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylic acids; fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, and mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, and nitric oxide donors.

[0113] The pills or tablets may also be coated with sugar, gastric or enteric materials. The injection may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. Furthermore, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, solubilizing agents, preservatives, etc. may be added.

[0114] The dosage when the pharmaceutical composition of the present invention is administered to mammals (e.g., humans, mice, rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs, etc.), particularly humans, varies depending on the symptoms, the patient's age, sex, weight, sensitivity, administration method, administration interval, type of active ingredient, and type of formulation, and is not particularly limited, but can be, for example, 30μg to 100g, 100μg to 500mg, or 100μg to 100mg, administered once or in divided doses. When administered by injection, 1μg / kg to 3000μg / kg or 3μg / kg to 1000μg / kg may be administered once or in divided doses, depending on the patient's weight.

[0115] The method for preventing or treating brain-related diseases using the peptide of the present invention can be carried out with reference to the description of the pharmaceutical composition above.

[0116] The pharmaceutical composition may be used as a preventive or therapeutic agent for neuromuscular diseases containing the above-mentioned complex.

[0117] Another embodiment disclosed in this specification relates to a drug for detecting brain-related diseases, which comprises the above-mentioned peptide, a salt thereof, or a solvate thereof.

[0118] Brain-related disease detection agents and detection kits The present invention also encompasses agents for detecting brain-related diseases that contain the peptides of the present invention. When used as detection agents, the peptides of the present invention may be detectably labeled. Examples of peptide labels include antibodies labeled with enzymes such as peroxidase and alkaline phosphatase; radioactive substances such as 125I, 131I, 35S, and 3H; fluorescent substances such as fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine isothiocyanate, and near-infrared fluorescent materials; and luminescent substances such as luciferase, luciferin, and aequorin. Antibodies labeled with nanoparticles such as gold colloids and quantum dots can also be detected. For example, brain-related diseases can be detected by preparing a complex between an antibody that binds to a specific target related to brain-related diseases and the peptides of the present invention, and then administering and detecting the complex. In immunoassays, the peptides of the present invention can also be labeled with biotin and detected by binding avidin or streptavidin labeled with an enzyme or the like. Among immunoassays, the ELISA method using enzyme labeling is preferred because it allows for simple and rapid antigen measurement. For example, an antibody is immobilized on a solid support, a sample is added and allowed to react, and then a labeled peptide of the present invention is added and allowed to react. After washing, the antibody is reacted with an enzyme substrate to develop color, and the absorbance is measured, thereby enabling the detection of brain-related diseases. After reacting the antibody immobilized on the solid support with the sample, an unlabeled peptide of the present invention may be added, and then an antibody against the peptide of the present invention may be enzyme-labeled and further added. When the enzyme is peroxidase, 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), etc. can be used as the enzyme substrate, and when the enzyme is alkaline phosphatase, p-nitrophenyl phosphate (NPP), etc. can be used.

[0119] In this specification, the term "solid phase carrier" is not particularly limited as long as it is a carrier capable of immobilizing an antibody, and examples include glass, metal, resin microtiter plates, substrates, beads, nitrocellulose membranes, nylon membranes, PVDF membranes, etc., and the target substance can be immobilized on these solid phase carriers according to known methods.

[0120] The test kit according to the present invention includes reagents and instruments necessary for the above detection (including, but not limited to, the peptide of the present invention, an antibody, a solid phase carrier, a buffer solution, an enzyme reaction stopping solution, a microplate reader, etc.).

[0121] Other embodiments disclosed in this specification may also be considered for use as kits for detecting brain-related diseases containing the above-mentioned brain-related disease detection drugs, or as tools for elucidating brain-related diseases and the various cellular functions and life phenomena associated with them.

[0122] This specification also provides use of a peptide for producing a medicament for preventing or treating a brain-related disease, in which the peptide may be any of those described above.

[0123] This specification also provides use of a peptide for the manufacture of a medicament for the prevention or treatment of a neuromuscular disease, in which case the peptide may be any of those described above.

[0124] The present specification also provides a method for preventing or treating brain-related diseases, comprising the step of administering an effective amount of a peptide, its pharmaceutically acceptable salt, or its solvate or complex to a human, non-human mammal, or avian subject. The peptide may be any of those described above, as appropriate. Examples of non-human mammals include non-human primates, pigs, cows, dogs, cats, horses, sheep, rats, and mice.

[0125] This specification also provides a method for preventing or treating brain-related diseases, comprising the step of administering to a subject, which is a human, non-human mammal, or bird, an effective amount of a peptide, a pharmaceutically acceptable salt thereof, or a solvate or complex thereof.

[0126] The abbreviations used herein, and particularly in the representative examples below, will be familiar to those of ordinary skill in the art. Some abbreviations used are as follows: Fmoc as 9-fluorenylmethyloxycarbonyl; HOAt as 1-hydroxybenzotriazole; HATU as O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; MeCN as acetonitrile; 1,8-diazabicyclo[5.4.0]-7-undecene as DBU; DIPEA as N,N-diisopropylethylamine; DODT as 3,6-dioxa-1,8-octan-dithiol; DMSO as dimethyl sulfoxide; DMF as N,N-dimethylformamide; Mtt as monomethyltrityl; Mmt as monomethoxytrityl; o-Ns as 2-nitrobenzenesulfonyl; TFA as trifluoroacetic acid; TIS as triisopropylsilane; Trityl as Trt milliliters (units) as mL; M as molar (unit); volume / volume as v / v; Adod as 12-aminododecanoic acid. [Example]

[0127] chemical synthesis All raw materials, building blocks, reagents, acids, bases, solid-phase resins, and solvents used in the chemical synthesis in the following examples were either commercially available or could be synthesized by those skilled in the art using organic chemistry techniques. Unless otherwise specified, commercially available amino acids containing protecting groups were used as they were.

[0128] Peptide chain elongation on solid-phase resins was carried out using the resins described in each example as starting materials under commonly used peptide coupling reaction conditions and Fmoc removal reaction conditions. The reactions were carried out using a CEM Liberty Blue automated peptide synthesizer according to the manufacturer's instructions. Common amino acids used are listed below, with side chain protecting groups indicated in parentheses.

[0129] Fmoc-Trp(Boc)-OH;Fmoc-Thr(tBu)-OH;Fmoc-N-Me-Gly-OH;Fmoc-Asp(OtBu)-OH;Fmoc-N-Me-Phe-OH;Fmoc-Ala-OH;Fmoc-N-Me-Ala-OH;Fmoc-His(Trt )-OH;Fmoc-Tyr(tBu)-OH;Fmoc-Val-OH;Fmoc-HydPro(tBu)-OH;Fmoc-Cys(Trt)-OH;Fmoc-Lys(Mtt)-OH;Fmoc-Ser(tBu)-OH;Fmoc-N-Me-Ser(tBu)-OH.

[0130] The chloroacetyl group was introduced by removing the Fmoc group from the α-amino group of the solid-phase resin carrying the Fmoc-protected peptide obtained in the previous step using the method described above, then adding chloroacetic acid (3 equivalents) to a DMF solution (0.5 M) of 3 equivalents of N,N'-diisopropylcarbodiimide and a DMF solution (0.5 M) of 3 equivalents of HOAt, and shaking the mixture at room temperature for 40 minutes.

[0131] To deprotect the side chain and cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and methylene chloride, respectively, and then dried under reduced pressure. Reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was then added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 150 minutes. The reaction solution was collected by filtration through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were collected through a frit and mixed with the filtrate. This filtrate was added to excess diethyl ether chilled to 0°C, resulting in the formation of a cloudy white precipitate. This mixture was centrifuged (9000 rpm, 3 min), and the solution was decanted. The resulting solid was washed again with a small amount of diethyl ether chilled to 0°C, and the resulting solid was used in the subsequent cyclization reaction.

[0132] The peptide cyclization reaction was carried out by dissolving the peptide in DMSO to a final concentration of 5 mM based on the molar amount of the solid-phase resin, adding 6 equivalents of triethylamine, and stirring at room temperature for approximately 16 hours. The resulting reaction solution was acidified with acetic acid and concentrated under reduced pressure using a Biotage® V-10 (Biotage Japan).

[0133] The resulting crude peptide was purified using reversed-phase preparative HPLC with a Waters AutoPurification System-SQD2 single quadruple mass spectrometer, and elution was performed while monitoring the m / z ions derived from the target compound. It was confirmed that the mass spectrum obtained in ESI-positive scan mode and the mass spectrum containing multiply charged ions calculated from the molecular formula of the target compound matched within the error range of the mass analyzer used. The purification conditions, including the column used, are shown in each example.

[0134] The purity of the chemically synthesized peptides of the present invention was determined by one of the following analytical methods. (Analysis conditions) Analysis conditions A Column: CORTECS® UPLC® C18 column (Nihon Waters), 90 Å, 1.6 μm, 2.1 x 100 mm Mobile phase: 0.025%TFA inMeCN / 0.025%TFA inH2O Temperature: 40℃ Gradient: 5-95% MeCN / 0.025% TFA in H2O in 5.56 min; linear gradient Flow rate 0.4mL / min Detection method: UV 220nm

[0135] Analysis conditions B Column: Kinetex EVO C18 2.6 μm, 2.1 ID x 150 mm, 100 Å (Phenomenex) Column temperature: 60℃ Mobile phase A: 0.025% TFA in H2O Mobile phase B:0.025%TFA in CH3CN Gradient: described in each example Flow rate: 0.25mL / min Detection: PDA (225 nm)

[0136] The structure of the chemically synthesized peptides was determined by ESI-MS(+) mass spectrometry, where the molecular weight was calculated based on the amino acids used in the target sequence and the building blocks used as needed. "ESI-MS(+)" refers to electrospray ionization mass spectrometry performed in positive ion mode. Detected masses were reported in m / z units. Compounds with molecular weights greater than approximately 1000 were frequently detected as doubly or triply charged ions.

[0137] Chemical synthesis of unique cyclic peptides that bind to hTfR hTfR-binding peptides were identified using the screening methods described in International Publication No. WO 2014 / 119600, International Publication No. WO 2012 / 033154, or International Publication No. WO 2007 / 066627. These peptides were chemically synthesized to confirm their binding activity to hTfR. The sequences of the synthesized peptides are shown in Table 1.

[0138] [Table 1]

[0139] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.6 mmol / g, 0.33 g) according to the general method described above, starting with the removal of Fmoc. The synthesis was performed using a CEM Liberty Blue HT solid-phase synthesizer, following the manufacturer's instructions. The condensation reaction was carried out at 75°C for 10 minutes. The Fmoc removal reaction was carried out in 20% piperidine in DMF at 75°C for 3 minutes. The chloroacetyl group was introduced by removing the Fmoc group from the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, followed by the addition of 5 equivalents of a 0.2 M DMF solution of chloroacetic acid, 5 equivalents of a 0.5 M DMF solution of HATU, and 10 equivalents of a 1 M DMF solution of DIPEA, followed by shaking at room temperature for 30 minutes. To deprotect the side chain and cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride and dried under reduced pressure. Next, a reagent cocktail (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction solution was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate. This filtrate was added to an excess of diethyl ether / hexane (1 / 1) chilled to 0°C, resulting in the formation of a cloudy white precipitate. This mixture was centrifuged (10,000 rpm, 1 min), and the solution was decanted. The resulting solid was washed again with a small amount of diethyl ether chilled to 0°C, dried, and used in the subsequent cyclization reaction. The peptide was dissolved in DMSO to a final concentration of 5 mM based on the molar amount of the solid-phase resin, and then 6 equivalents of triethylamine was added. The mixture was stirred at room temperature for approximately 16 hours. The resulting reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVac.

[0140] The resulting crude product was purified using the following conditions (column: Waters Xbridge® C18 5 μmμm® 50 × 150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (% B): 5-30% over 3 min, then 30-35% over 8 min, then 35-60% over 1 min; flow rate: 120 mL / min).

[0141] The target product was analyzed under the above-mentioned analytical conditions A or B, and its structure was confirmed by ESI-MS(+) in mass spectrometry. The ESI-MS(+) observed values ​​and the value of X when expressed as the number of protons added (M+XH)X+ are shown in Table 1. [Example]

[0142] Surface plasmon resonance (SPR) assay for evaluating the molecular interaction between human transferrin receptor (hTfR) and peptides The various synthesized peptides were tested for intermolecular interactions with the transferrin receptor (hTfR) by surface plasmon resonance (SPR) using the method described below. The specific test method is shown below.

[0143] [SPR measurement] An NTA sensor chip (Global Life Science Technologies Japan, Inc.) was inserted into a Biacore T200 (Global Life Science Technologies Japan, Inc.) and primed three times with running buffer: 10 mM HEPES pH 8.0 (Nacalai Tesque, Inc.), 150 mM NaCl (Nacalai Tesque, Inc.), 0.05% Tween 20 (Nacalai Tesque, Inc.), 0.1% BSA (Sigma-Aldrich), 1.0% DMSO (Fujifilm Wako Pure Chemical Industries, Ltd.). The NTA sensor chip was equilibrated at a flow rate of 30 μL / min. The eluate was then incubated with 350 mM EDTA solution at a flow rate of 10 μL / min for 60 seconds, followed by 0.5 mM NiCl solution (Kishida Chemical) at a flow rate of 10 μL / min for 60 seconds. The NTA sensor chip was then washed with 3 mM EDTA solution (Nacalai Tesque, Inc.) at a flow rate of 10 μL / min for 60 seconds. 50 μL of 60 mM EDC solution (Global Life Science Technologies Japan, Inc.) and 650 mM NHS solution (Global Life Science Technologies Japan, Inc.) were mixed and incubated at a flow rate of 10 μL / min for 420 seconds. 150 μL of a 3.2 μM hTfR solution was prepared by dilution with running buffer and incubated at a flow rate of 10 μL / min for 600 seconds to immobilize hTfR on an NTA sensor chip. After immobilization, capping was performed by incubating with 1.0 M ethanolamine solution (Global Life Science Technologies Japan, Inc.) at a flow rate of 10 μL / min for 420 seconds. A 10 mM peptide solution in DMSO was diluted with running buffer to a final peptide concentration of 10 μM, and peptide solutions of 100 nM, 50 nM, 25 nM, 10 nM, and 5 nM were prepared. Using the above samples, the kinetics of peptide binding to hTfR were measured by SPR. The kinetics evaluation model was Single Cycle Kinetics, and curve fitting was performed using Biacore T200 Evaluation Software Version 3.0 (Global Life Science Technologies Japan, Inc.).The resulting sensorgrams were subjected to curve fitting using the least squares method, and the KD values ​​were calculated to evaluate the binding of the peptides to hTfR. The KD values ​​calculated in this manner are shown in Table 1. A indicates a KD value of less than 1 nM, B indicates a KD value of 1 nM to less than 100 nM, C indicates a KD value of 100 nM to less than 1 μM, and D indicates a KD value of 1 μM or greater. The results demonstrated that hTfR No. 894 and cyclic peptides with similar amino acid sequences have significant binding ability to hTfR. The chemical structure of hTfR No. 894 is shown below.

[0144] [ka] [Example]

[0145] Synthesis of hTfR-binding peptide-PEG11-Vivotag750 conjugate (hTfR No. 894-vivotag750) A compound was synthesized for hTfR No. 894 in which the near-infrared fluorescent labeling substance Vivotag750 (VivoTag-S™ 750, PerkinElmer) was attached as the payload via a PEG11 linker (hTfR No. 894-vivotag750 or hTfR_000894_PEG11_(VivoTag)) (SEQ ID NO: 146). The chemical structure of the hTfR-binding peptide-PEG11 is shown below, and the indicated compound was synthesized by attaching Vivo-tag750 to this compound. Details are described below. [ka]

[0146] Synthesis of hTfR No. 894-PEG11-vivotag750 The chemical synthesis was carried out as follows: The target peptides were synthesized using Fmoc-NH-SAL-PEG-resin 1500-2000 Da (Watanabe Chemical, 0.38 mmol / g) according to standard procedures, starting with Fmoc removal. A CEM Liberty Blue solid-phase synthesizer was used, following the manufacturer's instructions. The condensation reaction was performed using HATU as the condensing agent, with a single reaction time of 10 min at 75 °C. However, the 11th and 12th residues were reacted twice for 20 min at 25 °C. The 13th and 14th residues were reacted twice for 10 min at 75 °C. The 15th residue was reacted once for 20 min at 25 °C. The 16th residue was reacted once for 60 min at 25 °C. The Fmoc removal reaction was performed with a 20% piperidine solution in DMF at 75 °C for 3 min. However, for residues 13 and 15, Fmoc removal was performed for 5 minutes at 25°C, followed by 10 minutes of reaction. Chloroacetyl group introduction was performed by adding 5 equivalents of a 0.2 M DMF solution of chloroacetic acid, 5 equivalents of a 0.5 M DMF solution of HATU, and 10 equivalents of a 1 M DMF solution of DIPEA to the resin obtained in the previous step and shaking at room temperature for 30 minutes. To deprotect the side chain and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride and dried under reduced pressure. Reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was then added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction mixture was collected by filtration through a frit. The solid phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through the frit and mixed with the filtrate described above. When this filtrate was added to an excess of a mixed solvent of diethyl ether and hexane cooled to 0°C, a cloudy white precipitate formed. This mixture was centrifuged (10,000 rpm, 1 min), and the supernatant was decanted. The solid was washed with ice-cold diethyl ether and then dried. The resulting solid was used in the subsequent cyclization reaction. The peptide was dissolved in DMSO to a final peptide concentration of 5 mM based on the molar number of the solid-phase resin, and then 6 equivalents of triethylamine were added. The mixture was shaken overnight at room temperature, concentrated under reduced pressure in a Savant Explorer SpeedVac, and purified by reverse-phase HPLC. The resulting peptide (26.1 mg, 9.30 μmol) was dissolved in DMSO / HO (9 / 1), and 0.91 equivalents of VivoTag-NHS and 4.5 equivalents of DIEA were added and stirred for 45 minutes. The reaction solution was quenched by adding AcOH.

[0147] The resulting reaction mixture was purified using the following conditions: column: XSelect CSH C18 5 μm 10 × 150 mm (Lot No. 147i362781); mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (%B): 7–32% over 3 minutes, then 32–37% over 8 minutes, then 37–60% over 1 minute; flow rate: 5 mL / min).

[0148] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 97.1%. Analysis condition B: Retention time = 13.20 min: Gradient (% B conc); 20-60% over 20 min, then 60-95% over 1 min, then 95% over 5 min. ESI-MS(+) Observed value m / z=1226.4(M+3H) 3+

[0149] Furthermore, the binding ability of the synthesized conjugate to hTfR was confirmed by SPR according to Example 2, and the result was KD=1.09 nM. [Example]

[0150] Synthesis of control peptide conjugate: hTfR No. 894FLIP-vivotag750 As a control for hTfR No. 894-vivotag750, a conjugate was synthesized in which a cyclic peptide with the following amino acid sequence, which is the reverse of the No. 894 sequence, was linked to vivotag750 via a PEG11 linker (referred to as hTfR No. 894FLIP-vivotag750 or FLIP_000894_PEG11_K_FITC). FLIP sequence: ClAc-Ala-MeTyr-Tyr-Arg-Arg-Ile-Ile-Tyr-Tyr-Asn-Trp-Val-Phe-Val-Cys (SEQ ID NO: 202)

[0151] Synthesis of hTfR No. 894FLIP-PEG11-vivotag750 The synthesis and purification of the peptide conjugate were carried out in the same manner as in Example 3, except that Sieber amide resin (Watanabe Chemical, 0.6 mmol / g, 0.33 g) was used as the starting resin. The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 95.5%. Analysis condition B: Retention time = 13.5 min: Gradient (% B conc); 20-60% over 20 min, then 60-95% over 1 min, then 95% over 5 min. ESI-MS(+) Observed value m / z=1226.4(M+3H) 3+ The N-terminus of the compound obtained as described above was labeled according to the Vivotag750 (VivoTag-S™ 750, PerkinElmer) protocol to obtain the title compound. Furthermore, when the binding ability of the synthesized conjugate to hTfR was confirmed by SPR according to Example 2, no binding to hTfR was observed. [Example]

[0152] Brain penetration evaluation test of hTfR No. 894-vivotag750 using hTfR-KI mice [Preparation of test solution] Hydroxypropyl-β-cyclodextrin (Wako Pure Chemical Industries, Ltd.) was dissolved in water to a concentration of 20 w / v % to prepare a 20% hydroxypropyl-β-cyclodextrin solution.

[0153] [Preparation of administration solution] hTfR No. 894-vivotag750 conjugate administration solution (No. 894 administration solution): 4.8 μL of dimethyl sulfoxide (DMSO, Sigma-Aldrich) was added to 6 μL of a 5 mM solution of the hTfR No. 894-vivotag750 conjugate synthesized in Example 2, followed by 66 μL of a 20% hydroxypropyl-β-cyclodextrin solution and mixing thoroughly. 48 μL of polyethylene glycol 400 was then added and mixed to prepare No. 894 administration solution. The hTfR No. 894-FITC conjugate concentration in No. 894 administration solution was 250 μM. hTfR No.894FLIP-vivotag750 conjugate administration solution (No.894NC administration solution): 66 μL of 20% hydroxypropyl-β-cyclodextrin solution was added to 6 μL of the 5 mM hTfR No.894FLIP-vivotag750 conjugate solution described above and mixed well. 48 μL of polyethylene glycol 400 was added and mixed to prepare the No.894NC administration solution. The hTfR No.894FLIP-vivotag750 conjugate concentration in the No.894FLIP administration solution was 250 μM.

[0154] [Generation of TfR-KI mice] Following the description in International Publication WO 2016-208695, a targeting vector was constructed containing a cDNA encoding a chimeric hTfR, whose intracellular domain is the amino acid sequence of mouse hTfR and whose extracellular domain is the amino acid sequence of human hTfR, and a DNA fragment containing a neomycin resistance gene flanked by loxP sequences at the 3' end of the cDNA. The targeting vector was then electroporated into mouse ES cells. After transfection, the mouse ES cells were selectively cultured in the presence of neomycin to select for mouse ES cells in which the targeting vector had been integrated into their chromosomes by homologous recombination. The resulting recombinant mouse ES cells were injected into 8-cell ICR mouse embryos (host embryos) and then transplanted into pseudopregnant recipient mice obtained by mating with vasectomized mice. The resulting offspring (chimeric mice) were examined for coat color and selected for individuals in which ES cells contributed efficiently to the formation of the organism, i.e., individuals with a high proportion of white hair relative to their total hair. This chimeric mouse was crossed with a C57BL6 / J mouse to generate F1 mice. White F1 mice were selected, and DNA extracted from their tails was analyzed. Mice in which the mouse transferrin receptor gene was heterozygously replaced with the chimeric hTfR on the chromosome were designated TfR-KI mice.

[0155] [Brain delivery evaluation test using TfR-KI mice] Next, we performed a brain penetration study using TfR-KI mice. Female, 12-week-old TfR-KI mice expressing hTfR were rapidly administered 100 μL of either the No. 894 or No. 894NC solution via the tail vein. One hour after administration, the mice were anesthetized with isoflurane and perfused with saline via the left ventricle for 4–5 minutes to remove blood. The tissues to be measured for fluorescence intensity (brain, heart, lung, liver, spleen, kidney, quadriceps, thymus, thoracic vertebrae, and femur) were then harvested and stored in saline to prevent drying. Fluorescence intensity was measured using an in vivo luminescence and fluorescence imaging system (IVIS Lumina III, PerkinElmer) and a filter set for the VivoTag 750 fluorescent dye, according to the manufacturer's instructions.

[0156] [Brain penetration evaluation results using hTfR-KI mice] The results of measuring the fluorescence intensity in each tissue are shown in Figures 1, 1-2, 1-3 and 2. In the figure, "#894" indicates the hTfRNo.894-vivotag750 conjugate administration group, and "#894FLIP" indicates the hTfRNo.894FLIP-vivotag750 conjugate administration group. The bar on the right side of each photograph indicates the average radiant efficiency ([p / sec / cm2 / sr] / [μW / cm2]). In Figure 1-1, (1) Brain shows a photograph of the brain, (2) Liver (Left lateral lobe) shows a photograph of the lateral left lobe of the liver, (3) Kidney shows a photograph of the kidney, (4) Lung shows a photograph of the lung, and (5) Thoracic vertebrae shows a photograph of the thoracic vertebrae. The color scale is (1), with the lowest value being 5.00 × 10 7 , with a maximum value of 1.3×10 8 For (2) and (3), the minimum value is 6.00 × 10 8 , with a maximum value of 2.4×10 10 For (3) and (4), the minimum value is 4.5 × 10 8 , with a maximum value of 5.4×10 9 In Figure 1-2, (1) Heart shows a photograph of the heart, (2) Spleen shows a photograph of the spleen, and (3) Thymus shows a photograph of the thymus. The color scale has a minimum value of 4.83 × 10 7 , with a maximum value of 4.8×10 9 Shows. In Figure 1-3, (1) Quadriceps shows a photograph of the quadriceps femoris, and (2) Femur shows a photograph of the femur. The color scale has a minimum value of 1.5 × 10 8 , with a maximum value of 1.5×10 9 Figure 2 shows an enlarged photograph of the brain shown in Figure 1-1(1). As shown in Figures 1-1, 1-2, 1-3, and 2, tissues in which stronger fluorescence was detected in the hTfR No. 894-vivotag750 conjugate-administered group than in the hTfR No. 894FLIP-vivotag750 conjugate-administered group were the kidney, thoracic vertebrae, heart, femur, quadriceps, and brain.Tissues in which stronger fluorescence was detected in the hTfR No. 894FLIP-vivotag750 conjugate-administered group than in the hTfR No. 894-vivotag750 conjugate-administered group were the liver and spleen.

[0157] In both groups, the strongest fluorescence was detected in the kidney, followed by the liver, suggesting that both test substances were primarily metabolized and excreted in the kidney and liver. Furthermore, stronger fluorescence was observed in the thoracic vertebrae, heart, femur, quadriceps, and brain of the No. 894-treated group compared with the No. 894NC-treated group. These tissues have relatively high TfR expression levels, suggesting that the difference in tissue distribution between the two compounds is due to the presence or absence of hTfR binding ability. This study confirmed that the hTfR-binding special cyclic peptide No. 894 crosses the BBB via binding to TfR in hTfR-KI mice and enters the brain, as well as various tissues, primarily muscle tissue. [Example]

[0158] Mouse brain localization test using a specific hTfR-binding cyclic peptide-fluorescently labeled probe conjugate [Preparation of a conjugate of the hTfR-binding special cyclic peptide No. 894 and the fluorescent substance FITC] A conjugate of hTfR-binding special cyclic peptide No. 894 and the fluorescent substance FITC was synthesized according to the protocol of the FITC labeling kit (Dojindo Laboratories) using hTfR No. 894-PEG11 synthesized in Example 3. The binding ability of the synthesized conjugate to hTfR was confirmed by SPR according to Example 2, and the KD was 0.28 nM.

[0159] [Single-dose experiment] A conjugate of the hTfR-binding special cyclic peptide No. 894 and the fluorescent substance FITC was administered to hTfR-KI mice in the same manner as in Example 5. The dose was adjusted to 3.7 mg / kg. One hour after administration, mice were anesthetized with isoflurane and perfused with saline through the left ventricle for 4–5 minutes to remove blood. The brains were then harvested for measurement of fluorescence intensity and stored in saline to prevent drying. This brain tissue was subjected to immunohistochemical staining using anti-FITC antibody (MLB Biosciences). Immunohistochemical staining of the anti-FITC antibody in the brain tissue was performed using a known method and observed under a fluorescence microscope. The results are shown in Figure 3. The red arrows in the figure indicate cerebral capillaries, and the arrowheads indicate Purkinje cells. (1) shows a photograph of the #894-FITC-treated group, and (2) shows a photograph of the control group not administered #894-FITC.

[0160] [Multi-dose experiment] A conjugate of the hTfR-binding special cyclic peptide No. 894 and the fluorescent substance FITC was administered to hTfR-KI mice in the same manner as the single dose described above, except that the dose was 3.7 mg / kg, administered every 10 minutes for a total of six times. The localization of the conjugate in brain tissue was confirmed by immunohistochemical staining. The results are shown in Figure 4. In the figure, the red arrow indicates a cerebral capillary, the arrowhead indicates a Purkinje cell, and the yellow arrow indicates a dendrite. (1) shows a photograph of the #894-FITC-administered group, and (2) shows a photograph of the control group that did not receive #894-FITC.

[0161] This study confirmed that the conjugate of hTfR-binding special cyclic peptide No. 894 and the fluorescent substance FITC was able to cross the BBB and enter the cerebellum at both doses. Furthermore, the results of multiple dose studies demonstrated that the conjugate reached neurons such as Purkinje cells. [Example]

[0162] SPR study to evaluate the intermolecular interactions between the transferrin receptor (hTfR) and hTfR No. 894 variant peptide, linker-added peptide, and payload conjugate [Synthesis of hTfR No. 894 variant and linker-added peptide] Peptides with sequences in which several amino acids were inserted, deleted, or substituted in the amino acid sequence of hTfR No. 894 (also referred to as variants) and peptides with various linkers were synthesized, and their binding ability to hTfR was similarly confirmed by SPR. Variant peptides were synthesized according to Example 1 unless otherwise specified in Examples 9 or 10. Linker-added peptides were synthesized according to Example 1, except that when the linker was PEG, Fmoc-NH-SAL-PEG resin (Watanabe Chemical) was used as the resin for peptide synthesis. Peptides with linkers of peptides, fatty acids, PEG, and their conjugates were also synthesized according to Example 1 unless otherwise specified in Examples 9 or 10. The KD values ​​determined from SPR measurements are shown in Tables 2 to 4. A indicates a KD value of less than 1 nM, B indicates a KD value of 1 nM to less than 100 nM, C indicates a KD value of 100 nM to less than 1 μM, and D indicates a KD value of 1 μM or greater. Table 2: Peptides or linker-added peptides

[0163] [Table 2-1]

[0164] [Table 2-2]

[0165] [Table 2-3]

[0166] [Table 2-4]

[0167] [Table 3-1] TIFF2026032056000010.tif237166

[0168] Table 3-2 TIFF2026032056000012.tif237166

[0169] Table 4-1 TIFF2026032056000014.tif176166

[0170] Table 4-2 TIFF2026032056000016.tif167166

[0171] Table 4-3 TIFF2026032056000018.tif164166

[0172] Table 4-4 TIFF2026032056000020.tif165166

[0173] Table 4-5 TIFF2026032056000022.tif164166

[0174] Table 4-6 TIFF2026032056000024.tif165166

[0175] Table 4-7 TIFF2026032056000026.tif165166

[0176] [Table 4-8] TIFF2026032056000028.tif167166

[0177] [Table 4-9] TIFF2026032056000030.tif207166

[0178] [Table 4-10] TIFF2026032056000032.tif194166

[0179] [Table 4-11] TIFF2026032056000034.tif191166

[0180] The results showed that hTfR No. 894, sequences in which several amino acids were inserted, deleted or substituted in its amino acid sequence, and peptides to which various linkers were attached had the ability to bind to hTfR. [Example]

[0181] [Cell culture] Human breast cancer cells BT-549 (Cosmobio) were cultured in RPMI-1640 medium (Thermo Fisher Scientific) containing 10% FBS and 2 mmol / L L-glutamine at 370°C in 5% CO2.

[0182] [Cell seeding] Collagen Type I (Corning) was diluted with 20 mmol / L acetic acid to a concentration of 50 μg / mL. A sterilized cover glass was placed in each well of a 24-well plate, and the diluted Collagen Type I solution was added. The plate was then incubated at 37°C for 1 hour. The Collagen Type I solution was removed, and the plate was washed three times with PBS. 1 × 10 per well was used. 5 Human breast cancer cells BT-549 were seeded and cultured overnight at 37°C and 5% CO2.

[0183] [Synthesis of various peptide conjugates] The samples used were hTfR_894_3m_PEG4dk5FAM (SEQ ID NO: 446), hTfR_894_variant03_PEG4dk5FAM (SEQ ID NO: 448), hTfR_894_variant61_PEG4dk5FAM (SEQ ID NO: 447), and Flip894_variant61_PEG4dk5FA (SEQ ID NO: 449)M as a negative control.

[0184] Synthesis of hTfR_894_3m_PEG4dk5FAM [ka]

[0185] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.47 mmol / g, 0.53 g) starting with the removal of Fmoc. The synthesis was performed using a CEM Liberty Blue HT automated synthesizer according to the manufacturer's instructions. For each residue, 1 eq of resin was used with 4.2 eq / 4 eq / 8 eq of Fmoc-AA / DIC / Oxyma pure, and the reaction was carried out at 75°C for 10 min. The second residue was reacted twice for 30 min at 75°C. The 11th and 12th residues were reacted twice for 20 min at 25°C. The 13th residue was reacted twice for 10 min at 75°C. The 15th residue was reacted for 20 min at 25°C. The Fmoc removal was carried out by reacting with a 20% piperidine solution in DMF at 75°C for 3 min. However, the removal of the Fmoc groups at residues 2 and 13 was performed by reacting for 5 minutes at 25°C, followed by a 10-minute reaction. To introduce the chloroacetyl group, the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step was pre-treated with the α-amino group removed as described above. Then, 10 eq of chloroacetic acid, 10 eq of DIPCI, and 10 eq of HOSu were stirred in DCM, and an equal volume of DMF was added to prepare a DCM / DMF solution of ClAcOSu. This solution was then added to the solid-phase resin and shaken at room temperature for 60 minutes. To deprotect the side chain and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, followed by drying under reduced pressure. Next, reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction solution was filtered through a frit. The solid resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were collected through the frit and mixed with the filtrate. This filtrate was added to an excess of diethyl ether / hexane (1 / 1) cooled to 0°C, resulting in the formation of a cloudy precipitate. This mixture was centrifuged (9000 rpm, 2 min), and the solution was decanted. The resulting solid was washed again with a small amount of diethyl ether cooled to 0°C, and then used in the subsequent cyclization reaction.The peptide cyclization reaction was carried out by dissolving the peptide in DMSO to a final concentration of 5 mM based on the molar number of the solid-phase resin, adding 5 equivalents of triethylamine, and shaking at room temperature for approximately 14 hours. F-FAM-NHS, prepared from 1.1 eq. of 5-FAM (Funakoshi), 1.2 eq. of EDC, and 1.2 eq. of HOSu, was added to the reaction solution and stirred at room temperature for 3 hours. The resulting reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVaC. The resulting crude intermediate peptide was purified using a WaterS Xbridge® C18 5 μmμm OBD® 50x150 mm column at 40°C with a gradient of 9-34% B over 3 min, followed by 34-39% B over 8 min, then 39-60% B over 1 min, at a flow rate of 120 mL / min. After lyophilization, the peptide was purified again using a COSMOSIL PBr 10x150 mm column at 40°C with a gradient of 21-46% B over 3 min, followed by 46-51% B over 8 min, then 51-60% B over 1 min, at a flow rate of 5 mL / min.

[0186] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 95.3%. Analysis condition B: Retention time = 4.47 min; Column: Kinetex EVO C18 2.6 μm μm 2.1 × 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in HO, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20-60% over 7.2 min, then 60-95% over 0.3 min, then 95% over 1.6 min; Flow rate: 0.5 mL / min. ESI-MS(+) Observed value m / z=944.42(M+3H) 3+

[0187] Synthesis of hTfR_894_variant03_PEG4dk5FAM, hTfR_894_variant61_PEG4dk5FAM, and Flip894_variant61_PEG4dk5FAM as a negative control F-FAM-NHS, which had been similarly prepared from 5-FAM, was added to hTfR_894_3m_PEG4, hTfR_894_variant03_PEG4, hTfR_894_variant61_PEG4, and Flip894_variant61_PEG4, which were synthesized and cyclized in the same manner as hTfR_894_3m_PEG4dk5FAM, to obtain the above-mentioned conjugates. The binding of each synthesized conjugate to hTfR was confirmed by SPR in the same manner as in Example 2. Only Flip894_variant91_PEG4 was not observed to bind to hTfR.

[0188] [Preparation of sample solution and addition to cells] The sample was diluted with a dilution medium (RPMI 1640 medium containing 0.5% bovine serum albumin and 20 μg / mL human transferrin holoform) to a concentration of 100 nmol / L. BT-549 cells were cultured overnight in a 24-well plate. After confirming adhesion to the cover glass, the cells were washed twice with RPMI 1640 medium. 500 μL / well of RPMI 1640 medium containing 0.5% bovine serum albumin was added and the plate was left to stand on ice for 15 minutes. 500 μL / well of the diluted sample solution was then added and the plate was left to stand at 37°C and 5% CO2 for 3 hours.

[0189] [Cell fixation, nuclear staining, and mounting] The sample solution was removed from the 24-well plate, and the BT-549 cells were washed three times with PBS. 500 μL of 4% paraformaldehyde in phosphate buffer (Fujifilm Wako Pure Chemical Industries, Ltd.) was added per well and incubated at room temperature for 15 minutes, followed by three washes with PBS. 500 μL of Hoechst 33342 (Thermo Fisher Scientific) diluted to 2 μg / mL in PBS was added per well and incubated at room temperature in the dark for 10 minutes, followed by three washes with PBS. The coverslips were removed from the 24-well plate, mounted on glass slides using Fluorescent Mounting Medium (Agilent), and incubated overnight at room temperature in the dark. Observations were performed using an inverted fluorescence microscope DMI6000B (Leica Microsystems) at wavelengths for FITC and DAPI detection. The results are shown in Figure 5. The scale bar in the figure indicates 50 μm. As can be seen in Figure 5, hTfR_894_3m_PEG4dk5FAM, hTfR_894_variant03_PEG4dk5FAM, and hTfR_894_variant61_PEG4dk5FAM were confirmed to be translocated into cells, but Flip894_variant91_PEG4, which contains a peptide that does not bind to hTfR, was not translocated into cells. These results demonstrate that hTfR_894 and its variants capable of binding to hTfR are translocated into cells via binding to hTfR. [Example]

[0190] The following peptides or linker-added peptides were synthesized.

[0191] [Example 9-1] Synthesis of 894_v01_PEG12 (SEQ ID NO: 102) [ka]

[0192] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.52 mmol / g, 0.19 g) starting with the removal of Fmoc. The synthesis was performed using a Biotage Syrol automated synthesizer according to the manufacturer's instructions. The condensation reaction was carried out twice for 20 minutes at 75°C. The introduction of the 15th and 16th residues was carried out at room temperature for 60 and 15 minutes, respectively. The PEG introduction reaction was carried out once, and the 11th amino acid was introduced three times. The Fmoc removal reaction was carried out in a DMF solution of piperazine (5%) and Oxima pure (0.2 M) at 50°C for 5 minutes, followed by another 15-minute reaction. The Fmoc removal of the 15th and 16th residues was carried out at 25°C for 5 minutes, followed by another 15-minute reaction. The introduction of chloroacetyl groups was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above, followed by adding 5 equivalents of ClAcOSu in DMF and shaking at room temperature for 60 minutes. To deprotect the side chain and cleavage the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, and then dried under reduced pressure. Reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was then added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction mixture was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were collected through a frit and mixed with the filtrate described above. The filtrate was added to an excess of diethyl ether / hexane (1 / 1) cooled to 0°C, resulting in a cloudy precipitate. The mixture was centrifuged (10,000 rpm, 1 min), and the solution was decanted. The resulting solid was washed again with a small amount of diethyl ether chilled to 0°C and used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO to a final concentration of 5 mM based on the molar number of the solid-phase resin, and then 10 equivalents of triethylamine was added and the mixture was shaken at room temperature for approximately 1 hour. The resulting reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVaC. Subsequently, the obtained solid phase resin was used to carry out deprotection of the side chain, cleavage from the solid phase resin, and cyclization reaction according to the general method described above. The resulting crude product was purified using the following conditions: column: WaterS Xbridge® C18 5 μmμm OBD® 50×150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (%B): 8-33% over 3 minutes, then 33-38% over 8 minutes, then 38-60% over 1 minute; flow rate: 120 mL / min).

[0193] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 91.5%. Analysis condition A: Retention time = 3.57 min: ESI-MS (+) Observed m / z = Theoretical value (M + 3H) 3+ Analysis condition B: Retention time = 12.8 min: Gradient (% B conc); 20-60% over 20 min, then 60-95% over 1 min, then 95% over 5 min. ESI-MS(+) Observed value m / z=899.6(M+3H) 3+

[0194] [Example 9-2] Synthesis of 894_v05_PEG8Me (SEQ ID NO: 43) [ka]

[0195] Cl-Trt(2-Cl) resin (1 g, 1.6 mmol, Watanabe Chemical Industry, 1.6 mmol / g) was swollen in DCM (10 mL, dehydrated, Nacalai Tesque) for 10 min, filtered, and washed twice with DCM (10 mL). To the swollen resin, a solution of Fmoc-CyS(CH2COOH)-PEG8Me (767 mg, 1 mmol) in DCM (10 mL) and DIEA (836 μL, 4.8 mmol, Watanabe Chemical Industry) were added sequentially and the mixture was shaken at room temperature for 35 min. After further addition of MeOH (1 mL, Kishida Chemical), the mixture was shaken for 10 minutes. The resin was then washed three times with DMF (10 mL, Kishida Chemical), three times with DCM (10 mL, Kishida Chemical), and three times with diethyl ether (10 mL, Kishida Chemical), followed by drying under reduced pressure to obtain Fmoc-CyS[CH2COO-Trt(2-Cl)-resin]-PEG8Me (1.686 g, 94%). The resulting resin was used to synthesize the target peptide, starting with the removal of the Fmoc group, using the general method described above. The synthesis was performed using a Biotage Syrol solid-phase synthesizer according to the manufacturer's instructions. Each residue was introduced using 1 eq of resin with Fmoc-AA-OH / HATU / DIEA (3.2 eq / 3 eq / 6.3 eq) in DMF at room temperature for 30 minutes, repeating the reaction twice. However, the introduction of the first, second, and fourth residues was performed three times for 60 minutes at room temperature. To introduce the third residue, the reaction was repeated twice for 60 minutes at 25°C. To introduce the ninth residue, the reaction was repeated three times for 30 minutes at room temperature. Fmoc removal was performed by reacting with 20% piperidine in DMF for 5 minutes at room temperature, followed by another 15-minute reaction. To introduce the chloroacetyl group, the Fmoc group on the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step was removed as described above. Then, 3 equivalents of a DMF solution (0.45 M) of chloroacetic acid, 3 equivalents of a DMF solution (0.43 M) of HCTU, and 3 equivalents of a DMF solution (1.57 M) of DIPEA were added to the solid-phase resin and shaken at room temperature for 30 minutes. To deprotect the side chains and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, and then dried under reduced pressure.Next, the reaction vessel containing the solid-phase resin was charged with a reagent cocktail of HFIP / DCM (1 / 4) and shaken at room temperature for 90 minutes. The reaction solution was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken with the cleavage cocktail, and the solution components were collected through a frit and mixed with the filtrate. This procedure was repeated three times. The filtrate was concentrated under reduced pressure using a GenevaC EZ-2 Elite and then added to excess diisopropyl ether chilled to 0°C, resulting in the formation of a cloudy white precipitate. This mixture was centrifuged (9000 rpm, 5 min), and the supernatant was decanted. The resulting solid was washed again with a small amount of diethyl ether chilled to 0°C and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. The peptide cyclization reaction was carried out by dissolving the peptide in DMF to a final concentration of 2.5 mM based on the molar ratio of the solid-phase resin. Then, 1.1 equivalents of a DMF solution of HATU (0.43 M) and 1.5 equivalents of DIPEA were added and the mixture was shaken at room temperature for approximately 1 hour. Three equivalents of acetic acid were added to the resulting reaction solution at room temperature, followed by concentration under reduced pressure using a GenevaC EZ-2 column. Ice-cold diisopropyl ether was added to the resulting residue, resulting in a white precipitate. This mixture was centrifuged (9000 rpm, 10 minutes), and the supernatant was decanted. The resulting solid was washed again with diethyl ether chilled to 0°C and dried under reduced pressure. Reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the resulting solid and the mixture was shaken at room temperature for 60 minutes. Excess ice-cold diisopropyl ether was added to produce a precipitate. The mixture was centrifuged (9000 rpm, 5 min), the supernatant was decanted, and the resulting solid was washed again with ice-cooled diethyl ether and then dried under reduced pressure.

[0196] The resulting crude product was purified using the following conditions: column: WaterS Xbridge® C18 5 μm® 30 × 150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (% B): 8-33% over 3 min, then 33-38% over 8 min, then 38-60% over 1 min; flow rate: 45 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 97.4%. Analysis condition B: Retention time = 11.93 min: Gradient (% B conc); 20-60% over 20 min, then 60-95% over 1 min, then 95% over 5 min ESI-MS(+) Observed value m / z=1211.6(M+2H) 2+

[0197] [Example 9-3] Synthesis of hTfR_894_E1_PEG12_(Hydrazide) (SEQ ID NO: 218) [ka]

[0198] The target peptide was synthesized using NH2NH-Trt(2-Cl)-resin (Watanabe Chemical, 0.78 mmol / g, 0.13 g) according to the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue HT solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. For each residue, Fmoc-AA-OH / HATU / DIEA (5.3 eq / 5 eq / 10 eq) was used per 1 eq of resin. The condensation reaction was carried out at 75°C for 10 min. The condensation reaction for the 11th and 12th amino acids was carried out twice for 30 min at 25°C. The 13th and 14th residues were carried out twice for 10 min at 75°C. The 15th residue was carried out once for 30 min at 25°C. The 16th residue was treated once for 120 minutes at 25°C using Fmoc-AA-OH / HATU / DIEA (3 eq / 3 eq / 6 eq). The basic conditions for Fmoc removal were a 3-minute reaction in 20% piperidine in DMF at 75°C. The Fmoc groups at residues 13 and 15 were removed by 5 minutes at 25°C, followed by a 10-minute reaction. The chloroacetyl group was introduced by removing the Fmoc group from the α-amino group of the Fmoc-protected peptide from the solid-phase resin, as described above, followed by stirring 10 eq of chloroacetic acid, 10 eq of DIPCI, and 10 eq of HOSu in DCM, adding an equal volume of NMP to the DCM to prepare a 0.2 M ClAcOSu solution. The solution was then added to the solid-phase resin and shaken at room temperature for 60 minutes. To deprotect the side chains and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride and then dried under reduced pressure. The reaction vessel containing the solid-phase resin was then charged with a reagent cocktail (a 92.5:2.5:2.5:2.5 volume ratio mixture of TFA / HO / TIS / DODT) and shaken at room temperature for 60 minutes. The reaction solution was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were collected through a frit and mixed with the filtrate. The filtrate was added to an excess of diethyl ether / hexane (1 / 1) cooled to 0°C, resulting in the formation of a cloudy precipitate. The mixture was centrifuged (9000 rpm, 2 min), and the solution was decanted.The resulting solid was washed again with a small amount of diethyl ether cooled to 0°C, dried, and used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO to a final concentration of 5 mM based on the molar number of the solid-phase resin, and then 5 equivalents of triethylamine were added and stirred at room temperature for approximately 14 hours. The reaction solution was quenched with acetic acid and concentrated under reduced pressure using a Biotage V-10.

[0199] The resulting crude product was purified using the following conditions (column: WaterS Xbridge® C18 5 μm® 30×150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (% B): 5-29% over 3 minutes, then 29-34% over 8 minutes, then 34-60% over 1 minute; flow rate: 45 mL / min).

[0200] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 77.0%. Analysis condition B: Retention time = 10.57 min; Column: Kinetex EVO C18 2.6 μm, 2.1 × 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 40 °C; Gradient (% B conc): 20 - 60% over 20 min, then 60 - 95% over 1 min, then 95-95% over 5 min; Flow rate: 0.25 mL / min. ESI-MS(+) Observed value m / z=1378.48(M+2H) 2+

[0201] [ Example 9-4 Synthesis of 894_3m_G_Azi (SEQ ID NO: 297) [ka]

[0202] Separately synthesized 894_3m_G (3.3 g, 1.39 mmol) was mixed with 1.1 eq of H-KN3-NH2 (316 mg, 1.52 mmol), 1.2 eq of HATU (632 mg, 1.66 mmol), and 5 eq of DIEA (1.21 mL, 6.93 mmol), all of which were synthesized by a known method, and the mixture was stirred at room temperature for 30 minutes.

[0203] The resulting mixture was purified using the following conditions: column: WaterS Xbridge® C18 5 μm® 50×150 mm; mobile phase: A=0.1% TFA in HO, B=0.1% TFA in MeCN; temperature: 40°C; gradient (%B): 7-7% over 2 min, then 7-32% over 1 min, then 32-37% over 8 min, then 37-60% over 1 min; flow rate: 20-20 mL / min over 1 min, then 20-120 mL / min over 1 min, then 120 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 97.1%. Analysis condition B: Retention time = 4.29 min; Column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in HO, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20-60% over 7.2 min, then 60-95% over 0.3 min, then 95-95% over 1.6 min; Flow rate: 0.5 mL / min. ESI-MS(+) Observed value m / z=1154.72(M+2H) 2+

[0204] [Example 9-5] Synthesis of 894_variant_39 (SEQ ID NO: 327) [ka]

[0205] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.47 mmol / g, 0.21 g) following the general procedure described above, starting with the removal of the Fmoc group. A CEM Liberty Blue HT solid-phase synthesizer was used, following the manufacturer's instructions. Each residue was introduced using 1 eq of resin and Fmoc-AA-OH / DIPCI / Oxima pure (5.3 eq / 10 eq / 5 eq). The reaction was repeated once for 3 minutes at 90°C in DMF. However, the introduction of the second and fourth residues was carried out twice for 30 minutes at 75°C. The ninth residue was reacted twice for 10 minutes at 90°C. The eleventh and twelfth residues were reacted twice for 15 minutes at 50°C. The thirteenth residue was reacted twice for 3 minutes at 90°C. The fifteenth residue was reacted once for 15 minutes at 50°C. The basic conditions for Fmoc removal were a 3-minute reaction with 20% piperidine in DMF at 75°C. However, the Fmoc removal of residues 2, 4, and 13 was repeated twice for 5 minutes at 25°C. The introduction of chloroacetyl groups was performed by removing the Fmoc group from the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above, followed by adding 5 eq of chloroacetic acid in DMF, 5 eq of HATU in DMF, and 10 eq of DIEA in DMF to the solid-phase resin and shaking at room temperature for 30 minutes. For side chain deprotection and cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, followed by drying under reduced pressure. Next, reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 60 minutes. The reaction solution was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were collected through a frit and mixed with the filtrate. This filtrate was added to an excess of a 1:1 diethyl ether / hexane mixture cooled to 0°C, resulting in the formation of a cloudy white precipitate. This mixture was centrifuged (9000 rpm, 1.5 min), and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether cooled to 0°C again and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction.The peptide was dissolved in DMSO to a final concentration of 5 mM based on the molar amount of the solid-phase resin, and then 10 equivalents of triethylamine was added. The mixture was shaken at room temperature for approximately 24 hours. The resulting reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVaC.

[0206] The resulting crude product was purified using the following conditions (column: WaterS Xbridge® C18 5 μm® 50×150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (% B): 11-36% over 3 minutes, then 36-41% over 8 minutes, then 41-60% over 1 minute; flow rate: 120 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 93.1%. Analysis condition B: Retention time = 12.76 min; Column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in HO, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20-60% over 20 min, then 60-95% over 1 min, then 95% over 5 min; Flow rate: 0.25 mL / min. ESI-MS(+) Observed value m / z=1196.12(M+2H) 2+

[0207] [Example 9-6] Synthesis of 894_variant_120 (SEQ ID NO: 353) [ka]

[0208] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.47 mmol / g, 0.43 g) following the general procedure described above, starting with the removal of the Fmoc group. A CEM Liberty Blue HT solid-phase synthesizer was used, following the manufacturer's instructions. Each residue was introduced using 1 eq of resin and 5 eq of Fmoc-AA-OH / HATU / DIEA (5 eq / 5 eq / 10 eq). The reaction was carried out once for 10 min at 75°C in DMF. The first and third residues were introduced once for 20 min at 75°C. The second and fourth residues were introduced twice for 30 min at 75°C. The ninth, tenth, and eleventh residues were introduced twice for 20 min at 75°C. The twelfth residue was introduced twice for 30 min at 25°C. The thirteenth residue was introduced twice for 10 min at 75°C. The 15th residue was reacted once for 30 minutes at 25°C. The basic conditions for Fmoc removal were a 3-minute reaction with 20% piperidine in DMF at 75°C. However, the Fmoc removal of the 2nd, 4th, and 13th residues was repeated twice for 5 minutes at 25°C. The chloroacetyl group was introduced by removing the Fmoc group from the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above, followed by adding 5 eq of ClAcOH in DMF, 5 eq of HATU in DMF, and 10 eq of DIEA in DMF to the solid-phase resin and shaking at 25°C for 30 minutes. The side chains were deprotected and the resin was cleaved from the solid-phase resin. The resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, and then dried under reduced pressure. Next, reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin, and the mixture was shaken at room temperature for 60 minutes. The reaction solution was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were collected through a frit and mixed with the filtrate described above. This filtrate was added to an excess of a 1 / 1 diisopropyl ether / hexane mixture cooled to 0°C, resulting in the formation of a cloudy precipitate. This mixture was centrifuged (9000 rpm, 2 min), and the solution was decanted.The resulting solid was washed again with a small amount of diethyl ether chilled to 0°C and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO to a final concentration of 5 mM based on the molar number of the solid-phase resin, and then 10 equivalents of triethylamine was added and the mixture was shaken at room temperature for approximately 16 hours. The resulting reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVaC.

[0209] The resulting crude product was purified using the following conditions: column: WaterS Xbridge® C18 5 μm® 50 × 150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (% B): 16–41% over 3 min, then 41–46% over 8 min, then 46–60% over 1 min; flow rate: 120 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 97.5%. Analysis condition B: Retention time = 6.07 min; Column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in HO, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20-60% over 7.2 min, then 60-95% over 0.3 min, then 95-95% over 1.6 min; Flow rate: 0.5 mL / min. ESI-MS(+) Observed value m / z=1036.90(M+2H) 2+

[0210] [Example 9-7] Synthesis of 894_variant_61 (SEQ ID NO: 354) [ka]

[0211] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.47 mmol / g, 0.21 g) by the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue HT solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's instructions. Each residue was introduced using Fmoc-AA-OH / DIPCI / Oxima pure (5.3 eq / 10 eq / 5 eq) in 90 ml of DMF for 1 eq of resin. ℃The reaction was carried out once for 3 minutes at 75°C. However, the second and fourth residues were reacted twice for 30 minutes at 75°C. The tenth and eleventh residues were reacted twice for 10 minutes at 90°C. The twelfth residue was reacted twice for 15 minutes at 50°C. The thirteenth residue was reacted twice for 3 minutes at 90°C. The fifteenth residue was reacted once for 15 minutes at 50°C. The basic condition for Fmoc removal was a reaction with a 20% piperidine solution in DMF at 75°C for 3 minutes. However, the Fmoc removal of the second, fourth, and thirteenth residues was repeated twice for 5 minutes at 25°C. The introduction of chloroacetyl groups was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. A 0.2 M ClAcOSu DCM / DMF solution was prepared by stirring 10 eq of chloroacetic acid, 5 eq of DIPCI, and 5 eq of HOSu in DCM and adding an equal volume of DMF to the DCM. This solution was then added to the solid-phase resin and shaken at room temperature for 60 minutes. To deprotect the side chains and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, then dried under reduced pressure. Reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was then added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction mixture was then collected by filtration through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate. This filtrate was added to an excess of a 1:1 diethyl ether / hexane (0°C) mixture, resulting in a cloudy precipitate. This mixture was centrifuged (9000 rpm, 2 min), and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether chilled to 0°C again and dried under reduced pressure. This solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO to a final concentration of 5 mM based on the molar amount of solid-phase resin, and then 5 eq of triethylamine was added. The mixture was shaken at room temperature for approximately 14 hours. The resulting reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVaC.

[0212] The resulting crude product was purified using the following conditions: column: WaterS Xbridge® C18 5 μm® 50 × 150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (% B): 15-40% over 3 min, then 40-45% over 8 min, then 45-60% over 1 min; flow rate: 120 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 87.5%. Analysis Condition B: Retention time = 16:50 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20-60% over 20 min, then 60-95% over 1 min, then 95% over 5 min; Flow rate: 0.25 mL / min ESI-MS(+) Observed value m / z=1160.58(M+2H) 2+

[0213] [Examples 9-8] Synthesis of 894_variant_14 (SEQ ID NO: 387) [ka]

[0214] The target peptide was synthesized using Fmoc-D-Glu(OtBu)-wang resin (Watanabe Chemical, 0.68 mmol / g, 184 mg) according to standard procedures, starting with the removal of the Fmoc group. A CEM Liberty Blue HT solid-phase synthesizer was used, following the manufacturer's instructions. The condensation reaction was carried out using HATU as the condensing agent, with two 10-minute cycles at 75°C. The second residue was reacted twice for 30 minutes at 75°C. The fourth residue was reacted twice for 30 minutes at 75°C. The sixth residue was reacted once for 10 minutes at 75°C. The seventh residue was reacted once for 10 minutes at 75°C. The eighth residue was reacted once for 10 minutes at 75°C. The tenth residue was reacted once for 10 minutes at 75°C. The eleventh residue was reacted twice for 30 minutes at 30°C. The 12th residue was reacted once for 30 minutes at 30°C. The 15th residue was reacted once for 30 minutes at 30°C. The Fmoc removal was performed by reacting with a 20% piperidine solution in DMF at 75°C for 3 minutes. However, the 2nd, 4th, and 13th residues were reacted twice for 5 minutes at room temperature. The chloroacetyl group was introduced by stirring chloroacetic acid (5 equivalents), DIPCI (5 equivalents), and HOSu (5 equivalents) in DCM, adding an equal volume of DMF to DCM to prepare a DCM / DMF solution of ClAcOSu. This solution was then added to the solid-phase resin obtained in the previous step and shaken at room temperature for 60 minutes. To deprotect the side chain and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride and then dried under reduced pressure. Next, reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction solution was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were collected through a frit and mixed with the filtrate. This filtrate was added to excess diethyl ether / hexane (3 / 1) chilled to 0°C, resulting in the formation of a cloudy precipitate. This mixture was centrifuged (9500 rpm, 1 min), and the supernatant was decanted and washed with diethyl ether chilled to 0°C. The resulting solid was used in the subsequent cyclization reaction.The peptide was dissolved in DMSO to a final concentration of 4 mM based on the molar amount of the solid-phase resin, and then 10 equivalents of triethylamine was added. The mixture was shaken at room temperature for approximately 16 hours. The resulting reaction solution was concentrated under reduced pressure using a GenevaC EZ-2 Elite.

[0215] The resulting reaction mixture was purified using the following conditions (column: WaterS Xbridge® C18 5 μm® 50 × 150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (%B): 11–36% over 3 minutes, then 36–41% over 8 minutes, then 41–60% over 1 minute; flow rate: 120 mL / min).

[0216] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 88.60%. Analysis condition B: Retention time = 13.03 min: Gradient (% B conc); 20-60% over 20 min, then 60-95% over 1 min, then 95% over 5 min. ESI-MS(+) Observed value m / z=1137.23(M+2H) 2+

[0217] [Example 9-9] Synthesis of 894_variant84 (SEQ ID NO: 390) [ka]

[0218] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.47 mmol / g, 0.21 g) by the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue HT solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's instructions. For the introduction of each residue, Fmoc-AA / DIPCI / Oxyma pure = 5.3 eq / 10 eq / 5 eq was used for 1 eq of resin, and 90% of the residue was dissolved in DMF. ℃The reaction was carried out once for 3 minutes at 75°C. However, the second and fourth residues were reacted twice for 45 minutes at 75°C. The 11th and 12th residues were reacted twice for 15 minutes at 50°C. The 13th residue was introduced twice for 3 minutes at 90°C. The basic conditions for Fmoc removal were a reaction with 20% piperidine in DMF at 75°C for 3 minutes. However, the Fmoc removal of the second, fourth, and 13th residues was carried out twice for 5 minutes at 25°C. After elongation to the first residue, the resin was suspended in DCM, Pd(PPh3)4 / PhSiH3 (0.2 eq / 10 eq) was added, and the mixture was shaken at room temperature for 1 hour. To introduce the chloroacetyl group, the solid-phase resin carrying the Fmoc-protected peptide obtained in the previous step was washed three times with DMF and three times with DCM. After removing the Fmoc group from the α-amino group as described above, 5 eq of chloroacetic acid in DMF, 5 eq of HATU in DMF, and 10 eq of DIEA in DMF were added and the mixture was shaken at room temperature for 30 minutes. To deprotect the side chain and cleavage the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, then dried under reduced pressure. Reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was then added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction mixture was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate described above. The filtrate was added to an excess of a 1:1 diethyl ether / hexane (1:1) mixture cooled to 0°C, resulting in a cloudy white precipitate. This mixture was centrifuged (10,000 rpm, 1 min), and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether cooled to 0°C again and dried under reduced pressure. This solid was used in the subsequent cyclization reaction. For the cyclization reaction, the peptide was dissolved in DMSO to a final concentration of 5 mM based on the molar number of the solid-phase resin, and then 10 equivalents of triethylamine were added and the mixture was shaken overnight at room temperature. The reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVaC. The resulting mixture was dissolved in DMSO (4 mL), and HOSu (10 eq) and EDC HCl (10 eq) were added. The mixture was stirred at room temperature for 2 hours.

[0219] The resulting crude product was purified using the following conditions: column: WaterS Xbridge® C18 5 μm® 30 × 150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (% B): 11–36% over 3 min, then 36–41% over 8 min, then 41–60% over 1 min; flow rate: 45 mL / min). The resulting cyclic peptide-NHS ester (25 mg, 11.3 μmol) was dissolved in DMF (225 μL), and Fmoc-MeK-OH hydrochloride (5 mg, 11.9 μmol) and DIEA (5.9 μL, 33.9 μmol) were added and stirred. After 1 hour, EtNH (5.9 μL, 56.5 μmol) was added to the reaction mixture and stirred. After 1 hour, the mixture was quenched with acetic acid. The resulting crude product was purified using the following conditions (column: WaterS Xbridge® C18 5 μm® 19×150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 60°C; gradient (%B): 8-33% over 3 minutes, then 33-38% over 8 minutes, then 38-60% over 1 minute; flow rate: 17 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 97.9%. Analysis Condition B: Retention time = 12.33 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B concentration): 20-60% over 20 min, then 60-95% over 1 min, then 95% over 5 min ESI-MS(+) Observed value m / z=1134.34(M+2H) 2+

[0220] [Examples 9-10] Synthesis of 894_variant89 (SEQ ID NO: 391) [ka]

[0221] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.47 mmol / g, 0.21 g) by the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue HT solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's instructions. Each residue was introduced using Fmoc-AA / DIPCI / Oxyma pure / DIEA (5.3 eq / 10 eq / 5 eq) in 90 ml of DMF per 1 eq of resin. ℃The reaction was carried out once for 3 minutes at 75°C. However, when introducing the second and fourth residues, the reaction was carried out twice for 45 minutes at 75°C. When introducing the 11th and 12th residues, the reaction was repeated twice for 15 minutes at 50°C. When introducing the 13th residue, the reaction was carried out twice for 3 minutes at 90°C. The basic conditions for Fmoc removal were a reaction with 20% piperidine in DMF at 75°C for 3 minutes. However, the Fmoc removal of the second, fourth, and 13th residues was carried out twice for 5 minutes at 25°C. After elongation to the first residue, the resin was suspended in DCM, Pd(PPh3)4 / PhSiH3 (0.2 eq / 10 eq) was added, and the mixture was shaken at room temperature for 1 hour. The solid-phase resin was washed three times with DMF and three times with DCM and then dried under reduced pressure. The resin (60 μmol) was suspended in DMF and HD-Glu(OtBu)-OtBu hydrochloride (71 mg, 0.24 mmol), 0.5 M Oxyma pure in DMF (0.48 mL, 0.24 mmol), DIPCI (37 μL, 0.24 mmol), and DIEA (42 μL, 0.24 mmol) were added. The mixture was incubated twice for 30 min at 75°C under microwave irradiation. The chloroacetyl group was introduced by removing the Fmoc group from the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. The mixture was then added with 5 eq of chloroacetic acid in DMF, 5 eq of HATU in DMF, and 10 eq of DIEA in DMF and shaken at room temperature for 30 min. The side chains were deprotected and the resin was cleaved from the solid-phase resin. The resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, followed by drying under reduced pressure. Next, reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction solution was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were collected through a frit and mixed with the filtrate. This filtrate was added to an excess of a 1 / 1 diethyl ether / hexane mixture cooled to 0°C, resulting in the formation of a cloudy white precipitate. This mixture was centrifuged (10,000 rpm, 1 min), and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether cooled to 0°C again and then dried under reduced pressure.The resulting solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO to a final concentration of 5 mM based on the molar number of the solid-phase resin, and then 10 eq of triethylamine was added and the mixture was shaken overnight at room temperature. The reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVaC.

[0222] The resulting crude product was purified using the following conditions: column: WaterS Xbridge® C18 5 μm® 50 × 150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (% B): 8-33% over 3 min, then 33-38% over 8 min, then 38-60% over 1 min; flow rate: 120 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 94.9%. Analysis condition B: Retention time = 11.81 min; Column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in HO, B = 0.025% TFA in MeCN; Temperature: 40 °C; Gradient (% B conc): 20-60% over 20 min, then 60-95% over 1 min, then 95% over 5 min; Flow rate: 0.25 mL / min. ESI-MS(+) Observed value m / z=1127.76(M+2H) 2+

[0223] [Examples 9-11] Synthesis of 894_variant_03 (sequence number 397) [ka]

[0224] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.47 mmol / g, 0.21 g) by the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue HT solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's instructions. Each residue was introduced using Fmoc-AA / DIPCI / Oxima pure (4.2 eq / 4 eq / 8 eq) in 75% DMF for 1 eq of resin. ℃The reaction was carried out once for 10 minutes at 75°C. However, the second and fourth residues were reacted twice for 30 minutes at 75°C. The 11th and 12th residues were reacted twice for 10 minutes at 90°C. The 13th residue was reacted twice for 10 minutes at 75°C. The 15th residue was reacted once for 20 minutes at 25°C. The basic conditions for Fmoc removal were a reaction with a 20% piperidine solution in DMF at 75°C for 3 minutes. However, the Fmoc removal of the second, fourth, and 13th residues was carried out for 5 minutes at 25°C, followed by a 10-minute reaction at 25°C. The introduction of chloroacetyl groups was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. A 0.2 M solution of ClAcOSu in DCM / DMF (0.2 M) was then prepared by stirring chloroacetic acid (10 equivalents), 10 equivalents of DIPCI, and 10 equivalents of HOSu in DCM and adding an equal volume of DMF to the DCM. This solution was then added to the solid-phase resin and shaken at room temperature for 60 minutes. To deprotect the side chains and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, then dried under reduced pressure. Reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was then added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction mixture was then collected by filtration through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate. This filtrate was added to an excess of a 1:1 diethyl ether / hexane (0°C) mixture, resulting in a cloudy precipitate. This mixture was centrifuged (9000 rpm, 2 min), and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether chilled to 0°C again and dried under reduced pressure. This solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in 50% MeCN aqueous solution to a final concentration of 5 mM based on the molar amount of solid-phase resin. Five equivalents of triethylamine were added, and the mixture was shaken at room temperature for approximately 14 hours. The resulting reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVaC.

[0225] The resulting crude product was purified using the following conditions (column: WaterS Xbridge® C18 5 μm® 50×150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (% B): 10-35% over 3 minutes, then 35-40% over 8 minutes, then 40-60% over 1 minute; flow rate: 120 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 98.2%. Analysis condition B: Retention time = 13.18 min; Column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in HO, B = 0.025% TFA in MeCN; Temperature: 40 °C; Gradient (% B conc): 20-60% over 20 min, then 60-95% over 1 min, then 95-95% over 5 min; Flow rate: 0.25 mL / min. ESI-MS(+) Observed value m / z=1072.49(M+2H) 2+

[0226] [Examples 9-12] Synthesis of 894_variant_31 (sequence number 400) [ka]

[0227] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.47 mmol / g, 0.21 g) by the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's instructions. For the introduction of each residue, Fmoc-AA / DIPCI / Oxima pure (4.2 eq / 4 eq / 8 eq) was used for 1 eq of resin, and 90% of the residue was dissolved in DMF. ℃The reaction was repeated twice for 3 minutes at 75°C. However, when introducing the second and fourth residues, the reaction was repeated twice for 45 minutes at 75°C. When introducing the third residue, the reaction was repeated twice for 30 minutes at 75°C. When introducing the seventh and eighth residues, the reaction was repeated once for 3 minutes at 90°C. When introducing the eleventh and twelfth residues, the reaction was repeated twice for 15 minutes at 50°C. When introducing the fifteenth and sixteenth residues, the reaction was repeated once for 15 minutes at 50°C. The basic condition for Fmoc removal was a reaction with a 20% piperidine solution in DMF at 75°C for 3 minutes. However, when introducing the second, fourth, and thirteenth residues, the reaction was repeated twice for 5 minutes at 25°C. The introduction of chloroacetyl groups was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. A 0.2 M solution of ClAcOSu in DCM / DMF (0.2 M) was then prepared by stirring chloroacetic acid (5 equiv.), 5 equiv. DIPCI, and 5 equiv. HOSu in DCM and adding an equal volume of DMF to the DCM. This solution was then added to the solid-phase resin and shaken at room temperature for 60 minutes. To deprotect the side chains and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, then dried under reduced pressure. Reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was then added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction mixture was then collected by filtration through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate. This filtrate was added to excess diisopropyl ether chilled to 0°C, resulting in a cloudy white precipitate. This mixture was centrifuged (9500 rpm, 1 min), and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether chilled to 0°C again and then dried under reduced pressure. This solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO to a final concentration of 4 mM based on the molar number of the solid-phase resin, and then 7 equivalents of triethylamine were added. The mixture was shaken at room temperature for approximately 12 hours. The resulting reaction solution was concentrated under reduced pressure using a GenevaC EZ-2 Elite.

[0228] The resulting crude product was purified using the following conditions (column: WaterS Xbridge® C18 5 μm® 50×150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (%B): 15-40% over 3 minutes, then 40-45% over 8 minutes, then 45-60% over 1 minute; flow rate: 120 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 95.4%. Analysis Condition B: Retention time = 12.64 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20-60% over 20 min, then 60-95% over 1 min, then 95% over 5 min; Flow rate: 0.25 mL / min ESI-MS(+) observed value m / z=1195.87(M+2H) 2+

[0229] [Examples 9-13] Synthesis of 894_variant_03_G4S2C (SEQ ID NO: 401) [ka]

[0230] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.73 mmol / g, 1.37 g) following the general method described above, starting with the removal of the Fmoc group. The synthesis was performed using a CEM Liberty Blue solid-phase synthesizer according to the manufacturer's instructions. For each residue, Fmoc-AA / HATU / DIEA (4.2 eq / 4 eq / 8 eq) was used per 1 eq of resin. The reaction was carried out once for 10 min in DMF at 75°C. The second and fourth residues were reacted twice for 30 min at 75°C. The 11th and 12th residues were reacted twice for 20 min at 25°C. The 13th residue was reacted twice for 10 min at 75°C. The 15th and 22nd residues were reacted once for 30 min at 25°C. The Fmoc removal was carried out by reacting the product with a 20% piperidine solution in DMF at 75°C for 3 min. However, the Fmoc removal of residues 2, 4, and 13 was carried out for 5 minutes at 25°C, followed by 10 minutes at room temperature. The introduction of chloroacetyl groups was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. Then, 10 eq of chloroacetic acid, 10 eq of DIPCI, and 10 eq of HOSu were stirred in DCM, and an equal volume of DMF was added to the DCM to prepare a 0.2 M ClAcOSu DCM / DMF solution. This solution was then added to the solid-phase resin and shaken at room temperature for 60 minutes. To deprotect the side chains and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, then dried under reduced pressure. Next, reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 90:2.5:2.5:5) was added to the reaction vessel containing the solid-phase resin, and the mixture was shaken at room temperature for 90 minutes. The reaction mixture was filtered through a frit. The solid resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were collected through a frit and mixed with the filtrate. This filtrate was added to an excess of a 1 / 1 diisopropyl ether / hexane mixture cooled to 0°C, resulting in the formation of a cloudy white precipitate. This mixture was centrifuged (9500 rpm, 1 min), and the solution was decanted and dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction.The peptide cyclization reaction was carried out by dissolving the peptide in DMSO to a final concentration of 5 mM based on the molar number of the solid-phase resin, adding 10 eq of triethylamine, and shaking at room temperature for approximately 15 hours. The resulting reaction solution was concentrated under reduced pressure using a GenevaC HT-12. The resulting mixture was dissolved in DMSO, added with 3 eq of silver acetate, and shaken for 3 hours. After adding 2 M DTT aqueous solution (11 eq), the mixture was centrifuged, and the supernatant was collected and concentrated under reduced pressure to obtain the crude product.

[0231] The resulting crude product was purified using the following conditions: column: WaterS Xbridge® C18 5 μm® 50 × 150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (% B): 11–11% over 3 min, then 11–36% over 3 min, then 36–41% over 8 min, then 41–60% over 1 min; flow rate: 120 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 94.8%. Analysis Condition B: Retention time = 4.31 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20-60% over 7.2 min, then 60-95% over 0.3 min, then 95-95% over 1.6 min; Flow rate: 0.5 mL / min ESI-MS(+) Observed value m / z=1324.94(M+2H) 2+

[0232] [Examples 9-14] Synthesis of 894_A_hgl_CyCloamide (SEQ ID NO: 408) [ka]

[0233] The target peptides were synthesized using Sieber amide resin (Watanabe Chemical, 0.47 mmol / g) according to standard procedures, starting with Fmoc removal. A CEM Liberty Blue HT solid-phase synthesizer was used, following the manufacturer's instructions. The condensation reaction was performed using DIPCI and Oxyma Pure as condensation agents, with a single reaction at 90°C for 3 minutes. For residues 11 and 12, the reaction was repeated twice for 15 minutes at 50°C. For residue 13, the reaction was repeated twice for 3 minutes at 90°C. The Fmoc removal was performed by reacting the residue with a 20% piperidine solution in DMF at 75°C for 3 minutes. For residue 13, the Fmoc removal was performed twice for 5 minutes at 25°C. The resulting resin was washed five times with DMF and three times with methylene chloride and dried under reduced pressure. Next, reagent cocktail A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was filtered and recovered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate described above. When this filtrate was added to an excess of a mixed solvent of diisopropyl ether and hexane cooled to 0°C, a cloudy white precipitate formed. This mixture was centrifuged (9000 rpm, 2 min), and the supernatant was decanted. The resulting solid was then washed with ice-cooled diethyl ether. The solid was washed with ether and then dried. The resulting solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMF to a final concentration of 5 mM based on the molar number of the solid-phase resin, and then 1.5 equivalents of HATU and 3 equivalents of triethylamine were added and the mixture was shaken at room temperature for approximately 1 hour. The resulting reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVaC.

[0234] The resulting crude product was purified using the following conditions: column: WaterS Xbridge® C18 5 μm® 30 × 150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (%B): 6-31% over 3 minutes, then 31-36% over 8 minutes, then 36-60% over 1 minute; flow rate: 45 mL / min).

[0235] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 96.8%. Analysis condition B: Retention time = 10.03 min: Gradient (% B conc); 20-60% over 20 min, then 60-95% over 1 min, then 95% over 5 min ESI-MS(+) Observed value m / z=1033.0(M+2H) 2+

[0236] [Examples 9-15] Synthesis of 894_BiCyCle_001 (SEQ ID NO: 409) [ka]

[0237] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.47 mmol / g, 0.53 mg) by the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue HT solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's instructions. Each residue was introduced using Fmoc-AA / HATU / DIEA (4.2 equivalents / 4 equivalents / 8 equivalents) per equivalent of resin, with 75% distillation in DMF. ℃ The reaction was carried out twice for 10 minutes at 75°C. However, the first, sixth, and tenth residues were reacted once for 10 minutes at 75°C. The second and third residues were reacted twice for 20 minutes at 75°C. The fourth residue was reacted twice for 30 minutes at 75°C. The eleventh and fourteenth residues were reacted once for 20 minutes at 75°C. The twelfth and fifteenth residues were reacted once for 30 minutes at 75°C. ℃The reaction was carried out once for 30 minutes at 75°C. The basic conditions for Fmoc removal were a 3-minute reaction with 20% piperidine in DMF at 75°C. However, the Fmoc removal of the 2nd, 4th, and 13th residues was carried out twice for 5 minutes at 25°C. The introduction of chloroacetyl groups was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. Then, 5 equivalents of chloroacetic acid, 5 equivalents of DIPCI, and 5 equivalents of HOSu were stirred in DCM, and an equal volume of DMF was added to prepare a 0.2 M ClAcOSu DCM / DMF solution. This solution was then added to the solid-phase resin and shaken at room temperature for 75 minutes. The side chains were deprotected and the resin was cleaved from the solid-phase resin. The resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, and then dried under reduced pressure. Next, reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction solution was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were collected through a frit and mixed with the filtrate. This filtrate was added to excess diisopropyl ether chilled to 0°C, resulting in a cloudy white precipitate. This mixture was centrifuged (9500 rpm, 1 min), and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether chilled to 0°C again and dried under reduced pressure. This solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in 5% aqueous DMSO to a final concentration of 4 mM based on the molar amount of the solid-phase resin, and 10 equivalents of triethylamine was added. The mixture was then shaken at room temperature for approximately 12 hours. The resulting reaction solution was concentrated under reduced pressure using a GenevaC EZ-2 Elite. The peptide was dissolved in DMF to a final concentration of 25 mM based on the molar amount of the solid-phase resin, and 1 equivalent of HATU and 3 equivalents of DIEA were added, followed by shaking at room temperature for approximately 30 minutes.

[0238] The resulting crude product was purified using the following conditions (column: WaterS Xbridge® C18 5 μm® 50 × 150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (% B): 11–36% over 3 min, then 36–41% over 8 min, then 41–60% over 1 min; flow rate: 120 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 94.8%. Analysis Condition B: Retention time = 13.78 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% B conc): 20-60% over 20 min, then 60-95% over 1 min, then 95-95% over 5 min; Flow rate: 0.25% ESI-MS(+) Observed value m / z=1072.26(M+2H) 2+

[0239] [Examples 9-16] Synthesis of 894_BiCyCle_006 (SEQ ID NO: 414) [ka]

[0240] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.47 mmol / g, 0.32 g) by the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue HT solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's instructions. Each residue was introduced using Fmoc-AA / DIPCI / Oxyma pure / DIEA (5.3 equivalents / 10 equivalents / 5 equivalents) in DMF for 1 equivalent of resin. ℃ The reaction was carried out once for 3 minutes under the conditions below. However, when the 2nd, 4th, 11th, and 13th residues were introduced, the reaction was carried out for 90 minutes. ℃The reaction was carried out twice for 3 minutes at 75°C. To introduce the 12th residue, the reaction was carried out twice for 15 minutes at 50°C. To introduce the 15th residue, the reaction was carried out once for 15 minutes at 50°C. The basic conditions for Fmoc removal were a 3-minute reaction with a 20% piperidine solution in DMF at 75°C. However, the Fmoc removal of the 2nd, 4th, and 13th residues was carried out twice for 5 minutes at 25°C. The solid-phase resin was washed sequentially with DMF and DCM, dried under reduced pressure, swollen with DCM, and then added with 10 equivalents of PhSiH3 and 0.2 equivalents of Pd(PPh3)4 and shaken at room temperature for 1 hour. The solid-phase resin was washed five times with DCM, five times with DMF, and three times with diethyl ether, and then dried. The solid-phase resin was swollen with NMP, added with 5 equivalents of HATU and 5 equivalents of DIEA, and shaken at room temperature for 30 minutes. After washing the resin, 5 equivalents of HATU and 5 equivalents of DIEA were added again and the mixture was shaken at room temperature for 30 minutes. The solid-phase resin was washed five times with DCM, five times with DMF, and three times with diethyl ether. To introduce the chloroacetyl group, the Fmoc group on the α-amino group of the Fmoc-protected peptide obtained in the previous step was removed using the method described above. Then, 5 equivalents of chloroacetic acid, 5 equivalents of DIPCI, and 5 equivalents of HOSu were stirred in DCM, and an equal volume of DMF was added to prepare a DCM / DMF solution of ClAcOSu (0.2 M). This solution was then added to the solid-phase resin and shaken at room temperature for 60 minutes. To deprotect the side chain and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, and then dried under reduced pressure. Next, reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction solution was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were collected through a frit and mixed with the filtrate. This filtrate was added to excess diisopropyl ether chilled to 0°C, resulting in the formation of a cloudy precipitate. This mixture was centrifuged (9500 rpm, 1 min), and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether chilled to 0°C again and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction.The peptide was dissolved in 5% DMSO to a final concentration of 3.3 mM based on the molar amount of the solid-phase resin, and then 7 equivalents of triethylamine were added. The mixture was shaken at room temperature for approximately 12 hours. The resulting reaction solution was concentrated under reduced pressure using a GenevaC EZ-2 Elite.

[0241] The resulting crude product was purified using the following conditions: (Column: WaterS Xbridge® C18 5 μm® 50 × 150 mm; Mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; Temperature: 40°C; Gradient (%B): 7–32% over 3 min, then 32–37% over 8 min, then 37–60% over 1 min; Flow rate: 120 mL / min) followed by lyophilization and further purification using the following conditions: (Column: WaterS Xbridge® C18 5 μm® 50 × 150 mm; Mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; Temperature: 40°C; Gradient (%B): 11–36% over 3 min, then 36–41% over 8 min, then 41–60% over 1 min; Flow rate: 120 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 89.0%. Analysis Condition B: Retention time = 3.67 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20-60% over 7.2 min, then 60-95% over 0.3 min, then 95-95% over 1.6 min; Flow rate: 0.5 mL / min ESI-MS(+) Observed value m / z=1055.13(M+2H) 2+

[0242] [Examples 9-17] Synthesis of 894_BiCyCle_012 (SEQ ID NO: 419) [ka]

[0243] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.47 mmol / g, 213 mg) by the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's instructions. The crude product was obtained in a similar manner to the syntheses of Bicycle_01 and Bicycle_06, except that Fmoc-W1aa(Allyl)-OH, Fmoc-Hgl(tBu)-OH, and Fmoc-Hly(Boc)-OH were used as amino acid starting materials.

[0244] The resulting crude product was purified using the following conditions (column: WaterS Xbridge® C18 5 μm® 19×150 mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 60°C; gradient (%B): 11-36% over 3 minutes, then 36-41% over 8 minutes, then 41-60% over 1 minute; flow rate: 17 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 94.8%. Analysis Condition B: Retention time = 4.76 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20-60% over 7.2 min, then 60-95% over 0.3 min, then 95% over 1.6 min ESI-MS(+) Observed value m / z=1093.23(M+2H) 2+

[0245] [Examples 9-18] Synthesis of 894_3m_G (SEQ ID NO: 421) [ka]

[0246] The target peptide was synthesized using Fmoc-Gly-Wang resin (Watanabe Chemical, 0.78 mmol / g, 1.31 g) following the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. For each residue, Fmoc-AA / HATU / DIEA (4.2 equiv / 4 equiv / 8 equiv) was used per equivalent of resin. The reaction was repeated twice for 15 min at 60 °C in DMF. The 15th residue was introduced with a single reaction at 25 °C for 30 min. The 11th and 12th residues were introduced with two 20-min reactions at 25 °C. The 10th residue was introduced with a single reaction at 60 °C for 15 min. The 7th residue was introduced with a single reaction at 60 °C for 15 min. The 5th residue was introduced with a single reaction at 60 °C for 15 min. To introduce the third residue, the reaction was carried out twice for 45 minutes at 60°C. The basic conditions for Fmoc removal were a 3-minute reaction with 20% piperidine in DMF at 75°C. However, the Fmoc removal of the second and 13th residues was carried out twice for 5 minutes at 25°C. To introduce the chloroacetyl group, the Fmoc group on the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step was removed as described above. Then, a 0.2 M solution of ClAcOSu in DCM / DMF (0.2 M) was prepared by stirring 5 equivalents of chloroacetic acid, 5 equivalents of DIPCI, and 5 equivalents of HOSu in DCM and adding an equal volume of DMF to the DCM. This solution was then added to the solid-phase resin and shaken at room temperature for 240 minutes. To deprotect the side chain and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, and then dried under reduced pressure. Next, reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were collected through a frit and mixed with the filtrate. When this filtrate was added to excess diisopropyl ether cooled to 0°C, a cloudy precipitate formed. This mixture was centrifuged (9500 rpm, 1 min), and the solution was decanted.The resulting solid was washed again with a small amount of diethyl ether cooled to 0°C and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO to a final concentration of 4 mM based on the molar number of the solid-phase resin, and then 10 equivalents of triethylamine was added and the mixture was shaken at room temperature for approximately 12 hours. The resulting reaction solution was concentrated under reduced pressure using a GenevaC EZ-2.

[0247] The resulting crude product was purified using the following conditions: column: WaterS Xbridge® C18 5 μm® 50 × 250 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 60 °C; gradient (%B): 0-0% over 5 min, then 0-4.2% over 2 min, then 4.2-28.6% over 3 min, then 28.6-33.7% over 15.5 min, then 33.7-60% over 1.5 min; flow rate: 18 mL / min for 5 min, then 18 mL / min-118 mL / min over 2 min, then 118 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 92.2%. Analysis condition B: Retention time = 11.52 min: Gradient (%B conc); 20-60% over 20 min, then 60-95% over 1 min, then 95% over 5 min ESI-MS(+) Observed value m / z=1078.24(M+2H) 2+

[0248] [Examples 9-19] Synthesis of 894_3m_G4S2C(ACNMe) (SEQ ID NO: 443) [ka]

[0249] Separately synthesized 894_3m_G4S2C (20 mg, 7.07 μmol) was dissolved in DMF (0.1 mL), and 4 equivalents of TEA (3.94 μL, 28.3 μmol) in DMF (39 μL) and 1.1 equivalents of 2-Iodo-N-Methylacetoamide (39.1 mg, 0.25 mmol) in DMF (15 μL) were added and stirred at room temperature for 1 hour.

[0250] The resulting mixture was purified using the following conditions: column: WaterS Xbridge® C18 5 μm® 50 × 150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 60 °C; gradient (% B): 6-31% over 3 min, then 31-36% over 8 min, then 36-60% over 1 min; flow rate: 17 mL / min. The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 97.3%. Analysis Condition B: Retention time = 3.53 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20-60% over 7.2 min, then 60-95% over 0.3 min, then 95-95% over 1.6 min; Flow rate: 0.5 mL / min ESI-MS(+) Observed value m / z=1337.32(M+2H) 2+

[0251] [Examples 9-20] Synthesis of 894_0263 (SEQ ID NO: 487) [ka]

[0252] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.48 mmol / g, 0.26 g) following the general procedure described above, starting with the removal of the Fmoc group. The synthesis was performed using a CEM Liberty Blue HT solid-phase synthesizer according to the manufacturer's instructions. Each residue was introduced using Fmoc-AA / DIPCI / Oxyma pure / DIEA (4.2 equiv / 8 equiv / 4 equiv) per equivalent of resin. The Fmoc group was removed from the α-amino group by the method described above. The chloroacetyl group was then introduced by adding 5 equiv of chloroacetic acid in DMF, 5 equiv of HATU in DMF, and 10 equiv of DIEA in DMF and shaking at 25°C for 30 minutes. To deprotect the side chain and cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, then dried under reduced pressure. Reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was then added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 60 minutes. The reaction solution was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were collected through a frit and mixed with the filtrate. This filtrate was added to an excess of a 1:1 diisopropyl ether / hexane mixture chilled to 0°C, resulting in the formation of a cloudy white precipitate. This mixture was centrifuged (9000 rpm, 2 min), and the solution was decanted. The resulting solid was washed again with a small amount of diethyl ether chilled to 0°C and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO to a final concentration of 5 mM based on the molar amount of solid-phase resin, and then 10 equivalents of triethylamine was added and the mixture was shaken overnight at room temperature. The reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVac. The resulting solid was dissolved in DMSO to a final peptide concentration of 5 mM based on the molar amount of solid-phase resin, and then MePEG4c (1.2 equivalents), HATU (1.1 equivalents), and DIEA (3 equivalents) were added and the mixture was shaken at room temperature for 1 hour.

[0253] The resulting crude product was purified using the following conditions (column: Waters Xbridge® C18 5 μm® 50×150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (% B): 13-38% over 3 minutes, then 38-43% over 8 minutes, then 43-60% over 1 minute; flow rate: 120 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 92.3%. Analysis Condition B: Retention time = 5.28 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20-60% over 7.2 min, then 60-95% over 0.3 min, then 95-95% over 1.6 min; Flow rate: 0.5 mL / min ESI-MS(+) Observed value m / z=1136.17(M+2H) 2+

[0254] The following peptide, linker and payload conjugates were synthesized: [Example 10-1] Synthesis of JCR_hTfR_000894_PEG11_K_FITC (SEQ ID NO: 147) [ka]

[0255] The target peptides were synthesized using Fmoc-NH-SAL-PEG-resin 1500-2000 Da (Watanabe Chemical, 0.38 mmol / g) according to standard procedures, starting with Fmoc removal. A CEM Liberty Blue solid-phase synthesizer was used, following the manufacturer's instructions. The condensation reaction was performed using HATU as the condensing agent, with a single reaction time of 10 min at 75 °C. However, the 11th and 12th residues were reacted twice for 20 min at 25 °C. The 13th and 14th residues were reacted twice for 10 min at 75 °C. The 15th residue was reacted once for 20 min at 25 °C. The 16th residue was reacted once for 60 min at 25 °C. The Fmoc removal reaction was performed with a 20% piperidine solution in DMF at 75 °C for 3 min. However, for residues 13 and 15, Fmoc removal was performed for 5 minutes at 25°C, followed by 10 minutes of reaction. Chloroacetyl group introduction was performed by adding 5 equivalents of a 0.2 M DMF solution of chloroacetic acid, 5 equivalents of a 0.5 M DMF solution of HATU, and 10 equivalents of a 1 M DMF solution of DIPEA to the resin obtained in the previous step and shaking at room temperature for 30 minutes. To deprotect the side chain and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride and dried under reduced pressure. Reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was then added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction mixture was collected by filtration through a frit. The solid phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through the frit and mixed with the filtrate described above. When this filtrate was added to an excess of a mixed solvent of diethyl ether and hexane cooled to 0°C, a cloudy white precipitate formed. This mixture was centrifuged (10,000 rpm, 1 min), and the supernatant was decanted. The solid was washed with ice-cold diethyl ether and then dried. The resulting solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO to a final concentration of 5 mM based on the molar number of the solid-phase resin, and then 6 equivalents of triethylamine were added and the mixture was shaken overnight at room temperature. 1.1 equivalents of a DMSO solution of FAM-OSu (0.71 M) was added to the resulting reaction solution and stirred for 30 minutes. The reaction solution was quenched with AcOH, and the solvent was concentrated under reduced pressure.

[0256] The crude product was purified using the following conditions: column: XSeleCt C18 30x150mm (Lot No. 151i3629811308); mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (%B): 6-31% over 3 minutes, then 31-36% over 8 minutes, then 36-60% over 1 minute; flow rate: 45mL / min).

[0257] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 97.1%. Analysis condition B: Retention time = 13.20 min: Gradient (% B conc); 20-60% over 20 min, then 60-95% over 1 min, then 95% over 5 min ESI-MS(+) Observed value m / z=793.2 Theoretical value 792.6 (M+4H) 4+

[0258] [Example 10-2] Synthesis of 894_v01_PEG12_C_Mal(FITC) (SEQ ID NO: 166) [ka]

[0259] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.6 mmol / g, 0.19 g) following the general procedure described above, starting with the removal of the Fmoc group. A CEM Liberty Blue HT solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. For each residue, Fmoc-AA / HATU / DIEA (5.3 equiv / 5 equiv / 10 equiv) was used per equivalent of resin. The reaction was carried out once for 10 minutes at 75°C in DMF. The second and thirteenth residues were reacted twice for 10 minutes at 75°C. The eleventh and twelfth residues were reacted twice for 20 minutes at 25°C. The fifteenth and seventeenth residues were reacted once for 30 minutes at 25°C. The Fmoc removal was carried out by reacting the product with a 20% piperidine solution in DMF at 75°C for 3 minutes. However, the Fmoc removal at residues 2, 13, 15, and 17 was performed at 25°C for 5 minutes, followed by 10 minutes at 25°C. The introduction of chloroacetyl groups was performed by removing the Fmoc group from the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above, followed by adding 5 equivalents of chloroacetic acid in DMF, 5 equivalents of HATU in DMF, and 10 equivalents of DIEA in DMF to the solid-phase resin and shaking at room temperature for 30 minutes. To deprotect the side chains and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, then dried under reduced pressure. Next, reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction solution was collected by filtration through a frit. The solid resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution was recovered through a frit and mixed with the filtrate. This filtrate was added to an excess of a 1:1 diethyl ether / hexane (1:1) mixture cooled to 0°C, resulting in a cloudy white precipitate. This mixture was centrifuged (10,000 rpm, 1 min), the solution was decanted, and the solid was washed with diethyl ether cooled to 0°C and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction.For the peptide cyclization reaction, the peptide was dissolved in DMSO to a final concentration of 5 mM based on the molar amount of solid-phase resin, and then 6 equivalents of triethylamine were added and the mixture was shaken overnight at room temperature. The resulting reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVaC. The resulting mixture was dissolved in DMSO, 3 equivalents of silver acetate was added, and the mixture was shaken for 3 hours. 10 equivalents of DTT were added, followed by centrifugation and the supernatant was collected.

[0260] The resulting crude intermediate peptide was purified using the following conditions: column: WaterS Xbridge® C18 5 μm® 50 × 150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (%B): 9–34% over 3 min, then 34–39% over 8 min, then 39–60% over 1 min; flow rate: 120 mL / min. The purified intermediate peptide (29.8 mg, 10.6 μmol) was dissolved in DMSO (424 μL), and 1.1 equivalents of fluorescein-5-maleimide and 5 equivalents of DIEA were added, followed by stirring for 1 hour and quenching with acetic acid. The crude product was purified using the following conditions: column: COSMOSIL PBr 10x150mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (%B): 23-48% over 3 min, then 48-53% over 8 min, then 53-60% over 1 min; flow rate: 5mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 82.8%. Analysis Condition B: Retention time = 13.93 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 40 °C; Gradient (% B conc): 20-60% over 20 min, then 60-95% over 1 min, then 95% over 5 min; Flow rate: 0.25 mL / min ESI-MS(+) Observed value m / z=1076.51 Theoretical value (M+2H) 2+

[0261] [Synthesis Example 10-3] Synthesis of 894_variant_61_G4S2C (SEQ ID NO: 358) [ka]

[0262] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.73 mmol / g, 1.37 g) by the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's instructions. Each residue was introduced using Fmoc-AA / HATU / DIEA (4.2 equivalents / 4 equivalents / 8 equivalents) per equivalent of resin, with 75% distillation in DMF. ℃The reaction was carried out once for 10 minutes at 75°C. However, the second and fourth residues were reacted twice for 30 minutes at 75°C. The 12th residue was reacted twice for 20 minutes at 25°C. The 13th residue was reacted twice for 10 minutes at 75°C. The 15th and 22nd residues were reacted once for 30 minutes at 25°C. The basic condition for Fmoc removal was to react with a 20% piperidine solution in DMF at 75°C for 3 minutes. However, the Fmoc removal of the fourth and 13th residues was carried out for 5 minutes at 25°C, followed by a 10-minute reaction at room temperature. The introduction of chloroacetyl groups was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. Then, 10 equivalents of chloroacetic acid, 10 equivalents of DIPCI, and 10 equivalents of HOSu were stirred in DCM, and an equal volume of DMF was added to the DCM to prepare a 0.2 M ClAcOSu solution in DCM / DMF. This solution was then added to the solid-phase resin and shaken at room temperature for 60 minutes. To deprotect the side chains and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, then dried under reduced pressure. Next, reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 90:2.5:2.5:5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction mixture was then collected by filtration through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution was recovered through a frit and mixed with the filtrate. This filtrate was added to an excess of a 1:1 diisopropyl ether / hexane (1:1) mixture cooled to 0°C, resulting in a cloudy precipitate. This mixture was centrifuged (9500 rpm, 1 min), the solution was decanted, and dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO to a final concentration of 5 mM based on the molar number of the solid-phase resin, and then 10 equivalents of triethylamine were added and the mixture was shaken at room temperature for approximately 15 hours. The reaction mixture was quenched with acetic acid and concentrated under reduced pressure using a GenevaC HT-12 column. The resulting mixture was dissolved in DMSO (20 mL), 3 equivalents of silver acetate was added, and the mixture was shaken for 3 hours. After adding 2 M DTT aqueous solution (11 equivalents), the mixture was centrifuged, the supernatant was collected, and the mixture was concentrated under reduced pressure to obtain the crude product.

[0263] The resulting crude product was purified using the following conditions: column: WaterS Xbridge® C18 5 μm® 50×250 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 60°C; gradient (%B): 6.7-6.7% over 5 min, then 6.7-10.3% over 2 min, then 10.3-35.8% over 3 min, then 35.8-40.8% over 15.5 min, then 40.8-60% over 1.5 min; flow rate: 18 mL / min-18 mL / min over 5 min, then 18 mL / min-118 mL / min over 2 min, then 118 mL / min. The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 94.8%. Analysis Condition B: Retention time = 5.45 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20-60% over 7.2 min, then 60-95% over 0.3 min, then 95-95% over 1.6 min; Flow rate: 0.5 mL / min ESI-MS(+) Observed value m / z=1289.65(M+2H) 2+

[0264] Peptides or linker-added peptides were synthesized as follows. The synthesized peptides and linker-added peptides are shown in Table 5, and the linkers are shown in Table 6.

[0265] [Table 5]

[0266] [Table 6-1]

[0267] [Table 6-2]

[0268] [Table 6-3]

[0269] [Synthesis Example 1-1] Synthesis of 894_3142 (SEQ ID NO: 547) [ka]

[0270] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.48 mmol / g, 0.52 g) starting with the removal of Fmoc. A CEM Liberty Blue was used as the automated synthesizer, and the synthesis was carried out according to the manufacturer's instructions. The synthesis was carried out in the same manner as in Example 1. The resulting crude product was purified using the following conditions: Column: WaterS Xbridge® C18 5 μm® 50×150 mm; Mobile phase: A=0.1% TFA in HO, B=0.1% TFA in MeCN; Temperature: 40° C.; Gradient (% B): 7-32% over 3 min, then 32-37% over 8 min, then 37-60% over 1 min; Flow rate: 120 mL / min.

[0271] The purity of the target substance was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 94.20%. Analysis condition B: Retention time = 4.12 min; Column: Kinetex EVO C18 2.6 μm, 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20 - 60% over 7.15 min, then 60 - 95% over 0.3 min, then 95-95% over 1.55 min; Flow rate: 0.5 mL / min ESI-MS(+) Observed value m / z=1147.70(M+2H) 2+

[0272] [Example 1-2] Synthesis of 894_3143 (SEQ ID NO: 548) [ka]

[0273] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.48 mmol / g, 0.52 g) starting with the removal of Fmoc. A CEM Liberty Blue HT was used as the automated synthesizer, and the synthesis was carried out according to the manufacturer's instructions. The synthesis was carried out in the same manner as in Example 1. The resulting crude product was purified using the following conditions (Column: WaterS Xbridge® C18 5 μm® 50×150 mm; Mobile phase: A=0.1% TFA in HO, B=0.1% TFA in MeCN; Temperature: 40° C.; Gradient (% B): 6-6% over 2 min, then 6-31% over 1 min, then 31-36% over 8 min, then 36-60% over 1 min; Flow rate: 20-20 mL / min over 1 min, then 20-120 mL / min over 1 min, then 120 mL / min).

[0274] The purity of the target substance was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions, and was found to be 97.61%. Analysis conditions: Retention time = 3.75 min; Column: Kinetex EVO C18 2.6 μm, 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20 - 60% over 7.15 min, then 60 - 95% over 0.3 min, then 95-95% over 1.55 min; Flow rate: 0.5 mL / min ESI-MS(+) Observed value m / z=1133.66(M+2H) 2+

[0275] [Examples 1-3] Synthesis of 894_3144 (SEQ ID NO: 549) [ka]

[0276] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.48 mmol / g, 0.52 g) starting with the removal of Fmoc. A CEM Liberty Blue was used as the automated synthesizer, and the synthesis was carried out according to the manufacturer's instructions. The synthesis was carried out in the same manner as in Example 1. The resulting crude product was purified using the following conditions (Column: WaterS Xbridge® C18 5 μm® 50×150 mm; Mobile phase: A=0.1% TFA in HO, B=0.1% TFA in MeCN; Temperature: 40° C.; Gradient (% B): 8-8% over 2 min, then 8-33% over 1 min, then 33-38% over 8 min, then 38-60% over 1 min; Flow rate: 20-20 mL / min over 1 min, then 20-120 mL / min over 1 min, then 120 mL / min).

[0277] The purity of the target substance was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions, and was found to be 97.52%. Analysis condition B: Retention time = 3.97 min; Column: Kinetex EVO C18 2.6 μm, 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20 - 60% over 7.15 min, then 60 - 95% over 0.3 min, then 95-95% over 1.55 min; Flow rate: 0.5 mL / min ESI-MS(+) Observed value m / z=1108.62(M+2H) 2+

[0278] [Examples 1-4] Synthesis of 894_3145 (SEQ ID NO: 550) [ka]

[0279] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.48 mmol / g, 0.52 g) starting with the removal of Fmoc. A CEM Liberty Blue was used as the automated synthesizer, and the synthesis was carried out according to the manufacturer's instructions. The synthesis was carried out in the same manner as in Example 1. The resulting crude product was purified using the following conditions: Column: WaterS Xbridge® C18 5 μm® 50×150 mm; Mobile phase: A=0.1% TFA in HO, B=0.1% TFA in MeCN; Temperature: 40° C.; Gradient (% B): 13-38% over 3 min, then 38-43% over 8 min, then 43-60% over 1 min; Flow rate: 120 mL / min.

[0280] The purity of the target substance was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions, and was found to be 98.02%. Analysis conditions: Retention time = 4.91 min; Column: Kinetex EVO C18 2.6 μm, 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20 - 60% over 7.15 min, then 60 - 95% over 0.3 min, then 95-95% over 1.55 min; Flow rate: 0.5 mL / min ESI-MS(+) Observed value m / z=1177.95(M+2H) 2+

[0281] [Examples 1-5] Synthesis of 894_3147 (SEQ ID NO: 551) [ka]

[0282] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.48 mmol / g, 0.52 g) starting with the removal of Fmoc. A CEM Liberty Blue was used as the automated synthesizer, and the synthesis was carried out according to the manufacturer's instructions. The synthesis was carried out in the same manner as in Example 1. The resulting crude product was purified using the following conditions: Column: WaterS Xbridge® C18 5 μm® 50×150 mm; Mobile phase: A=0.1% TFA in HO, B=0.1% TFA in MeCN; Temperature: 40° C.; Gradient (% B): 21-46% over 3 min, then 46-51% over 8 min, then 51-60% over 1 min; Flow rate: 120 mL / min.

[0283] The purity of the target substance was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions, and was found to be 96.34%. Analysis conditions: Retention time = 6.24 min; Column: Kinetex EVO C18 2.6 μm, 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20 - 60% over 7.15 min, then 60 - 95% over 0.3 min, then 95-95% over 1.55 min; Flow rate: 0.5 mL / min ESI-MS(+) Observed value m / z=1198.29(M+2H) 2+

[0284] [Examples 1-6] Synthesis of 894_3148 (SEQ ID NO: 552) [ka]

[0285] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.48 mmol / g, 0.52 g) starting with the removal of Fmoc. A CEM Liberty Blue was used as the automated synthesizer, and the synthesis was carried out according to the manufacturer's instructions. The synthesis was carried out in the same manner as in Example 1. The resulting crude product was purified using the following conditions: Column: WaterS Xbridge® C18 5 μm® 50×150 mm; Mobile phase: A=0.1% TFA in HO, B=0.1% TFA in MeCN; Temperature: 40° C.; Gradient (% B): 18-43% over 3 min, then 43-48% over 8 min, then 48-60% over 1 min; Flow rate: 120 mL / min.

[0286] The purity of the target substance was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions, and was found to be 96.03%. Analysis conditions: Retention time = 6.15 min; Column: Kinetex EVO C18 2.6 μm, 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20 - 60% over 7.15 min, then 60 - 95% over 0.3 min, then 95-95% over 1.55 min; Flow rate: 0.5 mL / min ESI-MS(+) Observed value m / z=1142.36(M+2H) 2+ [Synthesis Example 1-7] Synthesis of 894_variant_61_G_Azi (SEQ ID NO: 543) [ka]

[0287] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.47 mmol / g, 0.21 g) by the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue HT solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's instructions. Each residue was introduced using Fmoc-AA / HATU / DIEA (5.3 equivalents / 5 equivalents / 10 equivalents) in DMF at 75°C. ℃The reaction was carried out once for 10 minutes at 75°C. However, the second and fourth residues were reacted twice for 30 minutes at 75°C. The tenth, eleventh, and thirteenth residues were reacted twice for 10 minutes at 75°C. The twelfth residue was reacted twice for 20 minutes at 25°C. The fifteenth residue was reacted once for 20 minutes at 25°C. The basic condition for Fmoc removal was a reaction with a 20% piperidine solution in DMF at 75°C for 3 minutes. However, the Fmoc removal of the second, fourth, and thirteenth residues was carried out for 5 minutes at 25°C, followed by a 10-minute reaction at 25°C. The introduction of chloroacetyl groups was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. Then, 10 equivalents of chloroacetic acid, 10 equivalents of DIPCI, and 10 equivalents of HOSu were stirred in DCM, and an equal volume of DMF was added to the DCM to prepare a 0.2 M ClAcOSu solution in DCM / DMF. This solution was then added to the solid-phase resin and shaken at room temperature for 60 minutes. To deprotect the side chains and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, then dried under reduced pressure. Next, reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction mixture was then collected by filtration through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate. This filtrate was added to an excess of a 1:1 mixture of diethyl ether and hexane cooled to 0°C, resulting in a cloudy white precipitate. This mixture was centrifuged (9000 rpm, 2 min), and the solution was decanted and dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO to a final concentration of 5 mM based on the molar amount of solid-phase resin, and then 5 equivalents of triethylamine were added and the mixture was shaken at room temperature for approximately 14 hours. The resulting reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVaC.

[0288] The resulting crude product was purified using the following conditions: column: WaterS Xbridge® C18 5 μm® 50 × 150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (% B): 17-42% over 3 min, then 42-47% over 8 min, then 47-60% over 1 min; flow rate: 120 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 92.8%. Analysis Condition B: Retention time = 6.22 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20-60% over 7.2 min, then 60-95% over 0.3 min, then 95-95% over 1.6 min; Flow rate: 0.5 mL / min ESI-MS(+) Observed value m / z=1142.31(M+2H) 2+ [Synthesis Example 1-8] Synthesis of hTfR_894_3m_G_PEG4_gAbu(NHS) (a conjugate of the linker of SEQ ID NO: 635 and the peptide of SEQ ID NO: 296) [ka]

[0289] Using Fmoc-PEG4c-Abu-Alko resin (Watanabe Chemical, 1.00 mmol / g, 0.1 g) prepared by standard solid-phase synthesis, the C-terminal carboxylic acid peptide was synthesized starting with the removal of the Fmoc group according to the general method described above. The synthesis was performed using a CEM Liberty Blue solid-phase synthesizer according to the manufacturer's instructions. The chloroacetyl group was introduced by removing the Fmoc group from the α-amino group of the Fmoc-protected peptide from the solid-phase resin, as described above, followed by adding 0.2 M chloroacetic acid in DMF (5 equiv.), 0.5 M HATU in DMF (5 equiv.), and 1 M DIEA in DMF (10 equiv.) to the solid-phase resin and shaking at room temperature for 30 minutes. To deprotect the side chain and cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride and dried under reduced pressure. Next, a reagent cocktail (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction solution was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate. This filtrate was added to an excess of diethyl ether / hexane (1 / 1) chilled to 0°C, resulting in the formation of a cloudy white precipitate. This mixture was centrifuged (10,000 rpm, 1 min), and the solution was decanted. The resulting solid was washed again with a small amount of diethyl ether chilled to 0°C, dried, and used in the subsequent cyclization reaction. The peptide cyclization reaction was carried out by dissolving the peptide in DMSO to a final concentration of 5 mM based on the molar amount of solid-phase resin, adding 10 equivalents of triethylamine, and stirring at room temperature for approximately 17 hours. The reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVac. The resulting C-terminal carboxylic acid peptide was dissolved in DMSO / HO (9 / 1, 4 mL) to a final concentration of 5 mM based on the molar amount of solid-phase resin, and HOSu (230 mg, 20 equivalents) and EDC HCl (383.4 mg, 20 equivalents) were added and stirred.

[0290] The resulting crude product was purified using the following conditions (column: Waters Xbridge® C18 5 μm® 30×150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (%B): 9-34% over 3 minutes, then 34-39% over 8 minutes, then 39-60% over 1 minute; flow rate: 45 mL / min).

[0291] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 87.4%. Analysis Condition B: Retention time = 4.25 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 40 °C; Gradient (% B conc): 20-60% over 7.2 min, then 60-95% over 0.3 min, then 95-95% over 1.6 min; Flow rate: 0.5 mL / min ESI-MS(+) Observed value m / z=1292.86(M+2H) 2+ [Synthesis Example 1-9] Synthesis of 894_C12NH2 (conjugate of fatty acid linker and SEQ ID NO: 1) [ka]

[0292] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.53 mmol / g, 0.21 g) by the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's instructions. Each residue was introduced using Fmoc-AA / HATU / DIEA (5.3 equivalents / 5 equivalents / 10 equivalents) in DMF at 75°C for 1 equivalent of resin. ℃The reaction was carried out at 75°C for 10 minutes. However, the first, ninth, tenth, thirteenth, and fourteenth residues were reacted twice for 10 minutes at 75°C. The eleventh and twelfth residues were reacted twice for 20 minutes at 30°C. The 15th residue was introduced by a single reaction at 30°C for 20 minutes. The basic condition for Fmoc removal was a 3-minute reaction with a 20% piperidine solution in DMF at 75°C. However, the Fmoc removal of the 11th, 12th, 14th, and 15th residues was carried out at 25°C for 5 minutes, followed by a 10-minute reaction at 25°C. The introduction of chloroacetyl groups was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. A 0.2 M solution of ClAcOSu in DCM / DMF (0.2 M) was then prepared by stirring 5 equivalents of chloroacetic acid, 5 equivalents of DIPCI, and 5 equivalents of HOSu in DCM and adding an equal volume of DMF to the DCM. This solution was then added to the solid-phase resin and shaken at room temperature for 60 minutes. To deprotect the side chains and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, then dried under reduced pressure. Reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was then added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 60 minutes. The reaction mixture was then collected by filtration through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate. This filtrate was added to excess diisopropyl ether chilled to 0°C, resulting in a cloudy white precipitate. This mixture was centrifuged (6000 rpm, 4 min), and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether chilled to 0°C again and dried under reduced pressure. This solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO to a final concentration of 5 mM based on the molar amount of solid-phase resin, and then 5 equivalents of triethylamine were added and the mixture was shaken at room temperature for approximately 2 hours. The resulting reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVaC.

[0293] The resulting crude product was purified using the following conditions: column: WaterS Xbridge® C18 5 μm® 30 × 150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (% B): 11–36% over 3 min, then 36–41% over 3 min, then 41–60% over 1 min; flow rate: 45 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 96.3%. Analysis Condition B: Retention time = 12.61 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20-60% over 20 min, then 60-95% over 1 min, then 95% over 5 min; Flow rate: 0.25 mL / min ESI-MS(+) Observed value m / z=1140.56(M+2H) 2+

[0294] [Synthesis Example 1-10] Synthesis of 894_3m_G4 (a conjugate of a linker described in SEQ ID NO: 643 and a peptide described in SEQ ID NO: 296) [ka]

[0295] The target peptide was synthesized using Fmoc-Gly-Wang resin (Watanabe Chemical, 0.7 mmol / g, 1.43 g) by the general method described above, starting with the removal of the Fmoc group. The synthesis was performed using a CEM Liberty Blue solid-phase synthesizer according to the manufacturer's instructions. The condensation reaction was performed using HATU as the condensing agent, with a single reaction time of 10 minutes at 75°C. However, the introduction of the 15th residue was performed once at 25°C for 20 minutes. The introduction of the 13th residue was performed twice at 75°C for 10 minutes. The introduction of the second residue was performed twice at 75°C for 45 minutes. The Fmoc removal was performed by reacting the peptide with a 20% piperidine solution in DMF at 75°C for 3 minutes. However, the Fmoc removal of the second and 13th residues was performed by a 5-minute reaction time at 25°C, followed by a 10-minute reaction time. The introduction of chloroacetyl groups was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. Then, a solution of 4 equivalents of chloroacetic acid in 0.2 M DMF, 4 equivalents of DIPCI in 0.5 M DMF, and 4 equivalents of HOSu in 0.5 M DMF was added and the mixture was shaken at room temperature for 60 minutes. To deprotect the side chains and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, then dried under reduced pressure. Next, reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction mixture was collected by filtration through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate. This filtrate was added to excess diisopropyl ether cooled to 0°C, resulting in a cloudy white precipitate. This mixture was filtered, washed with diethyl ether, and dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO / IPA / HO (90 / 5 / 5) to a final concentration of 5 mM based on the molar amount of solid-phase resin. Five equivalents of triethylamine were added and the mixture was stirred at room temperature for approximately 16 hours. The resulting reaction solution was concentrated under reduced pressure using a GenevaC HT-12 column.

[0296] The resulting crude product was purified using the following conditions: column: WaterS Xbridge® C18 5 μm® 30 × 250 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (%B): 50-7% over 0.1 min, then 7-7% over 1.9 min, then 7-32% over 3 min, then 32-37% over 11 min, then 37-60% over 1 min; flow rate: 120 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 90.6%. Analysis condition B: Retention time = 11.34 min: Gradient (%B conc); 20-60% over 20 min, then 60-95% over 1 min, then 95% over 5 min ESI-MS(+) Observed value m / z=1163.59(M+2H) 2+

[0297] [Synthesis Example 1-11] Synthesis of 894_3m_G4S2_K(Mal) (a conjugate of a linker described in SEQ ID NO: 561 and a peptide described in SEQ ID NO: 296) [ka]

[0298] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.52 mmol / g, 2.4 g x 3) according to standard procedures, starting with the removal of Fmoc. A CEM Liberty Blue solid-phase synthesizer was used, following the manufacturer's instructions. The condensation reaction was performed using DIPCI and Oxyma Pure as condensation agents, with one reaction cycle at 75°C for 10 minutes. The 15th residue was reacted at 50°C for 20 minutes. The 3rd, 4th, 8th, 10th, 13th, and 14th residues were reacted twice at 75°C for 10 minutes. The 11th and 12th residues were reacted twice at 50°C for 20 minutes. The 2nd residue was reacted three times at 75°C for 60 minutes. The 1st residue was reacted twice at 75°C for 20 minutes. The basic conditions for Fmoc removal were a 3-minute reaction with a 20% piperidine solution in DMF at 75°C. However, for residues 15, 16, and 17, Fmoc removal was performed by reacting the residues at room temperature for 5 minutes, followed by another 3-minute reaction at 75°C. To introduce the chloroacetyl group, the Fmoc group on the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step was removed using the method described above. Then, a 0.15 M solution of ClAcOSu in DCM / DMF was prepared by stirring chloroacetic acid (5 equiv.), DIPCI (5 equiv.), and HOSu (5 equiv.) in DCM and adding an equal volume of DMF to the DCM. This solution was then added to the solid-phase resin and shaken at room temperature for 180 minutes. To deprotect the side chain and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride and then dried under reduced pressure. Next, reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were collected through a frit and mixed with the filtrate. When this filtrate was added to excess diisopropyl ether cooled to 0°C, a cloudy white precipitate formed. This mixture was filtered and washed with diethyl ether cooled to 0°C, and the resulting solid was used in the subsequent cyclization reaction.The peptide cyclization reaction was carried out by dissolving the peptide in DMSO to a final concentration of 4.9 mM based on the molar number of the solid-phase resin, adding 7 equivalents of triethylamine, and shaking at room temperature for approximately 1 hour. To the resulting reaction solution, 1.05 equivalents of SMCC was added and the mixture was shaken at room temperature for 1.5 hours. The resulting reaction solution was concentrated under reduced pressure using a GenevaC EZ-2 Elite.

[0299] The resulting crude product was purified using the following conditions: column: WaterS Xbridge® C18 5 μm® 50 × 250 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 60 °C; gradient (%B): 0-0% over 5.1 min, then 0-5% over 1.9 min, then 5-29% over 5 min, then 29-34% over 13.5 min, then 34-60% over 1.5 min; flow rate: 1 mL / min from 0-5.1 min, 1-119 mL / min from 5.1 to 7.0 min, then 119 mL / min).

[0300] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 94.1%. Analysis condition B: Retention time = 11.33 min: Gradient (%B conc); 20-60% over 20 min, then 60-95% over 1 min, then 95% over 5 min ESI-MS(+) Observed value m / z=1424.0(M+2H) 2+

[0301] [Synthesis Example 1-12] Synthesis of 894_3m_G4S2C (a conjugate of a linker of SEQ ID NO: 644 and a peptide of SEQ ID NO: 296) [ka]

[0302] Separately synthesized 894_3m_G (500 mg, 0.21 mmol) was dissolved in DMF (5 mL) and stirred at room temperature for 2.5 hours with 1.2 equivalents of H-Gly-Gly-Gly-Ser(OtBu)-Ser(OtBu)-CyS(Trt)-NH2 (207 mg, 0.25 mmol), 1.2 equivalents of EDC (39.1 mg, 0.25 mmol), and 1.2 equivalents of DIEA (35.8 mg, 0.25 mmol), all of which were synthesized by a known method. 0.24 equivalents of H-Gly-Gly-Gly-Ser(OtBu)-Ser(OtBu)-CyS(Trt)-NH2 (41.4 mg, 0.05 mmol), 0.24 equivalents of 7.8 mg, 0.05 mmol), and 0.24 equivalents of DIEA (7.2 mg, 0.05 mmol) were added and stirred at room temperature for 2 hours. After concentration using a Biotage V-10, the resulting mixture was incubated in TFA / TIS / HO / DODT (92.5 / 2.5 / 2.5 / 2.5) at room temperature for 75 minutes. The reaction mixture was added to excess diisopropyl ether precooled to 0°C, resulting in the formation of a cloudy white precipitate. The mixture was centrifuged (9000 rpm, 2 min), and the solution was decanted. The resulting solid was washed with diethyl ether precooled to 0°C, centrifuged (9000 rpm, 2 min), and the solution was decanted.

[0303] The resulting mixture was purified using the following conditions: column: WaterS Xbridge® C18 5 μm® 50 × 150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (% B): 5-30% over 3 min, then 30-35% over 8 min, then 35-60% over 1 min; flow rate: 120 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 96.1%. Analysis Condition B: Retention time = 3.52 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20-60% over 7.2 min, then 60-95% over 0.3 min, then 95-95% over 1.6 min; Flow rate: 0.5 mL / min ESI-MS(+) Observed value m / z=1301.89(M+2H) 2+

[0304] [Synthesis Example 1-13] Synthesis of 894_3m_GGRGRS_K(Mal) (a conjugate of a linker of SEQ ID NO: 573 and a peptide of SEQ ID NO: 296) [ka]

[0305] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.47 mmol / g, 532 mg) according to standard procedures, starting with the removal of Fmoc. A CEM Liberty Blue HT solid-phase synthesizer was used, following the manufacturer's instructions. The condensation reaction was carried out using HATU as the condensing agent, with two 10-minute cycles at 75°C. The second residue was reacted twice for 30 minutes at 75°C. The fifth, sixth, seventh, sixteenth, seventeenth, nineteenth, twenty-first, and twenty-second residues were reacted once for 10 minutes at 75°C. The eleventh residue was reacted twice for 15 minutes at 50°C. The twelfth, eighteenth, and twentyth residues were reacted once for 15 minutes at 50°C. The fifteenth residue was reacted once for 15 minutes at 50°C. The Fmoc removal was performed by reacting with a 20% piperidine solution in DMF at 75°C for 3 minutes. However, for residues 2 and 13, Fmoc removal was performed twice for 5 minutes at room temperature. The introduction of chloroacetyl groups was performed by stirring chloroacetic acid (5 equiv.), DIPCI (5 equiv.), and HOSu (5 equiv.) in DCM, adding an equal volume of DMF to DCM to prepare a 0.15 M ClAcOSu DCM / DMF solution. This solution was then added to the solid-phase resin obtained in the previous step and shaken at room temperature for 60 minutes. For side chain deprotection and cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride and dried under reduced pressure. Next, reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 150 minutes. The reaction mixture was filtered through a frit. The solid resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were collected through a frit and mixed with the filtrate. This filtrate was added to an excess of a mixed solvent of diethyl ether and hexane chilled to 0°C, resulting in a cloudy white precipitate. This mixture was centrifuged (9500 rpm, 1 min), and the supernatant was decanted and washed with diethyl ether chilled to 0°C. The resulting solid was used in the subsequent cyclization reaction.The peptide cyclization reaction was carried out by dissolving the peptide in DMSO (5% water content) to a final concentration of 5 mM based on the molar number of the solid-phase resin, adding 7 equivalents of triethylamine, and shaking at room temperature for approximately 3 hours. To the resulting reaction solution, 1.1 equivalents of SMCC was added and the mixture was shaken at room temperature for 3 hours. The resulting reaction solution was concentrated under reduced pressure using a GenevaC EZ-2 Elite.

[0306] The resulting crude product was purified using the following conditions: column: WaterS Xbridge® C18 5 μm® 50 × 250 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (% B): 5-29% over 3 min, then 29-34% over 8 min, then 34-60% over 1 min; flow rate: 120 mL / min).

[0307] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 95.4%. Analysis condition B: Retention time = 9.53 min: Gradient (%B conc); 20-60% over 20 min, then 60-95% over 1 min, then 95% over 5 min ESI-MS(+) Observed value m / z=1005.7(M+3H) 3+

[0308] [Synthesis Example 1-14] Synthesis of 894_3m_PEG12dk(Biotin) (a conjugate of the linker of SEQ ID NO:613 and the peptide of SEQ ID NO:296) [ka]

[0309] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.65 mmol / g, 0.54 g) starting with the removal of Fmoc. The synthesis was performed using a CEM Liberty Blue HT automated synthesizer according to the manufacturer's instructions. For each residue, 5.3 equivalents of Fmoc-AA / DIC / Oxyma pure (DIC) were used per equivalent of resin, followed by a 3-minute reaction at 90°C. The second residue was reacted twice for 30 minutes at 75°C. The 11th and 12th residues were reacted twice for 15 minutes at 50°C. The 13th residue was reacted twice for 3 minutes at 90°C. The 15th residue was reacted for 15 minutes at 50°C. The Fmoc removal was performed by reacting with a 20% piperidine solution in DMF at 75°C for 3 minutes. However, the removal of the Fmoc groups at residues 2 and 13 was carried out by two 5-minute reactions at 25°C. The introduction of chloroacetyl groups was carried out by adding 5 equivalents of ClAcOH in DMF, 5 equivalents of HATU in DMF, and 10 equivalents of DIEA in DMF to the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, after which the Fmoc group on the α-amino group was removed as described above, and shaking at room temperature for 30 minutes. To deprotect the side chains and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, followed by drying under reduced pressure. Next, reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction solution was collected by filtration through a frit. The solid resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution was recovered through a frit and mixed with the filtrate. This filtrate was added to an excess of diisopropyl ether / hexane (1 / 1) cooled to 0°C, resulting in a cloudy precipitate. This mixture was centrifuged (10,000 rpm, 1 min), and the solution was decanted. The resulting solid was washed again with a small amount of diethyl ether cooled to 0°C and used in the subsequent cyclization reaction.The peptide was dissolved in 5% DMSO containing 5% water to a final concentration of 5 mM based on the molar amount of the solid-phase resin, and then 10 equivalents of triethylamine was added. The mixture was shaken at room temperature for approximately 16 hours. The resulting reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVaC. The resulting crude intermediate peptide was purified using the following conditions: column: WaterS Xbridge® C18 5 μm OBD® 50 × 150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (% B): 5-30% over 3 min, then 30-35% over 8 min, then 35-60% over 1 min; flow rate: 120 mL / min). To a DMSO solution of the resulting intermediate peptide (22 mM, 160 μL), 1.3 equivalents of Biotin-NHS and 5 equivalents of DIEA were added and stirred at room temperature. After 2.5 hours, the reaction mixture was quenched with acetic acid. The resulting crude product was purified using the following conditions: column: WaterS Xbridge® C18 5 μm OBD® 19 × 150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 60 °C; gradient (% B): 10-35% over 3 min, then 35-40% over 8 min, then 40-60% over 1 min; flow rate: 17 mL / min.

[0310] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 98.7%. Analysis Condition B: Retention time = 4.30 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20-60% over 7.2 min, then 60-95% over 0.3 min, then 95% over 1.6 min; Flow rate: 0.5 mL / min ESI-MS(+) Observed value m / z=1017.76(M+3H) 3+

[0311] [Synthesis Example 1-15] Synthesis of 894_11K_3Me_PEG4C_KTrzMal (a conjugate of a linker of SEQ ID NO: 624 and a peptide of SEQ ID NO: 293) [ka]

[0312] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.52 mmol / g, 0.19 g) starting with the removal of Fmoc. The synthesis was performed using a Biotage SyroI automated synthesizer according to the manufacturer's instructions. For each residue, 3.2 equivalents of Fmoc-AA / DIC / Oxyma pure (DIC) were used per equivalent of resin, followed by two 20-minute reactions at 75°C. The 2nd, 13th, and 17th residues were reacted three times for 10 minutes at 75°C. The 15th residue was reacted twice for 15 minutes at 25°C. The 16th residue was reacted for 60 minutes at 25°C. Fmoc removal was performed by reacting the peptide with a 20% piperidine solution in DMF at 25°C for 5 minutes, followed by a 15-minute reaction. The introduction of chloroacetyl groups was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. Then, 5 equivalents of chloroacetic acid, 5 equivalents of DIPCI, and 5 equivalents of HOSu were stirred in DCM, and an equal volume of NMP was added to the DCM to prepare a 0.2 M DCM / NMP solution of ClAcOSu. This solution was then added to the solid-phase resin and shaken at room temperature for 90 minutes. To deprotect the side chains and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, followed by drying under reduced pressure. Next, reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 60 minutes. The reaction mixture was then collected by filtration through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate. This filtrate was added to an excess of diisopropyl ether / hexane (1 / 1) chilled to 0°C, resulting in a cloudy white precipitate. This mixture was centrifuged (9000 rpm, 3 min), and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether and hexane chilled to 0°C again, and then used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in 9% aqueous DMSO to a final concentration of 5 mM based on the molar amount of solid-phase resin, and 10 equivalents of triethylamine was added. The mixture was then shaken at room temperature for approximately 1.5 hours.The resulting reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVaC. The resulting crude intermediate peptide was purified using the following conditions: column: WaterS Xbridge® C18 5 μm OBD® 50 × 150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (% B): 9-34% over 3 min, then 34-39% over 8 min, then 39-60% over 1 min; flow rate: 120 mL / min. After lyophilization, 2 equivalents of aqueous CuSO4·5H2O (100 mM) and 10 equivalents of aqueous ascorbic acid (500 mM) were added to a DMF solution (15 mM) of the resulting intermediate peptide, followed by the addition of 2 equivalents of a DMF solution (100 mM) of N-propargylmaleimide and stirring at room temperature. The crude product was purified using the following conditions: column: COSMOSIL PBr 10x150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (%B): 17-42% over 3 min, then 42-47% over 8 min, then 47-60% over 1 min; flow rate: 5 mL / min).

[0313] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 82.7%. Analysis Condition B: Retention time = 11.37 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 40 °C; Gradient (% B conc): 20-60% over 7.2 min, then 60-95% over 0.3 min, then 95% over 1.6 min; Flow rate: 0.25 mL / min ESI-MS(+) Observed value m / z=1303.33(M+3H) 3+

[0314] [Synthesis Example 1-16] Synthesis of 894_variant_03_GKN3 (a conjugate of the linker of SEQ ID NO: 554 and SEQ ID NO: 397) [ka]

[0315] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.47 mmol / g, 0.21 g) by the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue HT solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's instructions. Each residue was introduced using Fmoc-AA / HATU / DIEA (4.2 eq / 4 eq / 8 eq) in 75% DMF for 1 eq of resin. ℃The reaction was carried out once for 10 minutes at 75°C. However, the second and fourth residues were reacted twice for 30 minutes at 75°C. The 11th and 12th residues were reacted twice for 20 minutes at 25°C. The 13th residue was reacted twice for 10 minutes at 75°C. The 15th and 22nd residues were reacted once for 30 minutes at 25°C. The basic conditions for Fmoc removal were a reaction with a 20% piperidine solution in DMF at 75°C for 3 minutes. However, the Fmoc removal of the second, fourth, and 13th residues was carried out for 5 minutes at 25°C, followed by a 10-minute reaction at room temperature. The introduction of chloroacetyl groups was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. Then, 5 eq. of chloroacetic acid, 5 eq. of DIPCI, and 10 eq. of HOSu were stirred in DCM, and an equal volume of DMF was added to the DCM to prepare a 0.2 M ClAcOSu DCM / DMF solution. This solution was then added to the solid-phase resin and shaken at room temperature for 30 minutes. To deprotect the side chains and cleave the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, then dried under reduced pressure. Next, reagent cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 20 minutes. The reaction mixture was then collected by filtration through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate. This filtrate was added to an excess of a 1:1 mixture of diethyl ether and hexane cooled to 0°C, resulting in a cloudy white precipitate. This mixture was centrifuged (10,000 rpm, 1 min), and the solution was decanted and dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in 5% aqueous DMSO to a final concentration of 5 mM based on the molar amount of solid-phase resin. Six equivalents of triethylamine were added and the mixture was shaken at room temperature for approximately 16 hours. The resulting reaction solution was concentrated under reduced pressure using a Savant Explorer SpeedVaC.

[0316] The resulting crude product was purified using the following conditions: column: WaterS Xbridge® C18 5 μm® 50 × 150 mm; mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (% B): 11–36% over 3 min, then 36–41% over 8 min, then 41–60% over 1 min; flow rate: 120 mL / min). The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B and was found to be 95.5%. Analysis condition B: Retention time = 4.84 min; Column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in HO, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20-60% over 7.2 min, then 60-95% over 0.3 min, then 95% over 1.6 min; Flow rate: 0.5 mL / min. ESI-MS(+) Observed value m / z=1178.06(M+2H) 2+

[0317] The novel amino acids were synthesized as follows. [Synthesis Example 2-1] (S)-2- [[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-3- Synthesis of (7-chloro-1-ethyl-1H-indol-3-yl)propanoic acid (W1Et7Cl)

[0318] [ka]

[0319] To a DMF solution (500 mL) of 7-chloro-3-iodo-1H-indole (18 g, 64.9 mmol), NaH (60 wt%, 8.7 g, 361 mmol) was added in portions at 0 °C. Subsequently, ethyl iodide (10.3 mL, 130.4 mmol) was added in portions at 0 °C, and the mixture was stirred at room temperature for approximately 16 hours. The reaction solution was diluted with water and extracted with ethyl acetate (3 × 200 mL). The combined organic layer was washed three times with water (100 mL), dried over anhydrous sodium sulfate, and filtered. The organic layer was concentrated, and the residue was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 1:5). Under a nitrogen atmosphere, iodine (1.6 g, 12.6 mmol) was added to a suspension of zinc (8 g, 122 mmol) in DMF (50 mL), followed by methyl (2R)-2-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-3-iodopropanoate (18.5 g, 41 mmol). The mixture was stirred at room temperature for 30 min. A solution of a portion of the product obtained above (15 g, 49 mmol) in DMF (300 mL) was added to the reaction mixture, followed by SPhos (0.84 g, 2.0 mmol) and Pd2(dba)3 (1.1 g, 1.2 mmol). The mixture was stirred at 50 °C for 4 h. The reaction was quenched with water, the solid was filtered off, and the filtrate was extracted four times with ethyl acetate (400 mL). The combined organic layer was washed four times with water (200 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated, and the resulting residue was purified by silica gel column chromatography (ethyl acetate:petroleum ether=2:8). Hydrogen chloride (60 g, 1.7 mol) was blown into a mixture of a portion of the obtained product (18 g, 35.8 mmol) and 1,4-dioxane (200 mL), and the mixture was stirred for 4 hours at 100° C. The reaction solution was concentrated, and the residue was purified by reverse-phase silica gel column chromatography (water:acetonitrile=100:0 to 0:100) to obtain the title compound. ESI-MS(+) Observed value m / z=489.10(M+H) +

[0320] [Synthesis Example 2-2] (S)-2-[[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-3- Synthesis of (2-((tert-butyloxycarbonyl)amino)pyridin-4-yl)propanoic acid (4Py6NH2)

[0321] [ka]

[0322] Under a nitrogen atmosphere, iodine (4.5 g, 17.7 mmol) was added to a suspension of zinc (5.8 g, 88.6 mmol) in DMF (200 mL), followed by the dropwise addition of a DMF solution (50 mL) of methyl (2R)-2-[[(9H-floren-9-ylmethoxy)carbonyl]amino]-3-iodopropanate (20 g, 44.32 mmol, 1.0 equiv.) at room temperature. The mixture was stirred at room temperature for 1 hour. Subsequently, 4-bromopyridin-2-amine (7.7 g, 44.3 mmol, 1 equiv.), Pd2(dba)3 (2.0 g, 2.2 mmol, 0.05 equiv.), and SPhos (1.8 g, 4.4 mmol, 0.1 equiv.) were added, and the mixture was stirred at 50 °C for approximately 16 hours. The reaction was quenched by the addition of 500 mL of water, and the solid was filtered off. The filtrate was extracted three times with ethyl acetate (300 mL). The combined organic extracts were washed three times with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The resulting residue was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 25:75). A portion of the product obtained above (10 g, 24.0 mmol) was dissolved in tertiary butyl alcohol (110 mL), BocO (6.3 g, 28.7 mmol), and NaI (4.3 g, 28.7 mmol) and stirred at room temperature for approximately 16 hours. The reaction mixture was concentrated, and the resulting residue was dissolved in 80 mL of ethyl acetate and washed three times with saturated brine (100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. To a solution of a portion of the obtained product (3 g, 5.8 mmol) in 2-propanol (30 mL), CaCl2 (6.4 g, 58.0 mmol) was added, followed by an aqueous solution (5 mL) of LiOH-HO (0.28 g, 11.6 mmol) at 0 °C. The mixture was stirred at room temperature for approximately 16 hours, diluted with 60 mL of water, and the solid was filtered off. The pH of the filtrate was adjusted to approximately 6 with aqueous citric acid and extracted three times with ethyl acetate (40 mL). The combined organic extracts were washed three times with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane:methanol = 92:8) to obtain the title compound. ESI-MS (+) observed value m / z = 504.15 (M+H). +

[0323] [Synthesis Example 2-3] (S)-2- [[(9H-Fluoren-9-ylmethoxy)carbonyl]amino]-3- Synthesis of (6-((tert-butyloxycarbonyl)amino)pyridin-3-yl)propanoic acid (3Py6NH2)

[0324] [ka]

[0325] The title compound was obtained in the same manner as in Example 1-x, except that 5-bromopyridin-2-amine was used instead of 4-bromopyridin-2-amine. ESI-MS (+) observed value m / z = 504.15 (M+H). +

[0326] [Synthesis Example 2-4] N2- [[(9H-fluoren-9-ylmethoxy)carbonyl]-N6-( Synthesis of 4-methylpiperazine-1-carbonyl)-L-lysine (KCOpipzMe)

[0327] [ka]

[0328] DIPEA (2.3 mL, 13.0 mmol) and triphosgene (1.23 g, 4.2 mmol) were added to 1-methylpiperazine (1.4 mL, 12.7 mmol) dissolved in dichloromethane (20 mL) at 0°C. The mixture was stirred at the same temperature for 1 hour and then concentrated. To the resulting residue, a dichloromethane solution (20 mL) of [(9H-fluoren-9-ylmethoxy)carbonyl]-L-lysine and DIPEA (2.8 mL, 16.3 mmol) was added at 0°C. The mixture was stirred at 0°C for approximately 16 hours. The reaction was quenched with saturated aqueous sodium bicarbonate, diluted with dichloromethane, and washed once with water, twice with 5% aqueous acetic acid, once with saturated aqueous sodium bicarbonate, and finally once with saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (dichloromethane:methanol=100:0-80:20) to give the title compound. ESI-MS(+) observed value m / z=495.40 (M+H). +

[0329] [Synthesis Example 2-5] N Alpha- [(9H-Fluoren-9-ylmethoxy)carbonyl]- Synthesis of 1-(2-amino-2-oxoethyl)-L-tryptophan (W1mCON)

[0330] [ka]

[0331] Under a nitrogen atmosphere, potassium tert-butoxide (3.7 g, 32.9 mmol) was added in several portions to a solution of N-Boc-tryptophan (5 g, 16.4 mmol) in DMF (30 mL) and THF (30 mL) at 0 °C. After stirring at the same temperature for 0.5 h, 2-chloroacetamide (1.5 g, 16.4 mmol) was added in several portions at 0 °C, and the mixture was stirred at room temperature for 3 h. Water (100 mL) was added to quench the reaction, followed by extraction three times with ethyl acetate (50 mL). The combined organic layer was washed three times with saturated brine (50 mL), dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated. The resulting residue was purified by silica gel column chromatography (ethyl acetate:petroleum ether = 90:10). A 2M solution of hydrogen chloride in ethyl acetate (30 mL) was added to a portion of the resulting product (6 g, 16.6 mmol) in ethyl acetate (30 mL), and the mixture was stirred at room temperature for approximately 16 hours. The resulting solid was collected by filtration and washed with ethyl acetate and diethyl ether. The resulting solid was dissolved in 1,4-dioxane (50 mL) and water (10 mL), and then 2,5-dioxypyrrolidin-1-yl-9H-fluoren-9-yl-methyl carbonate (5.9 g, 17.5 mmol) and sodium bicarbonate (5.9 g, 69.9 mmol) were added at 0°C. The mixture was stirred at room temperature for 3 hours and then diluted with 200 mL of water. The pH was adjusted to 5-6 with citric acid, and the mixture was extracted three times with ethyl acetate (60 mL). The combined organic extracts were washed three times with saturated brine (100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 92 / 8) to obtain the title compound. ESI-MS(+) observed value m / z = 484.25 (M+H) +

[0332] [Synthesis Example 2-6] Synthesis of Fmoc-Glu(d-Pro-O-allyl)-OH(Epryl2RCOO)

[0333] [ka]

[0334] HATU (2.7 g, 7.0 mmol), DIPEA (1.3 mL, 7.5 mmol), and allyl-D-prolinate (0.9 g, 5.80 mmol) were added to a DMF solution (10 mL) of 4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (2.5 g, 5.8 mmol), and the mixture was stirred at 0°C for 1 hour. The reaction solution was diluted with water and then extracted with a mixed solvent of ethyl acetate and hexane. The organic layer was washed four times with water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography. A portion of the product obtained in the previous reaction (2.6 g, 4.5 mmol) was stirred with dichloromethane / TFA (1:1, 30 mL) at room temperature for 3 hours. After concentrating the reaction solution, the residue was dissolved in dichloromethane and saturated aqueous sodium bicarbonate solution was added to adjust the pH to 8 or higher. Hydrochloric acid was then added to adjust the pH to 3-4, and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the title compound. ESI-MS (+) observed value m / z = 507.40 (M+H) +

[0335] [Synthesis Example 2-7] Synthesis of Fmoc-Asp(d-Pro-O-allyl)-OH(Dpryl2RCOO)

[0336] [ka] The title substance was obtained in the same manner as in Synthesis Example [1-x], except that 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutanoic acid was used instead of 4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid. ESI-MS (+) observed value m / z = 493.30 (M+H) +

[0337] [Synthesis Example 2-8] Synthesis of Fmoc-Lys(Gly-O-allyl)-OH (KaAC)

[0338] [ka]

[0339] A DMF solution (20 mL) of HATU (1.22 g, 3.1 mmol) was added to a DMF solution of tert-butyl(((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysinemethyl hydrochloride (1.2 g, 2.6 mmol), ((allyloxy)carbonyl)glycine (0.46 g, 2.9 mmol), and DIPEA (0.59 mL, 3.4 mmol) and stirred at 0°C for 1 hour. The mixture was diluted with a mixed solvent of ethyl acetate and hexane, and the organic layer was washed four times with water. The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate:hexane = 0:100-80:20). TFA (10 mL) was added to a dichloromethane solution (10 mL) of a portion of the product synthesized above (1.5 g, 2.6 mmol), and the mixture was stirred at room temperature for approximately 16 hours. The reaction solution was concentrated and subjected to azeotropic distillation with toluene three times. The resulting residue was washed with diisopropyl ether and dried under reduced pressure at 55°C to obtain the title compound. ESI-MS (+) observed value m / z = 510.40 (M+H). +

[0340] [Synthesis Example 2-9] N α Synthesis of -(((9H-fluoren-9-yl)methoxy)carbonyl)-1-(2-((tert-butyldimethylsilyl)oxy)ethyl)-L-tryptophan (Fmoc-W1EtOH-OH)

[0341] [ka]

[0342] To a mixture of N-Boc-tryptophan (20 g, 64.9 mmol) in THF (100 mL) and DMF (100 mL), potassium tert-butoxide (14.8 g, 69.0 mmol) was added at 0°C. After stirring for 15 minutes, 2-bromoethoxy(tert-butyl)dimethylsilane (16.5 g, 69.0 mmol) was added and the mixture was stirred at room temperature for approximately 16 hours. The reaction solution was adjusted to pH 6 with aqueous citric acid at 0°C and diluted with 200 mL of water. The reaction solution was extracted three times with ethyl acetate (100 mL), and the combined organic extracts were washed three times with saturated brine (100 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate:hexane = 1:5). Under a nitrogen atmosphere, 2,6-lutidine (20 mL, 172.9 mmol) was added to a solution of a portion of the obtained product (16 g, 34.6 mmol) in dichloromethane (150 mL), and TMSOTf (25 mL, 112 mmol) was added at 0 °C. The mixture was warmed to room temperature and stirred for 24 hours. The reaction solution was concentrated. Dioxane (300 mL) and water (150 mL) were added to the resulting mixture, followed by sodium bicarbonate (9.3 g, 110.35 mmol, 3.19 equiv.) and 9-fluorenylmethyl-N-succinimidyl carbonate (14.0 g, 41.4 mmol). The mixture was stirred at room temperature for approximately 16 hours. The solid was filtered, and the filtrate was adjusted to pH 7 with citric acid. The mixture was diluted with 200 mL of water and extracted three times with ethyl acetate (200 mL). The combined organic extracts were washed three times with saturated brine (200 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (ethyl acetate:hexane (1:15)) to obtain the title compound. ESI-MS (+) observed value m / z = 585.15 (M+H). + [Industrial Applicability]

[0343] This invention can be used in the pharmaceutical industry.

Claims

1. A peptide that binds to the transferrin receptor, A peptide consisting of any of the amino acid sequences of SEQ ID NOs: 203 to 448 and 450 to 552, which has a cyclic structure in which the N-terminal residue Ala or MeAla is linked to the C-terminal residue Cys, or A peptide consisting of an amino acid sequence in which a chloroacetyl group has been added to the N-terminal residue Ala or MeAla in any of the amino acid sequences of SEQ ID NOs: 203 to 448 and 450 to 552, and which forms a cyclic structure by bonding the N-terminal residue and the C-terminal residue Cys.

2. A conjugate comprising the peptide of claim 1 and a linker attached to said peptide.

3. A complex comprising the peptide of claim 1, a linker bound to the peptide, and a substance bound to the linker.

4. 3. The conjugate of claim 2, wherein the linker is: (i) a linker having a length of 1 to 15 amino acids and containing one or more glycines (Gly) or serines (Ser); (ii) a linker whose N-terminus is an optionally modified cysteine ​​(Cys) or an optionally modified lysine (Lys), or (iii) A conjugate in which the linker has a length of 1 to 5 amino acids and contains either or both of D-glutamic acid (de) and methylated glycine (MeG).

5. 3. The composite of claim 2, The linker is The conjugate is a PEG linker comprising polyethylene glycol (PEG) or a derivative of polyethylene glycol.

6. 6. The composite of claim 5, the PEG linker further comprises glycine (Gly), serine (Ser), glutamic acid (Glu), arginine (Arg), or lysine (Lys); Complex.

7. 3. The conjugate of claim 2, wherein the linker comprises: Polyethylene glycol (PEG), G linker, GS linker, Or a conjugate, wherein the conjugate is a linker having an amino acid sequence represented by any one of SEQ ID NOs: 201, 553 to 644.

8. 10. A peptide or complex according to claim 1, a complex according to claim 2, or a complex according to claim 3, which is capable of passing through the blood-brain barrier.

9. A peptide or complex according to claim 1, a complex according to claim 2, or a complex according to claim 3, which is directed towards muscle tissue.

10. A peptide or complex according to claim 1, a complex according to claim 2, or a complex according to claim 3, which has cell-permeability.

11. A peptide that binds to the transferrin receptor, A peptide consisting of any of the amino acid sequences of SEQ ID NOs: 2 to 157, 159 to 200, 203 to 448, and 450 to 552, which has a cyclic structure in which the N-terminal residue Ala or MeAla is linked to the C-terminal residue Cys, or a peptide consisting of an amino acid sequence in which a chloroacetyl group has been added to the N-terminal residue Ala or MeAla in any of the amino acid sequences of SEQ ID NOs: 2 to 157, 159 to 200, 203 to 448, and 450 to 552, and which forms a cyclic structure by bonding the N-terminal residue and the C-terminal residue Cys; A preventive or therapeutic agent for a brain-related disease or a neuromuscular disease, comprising a complex comprising a linker bound to the peptide and a substance bound to the linker, A preventive or therapeutic agent, wherein the substance is an active ingredient of the preventive or therapeutic agent for the brain-related disease or neuromuscular disease.

12. A peptide that binds to the transferrin receptor, A peptide consisting of any of the amino acid sequences of SEQ ID NOs: 2 to 157, 159 to 200, 203 to 448, and 450 to 552, which has a cyclic structure in which the N-terminal residue Ala or MeAla is linked to the C-terminal residue Cys, or When peptide A is a peptide consisting of an amino acid sequence in which a chloroacetyl group has been added to the N-terminal residue Ala or MeAla in any of the amino acid sequences of SEQ ID NOs: 2 to 157, 159 to 200, 203 to 448, and 450 to 552, and in which a cyclic structure is formed by bonding between the N-terminal residue and the C-terminal residue Cys, Peptide A, a conjugate comprising the peptide A and a linker bound to the peptide A; or A diagnostic agent for a brain-related disease or a neuromuscular disease, comprising a complex comprising the peptide A, a linker bound to the peptide A, and a substance bound to the linker.

13. A method for producing a preventive or therapeutic agent for brain-related diseases or neuromuscular diseases, comprising the step of obtaining the complex according to claim 3.

14. A peptide that binds to the transferrin receptor, A peptide consisting of any of the amino acid sequences of SEQ ID NOs: 2 to 157, 159 to 200, 203 to 448, and 450 to 552, which has a cyclic structure in which the N-terminal residue Ala or MeAla is linked to the C-terminal residue Cys, or A peptide consisting of an amino acid sequence in which a chloroacetyl group has been added to the N-terminal residue Ala or MeAla in any of the amino acid sequences of SEQ ID NOs: 2 to 157, 159 to 200, 203 to 448, and 450 to 552, and which forms a cyclic structure by bonding the N-terminal residue and the C-terminal residue Cys. A composition for delivering a substance to the brain, comprising:

15. A conjugate comprising a cyclic peptide for use in delivering a substance to the brain, The cyclic peptide is a peptide that binds to a transferrin receptor, A conjugate comprising a peptide having an amino acid sequence selected from any of SEQ ID NOs: 2 to 157, 159 to 200, 203 to 448, and 450 to 552, the peptide having a cyclic structure in which an N-terminal residue, Ala or MeAla, is bound to a C-terminal residue, Cys.

16. 16. The conjugate of claim 15, The conjugate, wherein the substance is delivered to the brain by transcytosis via the human transferrin receptor.

17. 16. The conjugate of claim 15, for crossing the blood-brain barrier and delivering a substance to the brain.

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