Human transferrin receptor binding peptides

Abstract

Provided is a peptide or the like that can cross the blood-brain barrier (BBB) by binding to human transferrin receptor (hTfR). A peptide or the like according to the present application has: the amino acid sequence recited in SEQ ID NO: 1 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys); or an amino acid sequence in which one or more and not more than 10 of the amino acid residues in the amino acid sequence recited in SEQ ID NO: 1 are substituted, deleted, added, and / or inserted.

Description

Technical Field

[0001] This invention relates to peptides that can bind to the human transferrin receptor (hTfR). This invention also relates to peptides that can cross the blood-brain barrier (BBB), peptides that are targeted to muscle tissue, and peptides that have cell-penetrating properties. Furthermore, this invention relates to methods for delivering any substance to the brain via such peptides and methods for delivering any substance to muscle tissue, etc. Background Technology

[0002] Except for a few regions containing periventricular organs (pineal gland, pituitary gland, area posterior, etc.), the capillaries supplying blood to most of the brain differ from those in other tissues such as muscles. The endothelial cells forming the brain's endothelium are interconnected through stable intercellular junctions. This hinders the passive transport of substances from the blood to the brain. While there are exceptions, substances with high lipid solubility or small molecular weights (below 200-500 Daltons) and electrical neutrality near physiological pH are difficult to migrate from the capillaries to the brain. This mechanism, which restricts the exchange of substances between the blood and the brain's tissue fluid via the capillary endothelium, is called the blood-brain barrier (BBB). Furthermore, the BBB restricts not only the brain but also the exchange of substances between the tissue fluid and blood of the central nervous system, including the brain and spinal cord. Through the presence of the BBB, most cells of the central nervous system are unaffected by fluctuations in the concentration of hormones, lymphocytes, and other substances in the blood, maintaining their biochemical homeostasis.

[0003] Various methods have been reported for modifying macromolecules to achieve affinity for transferrin receptors, membrane proteins present on the endothelial cells of capillaries in the brain, as methods for crossing the blood-brain barrier to allow macromolecules to reach the brain (Patent Documents 1-3). For example, Patent Document 1 describes a blood-brain barrier shuttle that has affinity for and can bind to the transferrin receptor.

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: Japanese Patent Publication No. 2015-528452

[0007] Patent Document 2: Japanese Patent Application Publication No. H06-228199

[0008] Patent Document 3: WO2016 / 208695

[0009] Patent Document 4: WO2019 / 151539 Summary of the Invention

[0010] The problem that the invention aims to solve

[0011] The purpose of the invention described in this specification is to provide a novel peptide that binds to the human transferrin receptor (hTfR).

[0012] Another objective of the invention is to further provide a peptide that can cross the blood-brain barrier (BBB), a peptide that is targeted to muscle tissue and can efficiently migrate to muscle tissue, and a peptide that is cell-penetrating.

[0013] Another objective of the invention is to provide various uses for the aforementioned novel peptides.

[0014] Technical means to solve the problem

[0015] The invention described in this specification relates to a peptide that binds to the transferrin receptor.

[0016] This peptide has: the amino acid sequence described in Serial No. 1 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys); or an amino acid sequence having 1 or more but less than 10 substitutions, deletions, additions and / or insertions of amino acid residues in the amino acid sequence described in Serial No. 1.

[0017] Invention efficacy

[0018] According to the invention described in this specification, as demonstrated by the examples, peptides that bind to human transferrin receptor (hTfR), peptides that can cross the blood-brain barrier (BBB), peptides that are targeted to muscle tissue, and peptides that are cell-penetrating can be provided. Attached Figure Description

[0019] Figure 1-1 It is a photograph with a displacement plot showing the results of fluorescence intensity measurements in various tissues.

[0020] Figure 1-2 It is a photograph with a displacement plot showing the results of fluorescence intensity measurements in various tissues.

[0021] Figure 1-3 It is a photograph with a displacement plot showing the results of fluorescence intensity measurements in various tissues.

[0022] Figure 2 The accompanying image is a photograph showing the results of fluorescence intensity measurements in a magnified brain.

[0023] Figure 3 This is a photograph with a replacement image showing the presence of a confirmatory test (single dose) result in a localized area of ​​the mouse brain.

[0024] Figure 4This is a photograph with a replacement image, showing the presence of confirmatory test (multi-dose) results in a localized area of ​​the mouse brain.

[0025] Figure 5 The attached image is a fluorescence microscope photograph showing the migration of human breast cancer cells. Detailed Implementation

[0026] For the purposes of this description, the accompanying drawings are used to illustrate the methods for carrying out the invention. The invention is not limited to the methods described below, but also includes suitable modifications that can be made to the following methods within the scope of understanding for those skilled in the art.

[0027] One of the inventions described in this specification relates to a peptide that binds to the transferrin receptor and can cross the blood-brain barrier.

[0028] transferrin receptor

[0029] The term "transferrin receptor" refers to a receptor contained in blood plasma that binds to transferrin, a protein that binds to iron ions, and has the function of being taken up into cells. Transferrin receptors are expressed on various cells, including reticulocytes, placental trophoblast cells, and lymphocytes, and are particularly suggested to be expressed on tumor cells. Furthermore, because transferrin receptors have the property of triggering endocytosis in cells through stimulation by the binding of iron ions in blood plasma, research is underway to use antibodies bound to transferrin receptors as direct-dose substances (DDS) to facilitate the passage of substances through the blood-brain barrier (BBB). In this specification, unless otherwise specified, human transferrin receptors are labeled as human TfR, hTfR, or TfR only.

[0030] peptides that bind to transferrin receptors

[0031] The term "binding to the transferrin receptor" (also known as "binding activity" or "affinity") refers to the specific binding of the transferrin receptor.

[0032] While affinity is expressed as an equilibrium constant (KD) for the dissociation of the transferrin receptor and the binding peptide, it is a measure of the binding strength between the transferrin receptor and the antigen-binding site on the binding peptide. As the value of KD decreases, the binding strength between the transferrin receptor and the binding peptide increases (in lieu of KD, affinity can also be expressed as 1 / KD, i.e., the affinity constant (KA)). As those skilled in the art will understand (e.g., based on further disclosures in this specification), affinity can be determined according to a well-known pattern depending on the specific antigen of the target. Binding activity is a measure of the binding strength between the transferrin receptor and the binding peptide. Binding activity relates 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.

[0033] The specific binding of the transferrin receptor to the binding peptide can be determined, for example, by competitive binding assays including surface plasmon resonance (SPR) analysis, Scatcherd analysis and / or radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competitive assay, as well as any suitable pattern of its various variants known in the art. Preferably, the affinity of the peptide of the present invention for the transferrin receptor is less than 100 nM, more preferably less than 50 nM KD.

[0034] It can cross the blood-brain barrier (BBB).

[0035] The ability 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 some point in time in any part of the brain after administration, or that insights can be obtained that can be inferred from the effects of the substance in the brain.

[0036] Brain-related diseases

[0037] Brain-related diseases refer to illnesses caused by abnormalities occurring in the brain, such as central nervous system (CNS) diseases. Examples of brain-related diseases are not limited but can include Alzheimer's disease, Parkinson's disease, prions, Huntington's disease, lysosomal storage diseases, CNS diseases, CNS tumors including brain tumors, cerebral ischemia, diseases associated with brain injury, traumatic CNS diseases, viral and bacterial CNS diseases, schizophrenia, depression, and other diseases that cause psychological impact.

[0038] It is targeted at muscle tissue.

[0039] Muscle tissue can be any of cardiac muscle, skeletal muscle, or smooth muscle. Cardiac or skeletal muscle tissue is particularly preferred. "Directional to muscle tissue" refers to the property of specifically and efficiently migrating to muscle tissue.

[0040] Neuromuscular diseases

[0041] Neuromuscular diseases refer to motor disorders such as decreased muscle strength caused by lesions in the nerves or muscles, such as the brain, spinal cord, and peripheral nerves. Examples of neuromuscular diseases are not limited, but can include spinocerebellar degeneration, amyotrophic lateral sclerosis (ALS), myasthenia gravis, muscular dystrophy, polymyositis, hereditary myopathy, muscular atrophy, drug-induced myopathy, acute heart failure, chronic heart failure, myocardial infarction, chronic fatigue syndrome, mitochondrial diseases, mitochondrial respiratory chain complex abnormalities, and Guillain-Barré syndrome.

[0042] Cell-penetrating peptides

[0043] Cell-permeable peptides, such as those described in Japanese Patent Nos. 6478632 and 6708770 (Cell-Permeable Peptides), are well known. 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, the target active ingredient can be delivered into cells; for example, nucleic acid pharmaceuticals can be delivered into cells.

[0044] peptides

[0045] Amino acids refer to the structure of multiple consecutive amino acids, and their meaning also includes polypeptides and proteins. Furthermore, in this case, the term "amino acid" includes not only naturally occurring amino acids that are translated into mRNA and incorporated into the peptide chain (natural amino acids), but also non-natural amino acids that can form part of a peptide chain through peptide binding (non-natural amino acids). Amino acids can be artificially synthesized or exist in nature.

[0046] Furthermore, in this case, the peptides and peptide-substance complexes of the present invention also include peptides that form a cyclic portion by post-synthesis cyclization (also called cyclic peptides), peptides obtained by further chemical modification of the peptides, peptide-substance complexes with peptides and substances bound to the peptides, and peptide-substance complexes bound together by linkers.

[0047] In this specification, a cyclic peptide refers to a peptide in which two amino acids separated by one or more amino acid residues in their amino acid sequence are linked together, thus forming a ring structure in whole or in part. Furthermore, the specific form of linkage between the two amino acids is not particularly limited, but cyclic peptides also include those formed through an amide bond between the carboxyl group of one amino acid and the amino group of another, a thioether bond between the carboxyl group of one amino acid and the thiol group of another, a thiol bond between the thiol groups of one amino acid and another, a lactam ring formation, or a macrocyclization reaction; or those with a lasso peptide structure, etc. However, when the two amino acids are linked by an amide bond, the amide bond is not limited to those formed by the linkage between the carboxyl group of one amino acid and the amino group of another, as long as the synthetic reaction results in linkage through an amide bond. The same applies to other linkage forms.

[0048] That is, in this case, a cyclic peptide can be defined as having a portion that forms a cyclic structure, or it can have a linear portion.

[0049] Furthermore, in this specification, there are cases where a portion of the amino acids is modified for peptide cyclization. This also includes amino acids whose portions have been modified. Examples include cyclization where a chloroacetyl group is added to the N-terminal amino acid and then binds to a cysteine ​​residue in the peptide. The amino acids in this case also include various (natural / non-natural) amino acids that have undergone chloroacetyl group addition.

[0050] Non-natural amino acids refer to compounds that possess the properties of amino acids, other than naturally occurring amino acids. Examples include, though not limited to, β-amino acids, γ-amino acids, L-amino acids; D-amino acids (also known as D-type amino acids); chemically modified amino acids such as amino acid variants and derivatives; and amino acids like leucine, β-alanine, and ornithine, which cannot be used as building blocks of proteins in vivo. Examples also include N-methyl amino acids, N-ethyl amino acids, D-amino acids, histidine-like amino acids, amino acids with extra methylene groups or aromatic rings in their side chains, and amino acid derivatives with carboxylic acid functional groups in their side chains replaced by sulfonic acid groups.

[0051] The following describes examples of non-natural amino acids and their abbreviations in this specification. Parentheses indicate the CAS reference number or the name of the company from which the amino acid was purchased; for newly synthesized examples, they indicate the synthesis example number. Furthermore, the CAS number indicates the monomer or protecting group of the non-natural amino acid, but this is not limited to specific amino acids. For example, structures in which one or more hydrogen atoms are replaced by alkyl groups are also considered specific amino acids. When hydrogen atoms are replaced by alkyl groups, the alkyl group is preferably methyl or ethyl, and more preferably methyl. In this specification, amino acids with "Me" or "N-Me-" preceding their name are N-methyl amino 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. Also, amino acids labeled with a single letter and preceded by "d" are represented as D-amino acids. For example, D-amino acids of alanine (Ala or A) are represented as da. Those without CAS numbers or sources of purchase can be purchased as general reagents. Furthermore, the following amino acids can be used in peptide synthesis by protecting the alpha amino group with Fmoc using known methods.

[0052] Yph(S)-2-amino-3-(4-phenoxyphenyl)propionic acid (CAS No.: 180414-93-1)

[0053] W7OMe(S)-2-amino-3-(7-methoxy-1H-indol-3-yl)propionic acid (CAS No.: 2416720-26-6)

[0054] W7N(S)-2-amino-3-(1H-pyrrolo[2,3-β]pyridin-3-yl)propionic acid (CAS No.: 737007-45-3)

[0055] W7F(S)-2-amino-3-(7-fluoro-1H-indol-3-yl)propionic acid (CAS No.: 1956434-65-3)

[0056] W6N(S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propionic acid (KISHIDA CHEMICAL Inc.)

[0057] W6F(S)-2-amino-3-(6-fluoro-1H-indol-3-yl)propionic acid (CAS No.: 908847-01-8)

[0058] W5OMe 5-Methoxy-L-Tryptophan (CAS No.: 460751-69-3)

[0059] W5F(S)-2-amino-3-(5-fluoro-1H-indol-3-yl)propionic acid (CAS No.: 908846-88-8)

[0060] W4OMe 4-Methoxy-L-Tryptophan (CAS No.: 1205553-56-5)

[0061] W4N(S)-2-amino-3-(1H-pyrrolo[3,2-β]pyridin-3-yl)propionic acid (CAS No.: 149818-23-5)

[0062] W4F(S)-2-((((9H-fluoren-9-yl)-methoxy)carbonyl)amino)-3-(1-(tert-butoxycarbonyl)-4-fluoro-1H-indol-3-yl)propionic acid (CAS No.: 2244532-65-6)

[0063] W4C(S)-2-amino-3-(4-chloro-1H-indol-3-yl)propionic acid (CAS No.: 2244532-68-9)

[0064] W2N(S)-2-amino-3-(1H-indol-3-yl)propionic acid (CAS No.: 2305185-20-8)

[0065] W1iPr 1-Isopropyl-L-Tryptophan (CAS No.: 1496563-42-8)

[0066] W1Et7Cl(S)-2-amino-3-(7-chloro-1-ethyl-1H-indol-3-yl)propionic acid (Synthetic Example 2-1)

[0067] W1Et 1-Ethyl-L-Tryptophan (CAS No.: 168471-23-6)

[0068] Tbg(S)-2-amino-3,3-dimethylbutyric acid (CAS No.: 33105-81-6)

[0069] pHPeG N-(4-hydroxyphenylethyl)glycine (CAS No.: 258332-56-8)

[0070] PeG N-(2-Phenylacetyl)-glycine (CAS No.: 540483-58-7)

[0071] NvaL-N-valine (CAS No.: 6600-40-4)

[0072] Nle N-α-chloroacetyl-L-norleucine (CAS No.: 688-12-0)

[0073] Nal2β-(2-naphthyl)L-alanine (CAS No.: 58438-03-2)

[0074] Nal1β-(1-Naphthyl)L-alanine (CAS No.: 2353616-32-5)

[0075] MeoBph N-α-methyl-2-phenyl-L-phenylalanine

[0076] MeNal2 N-α-methyl-β-(2-naphthyl)-L-alanine (CAS No.: 179385-30-9)

[0077] MeNal1 N-α-methyl-β-(1-naphthyl)-L-alanine (CAS No.: 1380327-68-3)

[0078] MemBph N-α-methyl-3-phenyl-L-phenylalanine

[0079] Hph L-High Phenylalanine (CAS No.: 943-73-7)

[0080] Hly(S)-2,7-Diaminoheptanoic acid (CAS No.: 498-56-6)

[0081] F4OMe(S)-2-amino-3-(4-methoxyphenyl)propionic acid (CAS No.: 7635-29-2)

[0082] F4G(4-guanidino)-L-phenylalanine (CAS No.: 59574-11-7)

[0083] F4F 4-Fluoro-L-phenylalanine (CAS No.: 1132-68-9)

[0084] F4C N-α-chloroacetyl-4-chloro-L-phenylalanine (CAS No.: 14173-39-8)

[0085] F3OMe(S)-2-amino-3-(3-methoxyphenyl)propionic acid (CAS No.: 98813-19-5)

[0086] F3F 3-Fluoro-L-phenylalanine (CAS No.: 19883-77-3)

[0087] F3C N-α-chloroacetyl-3-chloro-L-phenylalanine (CAS No.: 80126-51-8)

[0088] F2OMe(S)-2-amino-3-(2-methoxyphenyl)propionic acid (CAS No.: 206060-41-5)

[0089] F2C(S)-2-amino-3-(2-chlorophenyl)propionic acid (CAS No.: 198560-41-7)

[0090] MeF4OMe(s)-3-(4-methoxyphenyl)-2-(methylamino)propionic acid (CAS No.: 1260595-45-6)

[0091] MeF4F N-α-methyl-4-fluoro-L-phenylalanine (CAS No.: 1979176-87-8)

[0092] MeF3F N-α-methyl-3-fluoro-L-phenylalanine (CAS No.: 1820567-10-9)

[0093] MeF3C N-α-methyl-3-chlorofluoro-L-phenylalanine (CAS No.: 1446478-28-9)

[0094] MeBph N-α-methyl-4-phenyl-L-phenylalanine

[0095] Me4Py N-α-methyl-4-pyridyl-L-alanine

[0096] Me3Py N-α-methyl-3-pyridyl-L-alanine

[0097] dr D-arginine

[0098] dp D-proline

[0099] dc D-cysteine

[0100] dk D-lysine

[0101] Dap L-α,β-diaminopropionic acid (CAS No.: 515-94-6)

[0102] Dab(S)-4-amino-2-(2-chloroacetamide)butyric acid (CAS No.: 25691-37-6)

[0103] Cit 2-Amino-5-Ureaporic acid (CAS No.: 627-77-0)

[0104] Chaβ-cyclohexyl-L-alanine (CAS No.: 4441-50-3)

[0105] CeG N-(2-Carboxyethyl)-glycine (CAS No.: 174799-89-4)

[0106] Cbg(S)-2-amino-2-cyclobutylacetic acid (CAS No.: 1391630-31-1)

[0107] Cba cyclobutylalanine (CAS No.: 478183-62-9)

[0108] aMeYα-methyl-L-tyrosine (CAS No.: 658-48-0)

[0109] aMeWα-methyl-tryptophan (CAS No.: 153-91-3)

[0110] aMeKα-methyl-lysine (CAS No.: 111717-28-3)

[0111] aMeCα-methyl-L-cysteine ​​(CAS No.: 441317-73-3)

[0112] Aibα-methylalanine (CAS No.: 62-57-7)

[0113] Ahp / Alahp(S)-2-aminoheptanic acid (CAS No.: 1115-90-8)

[0114] Abu-L-α-aminobutyric acid (CAS No.: 1492-24-6)

[0115] A4paa(S)-2-amino-3-(1-(carboxymethyl)piperazin-4-yl)propionic acid (KISHIDACHEMICAL Inc.)

[0116] 5Ind(S)-2-amino-3-(1H-indol-5-yl)propionic acid (CAS No.: 1655518-66-3)

[0117] 4Py2NH2(S)-2-amino-3-(2-aminopyridin-4-yl)propionic acid (KISHIDACHEMICAL Inc.)

[0118] 4Py 4-pyridyl-L-alanine (CAS No.: 1956-21-4)

[0119] 3Py6NH2 2-Amino-3-(6-aminopyridin-3-yl)propionic acid (Synthetic Examples 2-3)

[0120] 3Py 3-pyridyl-L-alanine (CAS No.: 17470-24-5)

[0121] W1aa 1-(carboxymethyl)-L-tryptophan (CAS No.: 773823-50-0)

[0122] KCOpipzMe N6-(4-methylpiperazine-1-carbonyl)-L-lysine (KISHIDACHEMICAL Inc.)

[0123] W1mCON 1-(2-amino-2-oxoethyl)-L-tryptophan (Synthetic Examples 2-5)

[0124] W1EtOH 1-(2-hydroxyethyl)-L-tryptophan (Synthetic Examples 2-9)

[0125] 3Py6OMe(S)-2-amino-3-(6-methoxypyridin-3-yl)propionic acid (CAS No.: 1270317-99-1)

[0126] Epyrl2RCOO 2-((5-((R)-2-((allyloxy)carbonyl)pyrrolidin-1-yl)-5-oxopentanoic acid

[0127] Dpyrl2RCOO 2-((4-((R)-2-((allyloxy)carbonyl)pyrrolidin-1-yl)-4-oxobutyric acid (Examples 9-16)

[0128] MeF3COO 3-Carboxy-N-methyl-phenylalanine (CAS No.: 1499826-56-0)

[0129] 3Imp 2-amino-3-(imidazo[1,2-a]pyridin-3-yl)propionic acid (CAS No.: 2276942-95-9)

[0130] KaAc N6-glycyl-L-lysine (Synthetic Examples 2-8)

[0131] A1Me4pip 4-Amino-1-methylpiperazine-4-carboxylic acid (CAS No.: 15580-66-2)

[0132] Har N6-formamidinyl-L-lysine (CAS No.: 156-86-5)

[0133] Acpr(S)-2-amino-3-cyclopropylpropionic acid (CAS No.: 1492156-90-7)

[0134] Atb(S)-2-amino-4,4-dimethylvaleric acid (CAS No.: 1934633-35-8)

[0135] MeF35dC(S)-3-(3,5-dichlorophenyl)-2-(methylamino)propionic acid (CAS No.: 1542508-65-5)

[0136] Adod 12-aminododecanoic acid

[0137] Hly L-High Lysine

[0138] W5C 5-Chloro-L-Tryptophan

[0139] F3COO L-3-Carboxyphenylalanine

[0140] F3CON L-3-carbamoylphenylalanine

[0141] Hgl L-2-aminohexadic acid

[0142] Ndm N,N-dimethyl-L-asparagine

[0143] KN3 or LysN3 6-azido-L-ortholeucine

[0144] KAc N6-acetyl-L-lysine

[0145] dorn D-ornithine

[0146] NleL-Leucine

[0147] F3H 3-Hydroxy-L-phenylalanine

[0148] Yae O-(2-aminomethyl)-L-tyrosine

[0149] F4aao O-(2-Carboxymethyl)-L-Tyrosine

[0150] F4OEt O-ethyl-L-tyrosine

[0151] F34dOMe 3,4-Dimethoxy-L-phenylalanine

[0152] alT L-allethreonine

[0153] alI L-alloseleucine

[0154] MeK N-methyl-L-lysine

[0155] Tbg(S)-2-amino-3,3-dimethylbutyric acid

[0156] NvaL-N-valine

[0157] Abu(S)-(+)-2-aminobutyric acid

[0158] da D-alanine

[0159] Bph 4-Phenylon-L-phenylalanine

[0160] de D-glutamic acid

[0161] MeA N-methyl-L-alanine

[0162] PEG4c or PEG3 1-Amino-3,6,9,12-tetraoxapentadecano-15-carboxylic acid

[0163] MeR N-methyl-L-arginine

[0164] MeW N-methyl-L-tryptophan

[0165] PEG8c 1-Amino-3,6,9,12,15,18,21,24-octaoxaheptadecane-27-carboxylic acid

[0166] PEG12c, PEG11, or PEG12 1-Amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecoxane-39-carboxylic acid

[0167] Furthermore, novel synthetic amino acids are useful because they have the potential to add new functions to various peptides when manufacturing various peptide derivatives.

[0168] The peptide of the present invention has: the amino acid sequence described in Serial No. 1 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys); or an amino acid sequence having 1 or more and 10 or fewer amino acid residues in the amino acid sequence described in Serial No. 1, including substitutions, deletions, additions and / or insertions.

[0169] Target peptide sequence

[0170] The number of amino acids that are substituted, deleted, added, and / or inserted is between one and ten, with a lower limit of one. The upper limits are 10, 9, 8, 7, 6, 5, 4, 3, and 2, with a minimum of one. Preferably, the substitution of this amino acid is a preservative amino acid substitution.

[0171] Preservative amino acid replacement

[0172] The term "conservative amino acid substitution" refers to the substitution of an amino acid with functional equivalence or similarity. A conservative amino acid substitution in a peptide causes a static change to the peptide's amino acid sequence. For example, one or more amino acids with the same polarity act functionally equivalently, causing a static change to the peptide's amino acid sequence. Generally, substitutions within a certain group can be considered conservative in terms of both structure and function. However, as those skilled in the art will understand, the role of a particular amino acid residue can be determined by the significance of the molecule containing that amino acid in its three-dimensional structure. For example, cysteine ​​residues can be in their oxidized (disulfide) form, which is less polar than their reduced (thiol) form. Long aliphatic portions of arginine side chains can constitute structurally and functionally important features. Furthermore, side chains containing aromatic rings (tryptophan, tyrosine, phenylalanine) can contribute to ion-aromatic interactions or cation-pi interactions. In this case, even if amino acids with such side chains are replaced with amino acids belonging to the acidic or nonpolar groups, the structure and function can still be preserved. Residues such as proline, glycine, and cysteine ​​(in disulfide form) have the potential to have a direct effect on the stereostructure of the main chain and cannot be repeatedly replaced without structural deformation.

[0173] Preservative amino acid substitutions are as follows, including specific substitutions based on side chain similarity (Lehninger, Biochemistry, 2nd edition, revised, 1975, pp. 73-75: L. Lehninger, Biochemistry, 2nd edition, pp. 73-75, Worth Publisher, New York (1975)) and typical substitutions.

[0174] A preferred embodiment of this peptide comprises an amino acid sequence having one or more substitutions selected from the following group:

[0175] The first alanine residue in sequence number 1 is replaced by an aliphatic amino acid or a methylated aliphatic amino acid;

[0176] The second valine residue in sequence number 1 is replaced by a basic amino acid residue or a methylated basic amino acid residue.

[0177] The third phenylalanine residue in sequence number 1 is replaced by an aromatic amino acid residue, a methylated aromatic amino acid residue, an amino acid residue with an added aromatic ring, or an amino acid residue with an added fused ring.

[0178] The fourth valine residue in sequence number 1 is replaced by a methylated valine residue;

[0179] The fifth tryptophan residue in sequence number 1 is replaced by an aromatic amino acid residue, a methyltryptophan residue, a methylated aromatic amino acid residue, an amino acid residue with an added aromatic ring, or an amino acid residue with an added fused ring.

[0180] The sixth asparagine residue in sequence number 1 is replaced by a neutral amino acid or a methylated neutral amino acid;

[0181] The 7th and 8th tyrosine residues of sequence number 1 are replaced by aromatic amino acid residues, methylated aromatic amino acid residues, amino acid residues with added aromatic rings, or amino acid residues with added fused rings.

[0182] The 9th isoleucine residue of sequence number 1 is replaced by an aliphatic amino acid residue, a methylated aliphatic amino acid residue, or an amino acid residue with a branched structure.

[0183] The 10th isoleucine residue of sequence number 1 is replaced by any amino acid; and

[0184] The 11th serine residue in sequence number 1 was replaced by a neutral amino acid residue.

[0185] (1) Nonpolar amino acid group: alanine (hereinafter, marked "Ala" or only "A"), valine (hereinafter, marked "Val" or only "V"), leucine (hereinafter, marked "Leu" or only "L"), isoleucine (hereinafter, marked "Ile" or only "I"), proline (hereinafter, marked "Pro" or only "P"), phenylalanine (marked "Phe" or only "F"), tryptophan (hereinafter, marked "Trp" or only "W"), methionine (hereinafter, marked "Met" or only "M").

[0186] (2) Non-polarized amino acid groups: glycine (hereinafter, marked "Gly" or only "G"), serine (hereinafter, marked "S" or only "S"), threonine (hereinafter, marked "Thr" or only "T"), cysteine ​​(hereinafter, marked "Cys" or only "C"), tyrosine (hereinafter, marked "Tyr" or only "Y"), asparagine (hereinafter, marked "Asn" or only "N"), glutamine (hereinafter, marked "Gln" or only "Q").

[0187] (3) Acidic amino acid group: Aspartic acid (hereinafter, marked as "Asp" or only marked as "D"), glutamic acid (hereinafter, marked as "Glu" or only marked as "E").

[0188] (4) Basic amino acid group: Lysine (hereinafter, marked "Lys" or only "K"), Arginine (hereinafter, marked "Arg" or only "R"), Histidine (hereinafter, marked "His" or only "H").

[0189] Furthermore, amino acids that exist in nature can be divided into groups based on the properties of their common side chains, as follows.

[0190] (1) Hydrophobic amino acid group: Norleucine, Met, Ala, Val, Leu, Ile

[0191] (2) Neutral hydrophilic amino acid group: Cys, Ser, Thr, Asn, Gln

[0192] (3) Acidic amino acid group: Asp, Glu

[0193] (4) Basic amino acid group: His, Lys, Arg

[0194] (5) Groups of amino acids that affect the orientation of the main chain: Gly, Pro

[0195] (6) Aromatic amino acid group: Trp, Tyr, Phe

[0196] In addition, each group also contains non-natural amino acids such as N-methylated amino acids.

[0197] A preferred example of this peptide is a peptide having the amino acid sequence described in sequence number 2 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Arg-Arg-Tyr-MeTyr-Cys).

[0198] A preferred example of this peptide is one in which the amino acid sequence described in sequence number 2 has one or more substitutions selected from the following group:

[0199] The first alanine residue in sequence number 2 is replaced by an aliphatic amino acid or a methylated aliphatic amino acid;

[0200] The second valine residue in sequence number 2 is replaced by a basic amino acid residue or a methylated basic amino acid residue;

[0201] The third phenylalanine residue in sequence number 2 is replaced by an aromatic amino acid residue, a methylated aromatic amino acid residue, an amino acid residue with an added aromatic ring, or an amino acid residue with an added fused ring.

[0202] The fourth valine residue in sequence number 2 is replaced by a methylated valine residue;

[0203] The fifth tryptophan residue in sequence number 2 is replaced by an aromatic amino acid residue, a methylated aromatic amino acid residue, an amino acid residue with an added aromatic ring, or an amino acid residue with an added fused ring.

[0204] The sixth asparagine residue in sequence number 2 is replaced by a neutral amino acid or a methylated neutral amino acid;

[0205] The 7th and 8th tyrosine residues of sequence number 2 are replaced by aromatic amino acid residues, methylated aromatic amino acid residues, amino acid residues with added aromatic rings, or amino acid residues with added fused rings.

[0206] The 9th isoleucine residue of sequence number 2 is replaced by an aliphatic amino acid residue, a methylated aliphatic amino acid residue, or an amino acid residue with a branched structure.

[0207] The 10th isoleucine residue in sequence number 2 is replaced by any amino acid;

[0208] The 11th and 12th arginine residues in sequence number 2 are replaced by basic amino acid residues;

[0209] The 13th tyrosine residue in sequence number 2 is replaced by a hydrophilic amino acid residue;

[0210] The 14th methyltyrosine residue of sequence number 2 is replaced by a tyrosine residue, an aromatic amino acid residue, or a methylated aromatic amino acid residue; and

[0211] The 15th cysteine ​​residue in sequence number 2 was replaced by a methylated cysteine ​​residue.

[0212] Another preferred example of this peptide is that if peptide A is defined as containing the amino acid sequence described in sequence number 18 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Ser-Cys) from the 1st to the 10th amino acid, and the peptide length is 10 or more and 17 or less, then the peptide is the following:

[0213] Peptide A; or

[0214] In peptide A, there is a peptide having an amino acid sequence containing 1 or more but less than 6 amino acid residues that have been replaced, deleted, or inserted.

[0215] The preferred peptides in this specification are, like the peptides described above, peptides that can bind to the human transferrin receptor (hTfR). Furthermore, preferred examples are peptides that can cross the blood-brain barrier (BBB), peptides that are targeted to muscle tissue, or peptides that have cell-penetrating properties.

[0216] It can also be the following peptide: In peptide A, an amino acid sequence in which any one of the 2nd, 3rd, 5th, 8th and 10th amino acid residues of sequence number 18 is replaced.

[0217] The term "amino acid residue substitution" means that a specific amino acid residue is replaced with another amino acid residue that can be modified.

[0218] It can also be the following peptides: In peptide A,

[0219] The second amino acid residue of sequence number 18 is either a modifiable valine (Val) or a modifiable glutamic acid (Glu).

[0220] The third amino acid residue of sequence number 18 is phenylalanine (Phe), which can be modified.

[0221] The fifth amino acid residue of sequence number 18 is tryptophan (Trp), which can be modified.

[0222] The 8th amino acid residue of sequence number 18 is a modifiable tyrosine residue (Tyr).

[0223] The 10th amino acid residue of sequence number 18 is either isoleucine (Ile) or valine (Val), which can be modified.

[0224] The term "modifiable" refers to amino acids that can be modified or altered in a well-known manner. Examples of modifications include N-methylation, amino acid modifications (hereinafter referred to as such), modifications (conversions) to the D-type, and conversions to well-known derivatives of the amino acid.

[0225] The peptide is preferably 11 or more and 13 or less in length.

[0226] This peptide is further preferred to be a peptide having the following amino acid sequence.

[0227] The second amino acid residue of sequence number 18 could be Val or Glu.

[0228] The third amino acid residue of sequence number 18 is either Phe or MeF3C.

[0229] The fifth amino acid residue of sequence number 18 is either Trp or MeTrp.

[0230] The 8th amino acid residue of sequence number 18 is either Tyr or F4OMe.

[0231] The peptide with the 10th amino acid residue of sequence number 18 being either Ile or Val.

[0232] Furthermore, this peptide has the following amino acid sequence at the N-terminus of peptide A.

[0233] The 11th amino acid residue of sequence number 18 could be either Ser or His.

[0234] The peptide with the 12th amino acid residue of sequence number 18 being either Cys or Hgl.

[0235] Another preferred example of this peptide is that if peptide B is defined as containing the amino acid sequence described in sequence number 15 (Ala-Val-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Val-Pro-Arg-Asp-Cys) from the 1st to the 10th amino acid, and the peptide length is 10 or more and 19 or less, then the peptide is the following:

[0236] Peptide B; or

[0237] In peptide B, there is a peptide having an amino acid sequence containing 1 or more but less than 5 amino acid residues that have been replaced, deleted, or inserted.

[0238] It can also be the following peptide: In peptide B, an amino acid sequence in which any one of the 2nd, 3rd, 5th, 8th and 10th amino acid residues of sequence number 15 is replaced.

[0239] It can also be the following peptides: In peptide B,

[0240] The second amino acid residue of sequence number 15 is either a modifiable valine (Val) or a modifiable glutamic acid (Glu).

[0241] The third amino acid residue of sequence number 15 is either phenylalanine (Phe) or tryptophan (Trp), which can be modified.

[0242] The fifth amino acid residue of sequence number 15 is tryptophan (Trp), which can be modified.

[0243] The 8th amino acid residue of sequence number 15 is a modifiable tyrosine residue (Tyr).

[0244] The 10th amino acid residue of sequence number 15 is either isoleucine (Ile) or valine (Val) that can be modified.

[0245] The peptide length of these peptides can be 13 or more and 15 or less.

[0246] This peptide is further preferred to be a peptide having the following amino acid sequence.

[0247] The second amino acid residue of sequence number 15 could be Val or Glu.

[0248] The third amino acid residue of sequence number 15 is Phe, Trp, or MeF3C.

[0249] The fifth amino acid residue of sequence number 15 is either Trp or MeTrp.

[0250] The 8th amino acid residue of sequence number 15 is Tyr, Phe, or F4OMe.

[0251] The peptide with the 10th amino acid residue of sequence number 15 being either Ile or Val.

[0252] Furthermore, this peptide has the following amino acid sequence at the N-terminus of peptide B.

[0253] The 11th amino acid residue of sequence number 15 could be Phe.

[0254] The 12th amino acid residue of sequence number 15 is Arg.

[0255] The 13th amino acid residue of sequence number 15 is Glu, Asn, Asp, His, Gln, or MeTrp.

[0256] The 14th amino acid residue of sequence number 15 is a peptide of Cys.

[0257] Another preferred example of this peptide is that if peptide C is defined as containing the amino acid sequence described in sequence number 214 (MeA-Val-MeF3C-Val-MeW-Asn-Tyr-F4OMe-Ile-Ile-Arg-Arg-Phe-MeY-Cys) from the 1st to the 10th amino acid, and the peptide length is 11 or more and 19 or less, then the peptide is the following:

[0258] Peptide C; or

[0259] In peptide C, there is a peptide having an amino acid sequence containing 1 or more but less than 5 amino acid residues that have been replaced, deleted, or inserted.

[0260] It can also be the following peptide: in peptide C, an amino acid sequence in which any one of the 1st, 3rd, 5th and 8th amino acid residues of sequence number 214 is replaced.

[0261] It can also be the following peptides: In peptide C,

[0262] The first amino acid residue of sequence number 214 is either alanine (Ala) or glutamic acid (Glu), which can be modified.

[0263] The third amino acid residue of sequence number 214 is phenylalanine (Phe), which can be modified.

[0264] The fifth amino acid residue of sequence number 214 is tryptophan (Trp), which can be modified.

[0265] The 8th amino acid residue of sequence number 214 is phenylalanine (Phe), which can be modified.

[0266] These peptides are preferably 15 or more and 18 or less in length.

[0267] This peptide is further preferred to be a peptide having the following amino acid sequence.

[0268] The first amino acid residue of sequence number 214 may be Ala, Aib, Abu, Glu, Gly, Ser, Phe, Pro, or MeA, with Ala or MeA being particularly preferred.

[0269] The third amino acid residue of sequence number 214 is Phe, F3C, F2C, F2OMe, F4C, Cha, MeF, MeF35dC, MeF4F, MeF4Ome, MeNal1, Me3Py, Me4Py, Me3OMe, MeF3COO, MeF3F, Glu, Epyrl2RCOO, Dpyrl2RCOO, or MeF3C, with Phe, MeF, or MeF3C being particularly preferred.

[0270] The fifth amino acid residue of sequence number 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, with Trp or MeW being particularly preferred.

[0271] The 8th amino acid residue of sequence number 214 is Phe, Tyr, Typ, Ahp, MeY, F4OMe, 3Imp, 4Py, 3Py, 3Py6OMe, F3C, F3CON, F4C, F4aao, F4F, F4OEt, MeF34dOMe, Yae, Lys, Orn, or Nal1, with a peptide of Tyr or F4OMe being particularly preferred.

[0272] Furthermore, this peptide has the following amino acid sequence at the N-terminus of peptide C.

[0273] The 11th amino acid residue of sequence number 214 may be Arg, Ala, Asp, Gly, Glu, Lys, MeK, MeR, Dap, Dap, Abu, Aib, Hly, dorn, aMeK, A1Me4pip, KCOpipzMe, F4G, Nle, Nva, or Orn, with Lys or Arg being particularly preferred.

[0274] The 12th amino acid residue of sequence number 214 is Lys, Arg, dr, Tyr, F4G, Orn, Hly, da, Cit, Dap, or Dab, with Lys, Arg, or dr being particularly preferred.

[0275] The 13th amino acid residue of sequence number 214 is Ala, Phe, Asn, Tyr, or pHPeG, with Phe or Tyr being particularly preferred.

[0276] The 14th amino acid residue of sequence number 214 is MeTyr, Tyr, Phe, Ala, aMeY, Glu, Gly, Arg, Val, MeoBphMeBph, MeF, MemBph, MeNal1, MeNal2, MeoBph, MeW, or pHPeG, with Phe or MeW being particularly preferred.

[0277] The 15th amino acid residue of sequence number 214 is a peptide of Cys or Hgl.

[0278] Another preferred example of this peptide is that if peptide D is set to contain the amino acid sequence described in sequence number 219 (Ala-Glu-Phe-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Arg-Arg-Tyr-MeY-Cys) from the 1st to the 10th amino acid sequence and the peptide length is 11 or more and 19 or less, then the peptide is the following peptide:

[0279] Peptide D; or

[0280] In peptide D, there is a peptide having an amino acid sequence with 1 or more but less than 5 amino acid residues that have been replaced, deleted, or inserted.

[0281] It can also be the following peptide: In peptide D, an amino acid sequence in which any one of the 2nd, 3rd, 5th, 8th and 10th amino acid residues of sequence number 219 is replaced.

[0282] It can also be the following peptides: In peptide D,

[0283] The second amino acid residue of sequence number 219 is a modifiable valine (Val), a modifiable glutamic acid (Glu), a modifiable arginine (Arg), a modifiable lysine (Lys), a modifiable aspartic acid (Asp), or a modifiable phenylalanine (Phe).

[0284] The third amino acid residue of sequence number 219 is phenylalanine (Phe), which can be modified.

[0285] The fifth amino acid residue of sequence number 219 is tryptophan (Trp), which can be modified.

[0286] The 8th amino acid residue of sequence number 219 is a modifiable tyrosine residue (Tyr).

[0287] The 10th amino acid residue of sequence number 219 is a modifiable isoleucine (Ile), a modifiable glutamic acid (Glu), or a modifiable lysine (Lys).

[0288] These peptides are preferably 15 or more and 18 or less in length.

[0289] This peptide is further preferred to be a peptide having the following amino acid sequence.

[0290] The second amino acid residue of sequence number 219 may be Val, Glu, Ala, Arg, Lys, Asp, Phe, Dap, Har, Abu, Nva, AcPr, AtbAhp, or Hgl, with Gln or Val being particularly preferred.

[0291] The third amino acid residue of sequence number 219 is Phe, F3C, F2C, F2OMe, F4C, Cha, MeF, MeF35dC, MeF4F, MeF4Ome, MeNal1, Me3Py, Me4Py, Me3OMe, MeF3COO, MeF3F, Glu, Epyrl2RCOO, Dpyrl2RCOO, or MeF3C, with particular preference to be Phe, MeF, or MeF3C.

[0292] The fifth amino acid residue of sequence number 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, with Trp or MeW being particularly preferred.

[0293] The 8th amino acid residue of sequence number 219 is Phe, Tyr, Typ, Ahp, MeY, F4OMe, 3Imp, 4Py, 3Py, 3Py6OMe, F3C, F3CON, F4C, F4aao, F4F, F4OEt, MeF34dOMe, Yae, Lys, Orn, or Na1, with Tyr or F4OMe being particularly preferred.

[0294] The 10th amino acid residue of sequence number 219 is Ala, Abu, Accr, Ahp, Aib, alI, alT, Atb, Dab, Dap, dorn, Gln, Hly, Ile, Lys, KCOpipzMe, Leu, Nle, Nva, Pro, Arg, Ser, Thr, Tbg, Val, or Tyr, with a peptide of Ile or alI being particularly preferred.

[0295] Furthermore, this peptide is a peptide with the following amino acid sequence at the N-terminus of peptide C.

[0296] The 11th amino acid residue of sequence number 219 may be Arg, Ala, Asp, Gly, Glu, Lys, MeK, MeR, Dap, Dap, Abu, Aib, Hly, dorn, aMeK, A1Me4pip, KCOpipzMe, F4G, Nle, Nva, or Orn, with Lys or Arg being particularly preferred.

[0297] The 12th amino acid residue of sequence number 219 is Lys, Arg, dr, Tyr, F4G, Orn, Hly, da, Cit, Dap, or Dab, with Lys, Arg, or dr being particularly preferred.

[0298] The 13th amino acid residue of sequence number 219 is Ala, Phe, Asn, Tyr, or pHPeG, with Phe or Tyr being particularly preferred.

[0299] The 14th amino acid residue of sequence number 219 is MeTyr, Tyr, Phe, Ala, aMeY, Glu, Gly, Arg, Val, MeoBphMeBph, MeF, MemBph, MeNal1, MeNal2, MeoBph, MeW, or pHPeG, with Phe or MeW being particularly preferred.

[0300] The 15th amino acid residue of sequence number 219 is a peptide of Cys or Hgl.

[0301] Another preferred example of this peptide is that if peptide E is defined as containing the amino acid sequence described in sequence number 296 (Ala-Val-MeF-Val-Trp-Asn-Tyr-Tyr-Ile-Ile-Arg-Arg-Tyr-MeY-Cys) from the 1st to the 15th amino acid, and the peptide length is 15 or more and 18 or less, then the peptide is the following:

[0302] Peptide E; or

[0303] In peptide E, a peptide having an amino acid sequence with 1 or more but less than 5 amino acid residues that have been replaced, deleted, or inserted.

[0304] It can also be the following peptide: In peptide E, an amino acid sequence in which any one of the 3rd, 5th, 7th, 8th, 11th, 12th, or 13th amino acid residues of sequence number 296 is replaced.

[0305] It can also be the following peptides: In peptide E,

[0306] The third amino acid residue of sequence number 296 is phenylalanine (Phe), which can be modified.

[0307] The fifth amino acid residue of sequence number 296 is tryptophan (Trp), which can be modified.

[0308] The 7th amino acid residue of sequence number 296 is a tyrosine residue that can be modified (Tyr).

[0309] The 8th amino acid residue of sequence number 296 is a modifiable tyrosine residue (Tyr).

[0310] The 11th amino acid residue of sequence number 296 is either arginine (Arg) or alanine (Ala), which can be modified.

[0311] The 12th amino acid residue of sequence number 296 is either arginine (Arg) or lysine (Lys), which can be modified.

[0312] The 13th amino acid residue of sequence number 296 is either a modifiable tyrosine (Tyr) or a modifiable phenylalanine (Phe).

[0313] Furthermore, it can also be the following peptides: In peptide E,

[0314] The third amino acid residue of sequence number 296 is Phe, F3C, F2C, F2OMe, F4C, Cha, MeF, MeF35dC, MeF4F, MeF4Ome, MeNal1, Me3Py, Me4Py, Me3OMe, MeF3COO, MeF3F, Glu, Epyrl2RCOO, Dpyrl2RCOO, or MeF3C, with particular preference being Phe, MeF, or MeF3C.

[0315] The fifth amino acid residue of sequence number 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, with Trp or MeW being particularly preferred.

[0316] The 7th amino acid residue of sequence number 296 is Tyr, 3Py6OMe, Ala, Ahp, Phe, F3H, F4C, Na1, Arg, or Trp, with Tyr being particularly preferred.

[0317] The 8th amino acid residue of sequence number 296 is Phe, Tyr, Typ, Ahp, MeY, F4OMe, 3Imp, 4Py, 3Py, 3Py6OMe, F3C, F3CON, F4C, F4aao, F4F, F4OEt, MeF34dOMe, Yae, Lys, Orn, or Nal1, with Tyr or F4OMe being particularly preferred.

[0318] The 11th amino acid residue of sequence number 296 is Arg, Ala, Asp, Gly, Glu, Lys, MeK, MeR, Dap, Dap, Abu, Aib, Hly, dorn, aMeK, A1Me4pip, KCOpipzMe, F4G, Nle, Nva, or Orn, with Lys or Arg being particularly preferred.

[0319] The 12th amino acid residue of sequence number 296 is Lys, Arg, dr, Tyr, F4G, Orn, Hly, da, Cit, Dap, or Dab, with Lys, Arg, or dr being particularly preferred.

[0320] The 13th amino acid residue of sequence number 296 is Ala, Phe, Asn, Tyr or pHPeG, with Phe or Tyr being particularly preferred.

[0321] Furthermore, this peptide has the following amino acid sequence at the N-terminus of peptide C.

[0322] The 11th amino acid residue of sequence number 296 may be Arg, Ala, Asp, Gly, Glu, Lys, MeK, MeR, Dap, Dap, Abu, Aib, Hly, dorn, aMeK, A1Me4pip, KCOpipzMe, F4G, Nle, Nva, or Orn, with Lys or Arg being particularly preferred.

[0323] The 12th amino acid residue of sequence number 296 is Lys, Arg, dr, Tyr, F4G, Orn, Hly, da, Cit, Dap, or Dab, with Lys, Arg, or dr being particularly preferred.

[0324] The 13th amino acid residue of sequence number 296 is Ala, Phe, Asn, Tyr, or pHPeG, with Phe or Tyr being particularly preferred.

[0325] The 14th amino acid residue of sequence number 214 is MeTyr, Tyr, Phe, Ala, aMeY, Glu, Gly, Arg, Val, MeoBphMeBph, MeF, MemBph, MeNal1, MeNal2, MeoBph, MeW, or pHPeG, with Phe or MeW being particularly preferred.

[0326] The 15th amino acid residue of sequence number 296 is a peptide of Cys or Hgl.

[0327] Furthermore, it can also be the following peptides: In peptide E,

[0328] The third amino acid residue of sequence number 296 is phenylalanine (Phe), methylated phenylalanine (MeF), or N-α-methyl-N-α-chloroacetyl-3-chloro-L-phenylalanine (MeF3C).

[0329] The fifth amino acid residue of sequence number 296 is tryptophan (Trp) or methylated tryptophan (MeW).

[0330] The 7th amino acid residue of sequence number 296 is tyrosine (Tyr).

[0331] The 8th amino acid residue of sequence number 296 is either tyrosine (Tyr) or (S)-2-amino-3-(4-methoxyphenyl)propionic acid (F4OMe).

[0332] The 11th amino acid residue of sequence number 296 is either arginine (Arg) or lysine (Lys).

[0333] The 12th amino acid residue of sequence number 296 is either arginine (Arg) or D-arginine (dr).

[0334] The 13th amino acid residue of sequence number 296 is either tyrosine (Tyr) or phenylalanine (Phe).

[0335] Preferred examples of this peptide are peptides composed of any amino acid sequence from sequence number 3 to 200, or peptides composed of amino acid sequences in which the N-terminus is a chloroacetyl-Ala group.

[0336] The preferred example of this peptide is any of the peptides mentioned above, and it is a cyclic peptide.

[0337] A preferred example of this peptide is a peptide comprising the first to tenth amino acid sequences of any of the amino acid sequences described in sequence numbers 1-552 or a complex of an amino acid sequence and a linker, and wherein the amino acid sequence site has a cyclic structure.

[0338] A particularly preferred example of this peptide is a peptide consisting of the first to fifteenth amino acid sequences in a complex of an amino acid sequence and a linker as described in any of the sequence numbers 2, 9, 21-148, 159-200, 213-448, 450-552, and wherein the amino acid sequence sites have a cyclic structure.

[0339] Regarding cyclic peptides

[0340] This refers to a peptide in which two amino acids are combined, forming a ring structure, either entirely or partially. Furthermore, in this case, it also includes peptides in which amino acids form cross-linked structures, peptides that form cyclic structures through the formation of lactam rings or macrocyclization reactions, and peptides with a lasso-like structure. That is, in this case, a cyclic peptide only needs to have a portion forming a cyclic structure; it may also have a linear portion.

[0341] Generally, peptides exhibit poor metabolic stability in vivo and their large size makes them difficult to pass through cell membranes. To address this issue, methods for cyclizing peptides have been developed. Cycling peptides enhances protease resistance and metabolic stability without restricting conformational changes, thus implying increased rigidity and improved membrane permeability and affinity for target proteins.

[0342] cyclization method

[0343] The cyclization of peptides can be carried out using well-known methods.

[0344] While not limited to this, for example, by designing the peptide to contain two or more cysteine ​​residues, a cyclic structure can be formed via disulfide bonds after translation. Furthermore, following the method of Goto et al. (Y. Goto, et al. AcssChem. Biol. 3120-129 (2008)), peptides with a chloroacetyl group at the N-terminus can be synthesized using genetic code reprogramming techniques, and cysteine ​​residues can be incorporated into the peptide, thus enabling cyclization. Therefore, after translation, the thiol group automatically performs a nucleophilic attack on the chloroacetyl group, and the peptide cyclizes via a thioether bond. Cycling can also be achieved by incorporating combinations of other amino acids that bind to form a ring within the peptide using genetic code reprogramming techniques. Furthermore, peptides with a cyclic amide group at the N-terminus can also be cyclized by incorporating Hgl residues into the peptide. Thus, any well-known cyclization method can be used without particular restriction.

[0345] Preferred examples of this peptide are any of the peptides mentioned above, and it is a peptide composed of 15 amino acid residues.

[0346] peptide length

[0347] The number of amide bonds (number and length of amino acids) in the peptide and peptide site is not particularly limited, but it is preferred that the total number of amino acid residues (in the case where the substance bound to the peptide or the linker that binds the substance to the peptide contains amino acids, but does not contain such amino acids) is 20 residues or less. It is preferred that 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 it is more preferred that the number of amino acids is 19 or less, 18 or less, 17 or less, 16 or less, or 15 or less.

[0348] This specification describes an invention relating to a conjugate (complex). This complex comprises any one of the peptides described above, a linker bound to the peptide, and a substance bound to the linker. Preferably, it is a complex in which at least the substance can cross the blood-brain barrier. Alternatively, the entire complex can cross the blood-brain barrier. The complex is preferably directional towards muscle tissue. The complex is preferably capable of transporting at least the substance to muscle tissue. The complex is preferably cell-penetrating. This complex is preferably capable of transporting at least the substance to cells.

[0349] Examples of linkers are those with an amino acid length of 1 or more but less than 15, and which contain more than one glycine or serine.

[0350] A preferred example of this linker is that the N-terminus is a modifiable cysteine ​​(Cys) or a modifiable lysine (Lys).

[0351] Another example of a linker is an amino acid with a length of 1 or more and 5 or less, and which contains either or both of D-type glutamic acid (de) and methylated glycine (MeG).

[0352] A preferred example of this linker is a complex with a modified cysteine ​​(Cys) or a modified lysine (Lys) at the N-terminus.

[0353] Another example of a linker is a PEG linker that contains polyethylene glycol (PEG) or a derivative of PEG. PEG derivatives contain all well-known PEG linkers.

[0354] Preferably, the PEG linker further comprises glycine (Gly), serine (Ser), glutamic acid (Glu), arginine (Arg) or lysine (Lys).

[0355] A preferred example of this linker is a complex with a modified cysteine ​​(Cys) or a modified lysine (Lys) at the N-terminus.

[0356] Another example of a connector is a connector that has a sequence represented by any of the serial numbers 201, 553-644.

[0357] A preferred embodiment of this complex has a connector as follows:

[0358] Polyethylene glycol (PEG);

[0359] Peptide linkers composed of Gly or MeG, i.e., G linkers; peptide linkers composed of Gly or MeG and Ser, i.e., GS linkers; or

[0360] Linkers having an amino acid sequence represented by any of sequence numbers 201, 553-644.

[0361] In this specification, a linker (also called 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 can 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). Furthermore, it can be, for example, a complex of a chemical linker and a peptide linker. For example, it can be a linker structure having PEG and amino acid residues or a peptide moiety, as shown in Serial No. 616 or Serial No. 627.

[0362] Connectors can be those that disperse or separate due to environment or conditions, or those that maintain a stable structure.

[0363] Chemical linkers: In some embodiments, the linker may be a chemical linker. While not limited thereto, chemical linkers may include, for example, substituted or non-substituted alkylene groups, substituted or non-substituted heteroalkylene groups, substituted or non-substituted cycloalkylene groups, substituted or non-substituted heterocycloalkylene groups, substituted or non-substituted propyne groups, and / or substituted or non-substituted heteropropyl groups. Furthermore, the peptide and linker may be conjugated by a thioglycolic group, an amino group (ammonia), and / or a carbohydrate group or any suitable reactive group. Homodifunctional and heterodifunctional crosslinking agents (conjugators) can be derived from many commercial sources. Crosslinking agents may contain flexible arms, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms. Examples of crosslinking agents include BSS3 ([bis(sulfosuccinimide) octanoate]), NHSS / EDC (N-hydroxysuccinimide and N-ethyl-(dimethylaminopropyl)carbodiimide), sulfonic acid-EMCSS ([Ne-maleimide hexanoic acid] acylhydrazide, acylhydrazide, and SSATA (N-succinimide-SS-acetylacetic acid), etc.

[0364] A preferred example of a chemical linker is a PEG (Polyethylene glycol) linker. For example, a PEG linker can be a PEG linker composed of 1-24 ethylene glycol units.

[0365] Fatty acid linkers: Linkers can be fatty acid linkers induced by fatty acids and containing a divalent chemical moiety. For example, fatty acid linkers can be linkers with 12-aminododecanoic acid.

[0366] Peptide linker: The peptide linker contains at least one amino acid (e.g., a peptide with at least 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 40, or 50 amino acids). In a particular embodiment, the linker is one amino acid (e.g., any natural amino acid such as Cys). In other embodiments, a glycine-rich peptide, such as a peptide having the sequence [Gly-Gly-Gly-Gly-Ser]n (where n is 1, 2, 3, 4, 5, or 6), as described in U.S. Patent No. 7,271,149, is used. In another embodiment, a serine-rich peptide linker, such as that described in U.S. Patent No. 5,525,491, is used. Examples of serine-rich peptide linkers include those of the formula [XXXX-Gly]y (where at most two of the X are Thr, the other X are Ser, and y is 1-5) (e.g., Ser-Ser-Ser-Ser-Gly (where y is 2 or more)). In some cases, the linker is a single amino acid (e.g., any amino acid such as Gly or Ala).

[0367] The 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 comprises the aforementioned complex, and the aforementioned substance is the active ingredient.

[0368] The substance is intended to be delivered to the brain. The substance can be any substance desired by a person skilled in the art, as long as it is intended to be delivered to the brain. However, since the passage of the BBB is achieved through mechanisms such as endocytosis via binding to transferrin receptors and endocytic transport, excessively large substances that cannot be transported through these mechanisms are less desirable. Examples of such substances are not limited, but the following can be cited:

[0369] Compounds: This includes not only low-molecular-weight and medium-molecular-weight compounds, but also any compounds that can be introduced through cellular uptake mechanisms. Examples include well-known low-molecular-weight drugs.

[0370] Peptide: A peptide that can bind to a target in the body and exert a certain effect; for example, it can be a cyclic peptide.

[0371] RI: Low-molecular-weight or medium-molecular-weight compounds or antibodies labeled with radioactive isotopes, as long as they are compounds that can be labeled with radioactive isotopes. Examples include compounds used in PET scans.

[0372] Proteins: This includes any protein that performs useful functions in the body, such as antibodies and enzymes. Examples include enzymes used in enzyme supplementation therapy.

[0373] Nucleic acids: Any substance containing a base sequence, such as DNA or RNA. Examples include nucleic acid pharmaceuticals.

[0374] DDS: Can be DDS molecules such as liposomes or microcells. These DDS molecules may further contain compounds such as pharmaceuticals.

[0375] And, can be a complex of the aforementioned types.

[0376] The invention described in this specification relates to a method for manufacturing a preventive or therapeutic agent for brain-related diseases. This method is a method for manufacturing a preventive or therapeutic agent for brain-related diseases that includes the step of obtaining the aforementioned complex.

[0377] A preferred example of this method is a connector that is:

[0378] Polyethylene glycol (PEG);

[0379] G connector, GS connector; or

[0380] Linkers having an amino acid sequence represented by any of sequence numbers 201, 553-644.

[0381] The invention described in this specification relates to a diagnostic drug for brain-related diseases comprising the aforementioned complex.

[0382] The invention described in this specification is a preventive or therapeutic agent for neuromuscular diseases comprising the aforementioned complex. In this case, the substance is the active ingredient in the preventive or therapeutic agent for neuromuscular diseases. The invention described in this specification comprises the aforementioned complex. The invention described in this specification relates to a diagnostic drug for neuromuscular diseases comprising the aforementioned complex.

[0383] The peptides of the present invention can be manufactured by well-known peptide manufacturing methods such as chemical synthesis methods, including liquid-phase methods, solid-phase methods, hybridization of liquid-phase methods and solid-phase methods, and gene recombination methods.

[0384] Solid-phase methods include, for example, esterifying the hydroxyl groups of a resin with hydroxyl groups with the carboxyl group of the first amino acid (usually the C-terminal amino acid of the target peptide) protected by a protecting group. Esterification catalysts can be well-known dehydrating condensing agents such as 1-mesinesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIPCDI).

[0385] Next, the protecting group of the α-amino group of the first amino acid is removed, and a second amino acid with all functional groups except the carboxyl group of the main chain protected is added, activating the carboxyl group and causing the first and second amino acids to bind. Then, the α-amino group of the second amino acid is deprotected, and a third amino acid with all functional groups except the carboxyl group of the main chain protected is added, activating the carboxyl group and causing the second and third amino acids to bind. This process is repeated until a peptide of the target length is synthesized, at which point all functional groups are deprotected.

[0386] Examples of resins synthesized in the solid phase include Merrifield resin, MBHAresin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGAresin (Merck), and HMPA-PEGAresin (Merck). These resins can be used after cleaning with solvents such as dimethylformamide (DMF), 2-propanol, and chloromethane.

[0387] Examples of protecting groups for α-amino groups include benzyloxycarbonyl (Cbz or Z), tert-butoxycarbonyl (Boc), fluoreneoxycarbonyl (Fmoc), benzyl, allyl, allyloxycarbonyl (Alloc), etc.

[0388] 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.

[0389] The α-carboxyl group can be protected using methyl ester, ethyl ester, benzyl ester, tert-butyl ester, cyclohexyl ester, etc.

[0390] Other functional groups of amino acids include the hydroxyl groups of serine or threonine, which can be protected by benzyl or tert-butyl groups; and the hydroxyl groups of tyrosine, which can be protected by 2-bromobenzoxycarbonyl or tert-butyl groups. The amino group of the lysine side chain and the carboxyl group of glutamic acid or aspartic acid can be protected in the same way as the α-amino and α-carboxyl groups.

[0391] The activation of the carboxyl group can be achieved using condensing agents. Examples of condensing agents include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1-[bis(dimethylamino)methyl]-1H-benzotriazolium salt-3-oxide hexafluorophosphate (HBTU).

[0392] Peptide chains derived from resins can be cleaved by treatment with acids such as TFA or hydrogen fluoride (HF).

[0393] The manufacture of peptides by recombination (translation synthesis system) can be carried out using nucleic acids encoding the peptides of the present invention. The nucleic acids encoding the peptides of the present invention can be DNA or RNA.

[0394] The nucleic acid encoding the peptide of the present invention can be prepared using well-known methods or methods following those methods. For example, it can be synthesized using an automated synthesis apparatus. In order to insert the resulting DNA into a vector, a restriction enzyme recognition site can be added, or a base sequence encoding an amino acid sequence for cleaving the resulting peptide chain by an enzyme or the like can be incorporated.

[0395] As described 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 a membrane-permeable peptide.

[0396] To inhibit degradation by host-derived proteases, chimeric protein expression, which presents the target peptide as a chimeric peptide with other peptides, can also be used. In this case, the nucleic acid used can encode both the target peptide and the peptide bound to it.

[0397] Next, an expression vector is prepared using nucleic acids encoding the peptides of the present invention. The nucleic acid can be inserted downstream of the promoter of the expression vector, either directly, by digestion with restriction enzymes, or by adding a linker. Examples of vectors include plastids derived from *Escherichia coli* (pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pUC118, pBluescriptII, etc.), plastids derived from *Bacillus subtilis* (pUB110, pTP5, pC1912, pTP4, pE194, pC194, etc.), plastids derived from yeast (pSH19, pSH15, YEp, YRp, YIp, YAC, etc.), bacteriophages (e phage, M13 phage, etc.), viruses (retroviruses, vaccinia virus, adenovirus, adeno-associated virus (AAV), cauliflower mosaic virus, tobacco mosaic virus, baculovirus, etc.), and granules.

[0398] The promoter can be appropriately selected according to the type of host. When the host is an animal cell, promoters derived from SV40 (simian virus 40) or CMV (cytomegalovirus) can be used. When the host is Escherichia coli, trp promoters, T7 promoters, lac promoters, etc., can be used.

[0399] Nucleic acids encoding DNA replication origin (ori), selection markers (antibiotic resistance, nutritional requirements, etc.), enhancers, splicing signals, polyalpha addition signals, and tags (FLAG, HA, GST, GFP, etc.) can also be incorporated into the expression vector.

[0400] Next, appropriate host cells are transfected using the aforementioned expression vector. The host can be selected appropriately based on its relationship with the vector; for example, *Escherichia coli*, *Bacillus subtilis*, *Bacillus* species, yeast, insects or insect cells, and animal cells can be used. Animal cells can be, for example, HEK293T cells, CHO cells, COS cells, myeloma cells, HeLa cells, and Vero cells. Transformation can be performed according to the host species, following well-known methods such as lipid transfection, calcium phosphate transfection, electroporation, microinjection, and particle gun transfection. The target peptide is expressed by culturing the transfectants using conventional methods.

[0401] The purification of peptides from transgenic cultures involves obtaining a crude extract by: recovering the cultured cells, suspending them in an appropriate buffer, disrupting the cells through methods such as ultrasonic treatment or freeze-thaw cycles, followed by centrifugation and filtration. In cases where peptides are secreted in the culture medium, the supernatant is recovered.

[0402] Purification of crude extracts or culture supernatants can also be carried out by well-known methods or by following such methods (e.g., salting out, dialysis, ultrafiltration, gel filtration, SDS-PAGE, ion exchange chromatography, affinity chromatography, reversed-phase high-performance liquid chromatography, etc.).

[0403] The resulting peptides can also be converted from free bodies to salts or from salts to free bodies using well-known methods or by following those methods.

[0404] The translation synthesis system can be designed as a cell-free translation system. A cell-free translation system may contain, for example, ribosomal proteins, aminoacyl-tRNA synthetase (ARS), ribosomal RNA, amino acids, rRNA, GTP, ATP, translation initiation factor (IF), elongation factor (EF), release factor (RF), and ribosome recycling factor (RRF), as well as other factors required for translation. To improve performance efficiency, *E. coli* extract or wheat germ extract may also be added. Additionally, rabbit erythrocyte extract or insect cell extract may also be added.

[0405] In systems containing these components, proteins ranging from several hundred μg to several mg / mL can be produced by continuously supplying energy through dialysis. To simultaneously perform transcription from genetic DNA, systems incorporating RNA polymerase can also be used. Commercially available cell-free translation systems include the RTS-100 (registered trademark) from Roche Diagnostics, the PURESYSTEM from GeneFrontier, and the PURExpress InVitro Protein Synthesis Kit from NEW ENGLAND Biolabs, derived from *E. coli*. Systems using wheat germ extract include products from ZOEGENE and CellFree Sciences.

[0406] According to cell-free translation systems, expression products can be obtained in a high-purity form without the need for purification.

[0407] In cell-free translation systems, artificial aminoacyl-tRNAs, formed by linking (acylation) desired amino acids or hydroxy acids to tRNA, can be used to replace the aminoacyl-tRNAs synthesized by natural aminoacyl-tRNA synthetases. These aminoacyl-tRNAs can be synthesized using artificial ribonucleases.

[0408] Examples of this ribonuclease include flexizyme (H. Murakami, H. Saito, and H. Suga, (2003), Chemistry & Biology, Vol. 10, 655-662; H. Murakami, D. Kourouklis,andH.Suga,(2003),Chemistry&Biology,Vol.10,1077-1084;H.Murakami,A.Ohta,H.Ashigai,H.Suga(2006)Nature Methods 3,357-359"The flexizyme system: a highly flexible RNAaminoacylation tool for the synthesis of nonnatural peptides"; "Selectively charges amino acids activated by a water-friendly leaving group" (and WO2007 / 066627, etc.). Elastases are also known as the original elastase (Fx) and modified dinitrobenzyl elastase (dFx), enhanced elastase (eFx), amino elastase (aFx), etc.

[0409] By using tRNA generated by elastase and linking it to a desired amino acid or hydroxy acid, a desired codon can be associated with and translated from that amino acid or hydroxy acid. Specific amino acids can also be used as the desired amino acid. For example, the non-natural amino acid required for the aforementioned cyclization can also be introduced into the binding peptide using this method.

[0410] The chemical synthesis of macrocyclic peptides and their analogues of the present invention can be performed using various methods commonly used in the art, including staged solid-phase synthesis, semi-synthesis of peptide fragments supported by configurational relinking, and chemical linking. The synthesis of peptides and their analogues described in this specification is, for example, chemical synthesis using various solid-phase techniques as described in KJ Jensen, P.T. Shelton, S.L. Dedersen, Peptide Synthesis and Applications, 2nd Edition, Springer, 2013, etc. A preferred strategy is based on a combination of temporarily protecting the α-amino group and a Fmoc group that can be selectively removed by bases, and a protecting group that temporarily protects the side chain functional groups and is stable under Fmoc-removal conditions. Such general selection of peptide side chains is known from publications such as Peptide Synthesis and Applications, 2nd edition, GB Fields, RLNoble, Solid pHase Peptide Synthesis Utilizing 9-Fluorenylmethoxycarbonyl Amino Acids, Int. J. Peptide Protein Res. 35, 1990, 161-214. However, preferred peptide side chain protecting groups include Boc or Mtt groups for amino groups beginning with lysine, tert-butyl groups for carboxyl groups of glutamic acid or aspartic acid, and Trt and Mmt groups for thiol groups of cysteine.

[0411] The peptides and analogues described in this invention can be synthesized on the solid-phase resin and in a phased process. During synthesis, the α-amino protecting group of all amino acids and peptides used, including the C-terminal amino acid, must be selectively removed. Preferably, the solid-phase resin is used, and the C-terminal carboxyl group of the peptide appropriately protected by Fmoc or the C-terminal carboxyl group of the Fmoc-protected amino acid is activated by adding to an amino group on the solid-phase resin using a suitable reagent. Continuous elongation of the peptide chain can be achieved by sequentially repeating the removal of the N-terminal protecting group (Fmoc group) followed by the condensation of the protecting amino acid derivative, following the amino acid sequence of the target peptide. Furthermore, this allows the target peptide to be released in the final stage. For example, as conditions for its release, examples include Teixeira, WEBenckhuijsen, PEdeKoning, ARPMValentijn, JWDrijfhout, Protein Pept. Lett., 2002, 9, 379-385, etc. In TFA, as a scavenging agent, a TFA solution containing water / hydrosilane / thiol can be used to release it. A typical example is TFA / Water / TIS / DODT (volume ratio 92.5:2.5:2.5:2.5).

[0412] The synthesis of peptide analogs described in this specification can be carried out using a single- or multi-channel peptide synthesizer, such as the Liberty Blue synthesizer from CEM or the Syro I synthesizer from Biotage.

[0413] The activation of the carboxyl group can be carried out using condensing agents. Examples of condensing agents include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1-[bis(dimethylamino)methyl]-1H-benzotriazolium salt-3-oxide hexafluorophosphate (HBTU).

[0414] Another embodiment disclosed in this description relates to a pharmaceutical. This pharmaceutical contains the aforementioned peptides, pharmaceutically permissible salts or solvates thereof (for simplicity, these will also be referred to simply as peptides below). Preferably, this pharmaceutical contains an effective amount of the aforementioned peptides as its active ingredient.

[0415] In this instruction manual, the administration method of the pharmaceutical composition is not particularly limited; it can be administered orally or non-orally. Examples of non-oral administration include intramuscular injection, intravenous injection, subcutaneous injection, transdermal administration, and transmucosal administration (via nose, mouth, eye, lung, vagina, or rectum).

[0416] The aforementioned pharmaceutical ingredients can be modified in various ways due to the readily metabolizable and excreted nature of peptides. For example, the addition of polyethylene glycol (PEG) or sugar chains to peptides can increase their retention time in the blood and reduce their antigenicity. Furthermore, biodegradable polymers such as polylactic acid-ethylene glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared from unsaturated fatty acids, nanoparticles, and nanospheres can be used as sustained-release agents, and peptides can be included in these agents. In the case of transdermal administration, a weak current can be applied through the skin surface to allow it to penetrate the stratum corneum (iontophoresis).

[0417] The aforementioned pharmaceutical compositions can be formulated by directly using the active ingredient or by adding pharmaceutically permissible carriers, excipients, additives, etc. Examples of dosage forms include, for instance, liquids (e.g., injections), dispersants, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, lozenges, inhalers, ointments, eye drops, nasal drops, ear drops, and plasters.

[0418] Formulation can be carried out by using appropriate excipients, binders, disintegrants, lubricants, solubilizers, dissolution aids, colorants, flavorings, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, preservatives, antioxidants, etc., and by conventional methods.

[0419] Examples of ingredients that can be used in formulations include, but are not limited to, pharmaceutically permissible organic solvents such as purified water, saline, phosphate buffer, glucose, glycerol, and ethanol, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, silica, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, tragacanth, casein, agar, polyethylene glycol, diglycerides, glycerol, propylene glycol, petrolatum, paraffin, octyl dodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, and human serum albumin.

[0420] Given that peptides are difficult to absorb through mucous membranes, the aforementioned pharmaceutical ingredients may contain absorption enhancers to improve the absorption of poorly absorbed drugs. As such absorption enhancers, surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponins may be used; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylic acids; fatty acids such as hexanoic acid, decanoic acid, laurate acid, oleic acid, linoleic acid, and mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, and nitric oxide donors may be used.

[0421] Pills or tablets may also be coated with sugar, gastric-soluble, or enteric-soluble substances.

[0422] Injectable preparations may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. Furthermore, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, dissolving aids, preservatives, etc., may be added.

[0423] The dosage of the pharmaceutical composition of the present invention 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, susceptibility differences, administration method, administration interval, type of active ingredient, and type of formulation. While not particularly limited, for example, 30 μg-100 g, 100 μg-500 mg, or 100 μg-100 mg may be administered once or in several divided doses. In the case of injection, the dosage may be 1 μg / kg-3000 μg / kg or 3 μg / kg-1000 μg / kg, depending on the patient's weight, administered once or in several divided doses.

[0424] The methods for preventing or treating brain-related diseases using the peptides of the present invention can be implemented with reference to the description of the above-mentioned pharmaceutical composition.

[0425] For the prevention or treatment of neuromuscular diseases containing the above-mentioned complex, the above-mentioned pharmaceutical ingredients can be used.

[0426] Another embodiment disclosed in this specification relates to a diagnostic drug for brain-related diseases. This diagnostic drug for brain-related diseases comprises the aforementioned peptide, its salt, or a solvate thereof.

[0427] Drugs and reagent kits for detecting brain-related diseases

[0428] This invention also includes a brain-related disease detection drug containing the peptide of this invention. When used as a detection drug, the peptide of this invention can also be labeled as detectable. The peptide can be labeled, for example, using antibodies labeled with enzymes such as peroxidase and alkaline phosphatase, radioactive substances such as 125I, 131I, 35S, and 3H, fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine isothiocyanate, near-infrared fluorescent materials, luciferase, luciferin, jellyfish luminescent protein, etc. Alternatively, antibodies labeled with nanoparticles such as gold colloids and quantum dots can also be detected. For example, by creating a complex of an antibody that binds to a specific target related to brain-related diseases with the peptide of this invention, and then administering and detecting the complex, brain-related diseases can be detected.

[0429] Furthermore, in immunoassays, the peptides of the present invention can be labeled with biotin, allowing them to bind with avidin or other enzyme-labeled proteins such as enzymes for detection.

[0430] In immunoassays, enzyme-labeled ELISA methods are preferred due to their simplicity and speed in measuring antigens. For example, antibodies are immobilized on a solid-phase support, a sample is added and reacted, then a labeled peptide of the present invention is added and reacted again. After washing, it is reacted with an enzyme matrix to induce color development, and the absorbance is measured, thus enabling the detection of brain-related diseases. Alternatively, after reacting the antibody immobilized on the solid-phase support with the sample, an unlabeled peptide of the present invention can be added, or an antibody targeting the peptide of the present invention can be enzyme-labeled and further added.

[0431] When the enzyme is a peroxidase, enzyme substrates such as 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), and o-phenylenediamine (OPD) can be used; when the enzyme is an alkaline phosphatase, p-nitropheny phosphate (NPP) can be used.

[0432] In this specification, "solid support" is not specifically limited to any carrier capable of immobilizing antibodies. Examples include microplates, substrates, beads, nitrocellulose membranes, nylon membranes, PVDF membranes, etc., made of glass, metal, or resin. The target substance can be immobilized on such solid support using well-known methods.

[0433] The test kit of the present invention includes the reagents and instruments required for the above-mentioned tests (including the peptides, antibodies, solid-phase carriers, buffer solutions, enzyme reaction stop solutions, ELISA readers, etc. of the present invention, but not limited to these).

[0434] Another embodiment disclosed in this specification can also be considered as a brain-related disease detection kit containing the above-mentioned brain-related disease detection drug, a tool for detecting brain-related diseases and the various cellular functions or life phenomena associated with them.

[0435] This instruction manual also provides information on the use of peptides for manufacturing medicines for the prevention or treatment of brain-related diseases. In this case, the peptide may be any one of the above-mentioned peptides.

[0436] Furthermore, this instruction manual also provides information on the use of peptides for manufacturing medicines for the prevention or treatment of neuromuscular diseases. In this case, the peptide may be any one of the aforementioned types.

[0437] This specification also provides a method for the prevention or treatment of brain-related diseases, comprising the steps of administering an effective amount of a peptide, a pharmaceutically permissible salt thereof, a solvate thereof, or a complex thereof to a human, non-human mammal, or bird, i.e., the subject. The peptide may be any peptide described above. Examples of non-human mammals include primates other than humans, pigs, cattle, dogs, cats, horses, sheep, rats, and mice.

[0438] This instruction manual also provides a method for the prevention or treatment of brain-related diseases, comprising the steps of administering an effective amount of a peptide, its pharmaceutically permissible salt, or a solvate thereof, or a complex thereof to a human, non-human mammal, or bird, i.e., the subject.

[0439] The abbreviations used in this specification, especially in the following representative embodiments, are well known to those skilled in the art. Some of the abbreviations used are as follows:

[0440] Fmoc is 9-fluorenyloxycarbonyl;

[0441] HOAt is 1-hydroxybenzotriazole;

[0442] HATU is O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate;

[0443] MeCN is acetonitrile;

[0444] DBU is 1,8-diazabicyclo[5.4.0]-7-undecene;

[0445] DIPEA is N,N-diisopropylethylamine;

[0446] DODT is 3,6-dioxane-1,8-octane-dithiol;

[0447] DMSO stands for dimethyl sulfoxide.

[0448] DMF is N,N-dimethylformamide;

[0449] Mtt stands for monomethyltriphenylmethyl;

[0450] Mmt is monomethoxytriphenylmethyl;

[0451] Ns is 2-nitrobenzenesulfonic acid;

[0452] TFA stands for trifluoroacetic acid;

[0453] TIS stands for triisopropylsilane;

[0454] Trt stands for triphenylmethyl;

[0455] mL stands for milliliters (unit).

[0456] M is the molar concentration (unit);

[0457] v / v stands for volume / volume (volume / volume);

[0458] Adod is 12-aminododecanoic acid.

[0459] Example 1

[0460] Chemical synthesis

[0461] All raw materials, structural units, reagents, acids, bases, solid resins, and solvents used in the chemical synthesis in the following examples are commercially available products or synthesizable by those skilled in the art using organic chemical methods. Furthermore, amino acids containing protecting groups are commercially available products unless otherwise specified.

[0462] The elongation of peptide chains in the solid-phase resin was carried out using the resins described in the various examples as starting materials, under commonly used peptide coupling reaction conditions and Fmoc removal reaction conditions. The reaction was performed using an automated peptide synthesizer, namely CEM Liberty Blue, following the manufacturer's manual. Commonly used amino acids are listed below, with side-chain protecting groups indicated in parentheses.

[0463] 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.

[0464] The introduction of chloroacetyl groups was carried out as follows: For a solid resin containing the Fmoc-protected peptide obtained in the previous step, after removing the Fmoc group of the α-amino group using the method described, chloroacetic acid (3 equal volumes) was added to 3 equal volumes of DMF solution (0.5 M) of N,N'-diisopropylcarbodiimide and 3 equal volumes of DMF solution (0.5 M) of HOAt, and the mixture was shaken at room temperature for 40 minutes.

[0465] To remove the protection of the side chains and shear from the solid resin, firstly, the resin obtained after the step of introducing chloroacetyl groups was washed five times each with DMF and chloromethane, and then dried under reduced pressure. Next, in the reaction vessel already loaded with the solid resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 150 minutes. The reaction solution was recovered by glass flotation. The solid resin remaining in the reaction vessel was shaken again with the shearing mixture, and the solution components were recovered by glass flotation and mixed with the filtrate. Adding this filtrate to excess diethyl ether cooled to 0°C produced a turbid 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 cooled to 0°C, and the solid was used in the subsequent cyclization reaction.

[0466] The peptide cyclization reaction was carried out by dissolving the peptide in DMSO with a final concentration of 5 mM based on the molar amount of solid-phase resin, adding 6 equal volumes 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 Biotage V-10 (Biotage Japan).

[0467] As a purification method for the obtained crude and refined peptides, a Waters AutoPurification System-SQD2 single quadruple mass spectrometer was used with reversed-phase preparative HPLC to dissolve the target ion while monitoring its m / z value. It was confirmed that the mass spectra obtained in the ESI-positive scan mode were consistent with the mass spectra of the multivalent ions calculated from the molecular formula of the target ion within the error range of the mass analyzer used. Furthermore, the purification conditions including the column used are shown in the respective examples.

[0468] In this invention, the purity of the chemically synthesized peptides is determined using any of the following analytical methods.

[0469] (Analysis conditions)

[0470] Analysis condition A

[0471] Tube Column: CORTECS (registered trademark) UPLC (registered trademark) C18 column (Nihon Waters Company) 1.6μm, 2.1x100mm

[0472] Mobile phase: 0.025% TFA in MeCN / 0.025% TFA in H2O

[0473] Temperature: 40℃

[0474] Gradient: 5-95% MeCN / 0.025% TFA in H2O in 5.56 min; linear gradient

[0475] Flow rate: 0.4 mL / min

[0476] Detection method: UV 220nm

[0477] Analysis condition B

[0478] Column: Kinetex EVO C18 2.6μm, 2.1ID x 150mm (Phenomenex)

[0479] Column temperature: 60℃

[0480] Mobile phase A: 0.025% TFA in H2O

[0481] Mobile phase B: 0.025% TFA in CH3CN

[0482] Gradient: as described in each embodiment

[0483] Flow rate: 0.25 mL / min

[0484] Detection: PDA (225nm)

[0485] The structure of chemically synthesized peptides is determined by calculating the molecular weight, taking into account the amino acids used to follow the target sequence and the structural units required, and then confirming this molecular weight using ESI-MS(+) mass spectrometry. Furthermore, "ESI-MS(+)" indicates electrospray ionization mass spectrometry performed in positive ion mode. The detected mass is expressed in m / z units. Additionally, compounds with molecular weights approximately greater than 1000 are detected at high frequency as divalent or trivalent ions.

[0486] Chemical synthesis of special cyclic peptides that bind to hTfR

[0487] hTfR-binding peptides were identified using the screening methods described in International Publications WO2014 / 119600, WO2012 / 033154, or WO2007 / 066627. To confirm whether the peptides actually possess hTfR binding activity, chemical synthesis was performed. The sequences of the synthesized peptides are shown in Table 1.

[0488] [Table 1]

[0489]

[0490] Using Sieber amide resin (Watanabe Chemicals, 0.6 mmol / g, 0.33 g), the target peptide was synthesized following the general method described above, starting with the removal of Fmoc. A Liberty Blue HT solid-phase synthesizer from CEM was used as the solid-phase synthesizer, and the synthesis was performed according to the manufacturer's manual. Condensation reactions were performed at 75°C for 10 minutes as the baseline condition. De-Fmocization was performed at 75°C for 3 minutes in 20% pipeidine in DMF as the baseline condition. The introduction of chloroacetyl groups was carried out as follows: for a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, after removing the α-amino Fmoc group using the method described above, 5 equal volumes of a 0.2 M solution of chloroacetic acid in DMF, 5 equal volumes of a 0.5 M solution of HATU in DMF, and 10 equal volumes of a 1 M solution of DIPEA in DMF were added, and the mixture was shaken at room temperature for 30 minutes. To remove the protection of the side chains and shear from the solid resin, firstly, the resin obtained after the step of introducing chloroacetyl groups was washed five times with DMF and three times with chloromethane, and then dried under reduced pressure. Next, a reaction mixture (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 already loaded with the solid resin, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass flotation. The solid resin remaining in the reaction vessel was shaken again with the shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. Adding this filtrate to excess diethyl ether / hexane (1 / 1) cooled to 0°C produced a white turbid precipitate. This mixture was centrifuged (10000 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 for use in subsequent cyclization reactions. The peptide cyclization reaction was performed by dissolving the peptide in DMSO with the molar amount of solid resin as the reference, resulting in a final peptide concentration of 5 mM. Six equal volumes of triethylamine were then added, and the mixture was stirred at room temperature for approximately 16 hours. The resulting reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.

[0491] The crude product was purified under the following conditions (column: WaterS Xbridge (registered trademark) C185μmμm (registered trademark) 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 40℃; gradient (%B): 3 minutes 5-30%, then 8 minutes 30-35%, then 1 minute 35-60%; flow rate: 120mL / min).

[0492] The target analyte was analyzed under either analytical condition A or analytical condition B, and its structure was confirmed by ESI-MS(+) mass spectrometry. The obtained ESI-MS(+) observations and the value of X when the proton addition number (M+XH) in this case is displayed as X+ are shown in Table 1.

[0493] Example 2

[0494] Evaluation of intermolecular interactions between transferrin receptor (hTfR) and peptides by surface plasmon resonance (SPR) Price test

[0495] For the various synthesized peptides, the intermolecular interactions resulting from the surface plasmon resonance (SPR) of the peptide for the transferrin receptor (hTfR) were investigated using the methods shown below. The specific experimental methods are illustrated below.

[0496] [SPR Measurement]

[0497] The NTA sensor chip (Global Life Science Technologies Japan Co., Ltd.) was inserted into the Biacore T200 (Global Life Science Technologies Japan Co., Ltd.) and equilibrated three times using electrophoresis buffer: 10mM HEPES pH 8.0 (Nacalai Tesque Co., Ltd.), 150mM NaCl (Nacalai Tesque Co., Ltd.), 0.05% Tween 20 (Nacalai Tesque Co., Ltd.), 0.1% BSA (SIGMA-ALDRICH), and 1.0% DMSO (Fujifilm and Wako Pure Chemical Industries Co., Ltd.) at a flow rate of 30 μL / min. After reacting with 350mM EDTA solution at a flow rate of 10 μL / min for 60 seconds, and then with 0.5mM NiCl2 solution (KISHIDACHEMICAL) at a flow rate of 10 μL / min for 60 seconds, the NTA sensor chip was washed with 3mM EDTA solution (Nacalai Tesque Co., Ltd.) at a flow rate of 10 μL / min for 60 seconds. 50 μL each of 60 mM EDC solution (Global Life Science Technologies Japan) and 650 mM NHS solution (Global Life Science Technologies Japan) were mixed and reacted at a flow rate of 10 μL / min for 420 seconds. The mixture was then diluted with electrophoresis buffer to prepare 150 μL of a 3.2 μM hTfR solution, which was reacted at a flow rate of 10 μL / min for 600 seconds to immobilize hTfR on the NTA sensor chip. After immobilization, the mixture was reacted with 1.0 M ethanol-ammonia solution (Global Life Science Technologies Japan) at a flow rate of 10 μL / min for 420 seconds for capping. A 10 mM peptide solution was prepared in DMSO solution, and this final concentration was diluted with electrophoresis buffer to prepare 100 nM, 50 nM, 25 nM, 10 nM, and 5 nM peptide solutions. Using these samples, the peptide kinetics for hTfR were obtained by SPR measurement. The kinetic evaluation model was set as a Single Cycle, and curve fitting was performed using KineticsBiacore T200Evaluation Software Version 3.0 (Global Life Science Technologies Japan Co., Ltd.).For the obtained sensor maps, curve fitting was performed using the least squares method, and the binding of the peptide to hTfR was evaluated by calculating its KD value. For the calculated KD values, cases with a KD value less than 1 nM were labeled A, cases with a KD value greater than 1 nM but less than 100 nM were labeled B, cases with a KD value greater than 100 nM but less than 1 μM were labeled C, and cases with a KD value greater than 1 μM were labeled D, as shown in Table 1. The results showed that hTfR No. 894 and cyclic peptides with similar amino acid sequences exhibited significant binding ability to hTfR. The chemical structure of hTfR No. 894 is as follows.

[0498] [Chemistry 1]

[0499]

[0500] Example 3

[0501] Synthesis of hTfR-binding peptide-PEG11-Vivotag750 conjugate (hTfRNo.894-vivotag750)

[0502] For hTfR No. 894, a compound (represented as hTfR No. 894-vivotag750 or hTfR_000894_PEG11_(VivoTag)) (serial number 146) was synthesized by binding the near-infrared fluorescent tagging substance Vivotag 750 (VivoTag-S (trademark) 750, PerkinElmer) as the payload via a PEG11 linker. The chemical structure of the hTfR-binding peptide-PEG11 is as follows. The labeled compound was synthesized by binding this compound with Vivo-tag 750. Details are described below.

[0503] [Chemistry 2]

[0504]

[0505] Synthesis of hTfRNo.894-PEG11-vivotag750

[0506] Chemical synthesis is carried out as follows:

[0507] Using Fmoc-NH-SAL-PEG-resin 1500-2000 Da (Watanabe Chemicals, 0.38 mmol / g), the target peptide was synthesized following a standard method, starting with the removal of Fmoc. A Liberty Blue solid-phase synthesizer from CEM was used, following the manufacturer's manual. The basic conditions for the condensation reaction were set as follows: HATU was used as the condensing agent, and the reaction was carried out once at 75°C for 10 minutes. However, residues 11 and 12 were reacted twice at 25°C for 20 minutes. Residues 13 and 14 were reacted twice at 75°C for 10 minutes. Residue 15 was reacted once at 25°C for 20 minutes. Residue 16 was reacted once at 25°C for 60 minutes. Furthermore, the basic conditions for Fmoc removal were a reaction with a 20% piperidine DMF solution at 75°C for 3 minutes. However, for residues 13 and 15, Fmoc removal was performed by reacting the residue at 25°C for 5 minutes, followed by a 10-minute reaction. The introduction of chloroacetyl groups was carried out by adding 5 equal volumes of a 0.2M DMF solution of chloroacetic acid, 5 equal volumes of a 0.5M DMF solution of HATU, and 10 equal volumes of a 1M DMF solution of DIPEA to the solid-phase resin obtained in the previous step, followed by shaking at room temperature for 30 minutes. For deprotection of the side chains and shearing from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed 5 times with DMF and 3 times with chloromethane, and then dried under reduced pressure. Next, in the reaction vessel already loaded with the solid-phase resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by filtration through a glass frit filter. The remaining solid resin in the reaction vessel was shaken again with the shearing mixture, the solution components were recovered from the glass frit, and mixed with the filtrate. Adding this filtrate to excess diethyl ether and hexane mixed solvent cooled to 0°C produced a turbid precipitate. This mixture was centrifuged (10000 rpm, 1 min), and the supernatant was decanted. The resulting solid was washed with chilled diethyl ether and dried. The resulting solid was used in the subsequent cyclization reaction. The peptide cyclization reaction involved dissolving the peptide in DMSO at a final concentration of 5 mM based on the molar amount of solid resin, adding 6 equal volumes of triethylamine, shaking overnight at room temperature, concentrating under reduced pressure with Savant Explorer SpeedVac, and then purifying by reverse-phase HPLC. The obtained peptide (26.1 mg, 9.30 μmol) was dissolved in DMSO / H2O (9 / 1), and 0.91 μL of VivoTag-NHS and 4.5 μL of DIEA were added. The mixture was stirred for 45 minutes.Add AcOH to the reaction solution and quench it.

[0508] The resulting reaction solution was purified under the following conditions (column: XSelect CSH C18 5um 10x150mm (Lot No. 147i362781); mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40℃; gradient (%B): 7-32% for 3 minutes, 32-37% for 8 minutes, and 37-60% for 1 minute; flow rate: 5 mL / min).

[0509] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 97.1%.

[0510] Analysis condition B: Hold time = 13.20 minutes; Gradient (%B conc): 20 minutes for 20-60%, then 1 minute for 60-95%, then 5 minutes for 95%.

[0511] ESI-MS (+) observation m / z = 1226.4 (M+3H) 3+ .

[0512] Furthermore, following Example 2, the binding affinity of the synthesized conjugate to hTfR via SPR was confirmed to be KD = 1.09 nM.

[0513] Example 4

[0514] Synthesis of the peptide conjugate that forms the control group: hTfRNo.894FLIP-vivotag750

[0515] As the control group of hTfRNo.894-vivotag750, the following conjugate (represented as hTfRNo.894FLIP-vivotag750 or FLIP_000894_PEG11_K_FITC) is synthesized, which is a cyclic peptide having the following amino acid sequence that reverses the sequence of No.894 and is linked to vivotag750 via the PEG11 linker.

[0516] FLIP sequence: ClAc-Ala-MeTyr-Tyr-Arg-Arg-Ile-Ile-Tyr-Tyr-Asn-Trp-Val-Phe-Val-Cys (SEQ ID NO: 202).

[0517] Synthesis of hTfRNo.894FLIP-PEG11-vivotag750

[0518] 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.

[0519] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 95.5%.

[0520] Analysis condition B: Hold time = 13.5 minutes; Gradient (%B conc): 20 minutes for 20-60%, then 1 minute for 60-95%, then 5 minutes for 95%.

[0521] ESI-MS (+) observation m / z = 1226.4 (M+3H) 3+ .

[0522] The N-terminus of the compound obtained above is labeled according to the Vivotag 750 (VivoTag-S 750, PerkinElmer) procedure to obtain the title compound.

[0523] Furthermore, following Example 2, after confirming the binding ability of the synthesized conjugate with hTfR by SPR, no binding with hTfR was confirmed.

[0524] Example 5

[0525] The use of hTfR No. 894-vivotag750 in brain migration assessment experiments in hTfR-KI mice.

[0526] [Preparation of test solution]

[0527] Hydroxypropyl-β-cyclodextrin (Wako Pure Chemical Industries, Ltd.) was dissolved in water to a concentration of 20 w / v, thus creating a 20% hydroxypropyl-β-cyclodextrin solution.

[0528] [Preparation of the dosing solution]

[0529] hTfRNo.894-vivotag750 conjugate dosing solution (No. 894 dosing solution): To 6 mL of a 5 mM solution of hTfRNo.894-vivotag750 conjugate synthesized in Example 2, 4.8 μL of dimethyl sulfoxide (DMSO, Sigma-Aldrich) was added, followed by 66 μL of a 20% hydroxypropyl-β-cyclodextrin solution, and the mixture was carefully mixed. Then, 48 μL of polyethylene glycol 400 was added, and the mixture was used as No. 894 dosing solution. The concentration of hTfRNo.894-FITC conjugate in No. 894 dosing solution was 250 μM.

[0530] hTfRNo.894FLIP-vivotag750 conjugate dosing solution (No.894NC dosing solution): Add 66 μL of a 5 mM solution of the aforementioned hTfRNo.894FLIP-vivotag750 conjugate to a solution of 20% hydroxypropyl-β-cyclodextrin and mix carefully. Further add 48 μL of polyethylene glycol 400 to this mixture, which is then used as the No.894NC dosing solution. The concentration of hTfRNo.894FLIP-vivotag750 conjugate in the No.894FLIP dosing solution becomes 250 μM.

[0531] [The creation of TfR-KI mice]

[0532] Following the international publication WO2016-208695, a target vector was prepared. This vector contained a DNA fragment with a 3' arm containing a chimeric hTfR cDNA encoding an intracellular region of mouse hTfR and an extracellular region of human hTfR, along with a 5' arm and a 3' arm sequence of a neomycin resistance gene sandwiched by a loxP sequence. The prepared target vector was introduced into mouse ES cells via electroporation. The gene-introduced mouse ES cells were then selectively cultured in the presence of neomycin, selecting mouse ES cells in which the target vector was incorporated into the chromosome through homogeneous recombination. The resulting gene-recombined mouse ES cells were injected into 8-cell stage embryos (host embryos) of ICR mice and transplanted into pseudopregnant mice (fertilized mice) obtained by mating with mice that had undergone vasectomy. The resulting fetal mice (chimeric mice) were assessed for coat color, selecting ES cells that would contribute to the efficient formation of live individuals, i.e., individuals with a high proportion of white fur. This chimeric mouse was crossed with C57BL6 / J mice to obtain F1 mice. White F1 mice were selected, and DNA extracted from tail tissue was analyzed. Mice with the mouse transferrin receptor gene on their chromosomes heterologously replaced with chimeric hTfR were designated as TfR-KI mice.

[0533] [Brain migration assessment experiment using TfR-KI mice]

[0534] Next, a brain migration assessment experiment was conducted using TfR-KI mice. For female, 12-week-old TfR-KI mice exhibiting hTfR, 100 μL each of No. 894 or No. 894NC injection solution were rapidly administered into the tail vein. One hour after administration, the mice were anesthetized with isoflurane, and bloodletting was performed by perfusion of physiological saline into the left ventricle for 4-5 minutes. Subsequently, tissues (brain, heart, lung, liver, spleen, kidney, quadriceps femoris muscle, thymus, thoracic vertebrae, and femur) were collected for fluorescence intensity measurement and stored in physiological saline to prevent drying. Fluorescence intensity was measured using the In vivo chemiluminescence-fluorescence imaging system IVIS Lumina III (PerkinElmer) and the filter assembly for the VivoTag 750 fluorescent dye, following the operating instructions.

[0535] [Results of brain migration assessment using hTfR-KI mice]

[0536] The fluorescence intensity measurements in each tissue are expressed as follows: Figure 1-1 , Figure 1-2 , Figure 1-3 and Figure 2 .

[0537] The "#894" designation in the figure indicates the hTfRNo.894-vivotag750 conjugate injection group, and the "#894FLIP" designation indicates the hTfRNo.894FLIP-vivotag750 conjugate injection group. Furthermore, the bars on the right side of each image represent the average radiant efficiency (p / sec / cm²). 2 / sr] / [μW / cm 2 ]).

[0538] Figure 1-1 (1) Brain indicates a photograph of the brain, (2) Liver (Left lateral lobe) indicates a photograph of the left lateral lobe of the liver, (3) Kidney indicates a photograph of the kidney, (4) Lung indicates a photograph of the lung, and (5) Thoracicvertebrae indicates a photograph of the thoracic vertebrae. The color code for (1) indicates a minimum value of 5.00 × 10⁻⁶. 7 And the maximum value is 1.3 × 10 8 For (2) and (3), the minimum value is 6.00 × 10. 8 And the maximum value is 2.4 × 10 10 For (3) and (4), the minimum value is 4.5 × 10. 8 And the maximum value is 5.4 × 10 9 . Figure 1-2 (1) Heart represents a photograph of the heart, (2) Spleen represents a photograph of the spleen, and (3) Thymus represents a photograph of the thymus. Furthermore, the color scale indicates a minimum value of 4.83 × 10⁻⁶. 7 And the maximum value is 4.8 × 10 9 .

[0539] Figure 1-3 (1) Quadriceps represents a photograph of the quadriceps femoris, and (2) Femur represents a photograph of the femur. Furthermore, the color scale indicates a minimum value of 1.5 × 10⁻⁶. 8 And the maximum value is 1.5 × 10 9 . Figure 2 for Figure 1-1 (1) A magnified photograph of the brain.

[0540] Depend on Figure 1-1 , Figure 1-2 , Figure 1-3 and Figure 2It was found that the tissues showing stronger fluorescence in the hTfRNo.894-vivotag750 conjugate injection group than those in the hTfRNo.894FLIP-vivotag750 conjugate injection group were the kidney, thoracic vertebrae, heart, femur, quadriceps femoris muscle, and brain. The tissues showing stronger fluorescence in the hTfRNo.894FLIP-vivotag750 conjugate injection group than those in the hTfRNo.894-vivotag750 conjugate injection group were the liver and spleen.

[0541] The presence of the strongest fluorescence in the kidneys and the second strongest fluorescence in the liver in both groups indicates that the two tested substances are primarily metabolized and excreted through the kidneys and liver. Furthermore, the fluorescence in the thoracic vertebrae, heart, femur, quadriceps femoris muscle, and brain was stronger in the No. 894 group than in the No. 894NC group. Since these tissues exhibit higher levels of TfR expression, the difference in tissue distribution between the two groups is considered to be due to the presence or absence of hTfR binding capacity.

[0542] This experiment confirmed that the hTfR-binding specific cyclic peptide No. 894 in hTfR-KI mice binds to TfR, crosses the BBB, and migrates into the brain and various tissues, primarily muscle tissue.

[0543] Example 6

[0544] Confirmatory assay for the local presence of hTfR-binding specific cyclic peptide-fluorescently labeled probe conjugate in the mouse brain.

[0545] [Preparation of a conjugate of hTfR-binding specific cyclic peptide No. 894 and the fluorescent substance FITC]

[0546] A conjugate of the hTfR-binding specific cyclic peptide No. 894 and the fluorescent substance FITC was synthesized using hTfRNo. 894-PEG11 synthesized in Example 3, following the procedure of the FITC labeling kit (Dongjin Chemical Co., Ltd.). Furthermore, the synthesized conjugate was confirmed to have a KD of 0.28 nM after SPR binding assay, following Example 2.

[0547] [Single-dose trial]

[0548] Similar to Example 5, the conjugate of the hTfR-binding specific cyclic peptide No. 894 and the fluorescent substance FITC was administered to hTfR-KI mice. The dosage was 3.7 mg / kg.

[0549] One hour after administration, mice were anesthetized with isoflurane, and their left ventricles were perfused with physiological saline for 4-5 minutes for exsanguination. Subsequently, the brain tissue, with fluorescence intensity measured, was stored in physiological saline to prevent drying. Immunohistochemical staining with anti-FITC antibody (MLB Bioscience) was performed on this brain tissue. Immunohistochemical staining of the brain tissue with anti-FITC antibody was performed using well-known methods and observed using a fluorescence microscope. The results are presented as follows. Figure 3 In addition, the red arrows in the figure point to brain capillaries, and the arrowheads point to Purkinje cells. (1) shows the photo of the group that was given #894-FITC, and (2) shows the photo of the group that was not given #894-FITC as a control group.

[0550] [Multi-dose experiment]

[0551] Administer 3.7 mg / kg every 10 minutes for a total of 6 times. In addition, similar to the single-dose administration, hTfR-KI mice were given a conjugate of the hTfR-binding specific cyclic peptide No. 894 and the fluorescent substance FITC. The local presence of the conjugate in the brain tissue was confirmed by immunohistochemical staining. Results are presented as follows: Figure 4 In addition, the red arrows in the figure point to brain capillaries, the arrows point to Purkinje cells, and the yellow arrows point to dendrites. (1) shows the photo of the group that was given #894-FITC, and (2) shows the photo of the group that was not given #894-FITC as a control group.

[0552] This study confirmed that, in any dose trial, the conjugate of the hTfR-binding specific cyclic peptide No. 894 and the fluorescent substance FITC was introduced into the cerebellum via the BBB. Furthermore, in multiple dose trials, it was found that this conjugate reached neurons similar to Purkinje cells.

[0553] Example 7

[0554] The transferrin receptor (hTfR) and hTfR No. 894 variant peptides, linker addition peptides and... Evaluation test of intermolecular interactions of payload conjugates

[0555] [Synthesis of variants and linker addition peptides of hTfRNo.894]

[0556] Peptides with sequences containing insertions, deletions, or substitutions of several amino acids in the amino acid sequence of hTfR No. 894 (also called variants) and peptides bound to various linkers were synthesized, and the binding ability to hTfR by SPR was similarly confirmed. Variant peptides were synthesized according to Example 1 unless described in Example 9 or 10. Linker-added peptides were synthesized according to Example 1 unless the linker was PEG. In the case where the linker was PEG, Fmoc-NH-SAL-PEG resin (Watanabe Chemical) was used as the resin for peptide synthesis. Otherwise, the synthesis was carried out according to Example 1. Peptides added to linkers of peptides, fatty acids, PEG, and their complexes were also synthesized according to Example 1 unless described in Example 9 or 10. For the KD values ​​obtained by SPR measurements, cases with KD values ​​less than 1 nM are labeled A, cases with KD values ​​greater than 1 nM but less than 100 nM are labeled B, cases with KD values ​​greater than 100 nM but less than 1 μM are labeled C, and cases with KD values ​​greater than 1 μM are labeled D. The results are shown in Tables 2-4.

[0557] Table 2: Peptides or Linker Addition Peptides [Table 2-1]

[0558]

[0559] [Table 2-2]

[0560]

[0561] [Table 2-3]

[0562]

[0563] [Table 2-4]

[0564]

[0565] [Table 3-1]

[0566]

[0567]

[0568] [Table 3-2]

[0569]

[0570]

[0571] [Table 4-1]

[0572]

[0573]

[0574] [Table 4-2]

[0575]

[0576]

[0577] [Table 4-3]

[0578]

[0579]

[0580] [Table 4-4]

[0581]

[0582]

[0583] [Table 4-5]

[0584]

[0585]

[0586] [Table 4-6]

[0587]

[0588]

[0589] [Table 4-7]

[0590]

[0591]

[0592] [Table 4-8]

[0593]

[0594]

[0595] [Table 4-9]

[0596]

[0597]

[0598] [Table 4-10]

[0599]

[0600]

[0601] [Table 4-11]

[0602]

[0603]

[0604] The results show that peptides bound to hTfR No. 894, sequences with several amino acids inserted, deleted, or substituted, and various linkers exhibit the ability to bind to hTfR.

[0605] Example 8

[0606] [Cell Culture]

[0607] In the culture of human breast cancer cells BT-549 (COSMO BIO), RPMI-1640 medium (Thermo Fisher Scientific) containing 10% FBS and 2 mmol / L L-Glutamine was used. The culture was carried out at 370°C and 5% CO2.

[0608] [Cell seeding]

[0609] Collagen Type I (Corning) was diluted to 50 μg / mL using 20 mmol / L acetic acid. A sterile coverslip was placed in each well of a 24-well plate, and the diluted Collagen Type I solution was added. The plate was incubated at 37°C for 1 hour. The Collagen Type I solution was then removed, and the plate was washed three times with PBS. 1 × 10⁻⁶ seeds were seeded per well. 5 Individual breast cancer cells BT-549 were cultured overnight at 37°C and 5% CO2.

[0610] Synthesis of various peptide conjugates

[0611] hTfR_894_3m_PEG4dk5FAM (serial number 446), hTfR_894_variant03_PEG4dk5FAM (serial number 448), hTfR_894_variant61_PEG4dk5FAM (serial number 447), and Flip894_variant61_PEG4dk5FA (serial number 449)M as a negative control group were used as samples.

[0612] Synthesis of hTfR_894_3m_PEG4dk5FAM

[0613] [Chemistry 3]

[0614]

[0615] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.47 mmol / g, 0.53 g), starting with the removal of Fmoc. An automated synthesizer (CEM Liberty Blue HT) was used, following the manufacturer's manual. For the introduction of each residue, the following basic conditions were used: for 1 eq of resin, Fmoc-AA / DIC / Oxymapure = 4.2 eq / 4 eq / 8 eq was reacted at 75°C for 10 min. However, the second residue was reacted twice at 75°C for 30 min. The 11th and 12th residues were reacted twice at 25°C for 20 min. The 13th residue was reacted twice at 75°C for 10 min. The 15th residue was reacted at 25°C for 20 min. The de-Fmocization was performed under the following basic conditions: reacting with a 20% pipeidine DMF solution at 75°C for 3 min. However, the removal of the Fmoc groups at residues 2 and 13 was carried out by reacting at 25°C for 5 minutes, followed by a 10-minute reaction. The introduction of chloroacetyl groups was performed by removing the α-amino Fmoc groups from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, followed by stirring 10 eq of chloroacetic acid, 10 eq of DIPCI, and 10 eq of HOSu in DCM, adding an equal amount of DMF to the DCM, preparing a ClAcOSu DCM / DMF solution, adding the solid-phase resin, and shaking at room temperature for 60 minutes. Deprotection of the side chains and shearing from the solid-phase resin were performed by first washing the resulting resin 5 times with DMF and 3 times with chloromethane after the chloroacetyl group introduction step, followed by drying under reduced pressure. Next, in a reaction vessel already filled with solid resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass flotation. The solid resin remaining in the reaction vessel was shaken again with a shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. If this filtrate was added to excess diethyl ether / hexane (1 / 1) cooled to 0°C, a white turbid precipitate was formed. 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 the solid was used for subsequent cyclization reactions. The peptide cyclization reaction was performed by dissolving the peptide in DMSO with a final peptide concentration of 5 mM based on the molar amount of solid resin, adding 5 equal volumes of triethylamine, and shaking at room temperature for approximately 14 hours.F-FAM-NHS, adjusted by adding 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 the mixture was stirred at room temperature for 3 hours. The resulting reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.

[0616] The obtained crude intermediate peptide was purified under the following conditions (column: WaterS Xbridge (registered trademark) C185μm OBD (registered trademark) 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40℃; gradient (%B): 3 min 9-34%, then 8 min 34-39%, then 1 min 39-60%; flow rate: 120 mL / min). After freeze-drying, it was further purified under the following conditions (column: COSMOSIL PBr 10x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40℃; gradient (%B): 3 min 21-46%, then 8 min 46-51%, then 1 min 51-60%; flow rate: 5 mL / min).

[0617] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 95.3%.

[0618] Analytical conditions B: Hold time = 4.47 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60℃; Gradient (%B conc): 20-60% in 7.2 minutes, 60-95% in 0.3 minutes, and 95% in 1.6 minutes; Flow rate: 0.5 mL / min.

[0619] ESI-MS (+) observation m / z = 944.42 (M+3H) 3+ .

[0620] hTfR_894_variant03_PEG4dk5FAM, hTfR_894_variant61_PEG4dk5FAM and their effects Synthesis of Flip894_variant61_PEG4dk5FAM as the negative control group

[0621] The conjugates were synthesized and cyclized in the same manner as hTfR_894_3m_PEG4dk5FAM, including hTfR_894_3m_PEG4, hTfR_894_variant03_PEG4, hTfR_894_variant61_PEG4, and Flip894_variant61_PEG4, with the addition of F-FAM-NHS modified by 5-FAM.

[0622] Furthermore, for the various conjugates synthesized, binding with hTfR was confirmed using SPR in the same manner as in Example 2. Only Flip894_variant91_PEG4 was not confirmed to bind with hTfR.

[0623] [Preparation of the sample solution and its addition to the cells]

[0624] For sample dilution, use dilution medium (RPMI 1640 medium containing 0.5% bovine serum albumin and 20 μg / mL human transferrin). Dilute the sample to 100 nmol / L using the dilution medium.

[0625] After confirming that the BT-549 cells, which had been cultured overnight in the 24-well plate, were adhered to the coverslip, the plates were washed twice with RPMI 1640 medium. RPMI 1640 medium containing 0.5% bovine serum albumin was added at 500 μL / well, and the plates were incubated on ice for 15 minutes. Then, the diluted sample solution was added at 500 μL / well, and the plates were incubated at 37°C and 5% CO2 for 3 hours.

[0626] [Cell fixation, nuclear staining, mounting]

[0627] Remove the sample solution from the 24-well plate and wash BT-549 cells three times with PBS. Add 500 μL / well of 4% paraformaldehyde phosphate buffer (Fujifilm and Koden Chemical Co., Ltd.), incubate at room temperature for 15 minutes, and then wash three times with PBS. Next, add 500 μL / well of Hoechst 33342 (Thermo Fisher Scientific) diluted to 2 μg / mL with PBS, incubate at room temperature in the dark for 10 minutes, and then wash three times with PBS. Remove the coverslips from the 24-well plate, mount them on slides using Fluorescent Mounting Medium (Agilent Technologies), and incubate overnight in the dark at room temperature. Observation was performed using an inverted fluorescence microscope DMI6000B (Leica Microsystems) at the wavelengths used for FITC and DAPI detection. The results are presented as follows. Figure 5Furthermore, the linear scale bar in the figure represents 50 μm.

[0628] As by Figure 5 It was confirmed that while hTfR_894_3m_PEG4dk5FAM, hTfR_894_variant03_PEG4dk5FAM, and hTfR_894_variant61_PEG4dk5FAM were confirmed to migrate into cells, Flip894_variant91_PEG4, a peptide that does not bind to hTfR, did not migrate into cells. Therefore, these results indicate that hTfR_894 and its variants that bind to hTfR migrate into cells via hTfR binding.

[0629] Example 9

[0630] Synthesize the following peptides or linker addition peptides.

[0631] [Example 9-1]

[0632] Synthesis of 894_v01_PEG12 (serial number 102)

[0633] [Chemistry 4]

[0634]

[0635] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.52 mmol / g, 0.19 g), starting with the removal of Fmoc. The synthesis was performed using a Biotage SyroI automated synthesizer, following the manufacturer's manual. The basic conditions for the condensation reaction were two repeated reactions at 75°C for 20 minutes each. However, when introducing residues 15 and 16, the reactions were carried out at room temperature for 60 minutes and 15 minutes respectively. Furthermore, one condensation reaction was performed when introducing PEG, and three condensation reactions were performed when introducing the 11th amino acid residue. The basic conditions for Fmoc removal were a reaction in a DMF solution of piperazine (5%) and Oxima pure (0.2 M) at 50°C for 5 minutes, followed by a second 15 minutes of reaction. However, the removal of the Fmoc group from residues 15 and 16 was achieved by reacting at 25°C for 5 minutes, followed by a second 15 minutes of reaction. The introduction of chloroacetyl groups was carried out by removing the α-amino Fmoc groups from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above, followed by the addition of 5 equal volumes of a DMF solution of ClAcOSu and shaking at room temperature for 60 minutes. Deprotection of the side chains and shearing from the solid-phase resin were performed by first washing the resin obtained after the chloroacetyl introduction step with DMF five times and chloromethane three times, followed by drying under reduced pressure. Next, in a reaction vessel already filled with the solid-phase resin, a reaction mixture-A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass flotation. The remaining solid-phase resin in the reaction vessel was shaken again with the shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. Adding this filtrate to excess diethyl ether / hexane (1 / 1) cooled to 0°C resulted in a white 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 cooled to 0°C, and then used for the subsequent cyclization reaction. The peptide cyclization reaction was performed by dissolving the peptide in DMSO with the molar amount of solid resin as the basis and the final peptide concentration as 5 mM, adding 10 equal volumes of triethylamine, and shaking at room temperature for about 1 hour. The resulting reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.

[0636] Next, using the obtained solid resin, the side chains are deprotected and sheared and cyclized from the solid resin, following the general method described above.

[0637] The crude product was purified under the following conditions (column: WaterS Xbridge (registered trademark) C185μm OBD (registered trademark) 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 40℃; gradient (%B): 3 minutes 8-33%, then 8 minutes 33-38%, then 1 minute 38-60%; flow rate: 120mL / min).

[0638] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 91.5%.

[0639] Analysis condition A: Hold time = 3.57 minutes; ESI-MS (+) observed value m / z = theoretical value (M + 3H) 3+ .

[0640] Analysis condition B: Hold time = 12.8 minutes; Gradient (%B conc): 20 minutes for 20-60%, then 1 minute for 60-95%, then 5 minutes for 95%.

[0641] ESI-MS(+) observation m / z = 899.6(M+3H) 3+ .

[0642] [Example 9-2]

[0643] Synthesis of 894_v05_PEG8Me (serial number 43)

[0644] [Chemistry 5]

[0645]

[0646] Cl-Trt(2-Cl)resin (1 g, 1.6 mmol / L, Watanabe Chemical Industry, 1.6 mmol / g) was swelled in DCM (dehydrated, 10 mL, Nacalai tesque) for 10 minutes, and washed twice with filtered DCM (dehydrated, 10 mL). The swollen resin was then sequentially added to a DCM (dehydrated, 10 mL) solution of Fmoc-CyS(CH2COOH)-PEG8Me (767 mg, 1 mmol) and DIEA (836 μL, 4.8 mmol / L, Watanabe Chemical Industry), and shaken at room temperature for 35 minutes. Furthermore, after adding MeOH (1 mL, KISHIDA CHEMICAL) and shaking for 10 minutes, the resin was 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). Following washing, the resin was dried under reduced pressure to obtain Fmoc-CyS[CH2COO-Trt(2-Cl)-resin]-PEG8Me (1.686 g, 94%). Using the obtained resin, the target peptide was synthesized starting with the removal of the Fmoc group using the general method described above. The Biotage SyroI was used as the solid-phase synthesizer, and the synthesis was performed according to the manufacturer's manual. For each residue introduction, for 1 eq of resin, Fmoc-AA-OH / HATU / DIEA (3.2 eq / 3 eq / 6.3 eq) was used in DMF, and the reaction was repeated twice at room temperature for 30 minutes. However, the first, second, and fourth residues were introduced in three separate reactions at room temperature for 60 minutes each. The third residue was introduced in two repeated reactions at 25°C for 60 minutes each. The ninth residue was introduced in three separate reactions at room temperature for 30 minutes each. Furthermore, Fmoc removal was performed by reacting with a 20% piperidine DMF solution at room temperature for 5 minutes, followed by a further 15 minutes of reaction. The introduction of chloroacetyl groups was performed by removing the α-amino Fmoc group from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, followed by adding three equal volumes of a chloroacetic acid DMF solution (0.45 M), three equal volumes of an HCTU DMF solution (0.43 M), and three equal volumes of a DIPEA DMF solution (1.57 M) to the solid-phase resin and shaking at room temperature for 30 minutes. To remove the protection of the side chains and shear them from the solid resin, the resin obtained after the chloroacetyl introduction step was first washed five times with DMF and three times with chloromethane, and then dried under reduced pressure. Next, a reaction mixture of HFIP / DCM (1 / 4) was added to the reaction vessel already loaded with the solid resin, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was then recovered by glass frit filtration.The solid resin remaining in the reaction vessel was shaken with a shearing mixture, and the solution components were recovered from the glass frit. This process was repeated three times with the filtrate. After concentrating the filtrate under reduced pressure using a GenevaC EZ-2 Elite, the addition of excess diisopropyl ether cooled to 0°C resulted in a turbid precipitate. The 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 cooled to 0°C and dried under reduced pressure. The resulting solid was used for subsequent cyclization reactions. The peptide cyclization reaction involved dissolving the peptide in DMF with the molar amount of solid resin as the basis, resulting in a final peptide concentration of 2.5 mM. 1.1 equal volumes of HATU in DMF solution (0.43 M) and 1.5 equal volumes of DIPEA were added, and the mixture was shaken at room temperature for approximately 1 hour. 3 equal volumes of acetic acid were added to the resulting reaction solution at room temperature, followed by concentration under reduced pressure using a GenevaC EZ-2. Adding chilled diisopropyl ether to the resulting residue produces a white precipitate. Centrifuge the mixture (9000 rpm, 10 min) and decant the supernatant. Wash the resulting solid again with ether cooled to 0°C and dry under reduced pressure. Add reactant mixture A (a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) to the resulting solid and shake at room temperature for 60 min. Adding chilled excess diisopropyl ether produces a precipitate. Centrifuge the mixture (9000 rpm, 5 min) and decant the supernatant. Wash the resulting solid again with chilled ether and dry under reduced pressure.

[0647] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 30x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40℃; gradient (%B): 8-33% for 3 minutes, 33-38% for 8 minutes, and 38-60% for 1 minute; flow rate: 45 mL / min).

[0648] The purity of the target analyte was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 97.4%.

[0649] Analysis condition B: Hold time = 11.93 minutes; Gradient (%B conc): 20 minutes for 20-60%, then 1 minute for 60-95%, then 5 minutes for 95%.

[0650] ESI-MS (+) observation m / z = 1211.6(M+2H) 2+ .

[0651] [Example 9-3]

[0652] Synthesis of hTfR_894_E1_PEG12_(Hydrazide) (Serial No. 218)

[0653] [Chemistry 6]

[0654]

[0655] The target peptide was synthesized using NH2NH-Trt(2-Cl)-resin (Watanabe Chemicals, 0.78 mmol / g, 0.13 g), following the general method described above, starting with the removal of Fmoc. A CEM Liberty Blue HT solid-phase synthesizer was used as the solid-phase synthesizer, and the synthesis was performed according to the manufacturer's manual. For each residue introduction, the basic conditions were: Fmoc-AA-OH / HATU / DIEA (5.3 eq / 5 eq / 10 eq) for 1 eq of resin, with the condensation reaction carried out at 75°C for 10 min. For the introduction of the 11th and 12th amino acid residues, two condensation reactions were carried out at 25°C for 30 min each. The 13th and 14th residues were condensed twice at 75°C for 10 min each. The 15th residue was condensed once at 25°C for 30 min. The 16th residue was removed using Fmoc-AA-OH / HATU / DIEA (3eq / 3eq / 6eq) at 25°C for 120 minutes, once per reaction. The basic conditions for de-Fmocification were reaction in 20% pipeidine in DMF at 75°C for 3 minutes. However, the removal of the Fmoc group from residues 13 and 15 was carried out by reacting for 5 minutes at 25°C followed by a 10-minute reaction. The introduction of the chloroacetyl group was performed by removing the α-amino Fmoc group from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, followed by stirring 10 eq of chloroacetic acid, 10 eq of DIPCI, and 10 eq of HOSu in DCM, adding an equal amount of NMP to the DCM to prepare a ClAcOSu DCM / NMP solution (0.2M), adding the solid-phase resin, and shaking at room temperature for 60 minutes. To remove the side chain protection and shear from the solid resin, the resin obtained after the chloroacetyl introduction step was first washed five times with DMF and three times with chloromethane, and then dried under reduced pressure. Next, a reaction mixture (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 resin, and the mixture was shaken at room temperature for 60 minutes. The reaction solution was recovered by glass flotation. The remaining solid resin in the reaction vessel was shaken again with the shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. Adding this filtrate to excess diethyl ether / hexane (1 / 1) cooled to 0°C produced a white turbid 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 dried for use in subsequent cyclization reactions. The peptide cyclization reaction was carried out by dissolving the peptide in DMSO with the molar amount of solid resin as the basis and the final concentration of the peptide being 5 mM, followed by the addition of 5 equal volumes of triethylamine, and stirring at room temperature for about 14 hours.The reaction solution was rapidly cooled with acetic acid and then concentrated under reduced pressure using a Biotage V-10.

[0656] The crude product was purified under the following conditions (column: WaterS Xbridge (registered trademark) C18 5μm (registered trademark) 30x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 40℃; gradient (%B): 5-29% for 3 minutes, 29-34% for 8 minutes, and 34-60% for 1 minute; flow rate: 45 mL / min).

[0657] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 77.0%.

[0658] Analytical conditions B: Hold time = 10.57 minutes; Column: Kinetex EVO C18 2.6 μm, 2.1 x 150 mm. Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; temperature 40℃; gradient (%B conc) takes 20 minutes to reach 20–60%, then 1 minute to reach 60–95%, then 5 minutes to reach 95–95%; flow rate: 0.25 mL / min.

[0659] ESI-MS(+) observation m / z = 1378.48(M+2H) 2+ .

[0660] [Example 9-4]

[0661] Synthesis of 894_3m_G_Azi (serial number 297)

[0662] [Chemistry 7]

[0663]

[0664] For the separately synthesized 894_3m_G (3.3 g, 1.39 mmol), 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) synthesized by a well-known method were stirred at room temperature for 30 minutes.

[0665] The resulting mixture was purified under the following conditions (column: WaterS Xbridge C18 5μm 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 40℃; gradient (%B): 7-7% for 2 minutes, then 7-32% for 1 minute, then 32-37% for 8 minutes, then 37-60% for 1 minute; flow rate: 20-20 mL / min for 1 minute, then 20-120 mL / min for 1 minute, then 120 mL / min).

[0666] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 97.1%.

[0667] Analytical conditions B: Hold time = 4.29 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60℃; Gradient (% Bconc): 20-60% over 7.2 minutes, 60-95% over 0.3 minutes, and 95-95% over 1.6 minutes; Flow rate: 0.5 mL / min.

[0668] ESI-MS(+) observation m / z = 1154.72(M+2H) 2+ .

[0669] [Examples 9-5]

[0670] Synthesis of 894_variant_39 (serial number 327)

[0671] [Chemistry 8]

[0672]

[0673] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.47 mmol / g, 0.21 g) following 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 manual. For each residue introduction, for 1 eq of resin, Fmoc-AA-OH / DIPCI / Oxima pure (5.3 eq / 10 eq / 5 eq) was used in DMF, and the reaction was repeated once at 90 °C for 3 minutes. However, the introduction of the 2nd and 4th residues was repeated twice at 75 °C for 30 minutes. The 9th residue was repeated twice at 90 °C for 10 minutes. The 11th and 12th residues were repeated twice at 50 °C for 15 minutes. The 13th residue was repeated twice at 90 °C for 3 minutes. The 15th residue was repeated once at 50 °C for 15 minutes. Furthermore, the basic conditions for Fmoc removal were a reaction with a 20% pipeidine DMF solution at 75°C for 3 minutes. However, the Fmoc removal of residues 2, 4, and 13 was performed twice at 25°C for 5 minutes. The introduction of chloroacetyl groups was carried out by removing the α-amino Fmoc group from a 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, followed by shaking at room temperature for 30 minutes. Deprotection of the side chains and shearing from the solid-phase resin were performed by first washing the resin obtained after the chloroacetyl group introduction step with DMF five times and chloromethane three times, followed by drying under reduced pressure. Next, in a reaction vessel already filled with solid resin, reactant mixture A (a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 60 minutes. The reaction solution was recovered by glass flotation. The solid resin remaining in the reaction vessel was shaken again with a shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. If this filtrate was added to an excess ether / hexane (1 / 1) mixture cooled to 0°C, a white turbid precipitate was formed. This mixture was centrifuged (9000 rpm, 1.5 min), and the solution was decanted. The resulting solid was washed again with a small amount of ether cooled to 0°C and dried under reduced pressure. The resulting solid was used in a subsequent cyclization reaction. The peptide cyclization reaction was carried out by dissolving the peptide in DMSO with the molar amount of solid resin as the basis and the final concentration of the peptide being 5 mM, followed by the addition of 10 equal amounts of triethylamine and shaking at room temperature for about 24 hours.The resulting reaction solution was concentrated under reduced pressure using SavantExplorer SpeedVaC.

[0674] The crude product was purified under the following conditions (column: WaterS Xbridge (registered trademark) C18 5μm (registered trademark) 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 40℃; gradient (%B): 11-36% for 3 minutes, 36-41% for 8 minutes, and 41-60% for 1 minute; flow rate: 120 mL / min).

[0675] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 93.1%.

[0676] Analytical conditions B: Hold time = 12.76 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60℃; Gradient (%B conc): 20-60% over 20 minutes, 60-95% over 1 minute, and 95% over 5 minutes; Flow rate: 0.25 mL / min.

[0677] ESI-MS(+) observation m / z = 1196.12(M+2H) 2+ .

[0678] [Examples 9-6]

[0679] Synthesis of 894_variant_120 (serial number 353)

[0680] [Chemistry 9]

[0681]

[0682] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.47 mmol / g, 0.43 g) following 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 manual. For each residue introduction, for 1 eq of resin, Fmoc-AA-OH / HATU / DIEA (5 eq / 5 eq / 10 eq) was used in DMF, and the reaction was carried out once at 75°C for 10 minutes. However, the first and third residues were reacted once at 75°C for 20 minutes. The second and fourth residues were reacted once at 75°C for 30 minutes. The ninth, tenth, and eleventh residues were reacted twice at 75°C for 20 minutes. The twelfth residue was reacted twice at 25°C for 30 minutes. The 13th residue was reacted twice at 75°C for 10 minutes. The 15th residue was reacted once at 25°C for 30 minutes. The basic condition for Fmoc removal was reaction with a 20% pipeidine DMF solution at 75°C for 3 minutes. However, the Fmoc removal of the 2nd, 4th, and 13th residues was repeated twice at 25°C for 5 minutes. The introduction of chloroacetyl groups was performed by removing the α-amino Fmoc group from a 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. Deprotection of the side chains and shearing from the solid-phase resin were performed by first washing the resin obtained after the chloroacetyl group introduction step with DMF five times and chloromethane three times, followed by drying under reduced pressure. Next, in a reaction vessel already filled with solid resin, reactant mixture A (a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 60 minutes. The reaction solution was recovered by glass flotation. The solid resin remaining in the reaction vessel was shaken again with a shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. If this filtrate was added to excess diisopropyl ether / hexane (1 / 1) mixed solvent cooled to 0°C, a white turbid precipitate was formed. 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 dried under reduced pressure. The resulting solid was used in a subsequent cyclization reaction. The peptide cyclization reaction was carried out by dissolving the peptide in DMSO with the molar amount of solid resin as the basis and the final concentration of the peptide being 5 mM, followed by the addition of 10 equal amounts of triethylamine, and shaking at room temperature for about 16 hours.The resulting reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.

[0683] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 40℃; gradient (%B): 16-41% for 3 minutes, 41-46% for 8 minutes, and 46-60% for 1 minute; flow rate: 120 mL / min).

[0684] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 97.5%.

[0685] Analytical conditions B: Hold time = 6.07 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60℃; Gradient (%B conc): 20-60% over 7.2 minutes, 60-95% over 0.3 minutes, and 95-95% over 1.6 minutes; Flow rate: 0.5 mL / min.

[0686] ESI-MS(+) observation m / z = 1036.90(M+2H) 2+ .

[0687] [Examples 9-7]

[0688] Synthesis of 894_variant_61 (serial number 354)

[0689] [Chemistry 10]

[0690]

[0691] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.47 mmol / g, 0.21 g) following 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 manual. For each residue introduction, for 1 eq of resin, Fmoc-AA-OH / DIPCI / Oxima pure (5.3 eq / 10 eq / 5 eq) was used in DMF, and the reaction was carried out once at 90 °C for 3 minutes. However, the second and fourth residues were reacted twice at 75 °C for 30 minutes. The tenth and eleventh residues were reacted twice at 90 °C for 10 minutes. The twelfth residue was reacted twice at 50 °C for 15 minutes. The thirteenth residue was reacted twice at 90 °C for 3 minutes. The fifteenth residue was reacted once at 50 °C for 15 minutes. Furthermore, the basic conditions for Fmoc removal were a reaction with a 20% pipeidine DMF solution at 75°C for 3 minutes. However, the Fmoc removal of residues 2, 4, and 13 was performed twice at 25°C for 5 minutes. The introduction of chloroacetyl groups was carried out by removing the α-amino Fmoc group from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. This was achieved by stirring 10 eq of chloroacetic acid, 5 eq of DIPCI, and 5 eq of HOSu in DCM, adding an equal amount of DMF to prepare a ClAcOSu DCM / DMF solution (0.2 M), adding it to the solid-phase resin, and shaking at room temperature for 60 minutes. Deprotection of the side chains and shearing from the solid-phase resin were performed by first washing the resin obtained after the chloroacetyl group introduction step with DMF 5 times and chloromethane 3 times, followed by drying under reduced pressure. Next, in a reaction vessel already filled with solid resin, reactant mixture A (a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass flotation. The solid resin remaining in the reaction vessel was shaken again with a shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. If this filtrate was added to excess diethyl ether / hexane (1 / 1) mixed solvent cooled to 0°C, a white turbid precipitate was formed. 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 dried under reduced pressure. The resulting solid was used in a subsequent cyclization reaction. The peptide cyclization reaction was carried out by dissolving the peptide in DMSO with the molar amount of solid resin as the basis and the final concentration of the peptide being 5 mM, followed by the addition of 5 eq of triethylamine and shaking at room temperature for about 14 hours.The resulting reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.

[0692] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40℃; gradient (%B): 15-40% for 3 minutes, then 40-45% for 8 minutes, then 45-60% for 1 minute; flow rate: 120 mL / min).

[0693] The purity of the target analyte was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 87.5%.

[0694] Analytical conditions B: Hold time = 16.50 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60℃; Gradient (%B conc): 20-60% over 20 minutes, 60-95% over 1 minute, and 95% over 5 minutes; Flow rate: 0.25 mL / min.

[0695] ESI-MS(+) observation m / z = 1160.58(M+2H) 2+ .

[0696] [Examples 9-8]

[0697] Synthesis of 894_variant_14 (serial number 387)

[0698] [Chemistry 11]

[0699]

[0700] Using Fmoc-D-Glu(OtBu)-wang resin (Watanabe Chemicals, 0.68 mmol / g, 184 mg), the target peptide was synthesized following a standard method, starting with the removal of Fmoc. A CEM Liberty Blue HT solid-phase synthesizer was used, following the manufacturer's manual. The basic conditions for the condensation reaction were the use of HATU as the condensing agent and reaction twice at 75°C for 10 minutes each time. However, the second residue was reacted twice at 75°C for 30 minutes each time. The fourth residue was reacted twice at 75°C for 30 minutes each time. The sixth residue was reacted once at 75°C for 10 minutes each time. The seventh residue was reacted once at 75°C for 10 minutes each time. The eighth residue was reacted once at 75°C for 10 minutes each time. The tenth residue was reacted once at 75°C for 10 minutes each time. The eleventh residue was reacted twice at 30°C for 30 minutes each time. The 12th residue was reacted once at 30°C for 30 minutes. The 15th residue was reacted once at 30°C for 30 minutes. Furthermore, the basic condition for Fmoc removal was to react with a 20% pipeidine DMF solution at 75°C for 3 minutes. However, for the 2nd, 4th, and 13th residues, Fmoc removal was carried out twice at room temperature for 5 minutes. The introduction of chloroacetyl groups was performed by stirring chloroacetic acid (5 equal parts), DIPCI (5 equal parts), and HOSu (5 equal parts) in DCM, adding an equal amount of DMF to the DCM solution, adding this solution to the solid resin obtained in the previous step, and shaking at room temperature for 60 minutes. For the deprotection of the side chains and shearing from the solid resin, the resin obtained after the chloroacetyl group introduction step was first washed 5 times with DMF and 3 times with chloromethane, and then dried under reduced pressure. Next, in a reaction vessel already filled with solid resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass flotation. The solid resin remaining in the reaction vessel was shaken again with a shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. If this filtrate was added to excess diethyl ether / hexane (3 / 1) cooled to 0°C, a white turbid precipitate was formed. This mixture was centrifuged (9500 rpm, 1 min), the supernatant was decanted, and the mixture was washed with diethyl ether cooled to 0°C. The resulting solid was used for the subsequent cyclization reaction. The peptide cyclization reaction was performed by dissolving the peptide in DMSO with the molar amount of solid resin as the basis and the final peptide concentration as 4 mM, adding 10 equal volumes of triethylamine, and shaking at room temperature for approximately 16 hours. The resulting reaction solution was concentrated under reduced pressure using GenevaC EZ-2Elite.

[0701] The resulting reaction solution was purified under the following conditions (column: WaterS Xbridge (registered trademark) C185μm (registered trademark) 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 40℃; gradient (%B): 11-36% for 3 minutes, 36-41% for 8 minutes, and 41-60% for 1 minute; flow rate: 120mL / min).

[0702] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 88.60%.

[0703] Analysis condition B: Hold time = 13.03 minutes; Gradient (%B conc): 20 minutes for 20-60%, then 1 minute for 60-95%, then 5 minutes for 95%.

[0704] ESI-MS (+) observation m / z = 1137.23 (M+2H) 2+ .

[0705] [Examples 9-9]

[0706] Synthesis of 894_variant84 (serial number 390)

[0707] [Chemistry 12]

[0708]

[0709] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.47 mmol / g, 0.21 g) following 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 manual. For each residue introduction, for 1 eq of resin, Fmoc-AA / DIPCI / Oxyma pure = 5.3 eq / 10 eq / 5 eq was used in DMF, and the reaction was carried out once at 90°C for 3 minutes. However, the second and fourth residues were reacted twice at 75°C for 45 minutes. The eleventh and twelfth residues were reacted twice at 50°C for 15 minutes. The thirteenth residue was reacted twice at 90°C for 3 minutes. Furthermore, the basic condition for Fmoc removal was a reaction with a 20% pipeidine DMF solution at 75°C for 3 minutes. However, the removal of Fmoc from residues 2, 4, and 13 was performed twice at 25°C for 5 minutes. After elongation of even residue 1, the resin was suspended in DCM, and Pd(PpH3)4 / pHSiH3 (0.2 eq / 10 eq) was added, followed by shaking at room temperature for 1 hour. The introduction of chloroacetyl groups was carried out by washing the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step three times with DMF and three times with DCM, removing the α-amino Fmoc group using the method described above, and then adding 5 eq of chloroacetic acid in DMF, 5 eq of HATU in DMF, and 10 eq of DIEA in DMF, followed by shaking at room temperature for 30 minutes. Deprotection of the side chains and shearing from the solid-phase resin were performed by first washing the resin obtained after the chloroacetyl group introduction step five times with DMF and three times with chloromethane, followed by drying under reduced pressure. Next, in a reaction vessel already filled with solid resin, reactant mixture A (a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass flotation. The solid resin remaining in the reaction vessel was shaken again with a shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. If this filtrate was added to an excess ether / hexane (1 / 1) mixture cooled to 0°C, a white turbid precipitate was formed. 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 ether cooled to 0°C and dried under reduced pressure. The resulting solid was used in a subsequent cyclization reaction. The peptide cyclization reaction was carried out by dissolving the peptide in DMSO with the molar amount of solid resin as the basis and the final concentration of the peptide being 5 mM, then adding 10 equal amounts of triethylamine, and shaking overnight at room temperature.The reaction solution was concentrated under reduced pressure using 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.

[0710] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 30x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40℃; gradient (%B): 11-36% for 3 minutes, 36-41% for 8 minutes, and 41-60% for 1 minute; flow rate: 45 mL / min).

[0711] The obtained 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, Et2NH (5.9 μL, 56.5 μmol) was added to the reaction solution and stirred. After 1 hour, the solution was quenched with acetic acid.

[0712] The crude product was purified under the following conditions (column: WaterS Xbridge (registered trademark) C18 5μm (registered trademark) 19x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 60℃; gradient (%B): 8-33% for 3 minutes, 33-38% for 8 minutes, and 38-60% for 1 minute; flow rate: 17 mL / min).

[0713] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 97.9%.

[0714] Analytical conditions B: Hold time = 12.33 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60℃; Gradient (% Bconc): 20-60% for 20 minutes, then 60-95% for 1 minute, then 95% for 5 minutes.

[0715] ESI-MS(+) observation m / z = 1134.34(M+2H) 2+ .

[0716] [Examples 9-10]

[0717] Synthesis of 894_variant89 (serial number 391)

[0718] [Chemistry 13]

[0719]

[0720] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.47 mmol / g, 0.21 g) following 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 manual. For each residue introduction, for 1 eq of resin, Fmoc-AA / DIPCI / Oxyma pure / DIEA (5.3 eq / 10 eq / 5 eq) was used in DMF at 90°C for 3 minutes, with one reaction per reaction. However, the introduction of the second and fourth residues was performed twice at 75°C for 45 minutes. The introduction of the eleventh and twelfth residues was repeated twice at 50°C for 15 minutes. The introduction of the thirteenth residue was performed twice at 90°C for 3 minutes. Furthermore, the basic conditions for Fmoc removal were a reaction with a 20% pipeidine DMF solution at 75°C for 3 minutes, performed once. However, the removal of Fmoc from the 2nd, 4th, and 13th residues was performed twice at 25°C for 5 minutes. After elongation of even the 1st residue, the resin was suspended in DCM, and Pd(PpH3)4 / pHSiH3 (0.2 eq / 10 eq) was added, followed by shaking at room temperature for 1 hour. The solid resin was then washed three times with DMF and three times with DCM, and then dried under reduced pressure. 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 then microwaved and reacted twice at 75 °C for 30 minutes. The introduction of chloroacetyl groups was performed by removing the α-amino Fmoc group from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above, followed by the addition of 5 eq of chloroacetic acid in DMF, 5 eq of HATU in DMF, and 10 eq of DIEA in DMF, followed by shaking at room temperature for 30 minutes. Deprotection of the side chains and shearing from the solid-phase resin were first performed by washing the resin obtained after the chloroacetyl introduction step five times with DMF and three times with chloromethane, followed by drying under reduced pressure. Next, in the reaction vessel already filled with solid resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass frit filtration. The solid resin remaining in the reaction vessel was shaken again with the shearing mixture, the solution components were recovered by glass frit filtration, and then mixed with the filtrate.Adding this filtrate to an excess ether / hexane (1 / 1) mixture cooled to 0°C produces a white precipitate. The mixture is centrifuged (10,000 rpm, 1 min) and the solution is decanted. The resulting solid is washed again with a small amount of ether cooled to 0°C and dried under reduced pressure. The resulting solid is used in a subsequent cyclization reaction. The peptide cyclization reaction is performed by dissolving the peptide in DMSO at a final concentration of 5 mM based on the molar amount of solid resin, adding 10 eq of triethylamine, and shaking overnight at room temperature. The reaction solution is concentrated under reduced pressure using Savant Explorer SpeedVaC.

[0721] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40℃; gradient (%B): 8-33% for 3 minutes, 33-38% for 8 minutes, and 38-60% for 1 minute; flow rate: 120 mL / min).

[0722] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 94.9%.

[0723] Analytical conditions B: Hold time = 11.81 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 40℃; Gradient (%B conc): 20-60% over 20 minutes, 60-95% over 1 minute, and 95% over 5 minutes; Flow rate: 0.25 mL / min.

[0724] ESI-MS(+) observation m / z = 1127.76(M+2H) 2+ .

[0725] [Examples 9-11]

[0726] Synthesis of 894_variant_03 (serial number 397)

[0727] [Chemistry 14]

[0728]

[0729] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.47 mmol / g, 0.21 g) following 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 manual. For each residue introduction, for 1 eq of resin, Fmoc-AA / DIPCI / Oxima pure (4.2 eq / 4 eq / 8 eq) was reacted once in DMF at 75°C for 10 minutes. However, the second and fourth residues were reacted twice at 75°C for 30 minutes. The eleventh and twelfth residues were reacted twice at 90°C for 10 minutes. The thirteenth residue was reacted twice at 75°C for 10 minutes. The fifteenth residue was reacted once at 25°C for 20 minutes. Furthermore, the basic condition for Fmoc removal is to react with a 20% pipeidine DMF solution at 75°C for 3 minutes. However, the Fmoc removal of residues 2, 4, and 13 is carried out at 25°C for 5 minutes followed by a reaction at 25°C for 10 minutes. The introduction of chloroacetyl groups is performed by removing the α-amino Fmoc group from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. Chloroacetic acid (10 equal parts), DIPCI (10 equal parts), and HOSu (10 equal parts) are stirred in DCM, and an equal amount of DMF is added to prepare a ClAcOSu DCM / DMF solution (0.2 M). This solution is then added to the solid-phase resin and shaken at room temperature for 60 minutes. For deprotection of the side chains and shearing from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step is first washed 5 times with DMF and 3 times with chloromethane, and then dried under reduced pressure. Next, in a reaction vessel already filled with solid resin, reactant mixture A (a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass flotation. The solid resin remaining in the reaction vessel was shaken again with a shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. If this filtrate was added to excess diethyl ether / hexane (1 / 1) mixed solvent cooled to 0°C, a white turbid precipitate was formed. 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 dried under reduced pressure. The resulting solid was used in a subsequent cyclization reaction. The peptide cyclization reaction was carried out by dissolving the peptide in a 50% MeCN aqueous solution with the molar amount of solid resin as the basis and the final concentration of the peptide being 5 mM, followed by the addition of 5 equal volumes of triethylamine, and shaking at room temperature for about 14 hours.The resulting reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.

[0730] The crude product was purified under the following conditions (column: WaterS Xbridge (registered trademark) C18 5μm (registered trademark) 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 40℃; gradient (%B): 10-35% for 3 minutes, 35-40% for 8 minutes, and 40-60% for 1 minute; flow rate: 120mL / min).

[0731] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 98.2%.

[0732] Analytical conditions B: Hold time = 13.18 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 40℃; Gradient (%B conc): 20-60% over 20 minutes, 60-95% over 1 minute, and 95-95% over 5 minutes; Flow rate: 0.25 mL / min.

[0733] ESI-MS(+) observation m / z = 1072.49(M+2H) 2+ .

[0734] [Examples 9-12]

[0735] Synthesis of 894_variant_31 (serial number 400)

[0736] [Chemistry 15]

[0737]

[0738] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.47 mmol / g, 0.21 g) following the general method described above, starting with the removal of the Fmoc group. Liberty Blue from CEM was used as the solid-phase synthesizer, and the synthesis was performed according to the manufacturer's manual. For each residue introduction, for 1 eq of resin, Fmoc-AA / DIPCI / Oxima pure (4.2 eq / 4 eq / 8 eq) was used in DMF, and the reaction was repeated twice at 90°C for 3 minutes. However, the introduction of the 2nd and 4th residues was repeated twice at 75°C for 45 minutes. The 3rd residue was repeated twice at 75°C for 30 minutes. The 7th and 8th residues were repeated once at 90°C for 3 minutes. The introduction of the 11th and 12th residues was repeated twice at 50°C for 15 minutes. Residues 15 and 16 were reacted once at 50°C for 15 minutes. The basic condition for Fmoc removal was reaction with a 20% piperidine DMF solution at 75°C for 3 minutes. However, Fmoc removal of residues 2, 4, and 13 was repeated twice at 25°C for 5 minutes. The introduction of chloroacetyl groups was performed by removing the α-amino Fmoc group from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, followed by stirring chloroacetic acid (5 equal parts), DIPCI (5 equal parts), and HOSu (5 equal parts) in DCM, adding an equal amount of DMF to prepare a ClAcOSu DCM / DMF solution (0.2 M), adding it to the solid-phase resin, and shaking at room temperature for 60 minutes. To remove the side chain protection and shear from the solid resin, the resin obtained after the chloroacetyl introduction step was first washed five times with DMF and three times with chloromethane, then dried under reduced pressure. Next, in a reaction vessel already loaded with the solid resin, a reaction mixture-A (a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass flotation. The remaining solid resin in the reaction vessel was shaken again with the shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. Adding this filtrate to excess diisopropyl ether cooled to 0°C produced a white precipitate. 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 dried under reduced pressure. The resulting solid was used in subsequent cyclization reactions. The peptide cyclization reaction was carried out by dissolving the peptide in DMSO with the molar amount of solid resin as the basis and the final concentration of the peptide being 4 mM, followed by the addition of 7 equal volumes of triethylamine, and shaking at room temperature for about 12 hours.The resulting reaction solution was concentrated under reduced pressure using GenevaC EZ-2Elite.

[0739] The crude product was purified under the following conditions (column: WaterS Xbridge (registered trademark) C18 5μm (registered trademark) 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 40℃; gradient (%B): 15-40% for 3 minutes, then 40-45% for 8 minutes, then 45-60% for 1 minute; flow rate: 120mL / min).

[0740] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 95.4%.

[0741] Analytical conditions B: Hold time = 12.64 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60℃; Gradient (%B conc): 20-60% over 20 minutes, 60-95% over 1 minute, and 95% over 5 minutes; Flow rate: 0.25 mL / min.

[0742] ESI-MS(+) observation m / z = 1195.87(M+2H) 2+ .

[0743] [Examples 9-13]

[0744] Synthesis of 894_variant_03_G4S2C (serial number 401)

[0745] [Chemistry 16]

[0746]

[0747] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.73 mmol / g, 1.37 g) following the general method described above, starting with the removal of the Fmoc group. Liberty Blue resin from CEM was used as the solid-phase synthesizer, and the synthesis was performed according to the manufacturer's manual. For each residue introduction, for 1 eq of resin, Fmoc-AA / HATU / DIEA (4.2 eq / 4 eq / 8 eq) was used in DMF, and the reaction was carried out once at 75°C for 10 minutes. However, the second and fourth residues were reacted once at 75°C for 30 minutes. The eleventh and twelfth residues were reacted twice at 25°C for 20 minutes. The thirteenth residue was reacted twice at 75°C for 10 minutes. The fifteenth and twentieth residues were reacted once at 25°C for 30 minutes. Furthermore, the basic conditions for Fmoc removal were to react with a 20% pipeidine DMF solution at 75°C for 3 minutes. However, the Fmoc removal of residues 2, 4, and 13 was carried out at 25°C for 5 minutes, followed by a reaction at room temperature for 10 minutes. The introduction of chloroacetyl groups was performed by removing the α-amino Fmoc group from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. This was done by stirring 10 eq of chloroacetic acid, 10 eq of DIPCI, and 10 eq of HOSu in DCM, adding an equal amount of DMF to prepare a ClAcOSu DCM / DMF solution (0.2 M), adding it to the solid-phase resin, and shaking at room temperature for 60 minutes. For deprotection of the side chains and shearing from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed 5 times with DMF and 3 times with chloromethane, and then dried under reduced pressure. Next, in a reaction vessel already filled with solid resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 90:2.5:2.5:5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass flotation. The solid resin remaining in the reaction vessel was shaken again with a shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. If this filtrate was added to excess diisopropyl ether / hexane (1 / 1) mixed solvent cooled to 0°C, a white turbid precipitate was formed. This mixture was centrifuged (9500 rpm, 1 min), the solution was decanted, and dried under reduced pressure. The resulting solid was used for subsequent cyclization reactions. The peptide cyclization reaction was performed by dissolving the peptide in DMSO with a final peptide concentration of 5 mM based on the molar amount of solid 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, and 3 eq of silver acetate was added. The mixture was shaken for 3 hours. After adding 2M DTT aqueous solution (11 eq), the mixture was centrifuged, the supernatant was recovered, and the crude product was concentrated under reduced pressure.

[0748] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 40℃; gradient (%B): 11-11% for 3 minutes, 11-36% for 3 minutes, 36-41% for 8 minutes, and 41-60% for 1 minute; flow rate: 120 mL / min).

[0749] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 94.8%.

[0750] Analytical conditions B: Hold time = 4.31 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60℃; Gradient (% Bconc): 20-60% over 7.2 minutes, 60-95% over 0.3 minutes, and 95-95% over 1.6 minutes; Flow rate: 0.5 mL / min.

[0751] ESI-MS(+) observation m / z = 1324.94(M+2H) 2+ .

[0752] [Examples 9-14]

[0753] Synthesis of 894_A_hgl_CyCloamide (Serial No. 408)

[0754] [Chemistry 17]

[0755]

[0756] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.47 mmol / g) following standard procedures, starting with the removal of Fmoc. A CEM Liberty Blue HT solid-phase synthesizer was used, following the manufacturer's manual. The basic conditions for the condensation reaction were DIPCI and Oxyma Pure in the condensing agent, reacting once at 90°C for 3 minutes. However, residues 11 and 12 were reacted twice at 50°C for 15 minutes. Residue 13 was reacted twice at 90°C for 3 minutes. The basic conditions for Fmoc removal were reaction with a 20% piperidine DMF solution at 75°C for 3 minutes. However, for residue 13, Fmoc removal was performed twice at 25°C for 5 minutes. The resulting resin was washed five times with DMF and three times with chloromethane, and then dried under reduced pressure. Next, in a reaction vessel already filled with solid resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass flotation. The solid resin remaining in the reaction vessel was shaken again with a shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. If this filtrate was added to excess diisopropyl ether and hexane mixed solvent cooled to 0°C, a white turbid precipitate was formed. This mixture was centrifuged (9000 rpm, 2 min), and the supernatant was decanted. The resulting solid was washed with chilled diethyl ether and dried. The resulting solid was used in a subsequent cyclization reaction. The peptide cyclization reaction was performed by dissolving the peptide in DMF with a final peptide concentration of 5 mM based on the molar amount of solid resin, adding 1.5 equal parts HATU and 3 equal parts triethylamine, and shaking at room temperature for about 1 hour. The resulting reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.

[0757] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 30x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40℃; gradient (%B): 6-31% for 3 minutes, 31-36% for 8 minutes, and 36-60% for 1 minute; flow rate: 45 mL / min).

[0758] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 96.8%.

[0759] Analysis condition B: Hold time = 10.03 minutes; Gradient (%B conc): 20 minutes for 20-60%, then 1 minute for 60-95%, then 5 minutes for 95%.

[0760] ESI-MS(+) observation m / z = 1033.0(M+2H) 2+ .

[0761] [Examples 9-15]

[0762] Synthesis of 894_BiCyCle_001 (serial number 409)

[0763] [Chemistry 18]

[0764]

[0765] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.47 mmol / g, 0.53 mg) following 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 manual. For each residue introduction, Fmoc-AA / HATU / DIEA (4.2 / 4 / 8 equal parts) was used in DMF at 75°C for 10 minutes, with equal amounts of resin 1. However, residues 1, 6, and 10 were reacted once at 75°C for 10 minutes. Residues 2 and 3 were reacted twice at 75°C for 20 minutes. Residue 4 was reacted twice at 75°C for 30 minutes. Residues 11 and 14 were reacted once at 75°C for 20 minutes. Residues 12 and 15 were reacted once at 30°C for 30 minutes. The basic conditions for Fmoc removal were a reaction with a 20% piperidine DMF solution at 75°C for 3 minutes. However, Fmoc removal of residues 2, 4, and 13 was repeated twice at 25°C for 5 minutes. The introduction of chloroacetyl groups was performed by removing the α-amino Fmoc group from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, followed by stirring 5 equal parts chloroacetic acid, 5 equal parts DIPCI, and 5 equal parts HOSu in DCM, adding an equal amount of DMF to prepare a ClAcOSu DCM / DMF solution (0.2 M), adding it to the solid-phase resin, and shaking at room temperature for 75 minutes. To remove the side chain protection and shear from the solid resin, the resin obtained after the chloroacetyl introduction step was first washed five times with DMF and three times with chloromethane, then dried under reduced pressure. Next, in a reaction vessel already loaded with the solid resin, a reaction mixture-A (a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass flotation. The remaining solid resin in the reaction vessel was shaken again with the shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. Adding this filtrate to excess diisopropyl ether cooled to 0°C produced a white precipitate. 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 dried under reduced pressure. The resulting solid was used in subsequent cyclization reactions. The peptide cyclization reaction was carried out by dissolving the peptide in 5% aqueous DMSO with the molar amount of solid resin as the basis and the final concentration of the peptide being 4 mM, followed by the addition of 10 equal amounts of triethylamine and shaking at room temperature for about 12 hours.The resulting reaction solution was concentrated under reduced pressure using a GenevaC EZ-2 Elite. The resulting mixture was dissolved in DMF with the final peptide concentration set at 25 mM based on the number of moles of solid resin, and 1 equal volume of HATU and 3 equal volumes of DIEA were added. The mixture was then shaken at room temperature for approximately 30 minutes.

[0766] The crude product was purified under the following conditions (column: WaterS Xbridge (registered trademark) C18 5μm (registered trademark) 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 40℃; gradient (%B): 11-36% for 3 minutes, 36-41% for 8 minutes, and 41-60% for 1 minute; flow rate: 120 mL / min).

[0767] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 94.8%.

[0768] Analytical conditions B: Hold time = 13.78 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60℃; Gradient (% Bconc): 20-60% for 20 minutes, then 60-95% for 1 minute, then 95-95% for 5 minutes; Flow rate: 0.25%.

[0769] ESI-MS(+) observation m / z = 1072.26(M+2H) 2+ .

[0770] [Examples 9-16]

[0771] Synthesis of 894_BiCyCle_006 (serial number 414)

[0772] [Chemistry 19]

[0773]

[0774] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.47 mmol / g, 0.32 g) following 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 manual. For each residue introduction, an equal amount of Fmoc-AA / DIPCI / Oxyma pure / DIEA (5.3 / 10 / 5 equal amounts) was used in DMF at 90°C for 3 minutes, with one reaction per residue. However, the introduction of the 2nd, 4th, 11th, and 13th residues was performed twice at 90°C for 3 minutes. The introduction of the 12th residue was performed twice at 50°C for 15 minutes. The introduction of the 15th residue was performed once at 50°C for 15 minutes. Furthermore, the basic condition for Fmoc removal is a reaction with a 20% pipeidine DMF solution at 75°C for 3 minutes, performed once. However, the removal of Fmoc from the 2nd, 4th, and 13th residues is performed twice at 25°C for 5 minutes. The solid resin was sequentially washed with DMF and DCM and dried under reduced pressure. It was then swelled with DCM, and 10 equal parts of pHSiH3 and 0.2 equal parts of Pd(PpH3)4 were added, followed by shaking at room temperature for 1 hour. The solid resin was then washed 5 times with DCM, 5 times with DMF, and 3 times with ether, and dried. The solid resin was then swelled with NMP, and 5 equal parts of HATU and DIEA were added, followed by shaking at room temperature for 30 minutes. After washing the resin, 5 equal parts of HATU and DIEA were added again, followed by shaking at room temperature for 30 minutes. The solid resin was then washed 5 times with DCM, 5 times with DMF, and 3 times with ether. The introduction of chloroacetyl groups was performed by removing the α-amino Fmoc group from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. Then, 5 equal volumes of chloroacetic acid, 5 equal volumes of DIPCI, and 5 equal volumes of HOSu were stirred in DCM, and an equal volume of DMF was added to prepare a ClAcOSu DCM / DMF solution (0.2 M). This solution was added to the solid-phase resin and the mixture was shaken at room temperature for 60 minutes. For deprotection of the side chains and shearing from the solid-phase resin, the resin obtained after the chloroacetyl introduction step was first washed 5 times with DMF and 3 times with chloromethane, and then dried under reduced pressure. Next, in a reaction vessel already filled with solid-phase resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass frit filtration. The solid resin remaining in the reaction vessel is agitated again with the shearing mixture, the solution components are recovered from the glass frit, and then mixed with the filtrate.Adding this filtrate to excess diisopropyl ether cooled to 0°C produces a turbid precipitate. The mixture is centrifuged (9500 rpm, 1 min), and the solution is decanted. The resulting solid is washed again with a small amount of diethyl ether cooled to 0°C and dried under reduced pressure. The resulting solid is used in a subsequent cyclization reaction. The peptide cyclization reaction is performed by dissolving the peptide in 5% aqueous DMSO with the molar amount of solid resin as the basis, resulting in a final peptide concentration of 3.3 mM. Seven equal volumes of triethylamine are added, and the mixture is shaken at room temperature for approximately 12 hours. The resulting reaction solution is concentrated under reduced pressure using a GenevaC EZ-2 Elite.

[0775] The crude product obtained was purified under the following conditions (column: WaterS Xbridge C18 5μm 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40℃; gradient (%B): 7-32% for 3 minutes, 32-37% for 8 minutes, and 37-60% for 1 minute; flow rate: 120mL / min), then freeze-dried, and further purified under the following conditions. (Column: WaterS Xbridge (registered trademark) C18 5μm (registered trademark) 50x150mm; Mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; Temperature: 40℃; Gradient (%B): 11-36% for 3 minutes, 36-41% for 8 minutes, 41-60% for 1 minute; Flow rate: 120mL / min).

[0776] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 89.0%.

[0777] Analytical conditions B: Hold time = 3.67 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60℃; Gradient (% Bconc): 20-60% over 7.2 minutes, 60-95% over 0.3 minutes, and 95-95% over 1.6 minutes; Flow rate: 0.5 mL / min.

[0778] ESI-MS(+) observation m / z = 1055.13(M+2H) 2+ .

[0779] [Examples 9-17]

[0780] Synthesis of 894_BiCyCle_012 (serial number 419)

[0781] [Chemistry 20]

[0782]

[0783] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.47 mmol / g, 213 mg) and the general method described above, starting with the removal of the Fmoc group. Liberty Blue from CEM was used as the solid-phase synthesizer, and the synthesis was performed according to the manufacturer's manual. Fmoc-W1aa(Allyl)-OH, Fmoc-Hgl(tBu)-OH, and Fmoc-Hly(Boc)-OH were used as amino acid starting materials. The crude product was obtained using the same method as described in Bicycle_01 and Bicycle_06.

[0784] The crude product was purified under the following conditions (column: WaterS Xbridge (registered trademark) C18 5μm (registered trademark) 19x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 60℃; gradient (%B): 11-36% for 3 minutes, 36-41% for 8 minutes, and 41-60% for 1 minute; flow rate: 17 mL / min).

[0785] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 94.8%.

[0786] Analytical conditions B: Hold time = 4.76 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature 60℃; Gradient (% Bconc): 20-60% for 7.2 minutes, then 60-95% for 0.3 minutes, then 95% for 1.6 minutes.

[0787] ESI-MS(+) observation m / z = 1093.23(M+2H) 2+ .

[0788] [Examples 9-18]

[0789] Synthesis of 894_3m_G (serial number 421)

[0790] [Chemistry 21]

[0791]

[0792] 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. Liberty Blue from CEM was used as the solid-phase synthesizer, and the synthesis was performed according to the manufacturer's manual. For each residue introduction, Fmoc-AA / HATU / DIEA (4.2 / 4 / 8 equal parts) was used in DMF at 60°C for 15 minutes, with two repeated reactions relative to resin 1. However, the 15th residue was introduced once at 25°C for 30 minutes. The 11th and 12th residues were introduced twice at 25°C for 20 minutes. The 10th residue was introduced once at 60°C for 15 minutes. The 7th residue was introduced once at 60°C for 15 minutes. The introduction of the 5th residue was carried out once at 60°C for 15 minutes. The introduction of the 3rd residue was carried out twice at 60°C for 45 minutes. Furthermore, the basic condition for Fmoc removal was a reaction with a 20% piperidine DMF solution at 75°C for 3 minutes. However, the Fmoc removal of the 2nd and 13th residues was repeated twice at 25°C for 5 minutes. The introduction of the chloroacetyl group was performed by removing the α-amino Fmoc group from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, followed by stirring chloroacetic acid (5 equal parts), DIPCI (5 equal parts), and HOSu (5 equal parts) in DCM, adding an equal amount of DMF to prepare a ClAcOSu DCM / DMF solution (0.2 M), adding it to the solid-phase resin, and shaking at room temperature for 240 minutes. To remove the side chain protection and shear from the solid resin, the resin obtained after the chloroacetyl introduction step was first washed five times with DMF and three times with chloromethane, then dried under reduced pressure. Next, in a reaction vessel already loaded with the solid resin, a reaction mixture-A (a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass flotation. The remaining solid resin in the reaction vessel was shaken again with the shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. Adding this filtrate to excess diisopropyl ether cooled to 0°C produced a white precipitate. 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 dried under reduced pressure. The resulting solid was used in subsequent cyclization reactions.The peptide cyclization reaction was performed by dissolving the peptide in DMSO with the molar amount of solid-phase resin as the reference, resulting in a final peptide concentration of 4 mM. Then, 10 equal volumes of triethylamine were 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 reactor.

[0793] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 50x250mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 60℃; gradient (%B): 0-0% for 5 minutes, 0-4.2% for 2 minutes, 4.2-28.6% for 3 minutes, 28.6-33.7% for 15.5 minutes, 33.7-60% for 1.5 minutes; flow rate: 18 mL / min for 5 minutes, 18 mL / min-118 mL / min for 2 minutes, then 118 mL / min).

[0794] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 92.2%.

[0795] Analysis condition B: Hold time = 11.52 minutes; Gradient (%B conc): 20 minutes for 20-60%, then 1 minute for 60-95%, then 5 minutes for 95%.

[0796] ESI-MS(+) observation m / z = 1078.24(M+2H) 2+ .

[0797] [Examples 9-19]

[0798] Synthesis of 894_3m_G4S2C(ACNMe) (Serial No. 443)

[0799] [Chemistry 22]

[0800]

[0801] The separately synthesized 894_3m_G4S2C (20 mg, 7.07 μmol) was dissolved in DMF (0.1 mL), and 4 equal volumes of TEA (3.94 μL, 28.3 μmol) in DMF (39 μL) and 1.1 equal volumes of 2-Iodo-N-MethylaCetamide (39.1 mg, 0.25 mmol) in DMF (15 μL) were added. The mixture was stirred at room temperature for 1 hour.

[0802] The resulting mixture was purified under the following conditions (column: WaterS Xbridge (registered trademark) C185μm (registered trademark) 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 60℃; gradient (%B): 3 min 6-31%, then 8 min 31-36%, then 1 min 36-60%; flow rate: 17 mL / min).

[0803] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 97.3%.

[0804] Analytical conditions B: Hold time = 3.53 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60℃; Gradient (% Bconc): 20-60% over 7.2 minutes, 60-95% over 0.3 minutes, and 95-95% over 1.6 minutes; Flow rate: 0.5 mL / min.

[0805] ESI-MS(+) observation m / z = 1337.32(M+2H) 2+ .

[0806] [Examples 9-20]

[0807] Synthesis of 894_0263 (serial number 487)

[0808] [Chemistry 23]

[0809]

[0810] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.48 mmol / g, 0.26 g) following the general method described above, starting with the removal of the Fmoc group. A Liberty Blue HT solid-phase synthesizer from CEM was used as the solid-phase synthesizer, and the synthesis was performed according to the manufacturer's manual. For the introduction of each residue, Fmoc-AA / DIPCI / Oxyma pure / DIEA (4.2 / 8 / 4 equal volumes) were reacted with an equal volume of resin. Fmoc removal was carried out by reacting with a 20% pipeidine solution in DMF. The introduction of the chloroacetyl group was performed by removing the α-amino Fmoc group from the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above, followed by the addition of 5 equal volumes of chloroacetic acid in DMF, 5 equal volumes of HATU in DMF, and 10 equal volumes of DIEA in DMF, and shaking at 25°C for 30 minutes. To remove the protection of the side chains and shear from the solid resin, the resin obtained after the chloroacetyl introduction step was first washed five times with DMF and three times with chloromethane, and then dried under reduced pressure. Next, in a reaction vessel already loaded with the solid resin, a reaction mixture-A (a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 60 minutes. The reaction solution was recovered by glass flotation. The remaining solid resin in the reaction vessel was shaken again with the shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. Adding this filtrate to excess diisopropyl ether / hexane (1 / 1) mixed solvent cooled to 0°C produced a white turbid 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 dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. The peptide cyclization reaction involved dissolving the peptide in DMSO with the molar amount of solid resin as the reference, resulting in a final peptide concentration of 5 mM. Then, 10 equal volumes of triethylamine were added, and the mixture was shaken overnight at room temperature. The reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC. The resulting solid was then dissolved in DMSO with the molar amount of solid resin as the reference, resulting in a final peptide concentration of 5 mM. Then, 1.2 equal volumes of MePEG4c, 1.1 equal volumes of HATU, and 3 equal volumes of DIEA were added, and the mixture was shaken for 1 hour at room temperature.

[0811] The crude product was purified under the following conditions (column: WaterS Xbridge (registered trademark) C18 5μm (registered trademark) 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 40℃; gradient (%B): 13-38% for 3 minutes, 38-43% for 8 minutes, and 43-60% for 1 minute; flow rate: 120mL / min).

[0812] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 92.3%.

[0813] Analytical conditions B: Hold time = 5.28 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60℃; Gradient (% Bconc): 20-60% over 7.2 minutes, 60-95% over 0.3 minutes, and 95-95% over 1.6 minutes; Flow rate: 0.5 mL / min.

[0814] ESI-MS(+) observation m / z = 1136.17(M+2H) 2+ .

[0815] Synthesize the following complexes of peptides, linkers, and payloads.

[0816] [Example 10-1]

[0817] Synthesis of JCR_hTfR_000894_PEG11_K_FITC (Serial No. 147)

[0818] [Chemistry 24]

[0819]

[0820] Using Fmoc-NH-SAL-PEG-resin 1500-2000 Da (Watanabe Chemicals, 0.38 mmol / g), the target peptide was synthesized following a standard method, starting with the removal of Fmoc. Liberty Blue from CEM was used as the solid-phase synthesizer, and the process was performed according to the manufacturer's manual. The basic conditions for the condensation reaction were: using HATU as the condensing agent, reacting once at 75°C for 10 minutes. However, residues 11 and 12 were reacted twice at 25°C for 20 minutes. Residues 13 and 14 were reacted twice at 75°C for 10 minutes. Residue 15 was reacted once at 25°C for 20 minutes. Residue 16 was reacted once at 25°C for 60 minutes. Furthermore, the basic conditions for Fmoc removal were: reacting with a 20% pipeidine DMF solution at 75°C for 3 minutes. However, for residues 13 and 15, Fmoc removal was performed by reacting at 25°C for 5 minutes, followed by a 10-minute reaction. The introduction of chloroacetyl groups was carried out by adding 5 equal volumes of a 0.2M DMF solution of chloroacetic acid, 5 equal volumes of a 0.5M DMF solution of HATU, and 10 equal volumes of a 1M DMF solution of DIPEA to the solid-phase resin obtained in the previous step, followed by shaking at room temperature for 30 minutes. Deprotection of the side chains and shearing from the solid-phase resin were performed by first washing the resin obtained after the chloroacetyl group introduction step with DMF 5 times and chloromethane 3 times, followed by drying under reduced pressure. Next, in the reaction vessel already loaded with the solid-phase resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by filtration through a glass frit filter. The remaining solid resin in the reaction vessel was shaken again with the shearing mixture, the solution components were recovered from the glass frit, and mixed with the filtrate. Adding this filtrate to excess diethyl ether and hexane mixed solvent cooled to 0°C produced a turbid precipitate. This mixture was centrifuged (10000 rpm, 1 min), and the supernatant was decanted. The resulting solid was washed with chilled diethyl ether and dried. The resulting solid was used in the subsequent cyclization reaction. The peptide cyclization reaction involved dissolving the peptide in DMSO with a final concentration of 5 mM based on the molar amount of solid resin, adding 6 equal volumes of triethylamine, and shaking overnight at room temperature. 1.1 equal volumes of FAM-OSu DMSO solution (0.71 M) were added to the resulting reaction solution, and the mixture was stirred for 30 minutes. AcOH was added to the reaction solution for quenching, and the solvent was concentrated under reduced pressure.

[0821] The crude product was purified under the following conditions (column: XSeleCt C18 30x150mm (Lot No. 151i3629811308); mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 40℃; gradient (%B): 6-31% for 3 minutes, 31-36% for 8 minutes, and 36-60% for 1 minute; flow rate: 45 mL / min).

[0822] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 97.1%.

[0823] Analysis condition B: Hold time = 13.20 minutes; Gradient (%B conc): 20 minutes for 20-60%, then 1 minute for 60-95%, then 5 minutes for 95%.

[0824] ESI-MS (+) observed value m / z = 793.2, theoretical value 792.6 (M+4H) 4+ .

[0825] [Example 10-2]

[0826] Synthesis of 894_v01_PEG12_C_Mal(FITC) (Serial No. 166)

[0827] [Chemistry 25]

[0828]

[0829] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.6 mmol / g, 0.19 g) and following the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue HT solid-phase synthesizer was used as the synthesizer, and the synthesis was performed according to the manufacturer's manual. For each residue introduction, Fmoc-AA / HATU / DIEA (5.3 / 5 / 10 equivalents) was used in DMF at 75°C for 10 minutes, with an equal amount of resin 1. However, the second and thirteenth residues were reacted twice at 75°C for 10 minutes. The eleventh and twelfth residues were reacted twice at 25°C for 20 minutes. The fifteenth and seventeenth residues were reacted once at 25°C for 30 minutes. Furthermore, the basic condition for Fmoc removal was reaction with a 20% pipeidine solution in DMF at 75°C for 3 minutes. However, the removal of Fmoc from residues 2, 13, 15, and 17 was carried out at 25°C for 5 minutes, followed by a reaction at 25°C for 10 minutes. The introduction of chloroacetyl groups was performed by removing the α-amino Fmoc group from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, followed by adding 5 equal parts of chloroacetic acid in DMF, 5 equal parts of HATU in DMF, and 10 equal parts of DIEA in DMF, and shaking at room temperature for 30 minutes. Deprotection of the side chains and shearing from the solid-phase resin were first performed by washing the resin obtained after the chloroacetyl group introduction step with DMF 5 times and chloromethane 3 times, followed by drying under reduced pressure. Next, in a reaction vessel already filled with solid-phase resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass frit filtration. The solid resin remaining in the reaction vessel was shaken again with the shearing mixture, the solution components were recovered by glass frit, and mixed with the filtrate. If this filtrate was added to excess diethyl ether / hexane (1 / 1) mixed solvent cooled to 0°C, a turbid precipitate was formed. This mixture was centrifuged (10000 rpm, 1 min), the solution was decanted, the solid was washed with diethyl ether cooled to 0°C, and dried under reduced pressure. The resulting solid was used for subsequent cyclization reactions. The peptide cyclization reaction was performed by dissolving the peptide in DMSO with the molar amount of solid resin as the basis and the final peptide concentration as 5 mM, adding 6 equal volumes of triethylamine, and shaking overnight at room temperature. The resulting reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC. The resulting mixture was dissolved in DMSO, 3 equal volumes of silver acetate were added, and shaking was performed for 3 hours.After adding 10 equal amounts of DTT, centrifuge and recover the supernatant.

[0830] The crude intermediate peptides obtained were purified using the following conditions (column: WaterS Xbridge (registered trademark) C185μm (registered trademark) 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 40℃; gradient (%B): 3 min 9-34%, then 8 min 34-39%, then 1 min 39-60%; flow rate: 120 mL / min).

[0831] The purified intermediate peptide (29.8 mg, 10.6 μmol) was dissolved in DMSO (424 μL), and 1.1 equal amounts of fluorescein-5-cis-butenedimide and 5 equal amounts of DIEA were added. After stirring for 1 hour, the mixture was quenched with acetic acid.

[0832] The crude product was purified under the following conditions (column: COSMOSIL PBr 10x150 mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (%B): 23-48% for 3 minutes, 48-53% for 8 minutes, and 53-60% for 1 minute; flow rate: 5 mL / min).

[0833] The purity of the target analyte was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 82.8%.

[0834] Analytical conditions B: Hold time = 13.93 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 40℃; Gradient (% Bconc): 20-60% over 20 minutes, 60-95% over 1 minute, and 95% over 5 minutes; Flow rate: 0.25 mL / min.

[0835] ESI-MS(+) observation m / z = 1076.51 theoretical value (M+2H) 2+ .

[0836] [Synthesis Example 10-3]

[0837] Synthesis of 894_variant_61_G4S2C (serial number 358)

[0838] [Chemistry 26]

[0839]

[0840] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.73 mmol / g, 1.37 g) following the general method described above, starting with the removal of the Fmoc group. Liberty Blue from CEM was used as the solid-phase synthesizer, and the synthesis was performed according to the manufacturer's manual. For each residue introduction, Fmoc-AA / HATU / DIEA (4.2 / 4 / 8 equivalents) was used in DMF at 75°C for 10 minutes, with an equal amount relative to resin 1. However, the second and fourth residues were reacted once at 75°C for 30 minutes. The 12th residue was reacted twice at 25°C for 20 minutes. The 13th residue was reacted twice at 75°C for 10 minutes. The 15th and 22nd residues were reacted once at 25°C for 30 minutes. Furthermore, the basic condition for Fmoc removal is a reaction with a 20% pipeidine DMF solution at 75°C for 3 minutes. However, the Fmoc removal of the 4th and 13th residues is carried out at 25°C for 5 minutes, followed by a reaction at room temperature for 10 minutes. The introduction of chloroacetyl groups is performed by removing the α-amino Fmoc group from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. Then, 10 equal volumes of chloroacetic acid, 10 equal volumes of DIPCI, and 10 equal volumes of HOSu are stirred in DCM, and an equal amount of DMF is added to prepare a ClAcOSu DCM / DMF solution (0.2M). This solution is then added to the solid-phase resin and shaken at room temperature for 60 minutes. For the deprotection of the side chains and shearing from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step is first washed 5 times with DMF and 3 times with chloromethane, and then dried under reduced pressure. Next, in a reaction vessel already filled with solid resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 90:2.5:2.5:5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass flotation. The solid resin remaining in the reaction vessel was shaken again with a shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. If this filtrate was added to excess diisopropyl ether / hexane (1 / 1) mixed solvent cooled to 0°C, a white turbid precipitate was formed. This mixture was centrifuged (9500 rpm, 1 min), the solution was decanted, and dried under reduced pressure. The resulting solid was used for subsequent cyclization reactions. The peptide cyclization reaction was performed by dissolving the peptide in DMSO with a final peptide concentration of 5 mM based on the molar amount of solid resin, adding 10 equal volumes of triethylamine, and shaking at room temperature for approximately 15 hours. The reaction solution was quenched with acetic acid and concentrated under reduced pressure using a GenevaC HT-12.The resulting mixture was dissolved in DMSO (20 mL), and three equal volumes of silver acetate were added. The mixture was shaken for 3 hours. After adding 11 equal volumes of 2 M DTT aqueous solution, the mixture was centrifuged, and the supernatant was recovered and concentrated under reduced pressure to obtain the crude product.

[0841] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 50x250mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 60℃; gradient (%B): 6.7-6.7% for 5 minutes, 6.7-10.3% for 2 minutes, 10.3-35.8% for 3 minutes, 35.8-40.8% for 15.5 minutes, 40.8-60% for 1.5 minutes; flow rate: 18 mL / min for 5 minutes, 18 mL / min for 2 minutes, 118 mL / min, then 118 mL / min).

[0842] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 94.8%.

[0843] Analytical conditions B: Hold time = 5.45 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60℃; Gradient (% Bconc): 20-60% over 7.2 minutes, 60-95% over 0.3 minutes, and 95-95% over 1.6 minutes; Flow rate: 0.5 mL / min.

[0844] ESI-MS(+) observation m / z = 1289.65(M+2H) 2+ .

[0845] As shown below, synthetic peptides or linker-added peptides are produced. Furthermore, the synthesized peptides and linker-added peptides are listed in Table 5, and the linkers are listed in Table 6.

[0846] [Table 5]

[0847]

[0848] [Table 6-1]

[0849]

[0850] [Table 6-2]

[0851]

[0852] [Table 6-3]

[0853]

[0854] [Synthesis Example 1-1]

[0855] Synthesis of 894_3142 (serial number 547) [Chemistry 27]

[0856]

[0857] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.48 mmol / g, 0.52 g), starting with the removal of Fmoc. Liberty Blue from CEM was used as the automated synthesizer, and the synthesis was performed following the manufacturer's instructions. The synthesis was carried out in the same manner as in Example 1.

[0858] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40℃; gradient (%B): 7-32% for 3 minutes, 32-37% for 8 minutes, and 37-60% for 1 minute; flow rate: 120 mL / min).

[0859] The purity of the target compound was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions, and was 94.20%.

[0860] Analytical conditions B: Holding time = 4.12 minutes; Column: Kinetex EVO C18 2.6μm, 2.1x150mm; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60℃; Gradient (%B conc): 20–60% over 7.15 min, 60–95% over 0.3 min, 95–95% over 1.55 min; Flow rate: 0.5 mL / min.

[0861] ESI-MS(+) observation m / z = 1147.70(M+2H) 2+ .

[0862] [Examples 1-2]

[0863] Synthesis of 894_3143 (serial number 548)

[0864] [Chemistry 28]

[0865]

[0866] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 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 performed following the manufacturer's instructions. The synthesis was carried out in the same manner as in Example 1.

[0867] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40℃; gradient (%B): 6-6% for 2 minutes, then 6-31% for 1 minute, then 31-36% for 8 minutes, then 36-60% for 1 minute; flow rate: 20-20 mL / min for 1 minute, then 20-120 mL / min for 1 minute, then 120 mL / min).

[0868] The purity of the target compound was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions, and was 97.61%.

[0869] Analytical conditions: Hold time = 3.75 min; Column: Kinetex EVO C18 2.6 μm, 2.1 x 150 mm; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; temperature 60℃; gradient (% Bconc) takes 7.15 min for 20–60%, then 0.3 min for 60–95%, then 1.55 min for 95–95%; flow rate: 0.5 mL / min.

[0870] ESI-MS(+) observation m / z = 1133.66(M+2H) 2+ .

[0871] [Examples 1-3]

[0872] Synthesis of 894_3144 (serial number 549)

[0873] [Chemistry 29]

[0874]

[0875] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.48 mmol / g, 0.52 g), starting with the removal of Fmoc. Liberty Blue from CEM was used as the automated synthesizer, and the synthesis was performed following the manufacturer's instructions. The synthesis was carried out in the same manner as in Example 1.

[0876] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40℃; gradient (%B): 8-8% for 2 minutes, then 8-33% for 1 minute, then 33-38% for 8 minutes, then 38-60% for 1 minute; flow rate: 20-20 mL / min for 1 minute, then 20-120 mL / min for 1 minute, then 120 mL / min).

[0877] The purity of the target compound was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions, and was 97.52%.

[0878] Analytical conditions B: Hold time = 3.97 minutes; Column: Kinetex EVO C18 2.6μm, 2.1x150mm; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; temperature 60℃; gradient (%B conc) takes 7.15 min for 20–60%, then 0.3 min for 60–95%, then 1.55 min for 95–95%; flow rate: 0.5 mL / min.

[0879] ESI-MS(+) observation m / z = 1108.62(M+2H) 2+ .

[0880] [Examples 1-4]

[0881] Synthesis of 894_3145 (serial number 550)

[0882] [Chemistry 30]

[0883]

[0884] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.48 mmol / g, 0.52 g), starting with the removal of Fmoc. Liberty Blue from CEM was used as the automated synthesizer, and the synthesis was performed following the manufacturer's instructions. The synthesis was carried out in the same manner as in Example 1.

[0885] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40℃; gradient (%B): 13-38% for 3 minutes, 38-43% for 8 minutes, and 43-60% for 1 minute; flow rate: 120 mL / min).

[0886] The purity of the target compound was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions, and was 98.02%.

[0887] Analytical conditions: Hold time = 4.91 min; Column: Kinetex EVO C18 2.6 μm, 2.1 x 150 mm; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; temperature 60℃; gradient (%B conc) takes 7.15 min for 20–60%, then 0.3 min for 60–95%, then 1.55 min for 95–95%; flow rate: 0.5 mL / min.

[0888] ESI-MS (+) observation m / z = 1177.95 (M+2H) 2+ .

[0889] [Examples 1-5]

[0890] Synthesis of 894_3147 (serial number 551)

[0891] [Chemistry 31]

[0892]

[0893] The desired peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.48 mmol / g, 0.52 g), with Fmoc removal as the starting point. Liberty Blue from CEM was used as the automated synthesizer, and the synthesis was performed following the manufacturer's instructions. The synthesis was carried out in the same manner as in Example 1.

[0894] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40℃; gradient (%B): 21-46% for 3 minutes, 46-51% for 8 minutes, and 51-60% for 1 minute; flow rate: 120 mL / min).

[0895] The purity of the target compound was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions, and was 96.34%.

[0896] Analytical conditions: Hold time = 6.24 min; Column: Kinetex EVO C18 2.6 μm, 2.1 x 150 mm; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; temperature 60℃; gradient (%B conc) takes 7.15 min for 20–60%, then 0.3 min for 60–95%, then 1.55 min for 95–95%; flow rate: 0.5 mL / min.

[0897] ESI-MS(+) observation m / z = 1198.29(M+2H) 2+ .

[0898] [Examples 1-6]

[0899] Synthesis of 894_3148 (serial number 552)

[0900] [Chemistry 32]

[0901]

[0902] The desired peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.48 mmol / g, 0.52 g), with Fmoc removal as the starting point. Liberty Blue from CEM was used as the automated synthesizer, and the synthesis was performed following the manufacturer's instructions. The synthesis was carried out in the same manner as in Example 1.

[0903] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40℃; gradient (%B): 18-43% for 3 minutes, 43-48% for 8 minutes, and 48-60% for 1 minute; flow rate: 120 mL / min).

[0904] The purity of the target compound was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions, and was 96.03%.

[0905] Analytical conditions: Hold time = 6.15 min; Column: Kinetex EVO C18 2.6 μm, 2.1 x 150 mm; Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; temperature 60℃; gradient (%B conc) takes 7.15 min for 20–60%, then 0.3 min for 60–95%, then 1.55 min for 95–95%; flow rate: 0.5 mL / min.

[0906] ESI-MS(+) observation m / z = 1142.36(M+2H) 2+ .

[0907] [Synthesis Example 1-7]

[0908] Synthesis of 894_variant_61_G_Azi (serial number 543)

[0909] [Chemistry 33]

[0910]

[0911] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.47 mmol / g, 0.21 g) following 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 manual. For each residue introduction, an equal amount of Fmoc-AA / HATU / DIEA (5.3 / 5 / 10 equal amounts) was used in DMF at 75°C for 10 minutes. However, the second and fourth residues were reacted once at 75°C for 30 minutes. The tenth, eleventh, and thirteenth residues were reacted twice at 75°C for 10 minutes. The twelfth residue was reacted twice at 25°C for 20 minutes. The fifteenth residue was reacted once at 25°C for 20 minutes. Furthermore, the basic condition for Fmoc removal is to react with a 20% pipeidine DMF solution at 75°C for 3 minutes. However, the Fmoc removal of residues 2, 4, and 13 is carried out at 25°C for 5 minutes followed by a reaction at 25°C for 10 minutes. The introduction of chloroacetyl groups is performed by removing the α-amino Fmoc group from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. This is achieved by stirring 10 equal parts of chloroacetic acid, 10 equal parts of DIPCI, and 10 equal parts of HOSu in DCM, adding an equal amount of DMF to prepare a ClAcOSu DCM / DMF solution (0.2 M), adding it to the solid-phase resin, and shaking at room temperature for 60 minutes. For the deprotection of the side chains and shearing from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step is first washed 5 times with DMF and 3 times with chloromethane, and then dried under reduced pressure. Next, in a reaction vessel already filled with solid resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass flotation. The solid resin remaining in the reaction vessel was shaken again with a shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. If this filtrate was added to excess diethyl ether / hexane (1 / 1) mixed solvent cooled to 0°C, a white turbid precipitate was formed. This mixture was centrifuged (9000 rpm, 2 min), the solution was decanted, and dried under reduced pressure. The resulting solid was used for subsequent cyclization reactions. The peptide cyclization reaction was performed by dissolving the peptide in DMSO with a final peptide concentration of 5 mM based on the molar amount of solid resin, adding 5 equal volumes of triethylamine, and shaking at room temperature for approximately 14 hours. The resulting reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.

[0912] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40℃; gradient (%B): 17-42% for 3 minutes, 42-47% for 8 minutes, and 47-60% for 1 minute; flow rate: 120 mL / min).

[0913] The purity of the target analyte was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 92.8%.

[0914] Analytical conditions B: Hold time = 6.22 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60℃; Gradient (% Bconc): 20-60% over 7.2 minutes, 60-95% over 0.3 minutes, and 95-95% over 1.6 minutes; Flow rate: 0.5 mL / min.

[0915] ESI-MS(+) observation m / z = 1142.31(M+2H) 2+ .

[0916] [Synthesis Example 1-8]

[0917] hTfR_894_3m_G_PEG4_gAbu(NHS) (The linker recorded in serial number 635 is the same as that in serial number 296) Synthesis of peptide complexes

[0918] [Chemistry 34]

[0919]

[0920] Using Fmoc-PEG4c-Abu-Alko resin (Watanabe Chemicals, 1.00 mmol / g, 0.1 g) prepared according to general solid-phase synthesis, C-terminal carboxylic acid peptides were synthesized starting with the removal of Fmoc, following the general method described above. Liberty Blue from CEM was used as the solid-phase synthesizer, and the synthesis was performed according to the manufacturer's manual. The introduction of chloroacetyl groups was carried out by removing the α-amino Fmoc group from the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above, followed by adding 0.2 M chloroacetic acid in DMF (5 equal volumes), 0.5 M HATU in DMF (5 equal volumes), and 1 M DIEA in DMF (10 equal volumes) to the solid-phase resin, followed by shaking at room temperature for 30 minutes. Deprotection of the side chains and shearing from the solid-phase resin were performed by first washing the resin obtained after the chloroacetyl group introduction step with DMF five times and chloromethane three times, followed by drying under reduced pressure. Next, a reaction mixture (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 already filled with solid resin, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass flotation. The solid resin remaining in the reaction vessel was shaken again with a shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. If this filtrate was added to excess diethyl ether / hexane (1 / 1) cooled to 0°C, a white turbid precipitate was formed. This mixture was centrifuged (10,000 rpm, 1 min), and the solution was decanted. The resulting particles were washed again with a small amount of diethyl ether cooled to 0°C, and the resulting solid was dried for use in the subsequent cyclization reaction. The peptide cyclization reaction was carried out by dissolving the peptide in DMSO with a final peptide concentration of 5 mM based on the molar amount of solid resin, adding 10 equal volumes of triethylamine, and stirring at room temperature for approximately 17 hours. The reaction solution was concentrated under reduced pressure using Savant ExplorerSpeedVaC. The mixture was dissolved in DMSO / H2O (9 / 1, 4 mL) with HOSu (230 mg, 20 g equator) and EDC HCl (383.4 mg, 20 g equator) added and stirred, so that the final concentration of the C-terminal carboxylic acid peptide obtained was 5 mM based on the molar number of the solid phase resin.

[0921] The crude product was purified under the following conditions (column: WaterS Xbridge (registered trademark) C18 5μm (registered trademark) 30x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 40℃; gradient (%B): 3 minutes 9-34%, then 8 minutes 34-39%, then 1 minute 39-60%; flow rate: 45 mL / min).

[0922] The purity of the target analyte was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 87.4%.

[0923] Analytical conditions B: Hold time = 4.25 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 40℃; Gradient (% Bconc): 20-60% over 7.2 minutes, 60-95% over 0.3 minutes, and 95-95% over 1.6 minutes; Flow rate: 0.5 mL / min.

[0924] ESI-MS(+) observation m / z = 1292.86(M+2H) 2+ .

[0925] [Synthesis Example 1-9]

[0926] Synthesis of 894_C12NH2 (a complex of fatty acid linker and sequence number 1)

[0927] [Chemistry 35]

[0928]

[0929] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.53 mmol / g, 0.21 g) following the general method described above, starting with the removal of the Fmoc group. Liberty Blue from CEM was used as the solid-phase synthesizer, and the synthesis was performed according to the manufacturer's manual. For each residue introduction, Fmoc-AA / HATU / DIEA (5.3 / 5 / 10 equivalents) was used in DMF at 75°C for 10 minutes, with an equal amount relative to resin 1. However, residues 1, 9, 10, 13, and 14 were reacted twice at 75°C for 10 minutes. Residues 11 and 12 were reacted twice at 30°C for 20 minutes. Residue 15 was reacted once at 30°C for 20 minutes. Furthermore, the basic condition for Fmoc removal is to react with a 20% pipeidine DMF solution at 75°C for 3 minutes. However, the Fmoc removal of residues 11, 12, 14, and 15 is carried out at 25°C for 5 minutes followed by a 10-minute reaction at 25°C. The introduction of chloroacetyl groups is performed by removing the α-amino Fmoc group from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. Chloroacetic acid (5 equal parts), DIPCI (5 equal parts), and HOSu (5 equal parts) are stirred in DCM, and an equal amount of DMF is added to prepare a ClAcOSu DCM / DMF solution (0.2 M). This solution is then added to the solid-phase resin and shaken at room temperature for 60 minutes. For deprotection of the side chains and shearing from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step is first washed 5 times with DMF and 3 times with chloromethane, and then dried under reduced pressure. Next, in a reaction vessel already filled with solid resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 60 minutes. The reaction solution was recovered by glass flotation. The solid resin remaining in the reaction vessel was shaken again with a shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. If this filtrate was added to excess diisopropyl ether cooled to 0°C, a white turbid precipitate was formed. This mixture was centrifuged (6000 rpm, 4 min), and the solution was decanted. The resulting solid was washed again with a small amount of diethyl ether cooled to 0°C and dried under reduced pressure. The resulting solid was used for subsequent cyclization reactions. The peptide cyclization reaction was performed by dissolving the peptide in DMSO with a final peptide concentration of 5 mM based on the molar amount of solid resin, adding 5 equal volumes of triethylamine, and shaking at room temperature for approximately 2 hours. The resulting reaction solution was concentrated under reduced pressure using SavantExplorer SpeedVaC.

[0930] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 30x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40℃; gradient (%B): 11-36% for 3 minutes, 36-41% for 3 minutes, 41-60% for 1 minute; flow rate: 45 mL / min).

[0931] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 96.3%.

[0932] Analytical conditions B: Hold time = 12.61 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60℃; Gradient (%B conc): 20-60% over 20 minutes, 60-95% over 1 minute, and 95% over 5 minutes; Flow rate: 0.25 mL / min.

[0933] ESI-MS(+) observation m / z = 1140.56(M+2H) 2+ .

[0934] [Synthesis Example 1-10]

[0935] Synthesis of 894_3m_G4 (a complex of the linker described in sequence number 643 and the peptide in sequence number 296)

[0936] [Chemistry 36]

[0937]

[0938] The target peptide was synthesized using Fmoc-Gly-wang resin (Watanabe Chemicals, 0.7 mmol / g, 1.43 g) and following the general method described above, starting with the removal of the Fmoc group. Liberty Blue from CEM Corporation was used as the solid-phase synthesizer, and the synthesis was performed according to the manufacturer's manual. The basic conditions for the condensation reaction were: using HATU as the condensing agent, and conducting one reaction at 75°C for 10 minutes. However, when introducing the 15th residue, the reaction was conducted once at 25°C for 20 minutes. The 13th residue was introduced twice at 75°C for 10 minutes. The 2nd residue was introduced twice at 75°C for 45 minutes. Furthermore, the basic conditions for Fmoc removal were: reacting with a 20% pipeidine DMF solution at 75°C for 3 minutes. However, the Fmoc removal of the 2nd and 13th residues was carried out by reacting at 25°C for 5 minutes followed by a 10-minute reaction. The introduction of chloroacetyl groups was carried out by removing the α-amino Fmoc groups from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above, followed by the addition of four equal volumes of chloroacetic acid in DMF solution (0.2 M), four equal volumes of DIPCI in DMF solution (0.5 M), and four equal volumes of HOSu in DMF solution (0.5 M), and shaking at room temperature for 60 minutes. Deprotection of the side chains and shearing from the solid-phase resin were performed by first washing the resin obtained after the chloroacetyl introduction step with DMF five times and chloromethane three times, followed by drying under reduced pressure. Next, in a reaction vessel already loaded with the solid-phase resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and shaking was performed at room temperature for 90 minutes. The reaction solution was recovered by glass frit filtration. The remaining solid resin in the reaction vessel was agitated again with the shearing mixture, the solution components were recovered from the glass frit, and mixed with the filtrate. Adding this filtrate to excess diisopropyl ether cooled to 0°C produced a turbid precipitate. This mixture was filtered, washed with ether, and dried under reduced pressure. The resulting solid was used in a subsequent cyclization reaction. The peptide cyclization reaction was performed by dissolving the peptide in DMSO / IPA / H₂O (90 / 5 / 5) with the molar amount of solid resin as the basis and the final peptide concentration as 5 mM, followed by the addition of 5 equal volumes of triethylamine, and stirring at room temperature for approximately 16 hours. The resulting reaction solution was concentrated under reduced pressure using a GenevaC HT-12.

[0939] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 30x250mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 40℃; gradient (%B): 0.1 min 50-7%, then 1.9 min 7-7%, then 3 min 7-32%, then 11 min 32-37%, then 1 min 37-60%; flow rate: 120 mL / min).

[0940] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 90.6%.

[0941] Analysis condition B: Hold time = 11.34 minutes; Gradient (%B conc): 20 minutes for 20-60%, then 1 minute for 60-95%, then 5 minutes for 95%.

[0942] ESI-MS(+) observation m / z = 1163.59(M+2H) 2+ .

[0943] [Synthesis Example 1-11]

[0944] 894_3m_G4S2_K(Mal) (a complex of the linker described in sequence number 561 and the peptide in sequence number 296) synthesis

[0945] [Chemistry 37]

[0946]

[0947] Using Sieber amide resin (Watanabe Chemicals, 0.52 mmol / g, 2.4 g x 3), the target peptide was synthesized following a standard method, starting with the removal of Fmoc. Liberty Blue from CEM was used as the solid-phase synthesizer, following the manufacturer's manual. The basic conditions for the condensation reaction were: using DIPCI and Oxyma Pure as the condensing agent, and reacting once at 75°C for 10 minutes. However, residue 15 was reacted at 50°C for 20 minutes. Residues 3, 4, 8, 10, 13, and 14 were reacted twice at 75°C for 10 minutes. Residues 11 and 12 were reacted twice at 50°C for 20 minutes. Residue 2 was reacted three times at 75°C for 60 minutes. Residue 1 was reacted twice at 75°C for 20 minutes. Furthermore, the basic condition for Fmoc removal is to react with a 20% pipeidine DMF solution at 75°C for 3 minutes. However, for residues 15, 16, and 17, Fmoc removal is performed by reacting at room temperature for 5 minutes, followed by reacting at 75°C for 3 minutes. For the introduction of chloroacetyl groups, firstly, the α-amino Fmoc group is removed from the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described. Then, a ClAcOSu DCM / DMF solution (0.15 M) is prepared by stirring chloroacetic acid (5 equal volumes), DIPCI (5 equal volumes), and HOSu (5 equal volumes) in DCM, adding an equal amount of DMF to the DCM solution, and adding this solution to the solid-phase resin, followed by shaking at room temperature for 180 minutes. For the deprotection of the side chains and shearing from the solid-phase resin, firstly, the resin obtained after the chloroacetyl group introduction step is washed 5 times with DMF and 3 times with chloromethane, and then dried under reduced pressure. Next, in a reaction vessel already filled with solid resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass flotation. The solid resin remaining in the reaction vessel was shaken again with a shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. If this filtrate was added to excess diisopropyl ether cooled to 0°C, a turbid precipitate was formed. This mixture was filtered off, washed with ether cooled to 0°C, and the resulting solid was used for subsequent cyclization reactions. The peptide cyclization reaction was performed by dissolving the peptide in DMSO with the molar amount of solid resin as the basis and the final peptide concentration being 4.9 mM, adding 7 equal volumes of triethylamine, and shaking at room temperature for about 1 hour. 1.05 equal volumes of SMCC were added to the resulting reaction solution, and the mixture was shaken at room temperature for 1.5 hours.The resulting reaction solution was concentrated under reduced pressure using GenevaC EZ-2Elite.

[0948] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 50x250mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 60℃; gradient (%B): 5.1 min 0-0%, then 1.9 min 0-5%, then 5 min 5-29%, then 13.5 min 29-34%, then 1.5 min 34-60%; flow rate: 1 mL / min until 0-5.1 min, then 1-119 mL / min from 5.1 min to 7.0 min, then 119 mL / min).

[0949] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 94.1%.

[0950] Analysis condition B: Hold time = 11.33 minutes; Gradient (%B conc): 20 minutes for 20-60%, then 1 minute for 60-95%, then 5 minutes for 95%.

[0951] ESI-MS(+) observation m / z = 1424.0(M+2H) 2+ .

[0952] [Synthesis Example 1-12]

[0953] Synthesis of 894_3m_G4S2C (a complex of the linker described in sequence number 644 and the peptide in sequence number 296)

[0954] [Chemistry 38]

[0955]

[0956] The separately synthesized 894_3m_G (500 mg, 0.21 mmol) was dissolved in DMF (5 mL). Using 1.2 equal volumes of H-Gly-Gly-Gly-Ser(OtBu)-Ser(OtBu)-CyS(Trt)-NH2 (207 mg, 0.25 mmol), 1.2 equal volumes of EDC (39.1 mg, 0.25 mmol), and 1.2 equal volumes of DIEA (35.8 mg, 0.25 mmol), synthesized by a well-known method, the mixture was stirred at room temperature for 2.5 hours. Then, 0.24 equal volumes of H-Gly-Gly-Gly-Ser(OtBu)-Ser(OtBu)-CyS(Trt)-NH2 (41.4 mg, 0.05 mmol), 0.24 equal volumes of 7.8 mg, 0.05 mmol), and 0.24 equal volumes of DIEA (7.2 mg, 0.05 mmol) were added, and the mixture was stirred at room temperature for 2 hours. After concentration with Biotage V-10, the resulting mixture was reacted in a TFA / TIS / H2O / DODT mixture (92.5 / 2.5 / 2.5 / 2.5) at room temperature for 75 minutes. Adding excess diisopropyl ether, cooled to 0°C, to the reaction mixture resulted in a white precipitate. The mixture was centrifuged (9000 rpm, 2 min), and the solution was decanted. The resulting solid was washed with ether cooled to 0°C, centrifuged (9000 rpm, 2 min), and the solution was decanted.

[0957] The resulting mixture was purified under the following conditions (column: WaterS Xbridge (registered trademark) C185μm (registered trademark) 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 40℃; gradient (%B): 5-30% for 3 minutes, 30-35% for 8 minutes, and 35-60% for 1 minute; flow rate 120mL / min).

[0958] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 96.1%.

[0959] Analytical conditions B: Hold time = 3.52 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60℃; Gradient (% Bconc): 20-60% over 7.2 minutes, 60-95% over 0.3 minutes, and 95-95% over 1.6 minutes; Flow rate: 0.5 mL / min.

[0960] ESI-MS(+) observation m / z = 1301.89(M+2H) 2+ .

[0961] [Synthesis Example 1-13]

[0962] 894_3m_GGRGRS_K(Mal) (a complex of the linker recorded in sequence number 573 and the peptide in sequence number 296) Synthesis

[0963] [Chemistry 39]

[0964]

[0965] Using Sieber amide resin (Watanabe Chemicals, 0.47 mmol / g, 532 mg), the target peptide was synthesized following a standard method, starting with the removal of Fmoc. A CEM Liberty Blue HT solid-phase synthesizer was used, following the manufacturer's manual. The basic conditions for the condensation reaction were the use of HATU as the condensing agent and two cycles at 75°C for 10 minutes each. However, the second residue was reacted twice at 75°C for 30 minutes each. The fifth, sixth, seventh, sixteenth, seventeenth, nineteenth, twenty-first, and twenty-second residues were reacted once at 75°C for 10 minutes each. The eleventh residue was reacted twice at 50°C for 15 minutes each. The twelfth, eighteenth, and twentieth residues were reacted once at 50°C for 15 minutes each. The 15th residue was reacted once at 50°C for 15 minutes. Furthermore, the basic condition for Fmoc removal was reacting with a 20% pipeidine DMF solution at 75°C for 3 minutes. However, for the 2nd and 13th residues, Fmoc removal was performed twice at room temperature for 5 minutes each. The introduction of chloroacetyl groups was carried out by stirring chloroacetic acid (5 equal parts), DIPCI (5 equal parts), and HOSu (5 equal parts) in DCM, adding an equal amount of DMF to the DCM to prepare a ClAcOSu DCM / DMF solution (0.15 M), which was then added to the solid resin obtained in the previous step and shaken at room temperature for 60 minutes. Deprotection of the side chains and shearing from the solid resin were performed by first washing the resin obtained after the chloroacetyl group introduction step with DMF five times and chloromethane three times, followed by drying under reduced pressure. Next, in a reaction vessel already filled with solid resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 150 minutes. The reaction solution was recovered by glass flotation. The solid resin remaining in the reaction vessel was shaken again with a shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. If this filtrate was added to excess diethyl ether and hexane mixed solvent cooled to 0°C, a white turbid precipitate was formed. This mixture was centrifuged (9500 rpm, 1 min), the supernatant was decanted, and the mixture was washed with diethyl ether cooled to 0°C. The resulting solid was used for subsequent cyclization reactions. The peptide cyclization reaction was carried out by dissolving the peptide in DMSO (5% aqueous solution) with the molar amount of solid resin as the basis and the final peptide concentration as 5 mM, followed by the addition of 7 equal volumes of triethylamine, and shaking at room temperature for approximately 3 hours. Add 1.1 equal volume of SMCC to the resulting reaction solution and shake at room temperature for 3 hours. Concentrate the resulting reaction solution under reduced pressure using GenevaC EZ-2Elite.

[0966] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 50x250mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40℃; gradient (%B): 5-29% for 3 minutes, 29-34% for 8 minutes, and 34-60% for 1 minute; flow rate: 120 mL / min).

[0967] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 95.4%.

[0968] Analysis condition B: Hold time = 9.53 minutes; Gradient (%B conc): 20 minutes for 20-60%, then 1 minute for 60-95%, then 5 minutes for 95%.

[0969] ESI-MS(+) observation m / z = 1005.7(M+3H) 3+ .

[0970] [Synthesis Example 1-14]

[0971] 894_3m_PEG12dk(Biotin) (a complex of the linker recorded in sequence number 613 and the peptide in sequence number 296) Synthesis of (body)

[0972] [Chemistry 40]

[0973]

[0974] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.65 mmol / g, 0.54 g), starting with the removal of Fmoc. A CEM Liberty Blue HT was used as the automated synthesizer, and the synthesis was performed following the manufacturer's manual. For the introduction of each residue, the basic conditions were: using an equal volume of resin, Fmoc-AA / DIC / Oxymapure = 5.3 / 10 / 5 equal volumes, reacting at 90°C for 3 minutes. However, the second residue was reacted twice at 75°C for 30 minutes. The 11th and 12th residues were reacted twice at 50°C for 15 minutes. The 13th residue was reacted twice at 90°C for 3 minutes. The 15th residue was reacted at 50°C for 15 minutes. The basic conditions for Fmoc removal were: reacting with a 20% pipeidine DMF solution at 75°C for 3 minutes. However, the removal of the Fmoc groups at residues 2 and 13 was carried out by two reactions at 25°C for 5 minutes each. The introduction of chloroacetyl groups was performed by removing the α-amino Fmoc groups from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above, followed by the addition of 5 equal parts ClAcOHin DMF, 5 equal parts HATUin DMF, and 10 equal parts DIEA in DMF, followed by shaking at room temperature for 30 minutes. Deprotection of the side chains and shearing from the solid-phase resin were first performed by washing the resin obtained after the chloroacetyl introduction step with DMF 5 times and chloromethane 3 times, followed by drying under reduced pressure. Next, in a reaction vessel already loaded with solid-phase resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by glass frit filtration. The remaining solid resin in the reaction vessel was shaken again with the shearing mixture, the solution components were recovered from the glass frit, and mixed with the filtrate. Adding this filtrate to excess diisopropyl ether / hexane (1 / 1) cooled to 0°C produced a turbid precipitate. This mixture was centrifuged (10000 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 cyclization reaction was performed by dissolving the peptide in 5% aqueous DMSO with the molar amount of solid resin as the basis and the final peptide concentration as 5 mM, followed by the addition of 10 equal volumes of triethylamine, and shaking at room temperature for approximately 16 hours. The resulting reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.

[0975] The crude intermediate peptides obtained were purified using the following conditions (column: WaterS Xbridge (registered trademark) C185μm OBD (registered trademark) 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 40℃; gradient (%B): 3 minutes 5-30%, then 8 minutes 30-35%, then 1 minute 35-60%; flow rate: 120mL / min).

[0976] The obtained intermediate peptide was reacted with DMSO solution (22 mM, 160 μL) by adding 1.3 equal volumes of Biotin-NHS and 5 equal volumes of DIEA, and stirred at room temperature. After 2.5 hours, the reaction solution was quenched with acetic acid.

[0977] The crude product was purified under the following conditions (column: WaterS Xbridge (registered trademark) C18 5μm OBD (registered trademark) 19x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 60℃; gradient (%B): 10-35% for 3 minutes, 35-40% for 8 minutes, and 40-60% for 1 minute; flow rate: 17 mL / min).

[0978] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 98.7%.

[0979] Analytical conditions B: Hold time = 4.30 min; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60℃; Gradient (% Bconc): 20-60% over 7.2 minutes, 60-95% over 0.3 minutes, and 95% over 1.6 minutes; Flow rate: 0.5 mL / min.

[0980] ESI-MS(+) observation m / z = 1017.76(M+3H) 3+ .

[0981] [Synthesis Example 1-15]

[0982] 894_11K_3Me_PEG4C_KTrzMal (the linker recorded in sequence number 624 and the peptide in sequence number 293) Synthesis of complex

[0983] [Chemistry 41]

[0984]

[0985] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.52 mmol / g, 0.19 g), starting with the removal of Fmoc. The Biotage SyroI was used as the automated synthesizer, and the synthesis was performed following the manufacturer's instructions. For the introduction of each residue, the basic conditions were: using an equal volume of resin 1, Fmoc-AA / DIC / Oxyma pure = 3.2 / 3 / 6.3 equal volumes, and reacting twice at 75°C for 20 minutes. However, residues 2, 13, and 17 were reacted three times at 75°C for 10 minutes. Residue 15 was reacted twice at 25°C for 15 minutes. Residue 16 was reacted at 25°C for 60 minutes. De-Fmoc removal was performed by reacting with a 20% piperidine DMF solution at 25°C for 5 minutes followed by a 15-minute reaction. The introduction of chloroacetyl groups was performed by removing the α-amino Fmoc group from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. Then, 5 equal volumes of chloroacetic acid, 5 equal volumes of DIPCI, and 5 equal volumes of HOSu were stirred in DCM, and an equal volume of NMP was added to prepare a ClAcOSu DCM / NMP solution (0.2 M). This solution was added to the solid-phase resin and the mixture was shaken at room temperature for 90 minutes. For deprotection of the side chains and shearing from the solid-phase resin, the resin obtained after the chloroacetyl introduction step was first washed 5 times with DMF and 3 times with chloromethane, and then dried under reduced pressure. Next, in a reaction vessel already filled with solid-phase resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 60 minutes. The reaction solution was recovered by glass frit filtration. The remaining solid resin in the reaction vessel was shaken again with the shearing mixture, the solution components were recovered from the glass frit, and mixed with the filtrate. Adding this filtrate to excess diisopropyl ether / hexane (1 / 1) cooled to 0°C produced a turbid 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 and hexane cooled to 0°C, and then used for the subsequent cyclization reaction. The peptide cyclization reaction was performed by dissolving the peptide in 9% aqueous DMSO with the molar amount of solid resin as the basis and the final peptide concentration as 5 mM, followed by the addition of 10 equal volumes of triethylamine, and shaking at room temperature for approximately 1.5 hours. The resulting reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.

[0986] The crude intermediate peptides obtained were purified using the following conditions (column: WaterS Xbridge (registered trademark) C185μm OBD (registered trademark) 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature 40℃; gradient (%B): 3 min 9-34%, then 8 min 34-39%, then 1 min 39-60%; flow rate: 120 mL / min).

[0987] After freeze-drying, for the obtained intermediate peptide DMF solution (15 mM), add 2 equal volumes of CuSO4·5H2O aqueous solution (100 mM) and 10 equal volumes of ascorbic acid aqueous solution (500 mM), then add 2 equal volumes of N-propargyl maleic anhydride DMF solution (100 mM) and stir at room temperature.

[0988] The crude product was purified under the following conditions (column: COSMOSIL PBr 10x150 mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (% B): 17-42% for 3 minutes, 42-47% for 8 minutes, and 47-60% for 1 minute; flow rate: 5 mL / min).

[0989] The purity of the target analyte was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 82.7%.

[0990] Analytical conditions B: Hold time = 11.37 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 40℃; Gradient (% Bconc): 20-60% in 7.2 minutes, 60-95% in 0.3 minutes, 95% in 1.6 minutes; Flow rate: 0.25 mL / min.

[0991] ESI-MS(+) observation m / z = 1303.33(M+3H) 3+ .

[0992] [Synthesis Example 1-16]

[0993] 894_variant_03_GKN3 (a complex of the linker recorded in serial number 554 and serial number 397) become

[0994] [Chemistry 42]

[0995]

[0996] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.47 mmol / g, 0.21 g) following 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 manual. For each residue introduction, 1 eq of resin was used with Fmoc-AA / HATU / DIEA (4.2 eq / 4 eq / 8 eq) in DMF, and the reaction was carried out once at 75°C for 10 minutes. However, the second and fourth residues were reacted once at 75°C for 30 minutes. The eleventh and twelfth residues were reacted twice at 25°C for 20 minutes. The thirteenth residue was reacted twice at 75°C for 10 minutes. The fifteenth and twentieth residues were reacted once at 25°C for 30 minutes. Furthermore, the basic condition for Fmoc removal is to react with a 20% pipeidine DMF solution at 75°C for 3 minutes. However, the Fmoc removal of residues 2, 4, and 13 is carried out at 25°C for 5 minutes, followed by a reaction at room temperature for 10 minutes. The introduction of chloroacetyl groups is performed by removing the α-amino Fmoc group from a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above. This is done by stirring 5 eq of chloroacetic acid, 5 eq of DIPCI, and 10 eq of HOSu in DCM, adding an equal amount of DMF to the DCM, preparing a ClAcOSu DCM / DMF solution (0.2 M), adding it to the solid-phase resin, and shaking at room temperature for 30 minutes. For deprotection of the side chains and shearing from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step is first washed 5 times with DMF and 3 times with chloromethane, and then dried under reduced pressure. Next, in a reaction vessel already filled with solid resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 20 minutes. The reaction solution was recovered by glass flotation. The solid resin remaining in the reaction vessel was shaken again with a shearing mixture, the solution components were recovered by glass flotation, and mixed with the filtrate. If this filtrate was added to excess diethyl ether / hexane (1 / 1) mixed solvent cooled to 0°C, a white turbid precipitate was formed. This mixture was centrifuged (10,000 rpm, 1 min), the solution was decanted, and dried under reduced pressure. The resulting solid was used for subsequent cyclization reactions. The peptide cyclization reaction was performed by dissolving the peptide in 5% aqueous DMSO with the molar amount of solid resin as the basis and the final peptide concentration as 5 mM, followed by the addition of 6 equal volumes of triethylamine, and shaking at room temperature for approximately 16 hours. The resulting reaction solution was concentrated under reduced pressure using Savant Explorer SpeedVaC.

[0997] The crude product was purified under the following conditions (column: WaterS Xbridge C18 5μm 50x150mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40℃; gradient (%B): 11-36% for 3 minutes, 36-41% for 8 minutes, and 41-60% for 1 minute; flow rate: 120 mL / min).

[0998] The purity of the target analyte was calculated by the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and was 95.5%.

[0999] Analytical conditions B: Hold time = 4.84 minutes; Column: Kinetex EVO C18 2.6μm 2.1x150mm Mobile phase: A = 0.025% TFA in H2O, B = 0.025% TFA in MeCN; Temperature: 60℃; Gradient (% Bconc): 20-60% in 7.2 minutes, 60-95% in 0.3 minutes, and 95% in 1.6 minutes; Flow rate: 0.5 mL / min.

[1000] ESI-MS(+) observation m / z = 1178.06(M+2H) 2+ .

[1001] As follows, synthesize new amino acids.

[1002] [Synthesis example 2-1]

[1003] (S)-2- [[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-3- (7-Chloro-1-ethyl-1H-indol-3-yl)propionic acid Synthesis of (W1Et7Cl)

[1004] [Chemistry 43]

[1005]

[1006] A DMF solution (500 mL) of 7-chloro-3-iodo-1H-indole (18 g, 64.9 mmol) was prepared by adding NaH (60 wt%, 8.7 g, 361 mmol) in portions at 0 °C. Next, iodoethane (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 x 200 mL). The combined organic layers were washed three times with water (100 mL), dried over anhydrous sodium sulfate, and filtered. The organic layers were concentrated, and the residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:5).

[1007] Under nitrogen atmosphere, iodine (1.6 g, 12.6 mmol) was added to a DMF suspension (50 mL) of zinc (8 g, 122 mmol), followed by the addition of methyl(2R)-2-[[(9H-fluorene-9-ylmethoxy)carbonyl]amino]-3-iodopropionate (18.5 g, 41 mmol), and the mixture was stirred at room temperature for 30 minutes. To this reaction mixture, a portion (15 g, 49 mmol) of the resulting product in DMF (300 mL) was added, followed by the addition of SpHos (0.84 g, 2.0 mmol) and Pd2(dba)3 (1.1 g, 1.2 mmol), and the mixture was stirred at 50 °C for 4 hours. The reaction was stopped with water, the solid was filtered off, and the filtrate was extracted four times with ethyl acetate (400 mL). The combined organic layers were washed four times with water (200 mL). The organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The resulting residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 2:8).

[1008] A portion of the resulting product (18 g, 35.8 mmol) was mixed with 1,4-dioxane (200 mL), and hydrogen chloride (60 g, 1.7 mol) was blown in. The mixture was stirred at 100 °C for 4 hours. The reaction solution was concentrated, and the residue was purified by reverse-phase silica gel column chromatography (water:acetonitrile = 100:0-0:100) to obtain the title compound.

[1009] ESI-MS(+) observation m / z = 489.10(M+H) + .

[1010] [Synthesis example 2-2]

[1011] (S)-2- [[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-3- (2-((tert-butyloxycarbonyl)amino)pyridine- Synthesis of 4-yl)propionic acid (4Py6NH2)

[1012] [Chemistry 44]

[1013]

[1014] Under nitrogen atmosphere, iodine (4.5 g, 17.7 mmol) was added to a zinc (5.8 g, 88.6 mmol) suspension in 200 mL of DMF. Then, a DMF solution (20 g, 44.32 mmol, 1.0 mmol) was added dropwise at room temperature in 50 mL. The mixture was stirred at room temperature for 1 hour. Next, 4-bromopyridin-2-amino (7.7 g, 44.3 mmol, 1 mmol), Pd₂(dba)₃ (2.0 g, 2.2 mmol, 0.05 mmol), and SpHos (1.8 g, 4.4 mmol, 0.1 mmol) were added, and the mixture was stirred at 50 °C for approximately 16 hours. After the reaction was stopped by adding 500 mL of water, 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).

[1015] A portion (10 g, 24.0 mmol) of the product obtained above was added to 110 mL of tert-butanol, along with Boc₂O (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 solution 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.

[1016] A portion of the product (3 g, 5.8 mmol) was added to a solution of 2-propanol (30 mL), followed by the addition of CaCl2 (6.4 g, 58.0 mmol), and then an aqueous solution of LiOH-H2O (0.28 g, 11.6 mmol) (5 mL) was added 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 using an aqueous citric acid solution, and the extract was 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 (+) observation m / z = 504.15 (M+H) + .

[1017] [Synthesis example 2-3]

[1018] (S)-2- [[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-3- (6-((tert-butyloxycarbonyl)amino)pyridine- Synthesis of 3-yl)propionic acid (3Py6NH2)

[1019] [Chemistry 45]

[1020]

[1021] The title substance was obtained following synthetic example [1-x], using 5-bromopyridin-2-amine to replace 4-bromopyridin-2-amine, and by the same method. ESI-MS(+) observation m / z = 504.15 (M+H) + .

[1022] [Synthesis example 2-4]

[1023] N2- [[(9H-fluorene-9-ylmethoxy)carbonyl]-N6- (4-Methylpiperazine-1-carbonyl)-L-lysine Synthesis of (KCOpipzMe)

[1024] [Chemistry 46]

[1025]

[1026] In a solution of 1-methylpiperazine (1.4 mL, 12.7 mmol) dissolved in dichloromethane (20 mL), DIPEA (2.3 mL, 13.0 mmol) and triphosgene (1.23 g, 4.2 mmol) were added at 0 °C. After stirring at the same temperature for 1 hour, the mixture was concentrated. In the resulting residue, a dichloromethane solution (20 mL) of [(9H-fluorene-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. After stopping the reaction with a saturated sodium bicarbonate solution, the mixture was diluted with dichloromethane, washed once with water, twice with a 5% acetic acid solution, once with a saturated sodium bicarbonate solution, and finally once with saturated brine. The organic layer was dried with anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatography (dichloromethane:methanol = 100:0-80:20) to obtain the title compound. ESI-MS(+) observation m / z = 495.40 (M+H) + .

[1027] [Synthesis example 2-5]

[1028] Nα- [(9H-fluoren-9-ylmethoxy)carbonyl]- 1-(2-amino-2-oxoethyl)-L-tryptophan (W1mCON) synthesis

[1029] [Chemistry 47]

[1030]

[1031] Under nitrogen atmosphere, potassium tert-butoxide (3.7 g, 32.9 mmol) was added in portions to a mixed 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 hours, 2-chloroacetamide (1.5 g, 16.4 mmol) was added in portions at 0 °C, and the mixture was stirred at room temperature for 3 hours. After stopping the reaction by adding water (100 mL), the mixture was extracted three times with ethyl acetate (50 mL). The combined organic layers were 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).

[1032] A portion of the product (6 g, 16.6 mmol) was added to an ethyl acetate solution (30 mL) of hydrogen chloride in ethyl acetate (2 M, 30 mL), and the mixture was stirred at room temperature for approximately 16 hours. The resulting solid was filtered off and washed with ethyl acetate and diethyl ether. The solid was dissolved in 1,4-dioxane (50 mL) and water (10 mL), and 2,5-dioxypyrrolidone-1-yl-9H-fluorene-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. After adjusting the pH to 5-6 with citric acid, 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(+) observation m / z = 484.25 (M+H) + .

[1033] [Synthesis example 2-6]

[1034] Synthesis of Fmoc-Glu(d-Pro-O-allyl)-OH(Epryl2RCOO)

[1035] [Chemistry 48]

[1036]

[1037] In a DMF solution (10 mL) of 4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxovaleric acid (2.5 g, 5.8 mmol), HATU (2.7 g, 7.0 mmol), DIPEA (1.3 mL, 7.5 mmol), and allyl-D-proline (0.9 g, 5.80 mmol) were added, and the mixture was stirred at 0 °C for 1 hour. The reaction solution was diluted with water and extracted with a mixture 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.

[1038] A fraction (2.6 g, 4.5 mmol) of the product obtained from the reaction was stirred with dichloromethane / TFA (1:1, 30 mL) at room temperature for 3 hours. The reaction solution was concentrated, and the residue was dissolved in dichloromethane. A saturated aqueous sodium bicarbonate solution was added to adjust the pH to above 8. Hydrochloric acid was then added to adjust the pH to 3-4, and extraction was performed 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(+) observation m / z = 507.40 (M+H) + .

[1039] [Synthesis example 2-7]

[1040] Synthesis of Fmoc-Asp(d-Pro-O-allyl)-OH(Dpryl2RCOO)

[1041] [Chemistry 49]

[1042]

[1043] The title compound was synthesized following synthetic example [1-x], using 3-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutyric acid to replace 4-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxovalerate, and obtained by the same method. ESI-MS(+) observation m / z = 493.30 (M+H) + .

[1044] [Synthesis example 2-8]

[1045] Synthesis of Fmoc-Lys(Gly-O-allyl)-OH(KaAC)

[1046] [Transformation 50]

[1047]

[1048] A solution of tert-butyl(((9H-fluorene-9-yl)methoxy)carbonyl)-L-lysine methyl hydrochloride (1.2 g, 2.6 mmol), ((allyloxy)carbonyl)glycine (0.46 g, 2.9 mmol), and DIPEA (0.59 mL, 3.4 mmol) in DMF was added to a solution of HATU (1.22 g, 3.1 mmol) in DMF (20 mL), and the mixture was stirred at 0 °C for 1 hour. The organic layer was diluted with a mixture of ethyl acetate and hexane, and washed four times with water. The organic layer was dried over anhydrous sodium sulfate, concentrated, and the residue was purified by silica gel column chromatography (ethyl acetate:hexane = 0:100-80:20).

[1049] TFA (10 mL) was added to a portion (1.5 g, 2.6 mmol) of the synthesized product in a dichloromethane solution (10 mL), and the mixture was stirred at room temperature for approximately 16 hours. The reaction solution was concentrated by three azeotropic operations using toluene. The resulting residue was washed with diisopropyl ether and dried under reduced pressure at 55 °C to obtain the title compound. ESI-MS(+) observation m / z = 510.40 (M+H) + .

[1050] [Synthesis example 2-9]

[1051] N α -(((9H-fluorene-9-yl)methoxy)carbonyl)-1-(2-((tert-butyldimethylsilyl)oxy)ethyl)-L-color Synthesis of amino acids (Fmoc-W1EtOH-OH)

[1052] [Chemistry 51]

[1053]

[1054] Potassium tert-butoxide (14.8 g, 69.0 mmol) was added to a mixture of N-Boc-tryptophan (20 g, 64.9 mmol) in THF (100 mL) and DMF (100 mL) 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. At 0 °C, an aqueous citric acid solution was added to adjust the pH to 6, and the solution was 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).

[1055] Under nitrogen atmosphere, 2,6-dimethylpyridine (20 mL, 172.9 mmol) was added to a portion (16 g, 34.6 mmol) of the product in a solution of dichloromethane (150 mL), followed by TMSOTf (25 mL, 112 mmol) at 0 °C. The mixture was heated to room temperature and stirred for 24 hours. The reaction solution was concentrated. Dioxane (300 mL), water (150 mL), sodium bicarbonate (9.3 g, 110.35 mmol, 3.1 g equate), and 9-fluorenylmethyl-N-succinimide carbonate (14.0 g, 41.4 mmol) were added to the resulting mixture, and the mixture was stirred at room temperature for approximately 16 hours. The solid was filtered, and citric acid was added to the filtrate to adjust the pH to 7. The solution 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 with 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 (+) observation m / z = 585.15 (M+H) + .

[1056] Industrial availability

[1057] This invention can be applied to the pharmaceutical industry.

Claims

1. A transferrin receptor-binding peptide, characterized in that, A peptide with a cyclic structure is formed by the combination of N-terminal residues and C-terminal residues of a peptide moiety consisting of any amino acid sequence from sequence numbers 2-157 and 159-200; or The peptide moiety comprises an amino acid sequence consisting of an N-terminal residue of any amino acid sequence in sequences 2-157 and 159-200, wherein the N-terminal residue of the peptide moiety is combined with the C-terminal residue, namely Cys, to form a cyclic structure.

2. The peptide according to claim 1, characterized in that, It can cross the blood-brain barrier.

3. The peptide according to claim 1, characterized in that, It is targeted at muscle tissue.

4. The peptide according to claim 1, characterized in that, It has cell-penetrating properties.

5. A composite, characterized in that, It comprises: the peptide as described in claim 1, and a linker bound to the peptide.

6. A composite, characterized in that, It comprises: the peptide as described in claim 1, a linker bound to the peptide, and a substance bound to the linker.

7. The composite according to claim 6, characterized in that, The substance can cross the blood-brain barrier.

8. The composite according to claim 6, characterized in that, The linker has an amino acid length of 1 or more and 15 or less, and the linker contains one or more glycine or serine.

9. The composite according to claim 6, characterized in that, The N-terminus of the linker is a modifiable cysteine ​​(Cys) or a modifiable lysine (Lys).

10. The composite according to claim 6, characterized in that, The linker has an amino acid length of 1 to 5 and includes either or both of D-type glutamic acid and methylated glycine.

11. The composite according to claim 10, characterized in that, The N-terminus of the linker is a modifiable Cys or a modifiable lysine (Lys).

12. The composite according to claim 6, characterized in that, The connector is a PEG connector.

13. The composite according to claim 12, characterized in that, The PEG linker further comprises glycine (Gly), serine (Ser), glutamic acid (Glu), arginine (Arg), or lysine (Lys).

14. The composite according to claim 12 or 13, characterized in that, The N-terminus of the linker is a modifiable cysteine ​​(Cys) or a modifiable lysine (Lys).

15. The composite according to claim 6, characterized in that, The connector has a sequence represented by any one of serial numbers 201, 553-642.

16. The composite according to claim 6, characterized in that, The connector is: Polyethylene glycol (PEG); G connector, GS connector; or Linkers having an amino acid sequence represented by any of the sequence numbers 201, 553-644.

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