Human transferrin receptor binding peptides

Abstract

[Problem] To provide a novel peptide that binds to human transferrin receptor (hTfR), and various uses of the novel peptide. [Solution] A peptide comprising an amino acid sequence represented by Ala-Val-MeF3C-Val-W7N-Asn-3Py6NH2-F4OMe-Ile-Ile-Arg-Arg-4Py-MeTyr-Cys (SEQ ID NO: 1), or a pharmaceutically acceptable salt thereof, a complex comprising the peptide or the pharmaceutically acceptable salt thereof and a substance that has bound to a linker, and a composition comprising the peptide or the complex, and a method for producing a pharmaceutical or diagnostic composition.

Description

Technical Field

[0001] This invention relates to peptides that can bind to human transferrin receptor (hTfR). Background Art

[0002] With the exception of a few areas encompassing the periventricular organs (such as the pineal gland, pituitary gland, and the area postrema), the capillaries that supply blood to most tissues of the brain differ from those found in other tissues, such as muscle. The endothelial cells that form their endothelium are attached to each other through strong intercellular bonds. Consequently, the passive transport of substances from the blood to the brain is hindered. While there are exceptions, substances with high lipid solubility or low molecular weight (less than 200 to 500 Daltons) and electrical neutrality near physiological pH are difficult to transport from the capillaries to the brain. This mechanism, which restricts the exchange of substances between the blood and the brain's interstitial fluid via the capillary endothelium within the brain, is known as the blood-brain barrier (BBB). Furthermore, the BBB restricts the exchange of substances between the interstitial fluid and blood not only in the brain but also in the central nervous system, including the brain and spinal cord. The BBB protects most cells of the central nervous system from fluctuations in the concentrations of substances such as hormones and lymphokines in the blood, maintaining biochemical homeostasis.

[0003] As a method for allowing high-molecular-weight substances to reach the brain through the blood-brain barrier, various methods have been reported for modifying these high-molecular-weight substances so that they have an affinity for the transferrin receptor, a membrane protein present on endothelial cells of brain capillaries (Patent Documents 1 to 3). For example, as described in Patent Document 1, which describes a blood-brain barrier shuttle that has an affinity for and can bind to the transferrin receptor, substances that can bind to the transferrin receptor are known to have the potential to cross the blood-brain barrier.

[0004] Prior art literature

[0005] Patent Literature

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

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

[0008] Patent Document 3: WO2016 / 208695

[0009] Patent Document 4: WO2019 / 151539

[0010] Non-patent literature

[0011] Non-patent literature 1: PLOS ONE 2014, 9, e96340. Summary of the Invention

[0012] Problems to be solved by the invention

[0013] One object of the invention described in this specification is to provide a novel peptide that binds to human transferrin receptor (hTfR). Another object of the invention is to provide various uses of the novel peptide.

[0014] Technical means to solve problems

[0015] According to the present invention, one invention described in this specification relates to a peptide or a pharmaceutically acceptable salt thereof that binds to the transferrin receptor (hereinafter, these are collectively referred to as simply peptides).

[0016] A peptide having the following amino acid sequence:

[0017] The amino acid sequence of Ala-Val-MeF3C-Val-W7N-Asn-3Py6NH2-F4OMe-Ile-Ile-Arg-Arg-4Py-MeTyr-Cys (SEQ ID NO: 1), or

[0018] An amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1 having one or more substitutions selected from the following group:

[0019] (I) the second valine residue in SEQ ID NO: 1 is replaced by a polar amino acid;

[0020] (II) the MeF3C residue at position 3 of SEQ ID NO: 1 is substituted with an N-methyl amino acid having an aromatic ring or a heterocyclic ring in its side chain;

[0021] (III) the W7N residue in the fifth position of SEQ ID NO: 1 is replaced by tryptophan or N-methyltryptophan, which may have a substituent and may have the carbon atom on the indole ring replaced by an N atom;

[0022] (IV) substitution of the aspartic acid residue at position 6 of SEQ ID NO: 1 into Nmm;

[0023] (V) the 3Py6NH2 residue at position 7 of SEQ ID NO: 1 is substituted with an amino acid having an aromatic ring or a heterocyclic ring in its side chain that may have a substituent;

[0024] (VI) the F4OMe residue at position 8 of SEQ ID NO: 1 is substituted with an amino acid having an aromatic ring or a heterocyclic ring which may have a substituent in its side chain;

[0025] (VII) substitution of the 9th isoleucine residue in SEQ ID NO: 1 with Eva;

[0026] (VIII) the isoleucine residue at position 10 of SEQ ID NO: 1 is replaced by any amino acid;

[0027] (IX) the 11th arginine residue in SEQ ID NO: 1 is substituted with lysine which may have a substituent on its side chain or glutamine which may have a substituent on its side chain;

[0028] (X) the arginine residue at position 12 of SEQ ID NO: 1 is substituted with lysine, histidine, or glutamine, which may have a substituent on the side chain;

[0029] (XI) the 4Py residue at position 13 of SEQ ID NO: 1 is substituted with an amino acid having an aromatic ring or a heterocyclic ring which may have a substituent on its side chain, or with glutamic acid which may have a substituent on its side chain;

[0030] (XII) The 14th MeTyr residue in SEQ ID NO: 1 is substituted with an N-methyl amino acid having an optionally substituted aromatic or heterocyclic ring in its side chain.

[0031] Effects of the invention

[0032] According to the invention described in this specification, as actually demonstrated in the Examples, a peptide that binds to human transferrin receptor (hTfR) can be provided. DETAILED DESCRIPTION

[0033] The following describes the embodiment of the present invention. The present invention is not limited to the embodiment described below, and includes modifications made appropriately within the scope of understanding by those skilled in the art based on the embodiment described below.

[0034] One invention described in this specification relates to a peptide that binds to the transferrin receptor.

[0035] transferrin receptor

[0036] The transferrin receptor (also known as the transferrin receptor) is a receptor found in plasma that binds to transferrin, a protein that binds to iron ions, and has the function of being internalized into cells. It is also known to adopt a dimeric structure. The transferrin receptor is expressed on various cells, including reticulocytes, placental trophoblasts, and lymphocytes, and is particularly suggested to be expressed on tumor cells. Furthermore, because the transferrin receptor has the property of triggering cellular endocytosis through binding stimulation by iron ions in plasma, antibodies that bind to the transferrin receptor are used as DDS to study the passage of desired substances across the BBB. While transferrin receptors are known to exist in types I and II, type I (Gene ID: 7037) is preferred for the transferrin receptor used in the present invention. In this specification, unless otherwise specified, human transferrin receptors are referred to as human TfR, hTfR, or simply TfR.

[0037] Transferrin receptor-binding peptide

[0038] Binding to the transferrin receptor (also referred to as having binding activity or affinity) means specifically binding to the transferrin receptor.

[0039] Although affinity is expressed as the equilibrium constant (KD) for the dissociation of the transferrin receptor and the binding peptide, this is a measure of the binding strength between the transferrin receptor and the binding peptide. As the KD value decreases, the binding strength between the transferrin receptor and the binding peptide increases (alternatively, affinity can also be expressed as 1 / KD, i.e., the affinity constant (KA)). As will be understood by those skilled in the art (e.g., based on the further disclosures herein), affinity can be determined in a manner well known per se depending on the type and properties of the binding substance. Furthermore, 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.

[0040] The specific binding of the transferrin receptor to the binding peptide can be determined using any suitable format known in the art, including surface plasmon resonance (SPR) analysis, Scatchard analysis, and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assays, as described herein, as well as various variants thereof. Preferably, the affinity of the peptide of the present invention for the transferrin receptor is less than 100 nM, preferably less than 50 nM, more preferably less than 20 nM, and even more preferably less than 10 nM. Although not limited to this, it may be approximately 10 nM.－5 M~about 10 －9 M, or in another aspect about 10 －7 M or less, for example, 10 －7 M~10 －13 M, for example, can be 10 －9 M~10 －13 M. The present specification provides peptides having a transferrin receptor binding ability within the above-mentioned range and having various strengths of binding ability ranging from low to high.

[0041] peptides

[0042] A peptide is a structure consisting of multiple consecutive amino acids, and its meaning also includes polypeptides and proteins. Furthermore, in this case, amino acids include not only naturally occurring amino acids (natural amino acids) that are translated into mRNA within cells and incorporated into peptide chains, but also non-natural amino acids (unnatural amino acids) that can form part of a peptide chain through peptide binding. Amino acids can be artificially synthesized or exist in nature.

[0043] Furthermore, in the present invention, peptides formed by cyclization after synthesis to form a cyclic portion (also referred to as cyclic peptides) and peptides obtained by further chemically modifying such peptides are also encompassed by the peptides of the present invention.

[0044] In this specification, the so-called cyclic peptide refers to a peptide in which two amino acids separated by one or more amino acid residues in the amino acid sequence are bound to each other, thereby forming a ring in whole or in part. In addition, the bonding form of the two amino acids is not particularly limited, but cyclic peptides also include the following: an amide bond between the carboxyl group of one amino acid and the amino group of the other amino acid, a thiol bond between the carboxyl group of one amino acid and the thiol group of the other amino acid, a thiol bond between the thiol group of one amino acid and the thiol group of the other amino acid, a lactam ring formation or a macrocyclization reaction to form a ring structure; a lasso peptide-like structure, etc. However, when the two amino acids are bonded to each other through an amide bond, the amide bond is not limited to the one formed by the bonding of the carboxyl group of one amino acid to the amino group of the other amino acid, as long as the result of the synthesis reaction is bonded through an amide bond. The same applies to other bonding forms.

[0045] That is, in this case, a portion of the cyclic peptide may be a cyclic structure, and may also have a linear portion. Furthermore, a complex cyclic structure such as a bicyclic structure in which two amino acids contained in a cyclic peptide forming a single cyclic structure are further linked can also be obtained.

[0046] Furthermore, in this specification, there are cases where a portion of an amino acid is modified for peptide cyclization. The amino acids herein also include amino acids with such modified portions. For example, a chloroacetyl group is added to an amino acid at the N-terminus, and the amino acid is cyclized by binding to a cysteine ​​residue in a peptide. The amino acids herein also include various (natural / unnatural) amino acids to which a chloroacetyl group has been added.

[0047] Unnatural amino acids are compounds that possess the properties of amino acids other than natural amino acids. Examples include, but are not limited to, chemically modified amino acids such as β-amino acids, γ-amino acids, L-amino acids, D-amino acids (also known as D-amino acids), amino acid variants, and amino acid derivatives; and amino acids that cannot form protein components in vivo, such as norleucine, β-alanine, and ornithine. Other examples 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 in which the carboxylic acid functional group in the side chain is replaced by a sulfonic acid group.

[0048] The following are examples of unnatural amino acids and their abbreviations used in this specification. The CAS reference number or purchase source is indicated in parentheses, and for newly synthesized amino acids, the synthesis example number is indicated. Special amino acids are not limited to these. For example, structures in which one or more hydrogen atoms in such molecules are replaced by alkyl groups are also special amino acids. When hydrogen atoms are replaced by alkyl groups, the alkyl group is preferably a methyl group or an ethyl group, and more preferably a methyl group. Furthermore, in this specification, amino acids with Me or N-Me- listed before the amino acid name represent N-methyl amino acids unless otherwise specified. For example, the N-methylated amino acid of alanine (Ala or A) is represented as MeAla, N-MeAla, MeA, or N-MeA. Furthermore, amino acids with a d listed before the amino acid symbol marked with a letter 1 are represented as D-amino acids. For example, the D-amino acid of alanine (Ala or A) is represented as da. Those without a CAS number or purchase source can be purchased as general reagents. Furthermore, the following amino acids can be used in peptide synthesis by protecting the afaminyl group with Fmoc according to a known method.

[0049] W7N: L-7-azatryptophan (CAS No. 49758-35-2)

[0050] KCOpipzaa: N6-(4-(carboxymethyl)piperidin-1-carbonyl)-L-lysine (KISHIDACHEMICAL Co., Ltd.)

[0051] 4Py: L-4-pyridylalanine (CAS No. 37535-49-2)

[0052] PeG: N-phenylethylglycine (CAS No. 7738-38-7)

[0053] 3Py: 3-(3-pyridyl)-L-alanine (CAS No. 64090-98-8)

[0054] 3Py6NH2: (S)-2-amino-3-(6-aminopyridin-3-yl)propionic acid (CAS No. 1269968-61-7)

[0055] A4paa: (S)-2-amino-3-(1-(carboxymethyl)piperidin-4-yl)propionic acid (KISHIDACHEMICAL Co., Ltd.)

[0056] dkCOmeglumine: N6-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-D-lysine (Synthesis Example 1-7)

[0057] F3COO: (S)-3-(2-amino-2-carboxyethyl)benzoic acid (CAS No. 13861-02-4)

[0058] F3COO(allyl): (S)-3-(3-((allyloxy)carbonyl)phenyl)-2-aminopropionic acid (Synthesis Example 1-1)

[0059] F4aao: (S)-2-amino-3-(4-(carboxymethoxy)phenyl)propionic acid (CAS No. 24558-63-2)

[0060] F4COO(allyl): (S)-3-(4-((allyloxy)carbonyl)phenyl)-2-aminopropionic acid (Synthesis Example 1-2)

[0061] F4OMe: (S)-2-amino-3-(4-methoxyphenyl)propionic acid (CAS No. 6230-11-1)

[0062] KdMe: N6,N6-dimethyl-L-lysine (CAS No. 2259-86-1)

[0063] Kmor: (S)-2-amino-6-[ortho]phenoxyhexanoic acid (CAS No. 960135-14-2) (Synthesis Example 1-10)

[0064] KN3: N6-diazo-L-lysine (CAS No. 159610-92-1)

[0065] Me3Py: (S)-2-(methylamino)-3-(pyridin-3-yl)propanoic acid (CAS No. 2651172-69-7)

[0066] Me4Py: (S)-2-(methylamino)-3-(pyridin-4-yl)propanoic acid

[0067] MeA: Methyl-L-alanine (CAS No. 3913-67-5)

[0068] MeF: Methyl-L-phenylalanine (CAS No. 2566-30-5)

[0069] MeF3C: (S)-3-(3-chlorophenyl)-2-(methylamino)propanoic acid (CAS No. 2255324-91-3) MeF3COO: (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (CAS No. 1499826-56-0)

[0070] MeF3COO(allyl): (S)-3-(3-((allyloxy)carbonyl)phenyl)-2-(methylamino)propionic acid (Synthesis Example 1-3)

[0071] MeF4F: (S)-3-(4-fluorophenyl)-2-(methylamino)propionic acid (CAS No. 347851-71-2)

[0072] MeR: Methyl-L-arginine (CAS No. 2480-28-6)

[0073] MeW: Methyl-L-tryptophan (CAS No. 526-31-8)

[0074] MeY: Methyl-L-tyrosine (CAS No. 537-49-5)

[0075] QhEt: N5-(2-hydroxyethyl)-L-glutamic acid (CAS No. 2650-74-0) (Synthesis Example 1-4)

[0076] Qpipzaa: (S)-2-amino-5-(4-(carboxymethyl)piperidin-1-yl)-5-oxopentanoic acid (Synthesis Example 1-5)

[0077] W1aa: 1-(carboxymethyl)-L-tryptophan (CAS No. 773823-50-0)

[0078] W1aa(allyl): 1-(2-(allyloxy)-2-oxoethyl)-L-tryptophan (Synthesis Example 1-6)

[0079] W5C: (S)-2-amino-3-(5-chloro-1H-indol-3-yl)propionic acid (CAS No. 52448-15-4)

[0080] Ndm: N4,N4-dimethyl-L-aspartic acid (CAS No. 62937-43-3)

[0081] F4C: N-α-chloroacetyl-4-chloro-L-phenylalanine (CAS No.: 14173-39-8)

[0082] dr: D-arginine

[0083] dk: D-lysine

[0084] Aib: α-methylalanine (CAS number: 62-57-7)

[0085] Ahp / Alahp: (S)-2-aminoheptanoic acid (CAS No.: 1115-90-8)

[0086] MeF3COO(PEG4Me):(S)-3-(3-((2,5,8,11-tetraoxatridecan-13-yl)carbamoyl)phenyl)-2-(methylamino)propionic acid (Synthesis Example 1-11)

[0087] MeF4COO(PEG4Me):(S)-3-(4-((2,5,8,11-tetraoxatridecan-13-yl)carbamoyl)phenyl)-2-(methylamino)propionic acid (Synthesis Example 1-8)

[0088] F3COO(PEG4Me): (S)-3-(3-((2,5,8,11-tetraoxatridecan-13-yl)carbamoyl)phenyl)-2-aminopropionic acid (Synthesis Example 1-12)

[0089] K(Mecar): N6-(methoxycarbonyl)-L-lysine (CAS No.74761-45-8)

[0090] K(MePEG4c): (S)-20-amino-14-oxo-2,5,8,11-tetraoxa-15-azaheneicosane-21-oleic acid (CAS No. 1188295-19-3)

[0091] E(PEG1Me): N5-(2-methoxyethyl)-L-glutamic acid (CAS No.132432-96-3)

[0092] E(NHdPEG1Me): N5-(1,3-dimethoxypropane-2-yl)-L-glutamic acid

[0093] E(PEG4Me): (S)-18-amino-15-oxo-2,5,8,11-tetraoxa-14-azanonadecan-19-oleic acid (CAS No. 2234872-12-7)

[0094] E(PEG8Me): (S)-30-amino-27-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azatriunesco-31-oleic acid

[0095] E (Glucamine): N5-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)-L-glutamine (CAS No. 1956384-48-7)

[0096] MeF4COO(PEG8Me): (S)-3-(4-((2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)carbamoyl)phenyl)-2-(methylamino)propionic acid (Synthesis Example 1-13)

[0097] F3COO(PEG8Me): (S)-3-(3-((2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)carbamoyl)phenyl)-2-aminopropionic acid (Synthesis Example 1-9)

[0098] F4aao(PEG8Me): (S)-2-amino-3-(4-((2,7-oxo-2,5,8,11,14,17,20,23-octaoxa-26-azaoctacosan-28-yl)oxy)phenyl)propanoic acid

[0099] alI: L-alloisoleucine (CAS No. 1509-34-8)

[0100] da: D-alanine

[0101] de: D-glutamic acid

[0102] ds: D-serine

[0103] K(Mecar): N6-(methoxycarbonyl)-L-lysine (CAS No.74761-45-8)

[0104] PEG4c or PEG3: 1-amino-3,6,9,12-tetraoxapentadecan-15-oleic acid (CAS No. 663921-15-1)

[0105] PEG8c: 1-amino-3,6,9,12,15,18,21,24-octaoxacosanoic acid-27-oleic acid (CAS No. 756526-04-2)

[0106] PEG8Me: 2,5,8,11,14,17,20,23-octaoxapentacosane-25-amine (CAS No. 869718-81-0)

[0107] PEG12c or PEG11 or PEG12: 1-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecatrimona-39-oleic acid (CAS No. 1415408-69-3)

[0108] MePEG4c: 2,5,8,11-tetraoxatetradecane-14-oleic acid (CAS No. 67319-28-2)

[0109] PEG1Me: 2-methoxyethane-1-amine (CAS No. 109-85-3)

[0110] PEG4Me: 2,5,8,11-tetraoxotridecan-13-amine (CAS No. 85030-56-4)

[0111] NHdPEG1Me: 1,3-dimethoxypropane-2-amine (CAS No. 78531-29-0)

[0112] BCNOCO: ((1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-yl)methyl(2,5-dioxopyrrolidin-1-yl)carbonate (CAS No. 1426827-79-3)

[0113] K(BCNOCO): N6-(methyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl(((1R,8S,9s)-bicyclo[6,1,0]non-4-yn-9-yl)methoxy)carbonyl)-L-lysine (CAS No. N / A) 1493802-96-2)

[0114] C(AcNMe): S-(2-(methylamino)-2-oxoethyl)-L-cysteine

[0115] Cit: L-citrulline (CAS No. 372-75-8)

[0116] K(Ac): N6-acetyl-L-lysine (CAS No. 692-04-6)

[0117] K(C5Mal): N6-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)-L-lysine

[0118] PEG24c: 1-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosylpentadecan-75-oleic acid (CAS No. 2563873-76-5)

[0119] K(Maleimide): N6-(4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carbonyl)-L-lysine

[0120] F4aao (Glucamine): (S)-2-amino-3-(4-(2-oxo-2-(((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)propanoic acid

[0121] F4aao(pipzaa): (S)-2-amino-3-(4-(2-(4-(carboxymethyl)piperidin-1-yl)-2-oxoethoxy)phenyl)propanoic acid

[0122] YaeCOpipzaa: (S)-2-amino-3-(4-(2-(4-(carboxymethyl)piperidin-1-amide)ethoxy)phenyl)propionic acid

[0123] Har: N6-carbamimidoyl-L-lysine (CAS No. 156-86-5)

[0124] W1EtOH: 1-(2-hydroxyethyl)-L-tryptophan

[0125] Nmm: N4-methyl-L-aspartic acid (CAS No.7175-34-0)

[0126] Eva: (S)-2-amino-3-ethylvaleric acid (CAS No. 14328-49-5)

[0127] Mor: (S)-[Oral end] 3-oxo-1,2-dimethoxy-1-phenoxy-1-ol ...

[0128] Hpr: (S)-piperidine-2-carboxylic acid (CAS No. 3105-95-1)

[0129] Y26dF: (S)-2-amino-3-(2,6-difluoro-4-hydroxyphenyl)propionic acid (CAS No. 182756-57-6)

[0130] W1Me: 1-methyl-L-tryptophan (CAS No. 21339-55-9)

[0131] MeF4COO: (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid

[0132] Y3Me: (S)-2-amino-3-(4-hydroxy-3-methylphenyl)propionic acid (CAS No. 17028-03-4)

[0133] 3Imp: (S)-2-amino-3-(imidazo[1,2-a]pyridin-3-yl)propanoic acid (CAS No. 2276942-95-9)

[0134] 3Py6OMe: (S)-2-amino-3-(6-methoxypyridin-3-yl)propanoic acid (CAS No. 1270178-24-9)

[0135] 3Py6NHaa: (S)-2-amino-3-(6-((carboxymethyl)amino)pyridin-3-yl)propanoic acid

[0136] MeF4C: (S)-3-(4-chlorophenyl)-2-(methylamino)propionic acid (CAS No. 347851-70-1)

[0137] MeF4OMe: (S)-3-(4-methoxyphenyl)-2-(methylamino)propionic acid (CAS No. 52939-33-0)

[0138] MeF3OMe: (S)-3-(3-methoxyphenyl)-2-(methylamino)propionic acid

[0139] 3Py6Me: (S)-2-amino-3-(6-methylpyridin-3-yl)propionic acid (CAS No. 1270288-73-7)

[0140] 4Py2Me: (S)-2-amino-3-(2-methylpyridin-4-yl)propionic acid (CAS No. 1269969-41-6)

[0141] 4Py2OMe: (S)-2-amino-3-(2-methoxypyridin-4-yl)propanoic acid (CAS No. 1269920-00-4)

[0142] 4Py2NH2: (S)-2-amino-3-(2-aminopyridin-4-yl)propionic acid (CAS No. 1269969-46-1)

[0143] E(PEG8c): (S)-32-amino-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatricarboxane dioleic acid

[0144] F3COO(PEG8c): (S)-1-(3-(2-amino-2-carboxyethyl)phenyl)-1-oxo-5,8,11,14,17,20,23,26-octaoxa-2-azanonacosane-29-oleic acid

[0145] E(PEG4c): (S)-20-amino-17-oxo-4,7,10,13-tetraoxa-16-azaheneicosane dioleic acid

[0146] F3COO(PEG4c) or F3CONPEG4c: (S)-1-(3-(2-amino-2-carboxyethyl)phenyl)-1-oxo-5,8,11,14-tetraoxa-2-azaheptadecan-17-oleic acid

[0147] E(apa): N5-(3-aminopropyl)-L-glutamic acid

[0148] MeF4COO(PEG4c): (S)-1-(4-(2-amino-2-carboxyethyl)phenyl-1-oxo-5,8,11,14-tetraoxa-2-azaheptadecan-17-oleic acid

[0149] Furthermore, newly synthesized amino acids are useful because they have the potential to add new functions to various peptides during the production of various peptide derivatives.

[0150] The peptide of the present invention is preferably the following peptide (peptide A).

[0151] A peptide consisting of the following amino acid sequence:

[0152] Ala-Val-MeF3C-Val-W7N-Asn-3Py6NH2-F4OMe-Ile-Ile-Arg-Arg-4Py-MeTyr-Cys (SEQ ID NO: 1), or

[0153] A peptide consisting of an amino acid sequence having one or more substitutions selected from the following group in the amino acid sequence described in SEQ ID NO: 1:

[0154] (I) the second valine residue in SEQ ID NO: 1 is replaced by a polar amino acid;

[0155] (II) the MeF3C residue at position 3 of SEQ ID NO: 1 is substituted with an N-methyl amino acid having an aromatic ring or a heterocyclic ring in its side chain;

[0156] (III) the W7N residue in the fifth position of SEQ ID NO: 1 is replaced by tryptophan or N-methyltryptophan, which may have a substituent and may have the carbon atom on the indole ring replaced by an N atom;

[0157] (IV) substitution of the aspartic acid residue at position 6 of SEQ ID NO: 1 into Nmm;

[0158] (V) The 3Py6NH2 residue at position 7 of SEQ ID NO: 1 is substituted with an amino acid having an aromatic ring or a heterocyclic ring which may have a substituent in its side chain;

[0159] (VI) the F4OMe residue at position 8 of SEQ ID NO: 1 is substituted with an amino acid having an aromatic ring or a heterocyclic ring which may have a substituent in its side chain;

[0160] (VII) substitution of the 9th isoleucine residue in SEQ ID NO: 1 with Eva;

[0161] (VIII) the isoleucine residue at position 10 of SEQ ID NO: 1 is replaced by any amino acid;

[0162] (IX) the 11th arginine residue in SEQ ID NO: 1 is substituted with lysine which may have a substituent on its side chain or glutamine which may have a substituent on its side chain;

[0163] (X) the arginine residue at position 12 of SEQ ID NO: 1 is substituted with lysine, histidine, or glutamine, which may have a substituent on the side chain;

[0164] (XI) the 4Py residue at position 13 of SEQ ID NO: 1 is substituted with an amino acid having an aromatic ring or a heterocyclic ring which may have a substituent on its side chain, or with glutamic acid which may have a substituent on its side chain; and

[0165] (XII) The 14th MeTyr residue in SEQ ID NO: 1 is substituted with an N-methyl amino acid having an optionally substituted aromatic or heterocyclic ring in its side chain.

[0166] Furthermore, the above-mentioned options (I) to (XII) can be selected in any combination.

[0167] Furthermore, the peptides of the present invention may be composed of an amino acid sequence in which 1 to 3 amino acid residues have been substituted, deleted, added, or inserted into any of the above sequences. In such cases, the peptide is preferably one that has the ability to bind to hTfR. The peptide may be the peptide itself or in the form of a pharmaceutically acceptable salt (e.g., sodium salt, potassium salt).

[0168] Conservative amino acid substitutions

[0169] "Conservative amino acid substitution" refers to the substitution of an amino acid with a functionally equivalent or similar amino acid. Generally speaking, substitutions within a certain group are considered conservative with respect to structure and function. However, as one skilled in the art will appreciate, the role played by a particular amino acid residue can be determined by the three-dimensional structure of the molecule containing that amino acid. For example, cysteine ​​residues are less polar and can exist in an oxidized (disulfide) form compared to a reduced (thiol) form. The long aliphatic portion of the arginine side chain can be an important structural and functional feature. Furthermore, side chains containing aromatic rings (tryptophan, tyrosine, phenylalanine) can facilitate ion-aromatic interactions or cation-π interactions. In this context, even when amino acids with these side chains are substituted with amino acids belonging to the acidic or non-polar groups, structural and functional properties can still be preserved. Residues such as proline, glycine, and cysteine ​​(in disulfide form) may have a direct effect on the steric structure of the main chain and cannot be substituted repeatedly without causing structural changes.

[0170] Conservative amino acid substitutions include specific substitutions based on side chain similarity (Lehninger, Biochemistry, 2nd edition, 1975, pp. 73-75: L. Lehninger, Biochemistry, 2nd edition, pp. 73-75, Worth Publishers, New York (1975)) and typical substitutions as shown below.

[0171] Furthermore, the conservative amino acid substitution is preferably, for example, substitution of an amino acid with an amino acid belonging to the same group as the group in which natural amino acids are classified based on the properties of their common side chains, as follows.

[0172] (1) Hydrophobic (also called non-polar) amino acids: amino acids showing hydrophobicity (non-polarity), including alanine ("Ala" or also abbreviated as "A"), glycine ("Gly" or also abbreviated as "G"), valine ("Val" or also abbreviated as "V"), leucine ("Leu" or also abbreviated as "L"), isoleucine ("Ile" or also abbreviated as "I"), proline ("Pro" or also abbreviated as "P"), phenylalanine ("Phe" or also abbreviated as "F"), tryptophan ("Trp" or also abbreviated as "W"), tyrosine ("Tyr" or also abbreviated as "Y"), and methionine ("Met" or also abbreviated as "M").

[0173] Furthermore, hydrophobic amino acids can be further classified into the following groups.

[0174] Aliphatic amino acids: amino acids having fatty acids or hydrogen in their side chains, including Ala, Gly, Val, Ile, and Leu.

[0175] Aliphatic / branched-chain amino acids: amino acids with branched fatty acids in the side chains, including Val, Ile, and Leu.

[0176] Aromatic amino acids: amino acids with aromatic rings in their side chains, including Trp, Tyr, and Phe.

[0177] (2) Hydrophilic (also called polar) amino acids: Amino acids that exhibit hydrophilicity (polarity), including serine ("Ser" or abbreviated as "S"), threonine ("Thr" or abbreviated as "T"), cysteine ​​("Cys" or abbreviated as "C"), aspartic acid ("Asn" or abbreviated as "N"), glutamic acid ("Gln" or abbreviated as "Q"), aspartic acid ("Asp" or abbreviated as "D"), glutamic acid ("Glu" or abbreviated as "E"), lysine (also recorded as Lysine. "Lys" or abbreviated as "K"), arginine ("Arg" or abbreviated as "R"), and histidine ("His" or abbreviated as "H").

[0178] Furthermore, hydrophilic amino acids can be further classified into the following groups.

[0179] Acidic amino acids: amino acids with acidic side chains, including Asp, Glu,

[0180] Basic amino acids: amino acids with basic side chains, including Lys, Arg, and His.

[0181] Neutral amino acids: amino acids with neutral side chains, including Ser, Thr, Asn, Gln, and Cys.

[0182] Furthermore, Gly and Pro can also be classified as "amino acids that affect the direction of the main chain", and amino acids containing sulfur molecules in their side chains, Cys, and Met can also be classified as "sulfur-containing amino acids".

[0183] Furthermore, the group having an aromatic group in the side chain includes Trp, Tyr, and Phe.

[0184] In this specification, "amino acid" includes not only natural amino acids but also non-natural amino acids. Among non-natural amino acids, there are N-alkyl amino acids obtained by N-alkylation of the natural amino acids described above, and those in which the nitrogen forming the peptide bond is modified by a branched or unbranched low-alkyl group (for example, C1-C5, preferably C1-C3, more preferably C1). Among N-alkyl amino acids, N-ethyl amino acid, N-butyl amino acid or N-methyl amino acid is preferred, and N-methyl amino acid is more preferred. The so-called C4 or higher alkyl group refers to a straight chain or branched alkyl group with a carbon number of 4 or higher. Examples of C4 or higher alkyl groups are C 4－10 Alkyl, also C 4－6 Alkyl, also C 6－10 Alkyl groups. Furthermore, non-natural amino acids include chemically modified amino acids such as D-amino acids (also referred to as D-amino acids), β-amino acids, γ-amino acids, amino acid variants, and amino acid derivatives, as well as amino acids such as isoleucine and ornithine that cannot serve as building blocks for proteins in vivo. Furthermore, non-natural amino acids include amino acids in which functional groups have been added to the side chains of natural amino acids, or in which functional groups have been substituted with other functional groups (e.g., amino acids with substitutions or additions to the arylene or alkylene groups in the side chains; amino acids with increased carbon atoms in the arylene, alkylene, or alkyl groups in the side chains; amino acids with substitutions in the aromatic rings of the side chains; amino acid derivatives in which the carboxylic acid functional group in the side chain has been replaced by a sulfonic acid group; heterocyclic or condensed cyclized amino acids, etc.).

[0185] Furthermore, by adding or substituting functional groups or other structures onto the side chains of natural amino acids, properties different from those of natural amino acids can be imparted. That is, the aforementioned groups that classify natural amino acids based on their common side chain properties can include non-natural amino acids with similar side chain properties. For example, N-methylalanine (MeA), an amino acid formed by methylating the main chain nitrogen atom of alanine, an aliphatic amino acid, is a non-natural amino acid, but exhibits hydrophobicity (non-polarity) and can be classified as a hydrophobic amino acid. Thus, non-natural amino acids that exhibit the same side chain properties as an amino acid can also be included as targets for conservative amino acid substitutions.

[0186] In this specification, "non-limiting" and "in one aspect" can be used synonymously.

[0187] Non-limiting examples of conservative amino acid substitutions with non-natural amino acids include the following.

[0188] MeA: N-methylated amino acids, hydrophobic amino acid groups and aliphatic amino acid groups

[0189] MeF: N-methylated amino acids, hydrophobic amino acid groups and aromatic amino acid groups

[0190] MeW: N-methylated amino acids, hydrophobic amino acid groups and aromatic amino acid groups

[0191] MeY: N-methylated amino acid, hydrophobic amino acid group and aromatic amino acid group

[0192] MeR: N-methylated amino acids, hydrophilic amino acid group and basic amino acid group

[0193] da: D-type amino acids, hydrophobic amino acid groups and aliphatic amino acid groups

[0194] de: D-type amino acids, hydrophilic amino acid group and acidic amino acid group

[0195] dr: D-type amino acids, hydrophilic amino acid group and basic amino acid group

[0196] ds: D-type amino acids, hydrophilic amino acid group and neutral amino acid group

[0197] 4Py: basic amino acid group and aromatic amino acid group

[0198] 3Py: basic amino acid group and aromatic amino acid group

[0199] F4C: hydrophobic amino acid group and aromatic amino acid group

[0200] F4OMe: hydrophobic amino acid group and aromatic amino acid group

[0201] F4aao: hydrophobic amino acid group and aromatic amino acid group

[0202] F3COO: hydrophobic amino acid group and aromatic amino acid group

[0203] MeF3C: N-methylated amino acids, hydrophobic amino acid groups and aromatic amino acid groups

[0204] MeF4F: N-methylated amino acids, hydrophobic amino acid groups and aromatic amino acid groups

[0205] Me3Py: N-methylated amino acids, hydrophobic amino acid groups and aromatic amino acid groups

[0206] Me4Py: N-methylated amino acids, hydrophobic amino acid groups and aromatic amino acid groups

[0207] MeF3COO: N-methylated amino acids, hydrophobic amino acid groups and aromatic amino acid groups

[0208] MeF4COO: N-methylated amino acids, hydrophobic amino acid groups and aromatic amino acid groups

[0209] W5C: hydrophobic amino acid group and aromatic amino acid group

[0210] W1aa: hydrophobic amino acid group and aromatic amino acid group

[0211] W7N: basic amino acid group and aromatic amino acid group

[0212] W5C: hydrophobic amino acid group and aromatic amino acid group

[0213] 3Py6NH2: basic amino acid group and aromatic amino acid group

[0214] a1: hydrophobic amino acid group and aliphatic amino acid group

[0215] Aib: hydrophobic amino acid group and aliphatic amino acid group

[0216] Ahp: hydrophobic amino acid group and aliphatic amino acid group

[0217] A4paa: hydrophobic amino acid group and aliphatic amino acid group

[0218] Kmor: hydrophilic amino acid group

[0219] Kdme: hydrophilic amino acid group

[0220] KCOpipzaa: hydrophilic amino acid group

[0221] QhEt: hydrophilic amino acid group

[0222] W1EtOH: hydrophobic amino acid group and aromatic amino acid group

[0223] W1Me: hydrophobic amino acid group and aromatic amino acid group

[0224] 3Imp: basic amino acid group and aromatic amino acid group

[0225] Nmm: hydrophilic amino acid group

[0226] Y26dF: hydrophobic amino acid group and aromatic amino acid group

[0227] 3Py6NHaa: hydrophilic amino acid group and aromatic amino acid group

[0228] Y3Me: hydrophobic amino acid group and aromatic amino acid group

[0229] F4aao (Glucamine): Hydrophilic amino acid group and aromatic amino acid group

[0230] F4aao (pipzaa): hydrophilic amino acid group and aromatic amino acid group

[0231] YaeCOpipzaa: hydrophilic amino acid group and aromatic amino acid group

[0232] 3Py6OMe: basic amino acid group and aromatic amino acid group

[0233] Eva: Hydrophobic group and aliphatic / branched chain amino acid group

[0234] Har: hydrophilic amino acid group

[0235] Mor: hydrophilic amino acid group

[0236] Hpr: hydrophobic amino acid group

[0237] E (Glucamine): Hydrophilic amino acid group

[0238] E(PEG8Me): hydrophilic amino acid group

[0239] QPEG8Me: Hydrophilic amino acid group

[0240] E(PEG8c): Hydrophilic amino acid group

[0241] E(PEG4c): hydrophilic amino acid group

[0242] F3CONPEG4c: hydrophilic amino acid group and aromatic amino acid group

[0243] 3Py6Me: basic amino acid group and aromatic amino acid group

[0244] 4Py2Me: basic amino acid group and aromatic amino acid group

[0245] 4Py2OMe: basic amino acid group and aromatic amino acid group

[0246] 4Py2NH2: basic amino acid group and aromatic amino acid group

[0247] F3COO(PEG4c): hydrophilic amino acid group and aromatic amino acid group

[0248] F3COO(PEG8c): hydrophilic amino acid group and aromatic amino acid group

[0249] MeF4C: N-methylated amino acids, hydrophobic amino acid groups and aromatic amino acid groups

[0250] MeF4OMe: N-methylated amino acids, hydrophobic amino acid groups and aromatic amino acid groups

[0251] MeF3OMe: N-methylated amino acids, hydrophobic amino acid groups and aromatic amino acid groups

[0252] F4aao(PEG8Me): hydrophilic amino acid group and aromatic amino acid group

[0253] F3COO(PEG8Me): hydrophilic amino acid group and aromatic amino acid group

[0254] E(apa): basic amino acid group and hydrophilic amino acid group

[0255] MeF4COO(PEG4c): N-methylated amino acids, acidic amino acid groups and aromatic amino acid groups

[0256] D-amino acids can be classified as D-amino acids or according to the properties of their side chains. N-methyl amino acids can be classified as N-alkyl amino acids or according to the properties of the side chains of the original amino acids that are not N-methylated.

[0257] In this specification, "amino acids with positive charge" may be natural amino acids or non-natural amino acids. Examples of natural amino acids with positive charge are lysine, arginine and histidine. "Amino acids with positive charge" are amino acids with positive charge (e.g., NH3 + ) may be an amino acid, or may have various substituents.

[0258] Preferred examples of peptides in this specification are peptides that can bind to human transferrin receptor (hTfR), particularly TfR1, similarly to the above-mentioned peptides.

[0259] One aspect of the peptide of the present invention may be a peptide comprising an amino acid sequence that satisfies at least one of the following (I) to (V).

[0260] (I) The first residue in SEQ ID NO: 1 is an alanine residue.

[0261] (II) The fourth residue in SEQ ID NO: 1 is a valine residue.

[0262] (III) The sixth residue in SEQ ID NO: 1 is an asparagine residue.

[0263] (IV) The 7th position of SEQ ID NO: 1 is a tyrosine residue or a 3Py6NH2 residue.

[0264] (V) The ninth position of SEQ ID NO: 1 is an isoleucine residue.

[0265] Here, the alanine residue of (I) can also be a derivative thereof, the valine residue of (II) can also be a derivative thereof, the aspartic acid residue of (III) can also be a derivative thereof, the tyrosine residue or 3Py6NH2 residue of (IV) can also be a derivative thereof, and the isoleucine residue of (V) can also be a derivative thereof.

[0266] Furthermore, the above-mentioned options (I) to (V) can be selected in any combination, and the peptide may include an amino acid sequence that satisfies at least one of (I) to (V).

[0267] According to Example 3, amino acid residues (I) to (V) are believed to significantly influence binding to TfR1. Therefore, amino acid residues in SEQ ID NO: 1 that satisfy at least one, and preferably all, of the above-mentioned (I) to (V) can achieve strong binding to TfR1. The affinity of these peptides for the transferrin receptor is not limited, but can have a KD of less than 50 nM, more preferably less than 20 nM, and even more preferably less than 10 nM.

[0268] Furthermore, in one aspect of the peptide of the present invention provided herein, the fourth position of SEQ ID NO: 1 is a valine residue, and the Examples demonstrate that this is also an important residue for strong binding to TfR1.

[0269] The peptide may also have an amino acid sequence in which any of the 2nd, 3rd, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, and 14th amino acid residues in SEQ ID NO: 1 are substituted.

[0270] The term "amino acid residue is substituted" means that a specific amino acid residue is substituted with another amino acid residue that can be modified. Here, the amino acid residue substitution is preferably a conservative amino acid substitution.

[0271] The term "modifiable" means that known amino acid modifications or alterations can be made. Examples of modifications include N-methylation, amino acid modifications with the aforementioned abbreviations, modification (conversion) to the D-form, and conversion to known derivatives of the amino acid.

[0272] The phrase "may have a linker" means that the linker may be directly bonded to the amino acid residue. Examples of amino acids having a linker include K (MePEG4c) in which MePEG4c is bonded to the amino group on the side chain of a lysine residue, E (PEG1Me) in which PEG1Me is bonded to the carboxyl group on the side chain of a glutamic acid residue, F4aao (PEG8Me) in which PEG8Me is bonded to the carboxyl group on the side chain of F4aao, E (PEG8Me) in which PEG8Me is bonded to the carboxyl group on the side chain of a glutamic acid residue, E (PEG8c) in which PEG8c is bonded to the carboxyl group on the side chain of a glutamic acid residue, E (PEG4c) in which PEG8Me is bonded to the carboxyl group on the side chain of a glutamic acid residue, and QPEG8M in which PEG8Me is bonded to the amido group on the side chain of a glutamic acid residue. e. F3CONPEG4c in which PEG4c is bound to the acyl group of the side chain of F3CON, F3COO(PEG4Me) in which PEG4Me is bound to the carboxyl group of the side chain of F3COO, F3COO(PEG4c) in which PEG4c is bound to the carboxyl group of the side chain of F3COO, F3COO(PEG8c) in which PEG8c is bound to the carboxyl group of the side chain of F3COO, F3COO(PEG8Me) in which PEG8Me is bound to the carboxyl group of the side chain of F3COO, MeF3COO(PEG4Me) in which PEG4Me is bound to the carboxyl group of the side chain of MeF3COO, and a cysteine ​​residue in which PEG4c is bound to the carboxyl group of the C-terminus.

[0273] In one aspect of the present invention, the peptide may be a peptide having the following amino acid sequence (peptide B).

[0274] The aforementioned peptide may be composed of an amino acid sequence having one or more substitutions selected from the following:

[0275] (I) the second valine residue in SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of T and Kmor;

[0276] (II) the third MeF3C residue in SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of MeF and Me3Py;

[0277] (III) the W7N residue in the fifth position of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of W1aa, W, W1EtOH, W1Me, 3Imp, and MeW;

[0278] (IV) the 3Py6NH2 residue at position 7 of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of Y, Y26dF, 3Py6NHaa, Y3Me, and R;

[0279] (V) the F4OMe residue at position 8 of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of F4aao, F4aao(Glucamine), F4aao(pipzaa), YaeCOpipzaa, 3Py6Ome, 3Py6NHaa, 3Imp, Y, and F4aao(PEG8Me);

[0280] (VI) the isoleucine residue at position 10 of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of Har, Kmor, T, Mor, Hpr, and S;

[0281] (VII) the arginine residue at position 11 of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of Kmor, KCOpipzaa, K, E (glucamine), Q, Q which may have a linker on its side chain, E which may have a linker on its side chain, and E (apa);

[0282] (VIII) the arginine residue at position 12 of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of Kmor, KCOpipzaa, Q, and H;

[0283] (IX) the 4Py residue at position 13 of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of F3COO, F, F3CONPEG4c, Y3Me, 3Py6Me, 3Py6Ome, 3Py6NH2, 4Py2Me, 4Py2Ome, 4Py2NH2, 3Py, Y, F3COO which may have a linker, and E which may have a linker on its side chain; and

[0284] (X) The MeTyr residue at position 14 of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of MeF4COO, MeF3COO, MeF4C, MeF4OMe, MeF3C, MeF3OMe, Me4Py, Me3Py, and MeF4COO(PEG4c).

[0285] Furthermore, the above-mentioned options (I) to (X) can be selected in any combination.

[0286] Furthermore, the peptide of the present invention may be a peptide composed of an amino acid sequence in which 1 to 3 amino acid residues are substituted, deleted, added, or inserted into any of the above sequences. In such cases, the peptide is preferably a peptide having the ability to bind to hTfR.

[0287] Yet another aspect of the present invention is a peptide having the following amino acid sequence.

[0288] The peptides may be:

[0289] (I) the second amino acid residue of SEQ ID NO: 1 is V, T or K,

[0290] (II) the third amino acid residue of SEQ ID NO: 1 is MeF3C, MeF or Me3Py,

[0291] (III) the fifth amino acid residue of SEQ ID NO: 1 is W7N, W1aa, W, W1EtOH, W1Me, 3Imp or MeW,

[0292] (IV) the sixth amino acid residue of SEQ ID NO: 1 is N or Nmm,

[0293] (V) the 7th amino acid residue of SEQ ID NO: 1 is 3Py6NH2, Y, Y26dF, 3Py6NHaaY3Me or R,

[0294] (VI) the 8th amino acid residue of SEQ ID NO: 1 is F4Ome, F4aao, F4aao(Glucamine), F4aao(pipzaa), YaeCOpipzaa, 3Py6Ome, 3Py6NHaa, 3Imp, Y or F4aao(PEG8Me),

[0295] (VII) the ninth amino acid residue in SEQ ID NO: 1 is I or Eva,

[0296] (VIII) the 10th amino acid residue of SEQ ID NO: 1 is I, Har, Kmor, T, Mor, Hpr or S,

[0297] (IX) the 11th amino acid residue of SEQ ID NO: 1 is R, Kmor, KCOpipzaa, K, E(Glucamine), E(PEG8Me), Q, QPEG8Me, E(PEG8c), E(PEG4c) or E(apa),

[0298] (X) the 12th amino acid residue of SEQ ID NO: 1 is R, Kmor, KCOpipzaa, Q or H,

[0299] (XI) the 13th amino acid residue of SEQ ID NO: 1 is 4Py, F3COO, F, F3CONPEG4c, Y3Me, 3Py6Me, 3Py6OMe, 3Py6NH2, 4Py2Me, 4Py2OMe, 4Py2NH2, 3Py, F3COO(PEG4c), Y, E(PEG8c), F3COO(PEG8c), E(PEG4c) or F3COO(PEG8Me),

[0300] (XII) The 14th amino acid residue of SEQ ID NO: 1 is MeY, MeF4COO, MeF3COO, MeF4C, MeF4OMe, MeF3C, MeF3OMe, Me4Py, Me3Py or MeF4COO(PEG4c).

[0301] In addition, the above options (I) to (XII) can be selected in any combination.

[0302] Furthermore, according to the invention described in this specification, there is provided a peptide that binds to hTfR and whose binding ability changes depending on pH.

[0303] Although not limiting, in this specification, the phrase "peptide binding ability varies depending on pH" (also referred to herein as "having pH-dependent binding ability") means that the binding ability of the peptide to hTfR at at least two different pH values ​​is at least 2 times, 5 times, or preferably 10 times greater than the other. The binding ability can be affinity (expressed as KD) or dissociation rate (dissociation rate constant: expressed as koff). The temperature in this case can be, for example, 25°C or 37°C.

[0304] Furthermore, although not limiting, one of the two different pH values ​​may be acidic and the other neutral or physiological. Acidic pH is preferably pH 4 to 6.8, more preferably pH 5.5 to 6.8. Neutral pH is preferably pH 7 to 8, more preferably pH 7.3 to 7.5. Physiological pH refers to the pH under normal human physiological conditions and may be pH 7.3 to 7.4, pH 7.35 to 7.45, or pH 7.4.

[0305] Substances that bind to the transferrin receptor can cross the blood-brain barrier and reach the brain via endocytosis. In this case, factors that influence endocytosis include not only the ability of the substance to bind to the transferrin receptor but also the acidification of the endosome. It has been reported that endocytosis of antibodies whose binding ability to the transferrin receptor is reduced is promoted in acidified endosomes (Non-Patent Document 1). This suggests that endocytosis can be promoted when using substances with pH-dependent binding ability, such as those that strongly bind to the transferrin receptor at neutral pH but whose binding ability is reduced at acidic pH.

[0306] While not limiting, the peptides of the present invention include those having pH-dependent binding ability, such as dissociation rates at acidic pH that are at least 2 times, 5 times, or preferably 10 times greater than those at neutral or physiological pH. Peptides with this property are believed to strongly bind to transferrin receptors in the blood and be internalized into endosomes. Subsequently, within the acidified endosomes, their binding capacity to transferrin receptors decreases, promoting endocytic transport and enabling more efficient cross-blood-brain barrier delivery into the brain.

[0307] The peptide having pH-dependent binding ability as described above is a peptide having the amino acid sequence of the above-mentioned peptide A and / or peptide B, and preferably comprises an amino acid sequence that satisfies at least one of the following conditions and at least one of the following conditions A or B in the amino acid sequence described in SEQ ID NO: 1:

[0308] (I) the first position of SEQ ID NO: 1 is an alanine residue;

[0309] (II) the fourth position of SEQ ID NO: 1 is a valine residue;

[0310] (III) the sixth residue of SEQ ID NO: 1 is an aspartic acid residue;

[0311] (IV) the 7th position of SEQ ID NO: 1 is a tyrosine residue or a 3Py6NH2 residue;

[0312] (V) The ninth position of SEQ ID NO: 1 is an isoleucine residue.

[0313] A: At least two of the following must be met:

[0314] (a) the fifth residue of SEQ ID NO: 1 is a W7N residue or a 3Imp residue;

[0315] (b) The 7th residue of SEQ ID NO: 1 is 3Py6NH2 residue;

[0316] (c) The eighth residue of SEQ ID NO: 1 is F4aao (pipzaa);

[0317] (d) The 13th residue of SEQ ID NO: 1 is a 4Py residue.

[0318] B: (i) The 8th position of SEQ ID NO: 1 is a F4aao (pipzaa) residue, a F4aao (Glucamine) residue, a F4aao (PEG8Me) residue or a YaeCOpipzaa residue.

[0319] Here, the alanine residue of (I) can also be a derivative thereof, the valine residue of (II) can also be a derivative thereof, the aspartic acid residue of (III) can also be a derivative thereof, the tyrosine residue or 3Py6NH2 residue of (IV) can also be a derivative thereof, and the isoleucine residue of (V) can also be a derivative thereof.

[0320] Furthermore, the W7N residue or 3Imp residue in (a) may be a derivative thereof, the 3Py6NH2 residue in (b) may be a derivative thereof, the F4aao (pipzaa) residue in (c) may be a derivative thereof, and the 4Py residue in (d) may be a derivative thereof.

[0321] Furthermore, the F4aao(pipzaa) residue, F4aao(Glucamine) residue, F4aao(PEG8Me) residue or YaeCOpipzaa residue in (i) may also be derivatives thereof.

[0322] Furthermore, the options (I) to (V), (a) to (d), and (i) above can be selected in any combination.

[0323] However, it is preferred that the peptide having the amino acid sequence described in SEQ ID NO: 162 be excluded from any of the above peptides.

[0324] Preferred examples of the peptide of the present invention are peptides comprising the following amino acid sequences and having a cyclic structure at the amino acid sequence site: the amino acid sequence described in any one of SEQ ID NOs: 1-202, preferably SEQ ID NOs: 1-161 and 168-202, more preferably SEQ ID NOs: 1-149 and 168-197, and most preferably 1-68; or the 1st to 15th amino acid sequence of a combination of the amino acid sequence described in any one of SEQ ID NOs: 1-202, preferably SEQ ID NOs: 1-161 and 168-202, more preferably SEQ ID NOs: 1-149 and 168-197, and most preferably 1-68, and a linker.

[0325] Preferred examples of this peptide are any of the above-mentioned peptides, which are cyclic peptides.

[0326] A preferred example of this peptide is any of the above-mentioned peptides, wherein the first to fifteenth amino acid sequence sites have a cyclic structure.

[0327] A preferred example of this peptide is any of the above-mentioned peptides, which is a peptide consisting of an amino acid sequence in which the first amino acid at its N-terminus is chloroacetylated and the acetyl group is cyclized with the amino acid at the C-terminus, namely, cysteine.

[0328] About Cyclic Peptides

[0329] This refers to a peptide in which two amino acids are bonded, forming a ring in whole or in part. Furthermore, in this context, this also includes peptides in which amino acids form a cross-linked structure, peptides in which a ring structure is formed by lactam ring formation or macrocyclization, and peptides having a lasso-like structure. In other words, in this context, a cyclic peptide may also have a linear portion as long as a portion of the peptide forms a ring structure.

[0330] Peptides generally have poor metabolic stability in vivo and, due to their large size, have difficulty passing through cell membranes. To address this issue, peptide cyclization has been proposed. Cyclization improves protease resistance and metabolic stability without restricting conformational changes, suggesting increased rigidity, membrane permeability, and affinity for target proteins.

[0331] Cyclization method

[0332] The cyclization of the peptide can be carried out according to a well-known method.

[0333] Although not limited to this, for example, by designing a peptide to include 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. Acss Chem. Biol. 3, 120-129 (2008)), peptides with a chloroacetyl group at the N-terminus can be synthesized using genetic code reprogramming technology. Cyclization can also be achieved by placing a cysteine ​​residue in the peptide. Thus, after translation, the sulfhydryl group automatically undergoes nucleophilic attack on the chloroacetyl group, causing the peptide to cyclize via a thioether bond. Cyclization can also be achieved by placing other amino acid combinations that bind to form a ring within the peptide using genetic code reprogramming technology. Furthermore, peptides with a cyclic amide at the N-terminus can be synthesized by placing an Hgl residue in the peptide, which can also achieve cyclization. Thus, any well-known cyclization method can be used without particular limitation.

[0334] The peptide has a ring structure in which the N-terminal amino acid (the first amino acid residue) is bound to the cysteine ​​residue contained in the peptide. In one aspect, the peptide has a ring structure in which the N-terminal amino acid (the first amino acid residue) is bound to the 15th cysteine ​​residue contained in the peptide. In one aspect, the peptide has a ring structure in which the chloroacetylated N-terminal amino acid (the first amino acid residue) is bound to the 15th cysteine ​​residue contained in the peptide. The so-called "chloroacetylation" may be "haloacetylation" caused by other halogens. In addition, "acetylation" may be "acylation" caused by an acyl group other than an acetyl group.

[0335] In this specification, for the purpose of peptide cyclization, some amino acids may be modified. This also includes amino acids that have undergone such partial modification. For example, as described above, there are cases where a chloroacetyl group is added to an amino acid at the N-terminus, and the amino acid is cyclized by binding to a cysteine ​​residue in a peptide. Various amino acids (natural / unnatural) to which such chloroacetyl groups have been added are also included in the amino acids in this specification.

[0336] A preferred example of this peptide is any of the above-mentioned peptides, which is a peptide consisting of 15 amino acid residues.

[0337] Preferred examples of this peptide include any of the above peptides, wherein a linker is bound to the 8th, 11th, 13th, or 15th amino acid residue in the amino acid sequence. More preferably, the peptide has an amino acid sequence as set forth in any one of SEQ ID NOs: 1-202, more preferably SEQ ID NOs: 1-161 and 168-202, more preferably SEQ ID NOs: 1-149 and 168-197, and most preferably any one of SEQ ID NOs: 1-68.

[0338] Peptide length

[0339] The number of amide bonds (number of amino acids, length) in the peptide or peptide moiety is not particularly limited, but preferably the total amino acid residues (excluding amino acids in the case where the substance bound to the peptide or the linker binding the substance to the peptide contains such amino acids) are within 20 residues. The total amino acid residues are preferably 10 or more, 11 or more, 12 or more, 13 or more, or 14 or more, and preferably 19 or less, 18 or less, 17 or less, or 16 or less.

[0340] Another embodiment disclosed in this specification relates to a complex. This complex comprises any of the aforementioned peptides, a linker bound to the peptide, and a substance bound to the linker. Preferably, the complex is capable of binding to at least TfR.

[0341] The complex may be a complex formed by binding the C-terminus of a cysteine ​​(Cys) residue in any of the peptides to the aforementioned substance, or a complex formed by binding a linker bound to the C-terminus to the aforementioned substance. Furthermore, the complex may be a complex formed by binding a functional group at the end of a side chain of an amino acid residue in any of the peptides to the aforementioned substance, or a complex formed by binding a linker bound to the aforementioned functional group to the aforementioned substance.

[0342] Examples of linkers include those having an amino acid length of 1 or more and 15 or less, and containing one or more glycine (Gly) or serine (Ser).

[0343] Preferred examples of this linker include a modified cysteine ​​(Cys) or a modified lysine (Lys) at the N-terminus.

[0344] Another example of a linker is one having an amino acid length of not less than 1 and not more than 5, and containing either or both of D-glutamic acid (de) and methylated glycine (MeG).

[0345] Preferred examples of this linker include a modified cysteine ​​(Cys) or a modified lysine (Lys) at the N-terminus.

[0346] Another example of a linker is a PEG linker comprising polyethylene glycol (PEG) or a polyethylene glycol derivative. Polyethylene glycol derivatives include all known PEG linkers. Furthermore, a portion of the linker may be substituted with a functional group or have a functional group added thereto. Examples include, but are not limited to, methyl, amino, and azide groups.

[0347] Preferably, the PEG linker is PEG4c, PEG8c, PEG12c, PEG8Me, MePEG4c, PEG1Me, PEG4Me or NHdPEG1Me.

[0348] Furthermore, the linker may also contain a reactive functional group for bonding a desired substance. Examples of the reactive functional group include maleimide and hydrazide.

[0349] Another example of a linker is a sequence that is a portion of the linker sequence of SEQ ID NOs: 1-68 shown in Table 1-1. For example, in SEQ ID NO: 1, PEG4c is a linker, and in SEQ ID NO: 64, the sequence [G-KN3] starting from the amino acid residue described in the linker row, i.e., glycine, is a linker.

[0350] A preferred example of a peptide or complex bound by this linker is:

[0351] A peptide linker composed of polyethylene glycol (PEG), Gly or MeG is also called a G linker, a peptide linker composed of Gly or MeG and Ser is also called a GS linker, and a peptide linker composed of Gly and KN3 is also called a GKN3 linker.

[0352] In this specification, a linker (also referred to as a cross-linker) refers to a structure that further additively binds to the amino acid residues contained in the cyclic amino acid sequence of a peptide that binds to the transferrin receptor, or an intermolecular link between the peptide and a substance to be delivered to the TfR. It can be any linker known to the public or described in this specification. In specific embodiments, the linker is, for example, a chemical linker, a fatty acid linker, or a peptide linker (polypeptide linker). Furthermore, it can be a complex of a chemical linker and a peptide linker.

[0353] The linker may be dispersed or separated depending on the environment or conditions, or may maintain a stable structure.

[0354] Chemical linker: In some embodiments, the linker can be a chemical linker. Examples of chemical linkers, although not limited thereto, include substituted or unsubstituted alkylene groups, substituted or unsubstituted heteroalkylene groups, substituted or unsubstituted cycloalkylene groups, substituted or unsubstituted heterocycloalkylene groups, substituted or unsubstituted propyne groups, and / or substituted or unsubstituted heteroarylene groups. Furthermore, the peptide and linker can be conjugated via sulfhydryl groups, amino groups (amines), and / or carbohydrates, or any other suitable reactive groups. Homobifunctional and heterobifunctional crosslinkers (conjugating agents) are available from a variety of commercial sources. The crosslinker can include flexible arms, for example, having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 carbon atoms. Examples of cross-linking agents include BSS3 ([bis(sulfosuccinimidyl) suberate]), NHSS / EDC (N-hydroxysuccinimide and N-ethyl-(dimethylaminopropyl)carbodiimide), sulfo-EMCSS ([N-e-maleimidocaproic acid] hydrazide, hydrazide, and SSATA (N-succinimidyl-SS-acetylthioacetic acid).

[0355] A preferred example of a chemical linker is a PEG (Polyethylene glycol) linker. For example, the PEG linker may be a PEG linker consisting of 1 to 24 ethylene glycol units.

[0356] Fatty acid linker: The linker may be a fatty acid linker comprising a divalent chemical moiety induced by a fatty acid. For example, the fatty acid linker may be a linker comprising 12-aminododecanoic acid.

[0357] Peptide linker: The peptide linker comprises at least one amino acid (e.g., a peptide of at least 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 40, or 50 amino acids). In certain embodiments, the linker is a single amino acid (e.g., any 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 [X-X-X-X-Gly]y (wherein up to two of the X's are Thr, the remaining X's are Ser, and y is 1 to 5) (e.g., Ser-Ser-Ser-Ser-Gly (wherein y is 2 or greater)). In some embodiments, the linker is a single amino acid (e.g., any amino acid such as Gly or Ala).

[0358] Another embodiment disclosed herein comprises a peptide or complex bound to the aforementioned linker, wherein the aforementioned substance is an active ingredient. Another embodiment disclosed herein may be a complex comprising a substance bound to the peptide or linker. A "bound substance" may be any substance bound to the peptide or linker, and examples of bound substances include pharmaceuticals and pharmaceutical active ingredients.

[0359] The aforementioned substance can be any substance desired by a person skilled in the art as long as it is a substance that is intended to be delivered to TfR. Furthermore, when using the pH-dependent peptides of the present invention, the aforementioned substance can be a substance that is intended to be delivered to the brain. Examples of such substances include, but are not limited to, the following:

[0360] Compound: Not only low-molecular-weight compounds and medium-molecular-weight compounds, but any compound that can be introduced into cells through the cytosis mechanism may be used. Examples thereof include well-known low-molecular-weight drugs.

[0361] Peptide: A peptide that binds to a target in the body and exhibits a certain effect, and may be, for example, a cyclic peptide.

[0362] RI: Low-molecular-weight or medium-molecular-weight compounds or antibodies labeled with radioactive isotopes. Any compound that can be labeled with a radioactive isotope may be used. Examples include compounds for PET examination.

[0363] Protein: Any protein that exhibits a useful function in the body, such as an antibody or enzyme, may be used. Examples include enzymes used in enzyme supplementation therapy.

[0364] Nucleic acid: Any nucleic acid containing a base sequence such as DNA or RNA. Examples include nucleic acid pharmaceuticals.

[0365] DDS: It can be a DDS molecule such as liposomes or micelles. The DDS molecule can further contain compounds such as pharmaceuticals.

[0366] Furthermore, the aforementioned substances may be complexes of the aforementioned substances.

[0367] Another embodiment disclosed in this specification relates to a composition comprising any of the aforementioned peptides or a composition comprising any of the aforementioned complexes. The composition may also contain known materials other than the peptide or complex (e.g., a carrier, a solvent). Another embodiment disclosed in this specification relates to a method for producing a pharmaceutical or diagnostic composition. This method is a method for producing a preventive, therapeutic, or diagnostic agent comprising the step of obtaining the aforementioned complex.

[0368] Preferred examples of this method include linkers such as polyethylene glycol (PEG), G linkers, GS linkers, and GKN3 linkers. Alternatively, the linkers may be sequences corresponding to the linker portions of SEQ ID NOs: 1-68 shown in Table 1-1. Alternatively, the linkers may be sequences corresponding to the linker portions of SEQ ID NOs: 168-197 shown in Table 1-3.

[0369] Another embodiment disclosed herein relates to a pharmaceutical or diagnostic composition comprising a complex comprising the aforementioned peptide, or a complex of the peptide further bound to a linker. Preferably, the complex is a pharmaceutical or diagnostic composition for TfR-related diseases.

[0370] The peptide of the present invention can be produced by well-known peptide production methods such as chemical synthesis methods such as liquid phase methods, solid phase methods, hybridization methods combining liquid phase methods and solid phase methods, and gene recombination methods.

[0371] For example, the solid-phase method involves an esterification reaction between the hydroxyl group of a hydroxyl-containing resin and the carboxyl group of a first amino acid (typically the C-terminal amino acid of the target peptide) whose α-amino group is protected by a protecting group. Esterification catalysts include well-known dehydration condensation agents such as 1-trimethylbenzylsulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIPCDI).

[0372] Next, the protecting group on the α-amino group of the first amino acid is removed, and a second amino acid, in which all functional groups except the main chain carboxyl group are protected, is added. This carboxyl group is activated, allowing the first and second amino acids to bind. Furthermore, the α-amino group of the second amino acid is deprotected, and a third amino acid, in which all functional groups except the main chain carboxyl group are protected, is added. This carboxyl group is activated, allowing the second and third amino acids to bind. This process is repeated until all functional groups are deprotected when a peptide of the desired length is synthesized.

[0373] Examples of resins synthesized in the solid phase include Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGA resin (Merck), and HMPA-PEGA resin (Merck). These resins can be used after washing with a solvent such as dimethylformamide (DMF), 2-propanol, or methyl chloride.

[0374] Examples of the protecting group for the α-amino group include benzyloxycarbonyl (Cbz or Z), tert-butyloxycarbonyl (Boc), fluorenylmethyloxycarbonyl (Fmoc), benzyl, allyl, and allyloxycarbonyl (Alloc).

[0375] 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.

[0376] The α-carboxyl group can be protected by using methyl ester, ethyl ester, benzyl ester, tert-butyl ester, cyclohexyl ester or the like.

[0377] As other amino acid functional groups, the hydroxyl group of serine or threonine can be protected with a benzyl or tert-butyl group, and the hydroxyl group of tyrosine can be protected with a 2-bromobenzyloxycarbonyl or tert-butyl group. The amino group of the lysine side chain and the carboxyl group of glutamic acid or aspartic acid can be protected in the same manner as the α-amino and α-carboxyl groups.

[0378] The carboxyl groups can be activated using a condensing agent. Examples of the condensing agent 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-3-oxide hexafluorophosphate (HBTU).

[0379] The peptide chain from the resin can be cleaved by treating with an acid such as TFA or hydrogen fluoride (HF).

[0380] The production of peptides by genetic recombination (translational 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 may be DNA or RNA.

[0381] Nucleic acids encoding the peptides of the present invention can be prepared using well-known methods or methods that follow them. For example, they can be synthesized using an automated synthesizer. In order to insert the resulting DNA into a vector, a restriction enzyme recognition site can be added, or a nucleic acid having a base sequence encoding the amino acid sequence of the resulting peptide chain to be cleaved by an enzyme or the like can be incorporated.

[0382] 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 the membrane-permeable peptide.

[0383] In order to inhibit degradation by host-derived proteases, a chimeric protein expression method can be used to express the target peptide as a chimeric peptide with other peptides. In this case, as the nucleic acid, a nucleic acid encoding the target peptide and a peptide binding thereto can be used.

[0384] Next, an expression vector is prepared using the nucleic acid encoding the peptide of the present invention. The nucleic acid can be inserted downstream of the promoter of the expression vector directly, by digestion with a restriction enzyme, or by the addition of a linker. Examples of vectors include plasmids derived from Escherichia coli (pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pUC118, pBluescript II, etc.), plasmids derived from Bacillus subtilis (pUB110, pTP5, pC1912, pTP4, pE194, pC194, etc.), plasmids derived from yeast (pSH19, pSH15, YEp, YRp, YIp, YAC, etc.), bacteriophages (e phage, M13 phage, etc.), viruses (retroviruses, poxviruses, adenoviruses, adeno-associated viruses (AAV), cauliflower mosaic virus, tobacco mosaic virus, baculovirus, etc.), and adhesive plasmids.

[0385] The promoter can be appropriately selected depending on the host species. When the host is an animal cell, for example, a promoter derived from SV40 (simian virus 40) or a promoter derived from CMV (cytomegalovirus) can be used. When the host is Escherichia coli, the trp promoter, T7 promoter, lac promoter, etc. can be used.

[0386] The expression vector may also include nucleic acids encoding DNA replication origin (ori), selection markers (antibiotic resistance, nutrient requirements, etc.), enhancers, splicing signals, poly A addition signals, tags (FLAG, HA, GST, GFP, etc.), etc.

[0387] Next, appropriate host cells are transformed using the expression vector described above. The host can be appropriately selected based on its relationship with the vector; for example, Escherichia coli, Bacillus subtilis, Bacillus, yeast, insects or insect cells, animal cells, etc. can be used. Animal cells, for example, HEK293T cells, CHO cells, COS cells, myeloma cells, HeLa cells, and Vero cells can be used. Transformation can be performed using well-known methods such as lipofection, calcium phosphate method, electroporation, microinjection, and particle gun method, depending on the type of host. The target peptide is expressed by culturing the transformant according to conventional methods.

[0388] Peptides from transformant cultures are purified by obtaining a crude extract by recovering the cultured cells, suspending them in an appropriate buffer, disrupting them by sonication, freeze-thaw, or other methods, and then centrifuging and filtering them. If the peptide is secreted in the culture medium, the supernatant is recovered.

[0389] Purification from a crude extract or culture supernatant can also be performed by well-known methods or methods adapted therefrom (e.g., salting out, dialysis, ultrafiltration, gel filtration, SDS-PAGE, ion exchange chromatography, affinity chromatography, reversed-phase high-performance liquid chromatography, etc.).

[0390] The obtained peptide can also be converted from a free form to a salt, or from a salt to a free form, by a well-known method or a method in accordance therewith.

[0391] The translation synthesis system can be configured as a cell-free translation system. The cell-free translation system, for example, comprises ribosomal proteins, aminoacyl-tRNA synthetase (ARS), ribosomal RNA (rRNA), amino acids, 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, Escherichia coli extract or wheat germ extract can also be added. In addition, rabbit red blood cell extract or insect cell extract can also be added.

[0392] In a system including such a system, by continuously supplying energy using dialysis, it is possible to produce several 100 μg to several mg / mL of protein. In order to simultaneously perform transcription from gene DNA, a system including RNA polymerase can also be provided. As commercially available cell-free translation systems, as systems derived from Escherichia coli, the RTS-100 (registered trademark) of Roche Diagnostics, the PURESYSTEM of GeneFrontier, the PURExpress InVitro Protein Synthesis Kit of NEW ENGLAND Biolabs, etc. can be used. As systems using wheat germ extract, products of ZOEGENE and CellFree Sciences can be used.

[0393] If based on a cell-free translation system, the expression product can be obtained in a highly pure form without purification.

[0394] In cell-free translation systems, artificial aminoacyl-tRNAs, created by linking (acylating) tRNAs with the desired amino acid or hydroxy acid, can be used instead of aminoacyl-tRNAs synthesized by natural aminoacyl-tRNA synthetases. These aminoacyl-tRNAs can be synthesized using artificial ribonucleases.

[0395] 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 tRNA aminoacylation tool for the synthesis of nonnatural peptides"; N.Niwa,Y.Yamagishi,H.Murakami,H.Suga(2009)Bioorganic&Medicinal Chemistry Letters 19,3892-3894"A Elastase is also known as prototype elastase (Fx), and its modified dinitrobenzyl elastase (dFx), enhanced elastase (eFx), amino elastase (aFx), etc.

[0396] By using tRNA generated by elastase and conjugated to the desired amino acid or hydroxy acid, the desired codon can be linked to the desired amino acid or hydroxy acid and translated. Special amino acids can also be used as the desired amino acid. For example, the unnatural amino acids required for the aforementioned cyclization can also be introduced into the conjugated peptide using this method.

[0397] The chemical synthesis of the macrocyclic peptides and their mimetics of the present invention can be performed using a variety of methods commonly used in the art, including stepwise solid-phase synthesis, semisynthesis of peptide fragments via conformationally-assisted religation, and chemical ligation. The peptides and their mimetics described herein can be synthesized using various solid-phase techniques, such as those described in KJ Jensen, PT Shelton, SL Pedersen, Peptide Synthesis and Applications, 2nd Edition, Springer, 2013. A preferred strategy is to combine a temporarily protected α-amino group with an Fmoc group that can be selectively removed by a base, and a protecting group that temporarily protects the side chain functional group and is stable under Fmoc removal conditions. The selection of such general peptide side chains is known from the aforementioned Peptide Synthesis and Applications, 2nd edition, GB Fields, RL Noble, Solid pHase Peptide Synthesis Utilizing 9-Fluorenylmethoxycarbonyl Amino Acids, Int. J. Peptide Protein Res. 35, 1990, 161-214, etc., but preferred peptide side chain protecting groups include Boc or Mtt groups for amino groups headed by lysine, tert-butyl groups for carboxyl groups of glutamic acid or aspartic acid, and Trt and Mmt groups for thiol groups of cysteine.

[0398] The peptides and their mimetics described in the present invention can be synthesized on the aforementioned solid phase resin in a staged method. The α-amino protecting group of the C-terminal amino acid used and all amino acids and peptides used in the synthesis must be selectively removed during the synthesis process. Preferably, the aforementioned solid phase resin is used, and the C-terminal carboxyl group of a peptide appropriately protected by Fmoc or the C-terminal carboxyl group of an amino acid protected by Fmoc is made into an activated ester by an appropriate reagent, and then added to the amino group on the solid phase resin to start. The elongation of the continuous peptide chain can be achieved by repeating the removal of the N-terminal protecting group (Fmoc group) and then the condensation of the protected amino acid derivative in sequence, following the amino acid sequence of the target peptide. In addition, this can free the target peptide in the final stage. For example, conditions for dissociation include those described by Teixeira, WE Benckhuijsen, PE de Koning, ARP M Valentijn, JW Drijfhout, Protein Pept. Lett., 2002, 9, 379-385. In TFA, a TFA solution containing water / silyl hydride / thiol as a scavenger can be used to dissociate the molecule. A typical example is TFA / Water / TIS / DODT (volume ratio 92.5:2.5:2.5:2.5).

[0399] The synthesis of the peptidomimetics described herein 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.

[0400] Activation of the carboxyl group can be performed using a condensing agent. Examples of the condensing agent 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-3-oxide hexafluorophosphate (HBTU).

[0401] Another embodiment disclosed in this specification relates to a pharmaceutical composition. This pharmaceutical composition comprises the aforementioned peptide, a pharmaceutically acceptable salt or solvate thereof (hereinafter referred to simply as a peptide for simplicity), or a complex comprising the aforementioned peptide and a binding agent further bound to a linker. This pharmaceutical composition preferably contains an effective amount of the aforementioned peptide as an active ingredient.

[0402] In this specification, the administration method of the pharmaceutical composition is not particularly limited and can be administered orally or parenterally. Examples of parenterally administered pharmaceutical compositions include intramuscular injection, intravenous injection, subcutaneous injection, and other injections, transdermal administration, and transmucosal administration (nasal, oral, ocular, pulmonary, vaginal, and rectal).

[0403] The pharmaceutical compositions described above can be modified in various ways to account for the polypeptide's readily metabolized and excreted properties. For example, adding polyethylene glycol (PEG) or sugar chains to the polypeptide can increase its blood retention time and reduce its antigenicity. Furthermore, sustained-release bases such as polylactic acid-ethylene glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, and emulsions made from unsaturated fatty acids, nanoparticles, and nanospheres can be used, and the polypeptide can be incorporated into these. For transdermal administration, a weak electric current can be passed through the skin surface to allow the drug to penetrate the stratum corneum (iontophoresis).

[0404] The above-mentioned pharmaceutical compositions may be used as the active ingredient or may be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc. Examples of dosage forms include liquids (e.g., injections), dispersions, suspensions, tablets, pills, powders, suppositories, powders, granules, granules, capsules, syrups, lozenges, inhalants, ointments, eye drops, nasal drops, ear drops, and poultices.

[0405] Formulation can be carried out by conventional methods using, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubility aids, colorants, flavoring agents, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, preservatives, antioxidants, and the like as appropriate.

[0406] Examples of ingredients that can be used for formulation include, but are not limited to, purified water, saline, phosphate buffer, pharmaceutically acceptable organic solvents such as glucose, glycerol, and ethanol, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, silicon dioxide, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, gum tragacanth, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, lauryl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, and human serum albumin.

[0407] Given the difficulty of peptides being absorbed through the mucosa, the pharmaceutical composition may contain an absorption enhancer to improve the absorption of poorly absorbed drugs. Examples of such absorption enhancers include surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponins; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylates; fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, and mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, and nitric oxide donors.

[0408] Pills or lozenges may also be coated with sugar, gastric-soluble substances, or enteric-soluble substances.

[0409] Injections may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohol, etc. Furthermore, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, solubility aids, preservatives, etc. may be added.

[0410] The dosage of the pharmaceutical composition of the present invention when administered to mammals (e.g., humans, mice, rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs, etc.), especially humans, varies depending on the symptoms, the patient's age, sex, weight, sensitivity, administration method, administration interval, type of active ingredient, and type of formulation. Although not particularly limited, for example, 30 μg to 100 g, 100 μg to 500 mg, or 100 μg to 100 mg can be administered once or in divided doses. In the case of injection, 1 μg / kg to 3000 μg / kg or 3 μg / kg to 1000 μg / kg can be administered once or in divided doses depending on the patient's weight.

[0411] "TfR-related diseases" refer to diseases in which TfR is a therapeutic target protein, as well as diseases caused by cells or tissues expressing TfR. Furthermore, since TfR can cross the blood-brain barrier by binding to TfR, these diseases include brain diseases (diseases caused by abnormalities in the brain, such as central nervous system (CNS) diseases) and neuromuscular diseases (diseases characterized by movement disorders such as decreased muscle strength caused by lesions in nerves or muscles, such as the brain, spine, and peripheral nerves), as well as diseases with high TfR expression in muscle tissue.

[0412] TfR-related disease detection drugs and detection kits

[0413] The present invention also includes TfR-related disease detection drugs containing the peptides of the present invention. When used as detection drugs, the peptides of the present invention can also be labeled to be detectable. The labeling of the peptide can be, for example, antibodies labeled with enzymes such as peroxidase, alkaline phosphatase, radioactive substances such as 125I, 131I, 35S, 3H, fluorescent isothiocyanates, rose bengal, dansyl chloride, phycoerythrin, tetramethylrhodamine isothiocyanate, near-infrared fluorescent materials, fluorescent substances such as luciferase, fluorescein, aequorin, etc. Others can also detect antibodies labeled with nanoparticles such as gold colloids and quantum dots. For example, a complex is made with an antibody that binds to a specific target associated with a TfR-related disease and the peptide of the present invention, a complex having the labeled antibody or peptide of the present invention is made, and it is administered and detected, thereby detecting TfR-related diseases.

[0414] Furthermore, in immunoassays, the peptide of the present invention can be labeled with biotin and detected by binding it to avidin or avidin labeled with an enzyme or the like.

[0415] In immunoassays, the ELISA method using enzyme labeling is also preferred because it can measure antigens simply and quickly. For example, an antibody is fixed to a solid phase carrier, a sample is added and reacted, and then the labeled peptide of the present invention is added and allowed to react. After washing, it is reacted with an enzyme substrate to develop color, and the absorbance is measured, thereby detecting TfR-related diseases. After the antibody fixed to the solid phase carrier is reacted with the sample, an unlabeled peptide of the present invention is added, and the antibody against the peptide of the present invention is enzyme-labeled and further added.

[0416] When the enzyme is peroxidase, the enzyme substrate may include 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), etc. When the enzyme is alkaline phosphatase, p-nitrophenyl phosphate (NPP), etc. may be used.

[0417] In this specification, the term "solid phase carrier" is not particularly limited as long as it is a carrier that can fix antibodies. Examples include microporous plates, substrates, beads, nitrocellulose membranes, nylon membranes, PVDF membranes, etc. made of glass, metal, or resin. The target substance can be fixed on such solid phase carriers according to well-known methods.

[0418] The test kit of the present invention comprises the reagents and instruments required for the above-mentioned detection (including, but not limited to, the peptide of the present invention, antibodies, solid phase carriers, buffers, enzyme reaction stopping solution, microplate reader, etc.).

[0419] Another embodiment disclosed in this specification can also be considered to be used as a detection kit containing the above-mentioned TfR-related disease detection drug, a tool for discovering TfR-related diseases and various cell functions or life phenomena associated therewith.

[0420] This specification also provides for the use of peptides for the production of pharmaceutical or diagnostic peptides. In this case, the peptide can be any of the above-mentioned peptides.

[0421] This specification also provides a method for preventing or treating a TfR-related disease, comprising administering an effective amount of a peptide, a pharmaceutically acceptable salt thereof, a solvate thereof, or a complex thereof to a human, non-human mammal, or bird, i.e., a subject. The peptides described above can be used as appropriate. Examples of non-human mammals include non-human primates, pigs, cattle, dogs, cats, horses, sheep, rats, and mice.

[0422] This specification also provides a method for preventing or treating a TfR-related disease, comprising administering an effective amount of a peptide, a pharmaceutically acceptable salt thereof, or a solvate or complex thereof to a human, non-human mammal, or bird, i.e., a subject.

[0423] The present specification provides a method for testing a complex of a peptide and / or a substance comprising a peptide and a conjugate further bound to a linker.

[0424] a) Solubility in solvents;

[0425] b) binding ability to hTfR;

[0426] c) toxicity to cells and / or tissues;

[0427] d) toxicity to experimental animals,

[0428] A test method for at least one peptide and / or complex,

[0429] The aforementioned peptide has an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, inserted, and / or added to the amino acid sequence of the peptide of the present invention.

[0430] Furthermore, as one limitation of the aforementioned method, b) the binding ability to hTfR may be pH-dependent binding ability to hTfR.

[0431] The peptide of the present invention may be any peptide described in this specification, but in a preferred embodiment, may be a peptide having the amino acid sequence of peptide A and / or peptide B. Furthermore, it may be a peptide comprising an amino acid sequence described in SEQ ID NO: 1 that satisfies at least one of the following conditions:

[0432] (I) the first position of SEQ ID NO: 1 is an alanine residue;

[0433] (II) the fourth position of SEQ ID NO: 1 is a valine residue;

[0434] (III) the sixth residue of SEQ ID NO: 1 is an aspartic acid residue;

[0435] (IV) the 7th position of SEQ ID NO: 1 is a tyrosine residue or a 3Py6NH2 residue;

[0436] (V) The 9th residue of SEQ ID NO: 1 is an isoleucine residue,

[0437] Furthermore, the peptide may satisfy all of these requirements.

[0438] In addition, in the above, the alanine residue of (I) can also be a derivative thereof, the valine residue of (II) can also be a derivative thereof, the aspartic acid residue of (III) can also be a derivative thereof, the tyrosine residue or 3Py6NH2 residue of (IV) can also be a derivative thereof, and the isoleucine residue of (V) can also be a derivative thereof.

[0439] Furthermore, the above-mentioned options (I) to (V) can be selected in any combination.

[0440] Furthermore, the peptide of the present invention is not limited, but in one aspect, comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 1-202, preferably SEQ ID NOs: 1-161 and 168-202, more preferably SEQ ID NOs: 1-149 and 168-197, and most preferably SEQ ID NOs: 1-68. In one aspect, the peptide is a cyclic peptide comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1-202, preferably SEQ ID NOs: 1-161 and 168-202, more preferably SEQ ID NOs: 1-149 and 168-197, and most preferably SEQ ID NOs: 1-68. Although not limiting, one of ordinary skill in the art to which the present invention relates can appropriately select 1 to 3 amino acids from the amino acid sequence set forth in any of SEQ ID NOs: 1-202, preferably 1-161 and 168-202, to create a sequence in which the amino acids have been deleted, substituted, inserted, and / or added. In the aforementioned method, the number of amino acids deleted, substituted, inserted, and / or added is 1 to 3, preferably 1 to 2, and more preferably 1.

[0441] Furthermore, although not limited to deletion, substitution, insertion and / or addition of amino acid residues, substitution is preferred, and conservative amino acid substitution is more preferred.

[0442] The aforementioned peptide may also be a peptide comprising an amino acid sequence having at least 80% or more, preferably 85% or more, 86% or more, 87% or more, 88% or more, 90% or more, 91% or more, 92% or more, 93% or more, or 94% or more amino acid identity with the amino acid sequence possessed by the peptide of the present invention, or a peptide composed of the amino acid sequence.

[0443] The percent identity between two amino acid sequences can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity between two protein sequences can be determined by comparing sequence information based on an algorithm based on Needleman, SB and Wunsch, CD (J. Mol. Biol., 48:443-453, 1970) using the GAP computer program available from the University of Wisconsin Genetics Computer Group (UWGCG). Preferred default parameters for the GAP program include: (1) a scoring matrix, blosum62, as described by Henikoff, S. and Henikoff, JG (Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992); (2) a gap weight of 12; (3) a gap length weight of 4; and (4) no penalty for terminal gaps.

[0444] Other programs for sequence comparison that can be used by those of ordinary skill in the art to which the present invention belongs can also be used. The percentage of consistency can be determined by comparing with sequence information using the BLAST program described in, for example, Altschul et al. (Nucl. Acids. Res., 25, p. 3389-3402, 1997). The program can be used from the website of the National Center for Biotechnology Information (NCBI) or the DNA Data Bank of Japan (DDBJ) on the Internet. The various conditions (parameters) for consistency searches performed by the BLAST program are described in detail on the same website, and some of the settings can be appropriately changed, but the search is performed using the usual preset values. Alternatively, the % consistency of two amino acid sequences can also be determined using programs such as the genetic information processing software GENETYX Ver. 7 (manufactured by GENETYX), or the FASTA algorithm. In this case, the preset values ​​can be used for the search.

[0445] A person skilled in the art can use the amino acid sequence thus prepared as a basis to produce peptides for use in the test method of the present invention by known peptide production methods such as chemical synthesis methods such as the above-mentioned liquid phase method, solid phase method, and hybrid method combining liquid phase and solid phase methods, and genetic recombination methods.

[0446] Although not limited thereto, the aforementioned peptide having an amino acid sequence in which 1 to 3 amino acid residues have been deleted, substituted, inserted, and / or added (that is, the peptide subjected to the test described in the aforementioned test method) may be a peptide having the amino acid sequence of the aforementioned peptide A and / or peptide B, or may be a peptide comprising an amino acid sequence in the amino acid sequence described in SEQ ID NO: 1 that satisfies at least one of the following conditions:

[0447] (I) the first position of SEQ ID NO: 1 is an alanine residue;

[0448] (II) the fourth position of SEQ ID NO: 1 is a valine residue;

[0449] (III) the sixth residue of SEQ ID NO: 1 is an aspartic acid residue;

[0450] (IV) the 7th position of SEQ ID NO: 1 is a tyrosine residue or a 3Py6NH2 residue;

[0451] (V) The 9th residue of SEQ ID NO: 1 is an isoleucine residue,

[0452] Furthermore, a peptide that satisfies all of these conditions may be used. Such a peptide is believed to have a high ability to bind to hTfR.

[0453] In addition, in the above, the alanine residue of (I) can also be a derivative thereof, the valine residue of (II) can also be a derivative thereof, the aspartic acid residue of (III) can also be a derivative thereof, the tyrosine residue or 3Py6NH2 residue of (IV) can also be a derivative thereof, and the isoleucine residue of (V) can also be a derivative thereof.

[0454] Furthermore, the above-mentioned options (I) to (V) can be selected in any combination.

[0455] Furthermore, although not limited thereto, the peptide used in the aforementioned test method is preferably a peptide having the amino acid sequence of the aforementioned peptide A and / or peptide B, and further, a peptide comprising an amino acid sequence that satisfies at least one of the aforementioned (I) to (V) in the amino acid sequence described in SEQ ID NO: 1 and conforms to at least any one of the following A or B.

[0456] A: At least two of the following are met:

[0457] (a) the fifth residue of SEQ ID NO: 1 is a W7N residue or a 3Imp residue;

[0458] (b) The 7th residue of SEQ ID NO: 1 is 3Py6NH2 residue;

[0459] (c) The eighth residue of SEQ ID NO: 1 is F4aao (pipzaa);

[0460] (d) The 13th residue of SEQ ID NO: 1 is a 4Py residue.

[0461] B: (i) The 8th position of SEQ ID NO: 1 is a F4aao (pipzaa) residue, a F4aao (Glucamine) residue, a F4aao (PEG8Me) residue or a YaeCOpipzaa residue.

[0462] This peptide is believed to have a high binding affinity to hTfR and a pH-dependent binding ability.

[0463] In addition, in the above, the W7N residue or 3Imp residue in (a) can also be its derivatives, the 3Py6NH2 residue in (b) can also be its derivatives, the F4aao (pipzaa) residue in (c) can also be its derivatives, and the 4Py residue in (d) can also be its derivatives.

[0464] Furthermore, in the above, the F4aao(pipzaa) residue, F4aao(Glucamine) residue, F4aao(PEG8Me) residue or YaeCOpipzaa residue in (i) may also be derivatives thereof.

[0465] Furthermore, the options (I) to (V), (a) to (d), and (i) above can be selected in any combination.

[0466] However, it is preferred that the peptide having the amino acid sequence described in SEQ ID NO: 162 be excluded from any of the above peptides.

[0467] Regarding the aforementioned test methods, testing the solubility of a peptide in a solvent can be performed as a solubility measurement. When measuring solubility, the solvent is not limited and can be freely selected according to the intended purpose. Furthermore, the solubility measurement method can be appropriately selected from known methods depending on the type of solvent.

[0468] The test for hTfR binding ability can be performed by measuring the binding ability to hTfR. Although not limited to the above methods, known methods such as surface plasmon resonance (SPR) analysis, Scatchard analysis, and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assay can be preferably used. Furthermore, the pH-dependent binding ability to hTfR can be tested by measuring the binding ability to hTfR at various pH values ​​as described above.

[0469] Toxicity tests for cells and / or tissues can be known toxicity evaluation tests using cells and / or tissues, for example, methods performed in vitro. The cells and tissues can be those commonly used in toxicity evaluation tests for pharmaceuticals, without limitation.

[0470] The test method for toxicity in experimental animals can be a known toxicity evaluation test using experimental animals. The experimental animals are not particularly limited as long as they are commonly used, but examples include mice, rats, guinea pigs, gerbils, hamsters, ferrets, rabbits, dogs, cats, pigs, goats, horses, cattle, birds (e.g., chickens, quail, etc.), monkeys, and primates excluding humans (e.g., cynomolgus macaques, marmosets, rhesus monkeys, etc.).

[0471] The above-mentioned toxicity evaluation tests are not limited, but can be tests related to safety that are generally conducted in non-clinical trials of pharmaceuticals. Examples include general toxicity tests (single-dose toxicity test / repeated-dose toxicity test), genotoxicity tests (Ames test / chromosomal abnormality test / in vitro micronucleus test), carcinogenicity tests, reproductive toxicity tests (ICH-I, II, III), local irritation tests (eye irritation test, skin irritation test, etc.), other toxicity tests (skin sensitization test, phototoxicity test, antigenicity test), chemical analysis / in vivo analysis (TK / PK), etc.

[0472] 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:

[0473] Alexa Fluor (registered trademark) 647 is AF647;

[0474] Angstrom (unit) Chloroacetyl is ClAc;

[0475] Sulfo-Cy5-alkyne is Cy5SAlk;

[0476] Dichloromethane is DCM;

[0477] triisopropylsilyl is TIPS;

[0478] The tertiary butyl group is tBu;

[0479] Dithiothreitol is DTT;

[0480] Dimethyl sulfoxide is DMSO;

[0481] Trityl is Trt;

[0482] The tertiary butoxycarbonyl group is Boc;

[0483] N,N-dimethylformamide is DMF;

[0484] N,N-diisopropylethylamine is DIEA or DIPEA;

[0485] N,N'-diisopropylcarbodiimide is DIPCI;

[0486] Ethyl cyano(hydroxyimino)acetate was Oxyma Pure;

[0487] 3,6-dioxa-1,8-octane-dithiol is DODT;

[0488] 9-fluorenylmethyloxycarbonyl is Fmoc;

[0489] Gram (unit) is g;

[0490] O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate is HATU;

[0491] N-hydroxysuccinimide is HOSu;

[0492] High-speed liquid chromatography is HPLC;

[0493] Liquid chromatography mass spectrometry is called LC-MS or LC / MS

[0494] Milliliter (unit) is mL;

[0495] The mole (unit) is M;

[0496] Microliter (unit) is μL;

[0497] Millimole (unit) is mM;

[0498] Micromolar (unit) is μM;

[0499] Millimole (unit) is mmol;

[0500] Milligram (unit) is mg;

[0501] Acetonitrile is MeCN or CH3CN;

[0502] Minutes (unit) is min:

[0503] Millimeter (unit) is mm;

[0504] Micrometer (unit) is μm;

[0505] Nanometer (unit) is nm;

[0506] Nanomole (unit) is nM;

[0507] Succinimide is OSu;

[0508] Polyethylene glycol is PEG;

[0509] The unit of revolutions per minute is rpm;

[0510] The tertiary butyl group is tBu;

[0511] trifluoroacetic acid is TFA;

[0512] Triisopropylsilane is TIS;

[0513] Trityl is Trt or Tr;

[0514] 2,5,8,11-tetraoxatridecan-13-amine is H-PEG4Me;

[0515] 2,5,8,11,14,17,20,23-octaoxapentacosane-25-amine is H-PEG8Me (CAS No. 869718-81-0);

[0516] The amino acid is AA;

[0517] 7-((Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate is PyAOP;

[0518] 10-Camphorsulfonic acid is CSA;

[0519] N-(9-fluorenylmethoxycarbonyloxy)succinimide is Fmoc-OSu;

[0520] Tetrahydrofuran is THF;

[0521] N-(chloroacetoxy)succinimide is ClAcOSu;

[0522] ginseno(dibenzylideneacetone)dipalladium(0) is Pd2(dba)3;

[0523] 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl is SPhos;

[0524] 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is EDCI / HCl;

[0525] Tetrakis(triphenylphosphine)palladium(0) is Pd(PPh3)4;

[0526] 2-(piperidin-1-yl)acetic acid tert-butyl ester is H-pipzaa(tBu) and is Pd(PPh3)4 (CAS No. 827614-56-2);

[0527] Tert-Butyl 1-amino-3,6,9,12-tetraoxapentadecan-15-oate is H-PEG4c(tBu) (CAS No. 581065-95-4);

[0528] triethylammonium acetate is TEAA;

[0529] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-((allyloxy)carbonyl)phenyl)propanoic acid is Fmoc-F3COO(allyl)-OH (CAS No. 293141-52-1);

[0530] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(allyloxy)-5-oxopentanoic acid is Fmoc-E(allyl)-OH (CAS No. 133464-46-7);

[0531] (3-Aminopropyl)carbamic acid tertiary butyl ester is Boc-apa-H (CAS No. 75178-96-0);

[0532] Propane-1,3-diamine is apa (CAS No. 109-76-2);

[0533] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-3-(4-(allyloxy)carbonyl)phenyl)propanoic acid is Fmoc-MeF4COO(allyl)-OH (CAS No. 2973754-47-9).

[0534] Example 1

[0535] Chemical synthesis

[0536] All raw materials, building blocks, reagents, acids, bases, solid phase resins, and solvents used in the chemical syntheses in the following examples were either commercially available or synthesized using organic chemistry techniques by one of ordinary skill in the art. Furthermore, amino acids containing protecting groups were used as commercially available products unless otherwise specified.

[0537] Peptide chain extension on a solid-phase resin was performed using the resin described in each example as the starting material, using commonly used peptide coupling reaction conditions and Fmoc removal reaction conditions. The reactions were performed using a CEM Liberty Blue automated peptide synthesizer according to the manufacturer's manual. Common amino acids that can be used are listed below, with side chain protecting groups indicated in parentheses.

[0538] 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.

[0539] The introduction of the chloroacetyl group was carried out as follows: After removing the Fmoc group of the α-amino group by the above-mentioned method, chloroacetic acid (3 equivalents) was added to a DMF solution (0.5 M) of 3 equivalents of N,N'-diisopropylcarbodiimide and a DMF solution (0.5 M) of 3 equivalents of HOAt, and the mixture was shaken at room temperature for 40 minutes.

[0540] To deprotect the side chains and cleave them from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and five times with methyl chloride, then dried under reduced pressure. Next, the reaction mixture (cocktail A) (a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 150 minutes. The reaction solution was recovered by filtration through a glass frit. The solid-phase resin remaining in the reaction vessel was shaken again with the shearing mixture, and the solution fraction was recovered through the glass frit and mixed with the filtrate described above. When this filtrate was added to excess diethyl ether cooled to 0°C, a cloudy white precipitate formed. The mixture was centrifuged (9000 rpm, 3 minutes), and the solution was decanted. The resulting solid was washed again with a small amount of diethyl ether cooled to 0°C, and the resulting solid was used in the subsequent cyclization reaction.

[0541] The peptide cyclization reaction was performed by dissolving the peptide in DMSO to a final concentration of 5 mM based on the molar ratio of the solid phase resin. Six equivalents of triethylamine were then added and stirred at room temperature for approximately 16 hours. The resulting reaction solution was acidified with acetic acid and concentrated under reduced pressure using a Biotage (registered trademark) V-10 (Biotage Japan).

[0542] The crude peptide was purified using a Waters AutoPurification System-SQD2 single quadruple mass spectrometer with reversed-phase fractionation HPLC, eluting the target product while monitoring its m / z. The mass spectrum obtained using ESI-positive scanning mode was confirmed to be consistent with the mass spectrum of the multivalent ions calculated from the target product's molecular formula within the error range of the mass analyzer used. The purification conditions, including the column used, are shown in the Examples.

[0543] The structure of chemically synthesized peptides is determined by confirming the molecular weight calculated based on the amino acids used in the target sequence and the structural units used as needed using ESI-MS(+) mass spectrometry. "ESI-MS(+)" refers to electrospray ionization mass spectrometry performed in positive ion mode. Detected masses are expressed in "m / z" units. Compounds with a molecular weight greater than approximately 1000 are detected at a high frequency as divalent or trivalent ions. Analysis is performed using the following method (Analysis Conditions A and B) or the method described in the Examples.

[0544] Analytical methods

[0545] Analysis Condition A

[0546] Column: Kinetex EVO C18 2.6μm, 2.1IDx150mm, (Phenomenex)

[0547] Column temperature: 60°C

[0548] Mobile phase A: 0.025% TFA in water

[0549] Mobile phase B: 0.025% TFA in CH3CN

[0550] Gradient: 5-45% in 7.2 minutes

[0551] Flow rate: 0.5 mL / min

[0552] Detection: PDA (225 nm)

[0553] Analysis Condition B

[0554] Column: Kinetex EVO C18 2.6μm, 2.1IDx150mm, (Phenomenex)

[0555] Column temperature: 60°C

[0556] Mobile phase A: 0.025% TFA in water

[0557] Mobile phase B: 0.025% TFA in CH3CN

[0558] Gradient: 20-60% in 7.2 minutes

[0559] Flow rate: 0.5 mL / min

[0560] Detection: PDA (225 nm)

[0561] Example 2

[0562] Chemical synthesis of a specific cyclic peptide that binds to hTfR

[0563] Peptides with hTfR binding activity and hTfR-binding peptides with linkers were chemically synthesized. The sequences of the synthesized peptides are disclosed in Tables 1-1, 1-2, 2, and 3. The synthesized peptides were analyzed using the analytical conditions described in the respective Examples, and their structures were confirmed by ESI-MS(+) mass spectrometry. The resulting ESI-MS(+) observation values ​​and the value of X, expressed as the protonation number (M+XH)X+, are disclosed in Tables 1-1, 1-2, 1-3, 2-1, 2-2, and 3. The N-termini of all peptides were -NH2.

[0564] In addition, when there are differences between the sequences in the sequence listing and the sequences in the following tables, the sequences in the following tables are correct.

[0565] [Table 1-1]

[0566] [Table 1-1-1-1]

[0567]

[0568] [Table 1-1-1-2]

[0569]

[0570] [Table 1-1-2-1]

[0571]

[0572] [Table 1-1-2-2]

[0573]

[0574] [Table 1-1-3-1]

[0575]

[0576] [Table 1-1-3-2]

[0577]

[0578] [Table 1-2]

[0579] [Table 1-2-1-1]

[0580]

[0581] [Table 1-2-1-2]

[0582]

[0583] [Table 1-2-2-1]

[0584]

[0585] [Table 1-2-2-2]

[0586]

[0587] [Table 1-2-3-1]

[0588]

[0589] [Table 1-2-3-2]

[0590]

[0591] [Table 1-3]

[0592] [Table 1-3-1-1]

[0593]

[0594] [Table 1-3-1-2]

[0595]

[0596] [Table 1-3-2-1]

[0597]

[0598] [Table 1-3-2-2]

[0599]

[0600] [Table 2-1]

[0601] [Table 2-1-1]

[0602]

[0603] [Table 2-1-2]

[0604]

[0605] [Table 2-2]

[0606]

[0607] [Table 3]

[0608]

[0609] Example 3

[0610] Intermolecular Interactions between Transferrin Receptor (hTfR) and Peptides by Surface Plasmon Resonance (SPR) Evaluation test

[0611] The surface plasmon resonance (SPR)-induced intermolecular interaction of the various synthesized peptides with the transferrin receptor (hTfR) was tested by the following method. The specific test method is shown below.

[0612] [SPR measurement]

[0613] SPR measurements at pH 6.0

[0614] An NTA sensor chip (Cytiva) was inserted into a Biacore T200 (Cytiva), and three initial runs were performed using the electrophoresis buffer: HBS-P+, pH 6.0 (Cytiva). Equilibration was performed at a flow rate of 30 μL / min, and ligand immobilization was performed at a flow rate of 10 μL / min.

[0615] The NTA sensor chip surface was cleaned with 350mM EDTA solution. The carbomethyl dextran chains on the sensor chip were filled with nickel ions using a 500μM nickel chloride aqueous solution, followed by a wash with 3mM EDTA solution. 50μL of each of a 60mM EDC solution (Cytiva) and a 650mM NHS solution (Cytiva) were mixed and reacted at a flow rate of 10μL / min for 420 seconds. A 0.2μM hTfR solution (150μL) was prepared by diluting with 10mM acetic acid (pH 5.0) and reacting at a flow rate of 10μL / min for 420 seconds to immobilize hTfR on the NTA sensor chip. Furthermore, the recombinant human transferrin receptor (hTfR) described in Example 2 of WO2018 / 124121 was used. After immobilization, capping was performed by reacting with a 1.0 M aqueous ethanolamine solution (Cytiva) at a flow rate of 10 μL / min for 420 seconds. A 10 mM peptide solution was prepared in DMSO and diluted with electrophoresis buffer to a final concentration of 10 μM. Peptide solutions were then prepared at 250 nM, 125 nM, 62.5 nM, 25 nM, and 12.5 nM. Using these samples, SPR measurements were performed to determine the kinetics of the peptide on hTfR.

[0616] SPR measurements at pH 7.4

[0617] An NTA sensor chip (Cytiva) was inserted into a Biacore T200 (Cytiva) and subjected to three initial runs using a running buffer consisting of 10 mM HEPES pH 8.0 (nacalai tesque Co., Ltd.), 150 mM NaCl (nacalai tesque Co., Ltd.), 0.05% Tween 20 (nacalai tesque Co., Ltd.), 0.1% BSA (Sigma-Aldrich), and 1.0% DMSO (Fuji Film Wako Pure Chemical Industries, Ltd.). Equilibration was then performed at a flow rate of 30 μL / min. The reaction was then allowed to proceed with a 350 mM EDTA solution at a flow rate of 10 μL / min for 60 seconds, followed by a 0.5 mM NiCl₂ solution (Kishida Chemical) at a flow rate of 10 μL / min for 60 seconds. The NTA sensor chip was then washed with a 3 mM EDTA solution (nacalai tesque Co., Ltd.) at a flow rate of 10 μL / min for 60 seconds. After mixing 50 μL each of a 60 mM EDC solution (Cytiva) and a 650 mM NHS solution (Cytiva), the mixture was reacted at a flow rate of 10 μL / min for 420 seconds. 150 μL of a 0.5 μM hTfR dimer solution was prepared by diluting with electrophoresis buffer. This solution was reacted at a flow rate of 10 μL / min for 300 seconds, and the hTfR was immobilized on an NTA sensor chip. Furthermore, for the human transferrin receptor (hTfR), the recombinant hTfR described in Example 2 of WO2018 / 124121 was used. After immobilization, a 1.0 M aqueous ethanolamine solution (Cytiva) was reacted at a flow rate of 10 μL / min for 420 seconds for capping. A 10 mM peptide solution was prepared in DMSO and diluted with electrophoresis buffer to a final concentration of 10 μM. Then, 100 nM, 50 nM, 25 nM, 10 nM, and 5 nM peptide solutions were prepared. Using these samples, the kinetics of the peptide on hTfR were measured by SPR.

[0618] Alternatively, SPR measurement was performed at pH 7.4 using the following method.

[0619] A CM3 sensor chip (Cytiva) was inserted into a Biacore T200 (Cytiva), and three initial runs were performed using the electrophoresis buffer: HBS-P+, pH 7.4 (Cytiva). Equilibration was performed at a flow rate of 30 μL / min, and ligand immobilization was performed at a flow rate of 10 μL / min.

[0620] After mixing 50 μL each of 60 mM EDC solution (Cytiva) and 650 mM NHS solution (Cytiva), the mixture was reacted at a flow rate of 10 μL / min for 420 seconds. 150 μL of 20 μg / mg THE His Tag Antibody, mAb, Mouse (Gen Script) was prepared by diluting with 10 mM acetic acid solution (pH 5.0) and reacted at a flow rate of 10 μL / min for 420 seconds. The His Tag Antibody, mAb, Mouse (Gen Script) serving as the capture molecule was immobilized on a CM3 sensor chip. After immobilization, 1.0 M ethanolamine aqueous solution (Cytiva) was reacted at a flow rate of 10 μL / min for 420 seconds for capping. 300 nM hTfR was captured at a flow rate of 10 μL / min. A 10 mM peptide solution was prepared in DMSO and diluted with running buffer to a final concentration of 10 μM. Peptide solutions of 250 nM, 125 nM, 62.5 nM, 25 nM, and 12.5 nM were then prepared. The peptide samples were reacted for 120 seconds at a flow rate of 30 μL / min, followed by a 600-second delay. The kinetics of the peptide binding to hTfR were then determined by SPR measurement.

[0621] Measurement of koff and KD (nM)

[0622] The kinetic evaluation model was set to Single Cycle Kinetics, and curve fitting was performed using Biacore T200 Evaluation Software Version 3.0 (Cytiva). The resulting sensorgram was subjected to curve fitting using the least squares method, and the binding of the peptide to hTfR was evaluated by calculating its kon value, koff value, and KD value. The same results were shown in any of the above methods when measured at pH 7.4. The KD values ​​obtained in this way are disclosed in Tables 1-1, 1-2, 1-3, Tables 2-1, 2-2, and Table 3. In each table, LS stands for Low signal, which indicates that the TfR binding activity is low below the measurement threshold, and ND stands for No Data.

[0623] As a result, the synthesized special cyclic peptides listed in Tables 1-1, 1-2, 1-3 and Tables 2-1, 2-2 (and Table 3) were shown to have the ability to bind to hTfR.

[0624] Furthermore, for the specific cyclic peptide described in SEQ ID NO: 162 in Table 3, no signal was detected under the condition of pH 7.4.

[0625] Evaluation of pH dependence

[0626] The calculated koff and KD values ​​were then compared at pH 6.0 and 7.4 to determine pH dependence. The resulting koff and KD comparisons (pH 6.0 / 7.4) are shown in Tables 1-1 and 1-2.

[0627] The results showed that the synthesized special cyclic peptides listed in Tables 1-1, 1-2, and 1-3 showed pH-dependent binding to hTfR. Furthermore, the special cyclic peptides listed in Tables 2-1 and 2-2 also showed pH-dependence, as they exhibited TfR binding activity only at pH 6.0 or 7.4.

[0628] Example 4

[0629] The following peptides or linker-imparting peptides were synthesized.

[0630] [Example 4-1]

[0631] Synthesis of 894_0462 (SEQ ID NO: 1)

[0632] [Chemistry 1]

[0633]

[0634] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.48 mmol / g, 0.10 g) following the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue solid-phase synthesizer was used for synthesis according to the manufacturer's manual. For the introduction of each residue, the extension from residue 16 to residue 13 was performed using Fmoc-AA / DIPCI / Oxyma Pure (4.2 equiv. / 8 equiv. / 4 equiv.) per 1 equivalent of resin, with a single reaction at 75°C for 10 minutes in DMF. The extension from residue 12 to residue 1 was performed using Fmoc-AA / DIPCI / Oxyma Pure (5.3 equiv. / 15 equiv. / 7.5 equiv.) in DMF at 90°C for 3 minutes. However, the third residue was subjected to a second reaction at 75°C for 15 minutes. The 11th and 12th residues were reacted twice at 50°C for 15 minutes. The 13th residue was reacted twice at 75°C for 10 minutes. The 15th residue was reacted once at 50°C for 15 minutes. Furthermore, the Fmoc removal of the 16th to 13th residues was carried out by reacting with a 20% piperidine solution in DMF at 75°C for 3 minutes. The Fmoc removal of the 12th to 1st residue was carried out by reacting with a 10% pyrrolidine solution in DMF at 90°C for 1 minute. However, the Fmoc removal of the 2nd and 13th residues was carried out by reacting at 25°C for 1 minute and then reacting for 1 minute. Chloroacetyl group introduction was performed by removing the Fmoc group from the α-amino group of the Fmoc-protected peptide obtained in the previous step using the previously described method. Chloroacetic acid (approximately 10 equivalents), DIPCI (approximately 10 equivalents), and HOSu (approximately 10 equivalents) were then shaken in DCM for 60 minutes. DMF (equivalent to the DCM) was then added, followed by the addition of ClAcOSu (0.2 M) prepared in this manner, and the mixture was shaken at 25°C for 60 minutes. To deprotect the side chain and cleave it from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with chloromethane, followed by washing with diethyl ether and drying under reduced pressure. Next, reactant mixture A (3 mL, a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5 / 2.5 / 2.5 / 2.5) was added to the reaction vessel containing the solid-phase resin and the mixture was shaken at room temperature for 90 minutes. The reaction solution was recovered by filtration from the glass frit. The solid phase resin remaining in the reaction vessel was shaken again with the excision mixture, and the solution component was recovered from the glass frit and mixed with the filtrate described above. When this filtrate was added to an excess of a 1 / 1 diethyl ether / hexane mixture cooled to 0°C, a cloudy precipitate formed.The mixture was centrifuged (9000 rpm, 0°C, 2 min) and the solution was decanted. The obtained solid was washed again with a small amount of diethyl ether cooled to 0°C and dried under reduced pressure. The obtained solid was used for the subsequent cyclization reaction. The cyclization reaction of the peptide was carried out by dissolving it in DMSO / water / acetonitrile (1 / 1 / 1) in a manner such that the molar number of the solid phase resin was 5 mM based on the final concentration of the peptide, adding 10 equivalents of triethylamine and stirring at room temperature for 5 hours. After quenching the reaction solution with acetic acid, it was concentrated under reduced pressure using Genevac EZII-elite.

[0635] The crude product was purified using the following conditions (column: Waters Xbridge (registered trademark) C18 5μm 30x150mm; mobile phase: A = 0.1% TFA aqueous solution, B = 0.1% TFA in MeCN; temperature: 50°C; gradient (% B): 5-24% in 3 minutes, 24-29% in 8 minutes, and 29-60% in 1 minute; flow rate: 45 mL / min).

[0636] The purity of the target product was calculated from the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under analytical conditions and was 95.5%.

[0637] Analysis conditions: retention time = 4.88 minutes; column: Kinetex EVO C18 2.6 μm 2.1x150 mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% Bconc): 5-45% in 7.15 minutes, then 45-95% in 0.3 minutes, then 95-95% in 1.55 minutes; Flow rate: 0.5 mL / min

[0638] ESI-MS (+) observed value m / z = 793.93 (M+3H) 3+

[0639] [Example 4-2]

[0640] Synthesis of 894_0465 (SEQ ID NO: 4)

[0641] [Chemistry 2]

[0642]

[0643] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.48 mmol / g, 0.10 g) following the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue solid-phase synthesizer was used, and the synthesis was performed in the same manner as for 894_0462 according to the manufacturer's manual. The reaction with Fmoc-Kmor-OH was performed in DMF at 75°C for 10 minutes using Fmoc-Kmor-OH / HATU / Oxyma Pure (5.3 equiv / 5 equiv / 15 equiv).

[0644] The crude product was purified using the following conditions (column: Waters Xbridge (registered trademark) C18 5μm 30x150mm; mobile phase: A = 0.1% TFA aqueous solution, B = 0.1% TFA in MeCN; temperature: 50°C; gradient (% B): 5-24% in 3 minutes, 24-29% in 8 minutes, and 29-60% in 1 minute; flow rate: 45 mL / min).

[0645] The purity of the target product was calculated from the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under analytical conditions and was 90.1%.

[0646] Analysis conditions: retention time = 4.98 minutes; column: Kinetex EVO C18 2.6 μm 2.1x150 mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% Bconc): 5-45% in 7.15 minutes, then 45-95% in 0.3 minutes, then 95-95% in 1.55 minutes; Flow rate: 0.5 mL / min

[0647] ESI-MS (+) observed value m / z = 821.94 (M+3H) 3+

[0648] [Example 4-3]

[0649] Synthesis of 894_471 (SEQ ID NO: 10)

[0650] [Chemistry 3]

[0651]

[0652] Using Sieber amide resin (Watanabe Chemical, 0.48 mmol / g, 0.10 g), the target peptide was synthesized using the general method described above, starting with the removal of the Fmoc group. At this time, a CEM Liberty Prime solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's manual. For the introduction of each residue, Fmoc-AA / DIPCI / Oxyma Pure (5.3 equivalents / 20 equivalents / 7.5 equivalents) was used per equivalent of resin in DMF at 105°C for 1.5 minutes. However, for the second residue, the reaction was carried out twice at 75°C for 30 minutes. For the 12th residue, the reaction was carried out twice at 50°C for 15 minutes. For the 13th residue, the reaction was carried out once at 90°C for 10 minutes. For the 15th residue, the reaction was carried out once at 50°C for 15 minutes. Fmoc removal was performed by reacting a 25% pyrrolidine solution in DMF at 110°C for 1 minute. However, Fmoc removal of residues 2 and 13 was performed by reacting at 25°C for 1.5 minutes and then for another 1.5 minutes. Chloroacetyl group introduction was performed by removing the Fmoc group from the α-amino group of the Fmoc-protected peptide obtained in the previous step using the aforementioned method. Chloroacetic acid (approximately 5 equivalents), DIPCI (approximately 5 equivalents), and HOSu (approximately 5 equivalents) were then shaken in DCM for 60 minutes. DMF equal to the DCM was then added, followed by ClAcOSu (0.1 M) prepared in this manner, and the mixture was shaken at 25°C for 60 minutes. Side chain deprotection and cleavage from the solid-phase resin were performed by washing the resin obtained after the chloroacetyl group introduction step five times with DMF and three times with chloromethane, followed by washing with diethyl ether and drying under reduced pressure. Next, the reaction vessel containing the solid-phase resin was added with reactant mixture A (2 mL, a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5 / 2.5 / 2.5 / 2.5) and shaken at room temperature for 90 minutes. The reaction solution was recovered by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the excision mixture, and the solution components were recovered from the frit and mixed with the filtrate. When this filtrate was added to an excess of a 1 / 1 diethyl ether / hexane mixture cooled to 0°C, a cloudy precipitate formed. This mixture was centrifuged (9000 rpm, 0°C, 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 was carried out by dissolving the peptide in DMSO / water / isopropanol (90 / 5 / 5) so that the final concentration of the peptide was 5 mM based on the molar number of the solid phase resin, adding 10 equivalents of triethylamine, and stirring at room temperature for 5 hours.The reaction solution was concentrated under reduced pressure using a Genevac EZII-elite. The resulting mixture was dissolved in DMSO to a final peptide concentration of 12 mM based on the molar number of the solid phase resin. 1.1 equivalents of D-reduced glucosamine, 4 equivalents of DIEA, and 2 equivalents of PyAOP were added, and the mixture was stirred at room temperature for 60 minutes before being quenched with acetic acid.

[0653] The resulting mixture was purified using the following conditions (column: Waters Xbridge (registered trademark) C18 5μm 30x150mm; mobile phase: A = 0.1% TFA aqueous solution, B = 0.1% TFA in MeCN; temperature: 50°C; gradient (% B): 5-24% in 3 minutes, 24-29% in 8 minutes, and 29-60% in 1 minute; flow rate: 45 mL / min).

[0654] The purity of the target product was calculated from the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under analytical conditions and was 88.3%.

[0655] Analysis conditions: retention time = 5.12 minutes; column: Kinetex EVO C18 2.6 μm 2.1x150 mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% Bconc): 5-45% in 7.15 minutes, then 45-95% in 0.3 minutes, then 95-95% in 1.55 minutes; Flow rate: 0.5 mL / min

[0656] ESI-MS (+) observed value m / z = 839.31 (M+3H) 3+

[0657] [Example 4-4]

[0658] Synthesis of 894_473 (SEQ ID NO: 12)

[0659] [Chemistry 4]

[0660]

[0661] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.48 mmol / g, 0.10 g) according to the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Prime solid-phase synthesizer was used, and the synthesis was performed in the same manner as for 894_471 according to the manufacturer's manual. The condensation of the side chain carboxylates of the 8th and 11th residues with D-reduced glucosamine used 2.2 equivalents of D-reduced glucosamine, 8 equivalents of DIEA, and 4 equivalents of PyAOP.

[0662] The crude product was purified using the following conditions (column: Waters Xbridge (registered trademark) C18 5μm 30x150mm; mobile phase: A = 0.1% TFA aqueous solution, B = 0.1% TFA in MeCN; temperature: 50°C; gradient (% B): 5-19% in 3 minutes, 19-24% in 8 minutes, 24-60% in 1 minute; flow rate: 45 mL / min).

[0663] The purity of the target product was calculated from the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under analytical conditions and was 90.6%.

[0664] Analysis conditions: retention time = 4.48 minutes; column: Kinetex EVO C18 2.6 μm 2.1x150 mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% Bconc): 5-45% in 7.15 minutes, then 45-95% in 0.3 minutes, then 95-95% in 1.55 minutes; Flow rate: 0.5 mL / min

[0665] ESI-MS (+) observed value m / z = 908.38 (M+3H) 3+

[0666] [Examples 4-5]

[0667] Synthesis of 894_491 (SEQ ID NO: 22)

[0668] [Chemistry 5]

[0669]

[0670] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.59 mmol / g, 0.11 g) using the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue HT solid-phase synthesizer was used for synthesis according to the manufacturer's manual. For the introduction of each residue, the extension from residue 16 to residue 14 was performed using Fmoc-AA / DIPCI / Oxyma Pure (4.2 equiv. / 8 equiv. / 4 equiv.) in DMF at 75°C for 10 minutes per reaction, per equivalent of resin. The extension from residue 13 to residue 1 was performed using Fmoc-AA / DIPCI / Oxyma Pure (4.2 equiv. / 12 equiv. / 6 equiv.) in DMF at 90°C for 3 minutes per reaction. The reaction for the 11th residue was performed using Fmoc-E(allyl)-OH, and the reaction for the 8th residue was performed using Fmoc-F4aao(allyl)-OH. However, the reaction for the 2nd residue was performed twice at 75°C for 30 minutes. The reaction for the 13th residue was performed twice at 90°C for 3 minutes. The reaction for the 15th residue was performed once at 50°C for 15 minutes. Furthermore, Fmoc removal from residues 16 to 14 was performed by reacting with a 20% piperidine solution in DMF at 75°C for 3 minutes. Fmoc removal from residues 13 to 1 was performed by reacting with a 10% pyrrolidine solution in DMF at 90°C for 1 minute. However, Fmoc removal from residues 2 and 13 was performed by reacting at 25°C for 1 minute and then for 1 minute. After the peptide was elongated to the 11th residue in the solid-phase synthesis, 5 equivalents of Fmoc-Osu in DMF were added to 1 equivalent of the solid-phase resin obtained by removing the solid-phase synthesis base and shaken for 1 hour, followed by washing with DMF. The solid-phase resin was suspended in DCM, and 0.2 equivalents of tetrakis(triphenylphosphine)palladium(0) and 10 equivalents of phenylsilane were added to 1 equivalent of the resin. The mixture was shaken for 1 hour, followed by washing with DCM and DMF. The resin was suspended in DMF and reacted with H-PEG8Me / DIPCI / Oxyma Pure (4 equivalents / 8 equivalents / 4 equivalents) at 75°C for 20 minutes, based on 1 equivalent of the resin. The resulting solid-phase resin was washed with DMF, and the peptide was elongated using the above-mentioned method using a solid-phase synthesizer. After elongation to the 8th residue, 5 equivalents of Fmoc-Osu in DMF were added to 1 equivalent of the solid-phase resin obtained by removing the solid-phase synthesis base and shaken for 1 hour, followed by washing with DMF. The solid phase resin was suspended in DCM, and 0.2 equivalents of tetrakis(triphenylphosphine)palladium(0) and 10 equivalents of phenylsilane were added to 1 equivalent of the solid phase resin. The mixture was shaken for 1 hour and washed with DCM and DMF.The solid phase resin was suspended in DMF and reacted with H-Pipzaa(tBu) / DIPCI / Oxyma Pure (4 equivalents / 8 equivalents / 4 equivalents) per equivalent of the solid phase resin at 25°C for 60 minutes. The resulting solid phase resin was washed with DMF, and the peptide was elongated using the solid phase synthesizer using the above method. Chloroacetyl groups were introduced by reacting the solid phase resin, which had already retained the Fmoc-protected peptide obtained in the previous step, with a 10% pyrrolidine solution in DMF at 90°C for 1 minute to remove the Fmoc group from the α-amino group. After washing with DMF, chloroacetic acid (approximately 10 equivalents), DIPCI (approximately 10 equivalents), and HOSu (approximately 10 equivalents) were shaken in DCM for 60 minutes. Then, an equal amount of DMF was added to the DCM, followed by the addition of ClAcOSu (0.31 M) prepared in this manner, and the mixture was shaken at 25°C for 60 minutes. To deprotect the side chains and cleave them from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with chloromethane, then washed with diethyl ether and dried under reduced pressure. Next, the reaction mixture A (3 mL, a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5 / 2.5 / 2.5 / 2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction solution was recovered by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage mixture, and the solution fraction was recovered from the frit and mixed with the filtrate. This filtrate was added to an excess of a 1 / 1 diethyl ether / hexane mixture cooled to 0°C, resulting in a cloudy white precipitate. The mixture was centrifuged (9000 rpm, 0°C, 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 was performed by dissolving the peptide in acetonitrile / water (1 / 1) to a final concentration of 5 mM based on the molar ratio of the peptide to the solid phase resin. 10 equivalents of triethylamine was added and stirred at room temperature for 2 hours. The reaction solution was quenched with acetic acid and concentrated under reduced pressure using a Genevac EZ-IIelite.

[0671] The crude product was purified using the following conditions (column: Waters Xbridge (registered trademark) C18 5μm 30x150mm; mobile phase: A = 0.1% TFA aqueous solution, B = 0.1% TFA in MeCN; temperature: 50°C; gradient (% B): 5-30% in 3 minutes, 30-35% in 8 minutes, and 35-60% in 1 minute; flow rate: 45 mL / min).

[0672] The purity of the target product was calculated from the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under analytical conditions and was 92.7%.

[0673] Analysis conditions: retention time = 3.88 minutes; column: Kinetex EVO C18 2.6 μm 2.1x150 mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% Bconc): 20-60% in 7.15 minutes, then 60-95% in 0.3 minutes, then 95-95% in 1.55 minutes; Flow rate: 0.5 mL / min

[0674] ESI-MS (+) observed value m / z = 1493.80 (M+2H) 2+

[0675] [Examples 4-6]

[0676] Synthesis of 894_0552 (SEQ ID NO: 53)

[0677] [Chemistry 6]

[0678]

[0679] Using Sieber amide resin (Watanabe Chemical, 0.48 mmol / g, 0.10 g), the target peptide was synthesized by the general method described above, starting with the removal of the Fmoc group. At this time, a CEM Liberty Prime solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's manual. For the introduction of each residue, Fmoc-AA / DIPCI / Oxyma Pure (4.2 equivalents / 16 equivalents / 6 equivalents) was used per equivalent of resin, and the reaction was carried out once in DMF at 105°C for 1.5 minutes. However, for the second residue, the reaction was carried out twice at 75°C for 30 minutes. For the 12th residue, the reaction was carried out twice at 50°C for 15 minutes. For the 13th residue, the reaction was carried out twice at 90°C for 10 minutes. For the 15th residue, the reaction was carried out once at 50°C for 15 minutes. The fifth residue was removed from the solid-phase resin synthesizer and reacted with Fmoc-AA / HATU / DIEA (4 equivalents / 4 equivalents / 8 equivalents) at 25°C for 60 minutes with shaking. After washing the solid-phase resin with DMF, peptide elongation was continued using the solid-phase synthesizer. Fmoc removal was performed by reacting with a 4% pyrrolidine and 83 mM Oxyma Pure solution in DMF at 110°C for 1 minute. However, Fmoc removal of the second and 13th residues was performed by reacting with a 10% pyrrolidine solution in DMF at 25°C for 1.5 minutes and then for another 1.5 minutes. Chloroacetyl groups were introduced by removing the Fmoc group from the α-amino group of the solid-phase resin holding the Fmoc-protected peptide obtained in the previous step using the aforementioned method. Then, a DMF solution of N-(chloroacetoxy)succinimide (approximately 5 equivalents) was added and the mixture was shaken at 25°C for 60 minutes. To deprotect the side chains and cleave them from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with chloromethane, then washed with diethyl ether and dried under reduced pressure. Next, reactant mixture A (3 mL, a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5 / 2.5 / 2.5 / 2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction solution was recovered by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage mixture, and the solution fraction was recovered from the frit and mixed with the filtrate. This filtrate was added to an excess of a 1 / 1 diisopropyl ether / hexane mixture cooled to 0°C, resulting in a cloudy precipitate. The mixture was centrifuged (9000 rpm, 0°C, 2 min), and the solution was decanted. The obtained solid was washed again with a small amount of diethyl ether cooled to 0° C. and then dried under reduced pressure. The obtained solid was used in the subsequent cyclization reaction.The peptide cyclization reaction was performed by dissolving the peptide in acetonitrile / water (1 / 1) to a final concentration of 2.5 mM based on the molar ratio of the solid phase resin. 10 equivalents of triethylamine were added, and the mixture was stirred at room temperature for 16 hours before being quenched with acetic acid. The reaction solution was concentrated under reduced pressure using a Genevac EZII-elite.

[0680] The crude product was purified using the following conditions (column: Waters Xbridge (registered trademark) C18 5μm 30x150mm; mobile phase: A = 0.1% TFA aqueous solution, B = 0.1% TFA in MeCN; temperature: 50°C; gradient (% B): 5-29% in 3 minutes, 29-34% in 8 minutes, and 34-60% in 1 minute; flow rate: 45 mL / min).

[0681] The purity of the target product was calculated from the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under analytical conditions and was 92.1%.

[0682] Analysis conditions: retention time = 3.23 minutes; column: Kinetex EVO C18 2.6 μm 2.1x150 mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% Bconc): 20-60% in 7.15 minutes, then 60-95% in 0.3 minutes, then 95-95% in 1.55 minutes; Flow rate: 0.5 mL / min

[0683] ESI-MS (+) observed value m / z = 818.33 (M+3H) 3+

[0684] [Examples 4-7]

[0685] Synthesis of 894_0436 (SEQ ID NO: 144)

[0686] [Chemistry 7]

[0687]

[0688] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.48 mmol / g, 0.21 g) using the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue solid-phase synthesizer was used for synthesis according to the manufacturer's manual. For the introduction of each residue, Fmoc-AA / DIPCI / Oxyma Pure (5.3 equiv / 10 equiv / 5 equiv) was used per equivalent of resin in DMF at 90°C for 10 minutes, with one reaction. Fmoc-F3COO(allyl)-OH was used for the reaction of the 13th residue. However, the second residue was reacted twice at 75°C for 30 minutes. The fifth, sixth, and seventh residues were reacted at 90°C for 3 minutes. The 12th residue was reacted twice at 50°C for 15 minutes. The 13th residue was reacted twice at 90°C for 3 minutes. The 14th and 16th residues were reacted at 90°C for 3 minutes. The 15th residue was reacted at 50°C for 15 minutes. Furthermore, Fmoc removal was carried out up to the second residue by reacting with a 10% pyrrolidine DMF solution at 90°C for 1 minute. However, Fmoc removal of the second and 13th residues was carried out by reacting at 25°C for 1 minute and then reacting for 1 minute. 1 equivalent of the obtained solid phase resin was shaken with 5 equivalents of a DMF solution of Fmoc-Osu for 1 hour and washed with DMF. The solid phase resin was suspended in DCM, and 0.2 equivalents of tetrakis(triphenylphosphine)palladium(0) and 10 equivalents of phenylsilane were added to 1 equivalent of the resin, shaken for 1 hour, and washed with DCM and DMF. The resulting solid-phase resin was suspended in DMF and reacted with H-PEG4c(tBu) / DIPCI / Oxyma Pure (4 equivalents / 8 equivalents / 4 equivalents) per equivalent of the solid-phase resin at 75°C for 30 minutes, followed by washing with DMF. Chloroacetyl groups were introduced by reacting the solid-phase resin, already holding the Fmoc-protected peptide obtained in the previous step, with a 20% piperidine solution in DMF twice at 25°C for 5 minutes to remove the Fmoc group from the α-amino group. After washing with DMF, chloroacetic acid (approximately 5 equivalents), DIPCI (approximately 5 equivalents), and HOSu (approximately 5 equivalents) were shaken in DCM for 60 minutes. Then, an equal amount of DMF was added to the DCM, followed by the addition of ClAcOSu (0.05 M) prepared in this manner, and the mixture was shaken at 25°C for 60 minutes. For the deprotection of the side chain and the cleavage from the solid phase resin, the resin obtained after the chloroacetyl group introduction step was first washed with DMF five times and chloromethane three times, then washed with diethyl ether and dried under reduced pressure.Next, the reaction vessel containing the solid-phase resin was added with reactant mixture A (4 mL, a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5 / 2.5 / 2.5 / 2.5) and shaken at room temperature for 60 minutes. The reaction solution was recovered by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the excision mixture, and the solution components were recovered from the frit and mixed with the filtrate. When this filtrate was added to an excess of a 1 / 1 diisopropyl ether / hexane mixture cooled to 0°C, a cloudy precipitate formed. This mixture was centrifuged (9000 rpm, 2 minutes), 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 was performed by dissolving the peptide in DMSO / water (9 / 1) to a final concentration of 5 mM based on the molar ratio of the solid phase resin. Ten equivalents of triethylamine were then added and stirred at room temperature for 15 hours. The reaction solution was quenched with acetic acid and concentrated under reduced pressure using a Genevac HT-12.

[0689] The crude product was purified using the following conditions (column: Waters Xbridge (registered trademark) C18 5μm 50x250mm; mobile phase: A = 0.1% TFA in water, B = 0.1% TFA in MeCN; temperature: 50°C; gradient (% B): 0-0% in 4.9 minutes, 0-4.2% in 2 minutes, 4.2-29.7% in 3 minutes, 29.7-34.7% in 15 minutes, and 34.7-60% in 3 minutes; flow rate: 18-18 mL / min in 4.9 minutes, then 18-118 mL / min in 2 minutes, then 118 mL / min).

[0690] The purity of the target product was calculated from the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under analytical conditions and was 97.8%.

[0691] Analysis conditions: retention time = 3.91 minutes; column: Kinetex EVO C18 2.6 μm 2.1x150 mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% Bconc): 20-60% in 7.15 minutes, then 60-95% in 0.3 minutes, then 95-95% in 1.55 minutes; Flow rate: 0.5 mL / min

[0692] ESI-MS (+) observed value m / z = 895.81 (M+3H) 3+

[0693] [Examples 4-8]

[0694] Synthesis of 894_0438 (SEQ ID NO: 146)

[0695] [Chemistry 8]

[0696]

[0697] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.48 mmol / g, 0.21 g) using the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue solid-phase synthesizer was used, and synthesis was performed in the same manner as for 894_0436 according to the manufacturer's manual. For the solid-phase synthesis, Fmoc-E(allyl)-OH was used for the reaction at residue 11, and Fmoc-F3COO(allyl)-OH was used for the reaction at residue 13. The condensation of the side-chain carboxylic acids at residues 11 and 13 with H-PEG4c(tBu) was performed using H-PEG4c(tBu) / DIPCI / Oxyma Pure (8 equiv / 16 equiv / 8 equiv).

[0698] The crude product was purified using the following conditions (column: Waters Xbridge (registered trademark) C18 5μm 30x150mm; mobile phase: A = 0.1% TFA in water, B = 0.1% TFA in MeCN; temperature: 50°C; gradient (% B): 2.2-2.2% in 4.9 minutes, 2.2-6.0% in 1 minute, 6.0-31.6% in 3 minutes, 31.6-36.7% in 9 minutes, and 36.7-60% in 1 minute; flow rate: 9-9 mL / min in 4.9 minutes, then 9-44 mL / min in 1 minute, then 44 mL / min).

[0699] The purity of the target product was calculated from the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under analytical conditions and was 95.7%.

[0700] Analysis conditions: retention time = 4.28 minutes; column: Kinetex EVO C18 2.6 μm 2.1x150 mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% Bconc): 20-60% in 7.15 minutes, then 60-95% in 0.3 minutes, then 95-95% in 1.55 minutes; Flow rate: 0.5 mL / min

[0701] ESI-MS (+) observed value m / z = 963.85 (M+3H) 3+

[0702] [Examples 4-9]

[0703] Synthesis of 894_3426 (SEQ ID NO: 67)

[0704] [Chemistry 9]

[0705]

[0706] Using Sieber amide resin (Watanabe Chemical, 0.54 mmol / g, 0.93 g), the target peptide was synthesized using the general method described above, starting with the removal of the Fmoc group. At this time, a CEM Liberty Blue solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's manual. For the introduction of each residue, Fmoc-AA / DIPCI / Oxyma Pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used per equivalent of resin, and the reaction was carried out once in DMF at 75°C for 10 minutes. However, for the second residue, the reaction was carried out twice at 75°C for 30 minutes. For the eighth, ninth, and thirteenth residues, the reaction was carried out twice at 75°C for 10 minutes. For the twelfth residue, the reaction was carried out twice at 50°C for 15 minutes. For the fifteenth residue, the reaction was carried out once at 50°C for 15 minutes. The fifth residue was removed from the solid phase resin synthesizer and reacted with Fmoc-AA / HATU / DIEA (5 equivalents / 5 equivalents / 10 equivalents) at 40°C for 60 minutes under shaking. After washing the solid phase resin with DMF, the peptide was continuously extended using the solid phase synthesizer. Fmoc removal was carried out by reacting with a 10% pyrrolidine DMF solution at 75°C for 3 minutes. However, Fmoc removal of the second and 13th residues was carried out by reacting at 25°C for 5 minutes and then for 5 minutes. The introduction of the chloroacetyl group was carried out by removing the Fmoc group of the α-amino group from the solid phase resin holding the Fmoc-protected peptide obtained in the previous step using the above-mentioned method, adding a DMF solution of N-(chloroacetyloxy)succinimide (about 10 equivalents), and shaking at 25°C for 60 minutes. To deprotect the side chains and cleave them from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with chloromethane, then washed with diethyl ether and dried under reduced pressure. Next, reactant mixture A (30 mL, a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5 / 2.5 / 2.5 / 2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction solution was recovered by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage mixture, and the solution fraction was recovered from the frit and mixed with the filtrate. This filtrate was added to an excess of a 1 / 1 diethyl ether / hexane mixture cooled to 0°C, resulting in a cloudy precipitate. The mixture was centrifuged (9000 rpm, 0°C, 2 min), and the solution was decanted. The obtained solid was washed again with a small amount of diethyl ether cooled to 0° C. and then dried under reduced pressure. The obtained solid was used in the subsequent cyclization reaction.The peptide cyclization reaction was performed by dissolving the peptide in DMSO / water (9 / 1) at a final concentration of 5 mM based on the molar ratio of the solid phase resin. Ten equivalents of triethylamine were then added and stirred at room temperature for 2 hours. The reaction solution was quenched with acetic acid and concentrated under reduced pressure using a Genevac HT-12.

[0707] The crude product was purified using the following conditions (column: Waters Xselect CSHPrepC18 5μm 50x250mm; mobile phase: A = 20mM TEAA in water, B = 0mM TEAA in MeCN, C = 200mM TEAA in water, D = MeCN; temperature: 50°C; gradient (% A): 0.1-0.1% in 5 minutes, 0.1-71.1% in 0.1 minutes, then (100-% B), gradient (% B): 0-0% in 5 minutes, 0-28.9% in 0.1 minutes, 28.9-30.7% in 1.9 minutes, 30.7-30.7% in 3 minutes, 30.7-35.8 in 15.5 minutes). %, 35.8-60% in 1.5 minutes, 60-90% in 4 minutes, gradient (% C): 71.0-71.0% in 5 minutes, 71.0-0% in 0.1 minute, then 0%, gradient (% D): 28.9-28.9% in 5 minutes, 28.9-0% in 0.1 minute, then 0%; flow rate: 18-18 mL / min in 5.1 minutes, then 18-118 mL / min in 1.9 minutes, then 118 mL / min).

[0708] The purity of the target product was calculated from the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under analytical conditions and was 95.7%.

[0709] Analysis conditions: retention time = 3.58 minutes; column: Kinetex EVO C18 2.6 μm 2.1x150 mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% Bconc): 20-60% in 7.15 minutes, then 60-95% in 0.3 minutes, then 95-95% in 1.55 minutes; Flow rate: 0.5 mL / min

[0710] ESI-MS (+) observed value m / z = 1208.36 (M+2H) 2+

[0711] [Examples 4-10]

[0712] Synthesis of 894_3427 (SEQ ID NO: 66)

[0713] [Chemistry 10]

[0714]

[0715] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.54 mmol / g, 0.46 g x 2) using the general method described above, starting with the removal of the Fmoc group. At this time, a CEM Liberty Blue solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's manual. For the introduction of each residue, Fmoc-AA / DIPCI / Oxyma Pure (4.2 eq / 8 eq / 4 eq) was used per 1 equivalent of resin, and the reaction was carried out once in DMF at 90°C for 3 minutes. However, the 15th residue was reacted once at 50°C for 15 minutes. The 13th residue was reacted twice at 90°C for 3 minutes. The 11th and 12th residues were reacted twice at 50°C for 15 minutes. The 9th residue was reacted twice at 90°C for 10 minutes. The 8th residue was reacted once at 90°C for 10 minutes. The second residue was reacted twice at 75°C for 30 minutes. Furthermore, Fmoc removal was performed by reacting a 10% pyrrolidine solution in DMF at 90°C for 1 minute. However, Fmoc removal of the second and 13th residues was performed by reacting at 25°C for 1 minute and then for 1 minute. The introduction of the chloroacetyl group was performed by removing the Fmoc group of the α-amino group from the solid-phase resin holding the Fmoc-protected peptide obtained in the previous step using the aforementioned method, adding N-(chloroacetoxy)succinimide (approximately 10 equivalents), and shaking at 25°C for 60 minutes. To deprotect the side chain and cleave it from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with chloromethane, then washed with diethyl ether and dried under reduced pressure. Next, the reaction vessel containing the solid-phase resin was added with reactant mixture A (33 mL, a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5 / 2.5 / 2.5 / 2.5) and shaken at room temperature for 90 minutes. The reaction solution was recovered by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the excision mixture, and the solution components were recovered from the frit and mixed with the filtrate. When this filtrate was added to an excess of a 1 / 1 diethyl ether / hexane mixture cooled to 0°C, a cloudy precipitate formed. This mixture was centrifuged (9000 rpm, 0°C, 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 was carried out by dissolving the peptide in DMSO / water (9 / 1) so that the final concentration of the peptide was 5 mM based on the molar number of the solid phase resin, adding 10 equivalents of triethylamine, and stirring at room temperature for 2 hours.The reaction solution was quenched with 15 equivalents of acetic acid and then concentrated under reduced pressure using Genevac HT-12.

[0716] The crude product was purified using the following conditions (column: Waters Xbridge (registered trademark) C18 5μm 50x150mm; mobile phase: A = 0.1% TFA in water, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (%B): 5-5% over 2 minutes, 5-23% over 1 minute, 23-28% over 8 minutes, and then 28-60% over 1 minute; flow rate: 20-20 mL / min over 1 minute, then 20-120 mL / min over 1 minute, and then 120 mL / min).

[0717] The purity of the target product was calculated from the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under analytical conditions and was 93.3%.

[0718] Analysis conditions: retention time = 5.11 minutes; column: Kinetex EVO C18 2.6 μm 2.1x150 mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% Bconc): 5-45% in 7.15 minutes, then 45-95% in 0.3 minutes, then 95-95% in 1.55 minutes; Flow rate: 0.5 mL / min

[0719] ESI-MS (+) observed value m / z = 781.65 (M+3H) 3+

[0720] [Example 4-11]

[0721] Synthesis of 894_3428 (SEQ ID NO: 68)

[0722] [Chemistry 11]

[0723]

[0724] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.54 mmol / g, 0.46 g x 2) according to the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue solid-phase synthesizer was used for synthesis according to the manufacturer's manual. For the introduction of each residue, Fmoc-AA / DIC / Oxyma Pure (4.2 equiv. / 8 equiv. / 4 equiv.) was used per equivalent of resin, and the reaction was carried out once in DMF at 90°C for 3 minutes. However, the 15th residue was reacted once at 50°C for 15 minutes. The 13th residue was reacted twice at 90°C for 3 minutes. The 1st, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, and 11th residues were reacted once at 40°C for 30 minutes. The second residue was reacted twice at 75°C for 30 minutes. Fmoc removal was performed by reacting with a 10% pyrrolidine solution in DMF at 90°C for 1 minute. However, Fmoc removal of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, and thirteenth residues was performed by reacting at 25°C for 1 minute and then for 1 minute. Chloroacetyl group introduction was performed by removing the Fmoc group from the α-amino group of the solid phase resin holding the Fmoc-protected peptide obtained in the previous step using the aforementioned method, then adding N-(chloroacetoxy)succinimide (approximately 10 equivalents) and shaking at 25°C for 60 minutes. To deprotect the side chains and cleave them from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with chloromethane, then washed with diethyl ether and dried under reduced pressure. Next, reactant mixture A (30 mL, a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5 / 2.5 / 2.5 / 2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction solution was recovered by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage mixture, and the solution fraction was recovered from the frit and mixed with the filtrate. This filtrate was added to an excess of a 1 / 1 diethyl ether / hexane mixture cooled to 0°C, resulting in a cloudy precipitate. The mixture was centrifuged (9000 rpm, 0°C, 2 min), and the solution was decanted. The obtained solid was washed again with a small amount of diethyl ether cooled to 0° C. and then dried under reduced pressure. The obtained solid was used in the subsequent cyclization reaction.The peptide cyclization reaction was performed by dissolving the peptide in DMSO / water (9 / 1) to a final concentration of 5 mM based on the molar ratio of the solid phase resin. Then, 10 equivalents of triethylamine were added and stirred at room temperature for 2 hours. The reaction solution was quenched with 15 equivalents of acetic acid and concentrated under reduced pressure using a Genevac HT-12.

[0725] The crude product was purified using the following conditions (column: Waters Xbridge (registered trademark) C18 5μm 50x150mm; mobile phase: A = 0.1% TFA in water, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (%B): 5-5% over 2 minutes, 5-23% over 1 minute, 23-28% over 8 minutes, and then 28-60% over 1 minute; flow rate: 20-20 mL / min over 1 minute, then 20-120 mL / min over 1 minute, and then 120 mL / min).

[0726] The purity of the target product was calculated from the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under analytical conditions and was 97.3%.

[0727] Analysis conditions: retention time = 5.15 minutes; column: Kinetex EVO C18 2.6 μm 2.1x150 mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% Bconc): 5-45% in 7.15 minutes, then 45-95% in 0.3 minutes, then 95-95% in 1.55 minutes; Flow rate: 0.5 mL / min

[0728] ESI-MS (+) observed value m / z = 809.62 (M+3H) 3+

[0729] [Example 4-12]

[0730] Synthesis of 894_3484 (SEQ ID NO: 195)

[0731] [Chemistry 12]

[0732]

[0733] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.60 mmol / g, 0.83 g) using the general method described above, starting with the removal of the Fmoc group. At this time, a CEM Liberty Blue HT solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's manual. For the introduction of each residue, Fmoc-AA / DIPCI / Oxyma Pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used per equivalent of resin, and the reaction was carried out 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 13th residue was reacted twice at 75°C for 10 minutes using Fmoc-F3COO(allyl)-OH. The 15th residue was reacted once at 50°C for 15 minutes. Fmoc removal was performed by reacting with a 10% pyrrolidine solution in DMF twice at room temperature for 5 minutes. However, Fmoc removal of the third and fifth residues was performed by reacting for 5 minutes and then for 10 minutes. Fmoc removal of the 14th, 15th, 16th, and 17th residues was performed by reacting at 75°C for 3 minutes. The solid-phase resin removed from the solid-phase synthesis base was suspended in DCM / HFIP (99 / 1). 0.2 equivalents of tetrakis(triphenylphosphine)palladium(0) and 10 equivalents of phenylsilane were added per equivalent of the resin, shaken for 1 hour, and washed with DCM and DMF. The resin was suspended in DMF and reacted with H-PEG4c(tBu) / HATU / DIEA (4.2 equivalents / 4 equivalents / 8 equivalents) per equivalent of the resin at room temperature for 30 minutes. Chloroacetyl groups were introduced by reacting the solid-phase resin holding the Fmoc-protected peptide obtained in the previous step with a 10% pyrrolidine-containing DMF solution at room temperature for 2 minutes to remove the α-amino Fmoc group. After washing with DMF, the resin was shaken with a DMF solution of N-(chloroacetyloxy)succinimide (approximately 10 equivalents) for 60 minutes. To deprotect the side chain and cleave it from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with chloromethane, followed by washing with diethyl ether and drying under reduced pressure. Next, reactant mixture A (25 mL, a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5 / 2.5 / 2.5 / 2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 40 minutes. The reaction solution was recovered by filtration through the frit. The solid phase resin remaining in the reaction vessel was shaken again with the excision mixture, and the solution component was recovered from the frit and mixed with the filtrate. When this filtrate was added to an excess of diethyl ether / hexane (1 / 1) mixed solvent cooled to 0°C, a white turbid precipitate was formed.The mixture was centrifuged (9000 rpm, 0°C, 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 was performed by dissolving it in DMSO / acetonitrile / water (1 / 1 / 1) so that the final concentration of the peptide was 5 mM based on the molar number of the solid phase resin. 10 equivalents of triethylamine was added and stirred at room temperature for 2 hours. The reaction solution was quenched with acetic acid and concentrated under reduced pressure using a Genevac EZ-II elite.

[0734] The crude product was purified using the following conditions (column: Waters Xselect CSH (registered trademark) C18 5μm 50x250mm; mobile phase: A = 0.1% TFA in water, B = 0.1% TFA in MeCN; temperature: 50°C; gradient (% B): 1.1% in 5 minutes, 1.1-5.2% in 2 minutes, 5.2-30.7% in 3 minutes, 30.7-35.8% in 15.5 minutes, and 35.8-60% in 1.5 minutes; flow rate: 18 mL / min in 8 minutes, 18-118 mL / min in 2 minutes, and 118 mL / min thereafter).

[0735] The purity of the target product was calculated from the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under analytical conditions and was 96.4%.

[0736] Analysis conditions: retention time = 4.10 minutes; column: Kinetex EVO C18 2.6 μm 2.1x150 mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% Bconc): 20-60% in 7.15 minutes, then 60-95% in 0.3 minutes, then 95-95% in 1.55 minutes; Flow rate: 0.5 mL / min

[0737] ESI-MS (+) observed value m / z = 1292.43 (M+2H) 2+

[0738] [Example 4-13]

[0739] Synthesis of 894_3486 (SEQ ID NO: 196)

[0740] [Chemistry 13]

[0741]

[0742] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.60 mmol / g, 0.41 g x 2 batches) using the general method described above, starting with the removal of the Fmoc group. Synthesis was performed using CEM's Liberty Blue solid-phase synthesizer according to the manufacturer's manual. For the introduction of each residue, Fmoc-AA / DIPCI / Oxyma Pure (4.2 eq / 8 eq / 4 eq) was used per 1 equivalent of resin in DMF at 90°C for one reaction for 3 minutes. However, the reaction for the second residue was performed at 75°C for two reactions for 30 minutes. The introduction of the seventh residue was performed at 40°C for one reaction for 30 minutes using Fmoc-AA / HATU / DIEA (4.2 eq / 4 eq / 8 eq). The ninth residue was introduced at 90°C for two reactions for 10 minutes. The 10th residue was reacted once at 90°C for 10 minutes. The 12th residue was reacted twice at 50°C for 15 minutes. The 13th residue was reacted twice at 90°C for 10 minutes. The 15th residue was reacted once at 50°C for 15 minutes. Furthermore, Fmoc removal was performed by reacting with a 10% pyrrolidine solution in DMF at 90°C for 1 minute. However, Fmoc removal of the 2nd, 4th, 9th, and 13th residues was performed by reacting with a 10% pyrrolidine solution in DMF at 25°C for 1 minute, followed by another reaction for 1 minute. Chloroacetyl groups were introduced by mixing the solid phase resin holding the Fmoc-protected peptide obtained in the previous step, removing the Fmoc group from the α-amino group using the aforementioned method, and then adding a DMF solution of N-(chloroacetyloxy)succinimide (approximately 5 equivalents) and shaking at 25°C for 60 minutes. To deprotect the side chains and cleave them from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with chloromethane, then washed with diethyl ether and dried under reduced pressure. Next, reactant mixture A (25 mL, a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5 / 2.5 / 2.5 / 2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 60 minutes. The reaction solution was recovered by filtration from the frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage mixture, and the solution fraction was recovered from the frit and mixed with the filtrate. This filtrate was added to an excess of a 1 / 1 diisopropyl ether / hexane mixture cooled to 0°C, resulting in a cloudy precipitate. The mixture was centrifuged (9000 rpm, 0°C, 2 min), and the solution was decanted. The obtained solid was washed again with a small amount of diethyl ether cooled to 0° C. and then dried under reduced pressure. The obtained solid was used in the subsequent cyclization reaction.The peptide cyclization reaction was performed by dissolving the peptide in DMSO / water (9 / 1) to a final concentration of 2.8 mM based on the molar ratio of the solid phase resin. Ten equivalents of triethylamine were added, and the mixture was stirred at room temperature for 3 hours before being quenched with acetic acid. The reaction solution was concentrated under reduced pressure using a Genevac HT-12.

[0743] The crude product was purified using the following conditions (column: Waters Xselect CSH (registered trademark) C18 5μm 50x250mm; mobile phase: A = 0.1% TFA in water, B = 0.1% TFA in MeCN; temperature: 50°C; gradient (%B): 0% in 5 minutes, 0-4.2% in 2 minutes, 4.2-25.6% in 3 minutes, 25.6-30.7% in 15.5 minutes, and 30.7-60% in 1.5 minutes; flow rate: 18 mL / min in 8 minutes, 18-118 mL / min in 2 minutes, and then 118 mL / min).

[0744] The purity of the target product was calculated from the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under analytical conditions and was 94.9%.

[0745] Analysis conditions: retention time = 3.02 minutes; column: Kinetex EVO C18 2.6 μm 2.1x150 mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% Bconc): 20-60% in 7.15 minutes, then 60-95% in 0.3 minutes, then 95-95% in 1.55 minutes; Flow rate: 0.5 mL / min

[0746] ESI-MS (+) observed value m / z = 1161.72 (M+2H) 2+

[0747] [Example 4-14]

[0748] Synthesis of 894_3488 (SEQ ID NO: 193)

[0749] [Chemistry 14]

[0750]

[0751] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.60 mmol / g, 0.83 g) according to the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue HT solid-phase synthesizer was used for synthesis according to the manufacturer's manual. For the introduction of each residue, Fmoc-AA / DIPCI / Oxyma Pure (4.2 equiv. / 8 equiv. / 4 equiv.) was used per equivalent of resin in DMF, with a single reaction at 75°C for 10 minutes. However, for the second and fourth residues, two reactions were performed at 75°C for 30 minutes. For the 12th residue, two reactions were performed at 50°C for 20 minutes. For the 13th residue, Fmoc-F3COO(allyl)-OH was used, with two reactions at 75°C for 30 minutes. For the 15th residue, one reaction was performed at 50°C for 20 minutes. Fmoc removal was performed by reacting with a 10% pyrrolidine solution in DMF at 75°C for 3 minutes. However, Fmoc removal of the second, fourth, and 13th residues was performed by reacting at room temperature for 5 minutes and then for 5 minutes. The solid-phase resin obtained was shaken for 60 minutes with 5 equivalents of a DCM solution of Fmoc-Osu per equivalent of the obtained solid-phase resin, and then washed with DMF. The solid-phase resin obtained by removing the solid-phase resin from the solid-phase synthesis base was suspended in DCM, and 0.2 equivalents of tetrakis(triphenylphosphine)palladium(0) and 10 equivalents of phenylsilane were added per equivalent of the resin. The mixture was shaken for 1 hour and washed with DCM and DMF. The resin was suspended in DMF and reacted with H-PEG4c(tBu) / DIC / Oxyma Pure (4 equivalents / 8 equivalents / 4 equivalents) per equivalent of the resin at 75°C for 30 minutes. Chloroacetyl groups were introduced by reacting the solid-phase resin holding the Fmoc-protected peptide obtained in the previous step with a 10% pyrrolidine solution in DMF at room temperature for 5 minutes to remove the Fmoc group from the α-amino group. After washing with DMF, the resin was shaken for 60 minutes with a solution of N-(chloroacetyloxy)succinimide (approximately 5 equivalents) in DMF / DCM (1 / 1). To deprotect the side chain and cleave it from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with chloromethane, followed by washing with diethyl ether and drying under reduced pressure. Next, reactant mixture B (20 mL, a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 90 / 2.5 / 2.5 / 5.0) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 60 minutes. The reaction solution was recovered by filtration through the frit. The solid phase resin remaining in the reaction container is shaken again with the excision mixture, and the solution component is recovered from the frit and mixed with the aforementioned filtrate.If this filtrate is added to an excess of a mixed solvent of diethyl ether / hexane (1 / 1) cooled to 0°C, a white turbid precipitate is produced. The mixture is centrifuged (9000 rpm, 0°C, 2 min) and the solution is decanted. The obtained solid is washed again with a small amount of diethyl ether cooled to 0°C and dried under reduced pressure. The obtained solid is used for the subsequent cyclization reaction. The cyclization reaction of the peptide is carried out by dissolving it in 1% triethylamine containing acetonitrile / water (1 / 1) in such a way that the final concentration of the peptide is 5 mM based on the molar number of the solid phase resin, and stirring it at room temperature for 1 hour. After quenching the reaction solution with acetic acid, it is concentrated under reduced pressure using Genevac HT-12.

[0752] The crude product was purified using the following conditions (column: Waters Xselect CSH (registered trademark) C18 5μm 50x250mm; mobile phase: A = 0.1% TFA in water, B = 0.1% TFA in MeCN; temperature: 50°C; gradient (% B): 1.1% in 5 minutes, 1.1-5.2% in 2 minutes, 5.2-30.7% in 3 minutes, 30.7-35.8% in 15.5 minutes, and 35.8-60% in 1.5 minutes; flow rate: 18 mL / min in 8 minutes, 18-118 mL / min in 2 minutes, and 118 mL / min thereafter).

[0753] The purity of the target product was calculated from the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under analytical conditions and was 94.0%.

[0754] Analysis conditions: retention time = 4.05 minutes; column: Kinetex EVO C18 2.6 μm 2.1x150 mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% Bconc): 20-60% in 7.15 minutes, then 60-95% in 0.3 minutes, then 95-95% in 1.55 minutes; Flow rate: 0.5 mL / min

[0755] ESI-MS (+) observed value m / z = 1299.41 (M+2H) 2+

[0756] [Example 4-15]

[0757] Synthesis of 894_0640 (SEQ ID NO: 172)

[0758] [Chemistry 15]

[0759]

[0760] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.60 mmol / g, 1.67 g) using the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue solid-phase synthesizer was used for synthesis according to the manufacturer's manual. For the introduction of each residue, Fmoc-AA / DIPCI / Oxyma Pure (4.2 equiv. / 8 equiv. / 4 equiv.) was used per equivalent of resin in DMF at 90°C for 10 minutes, with one reaction performed until the 12th residue. The 12th residue was introduced by two reactions at 50°C for 20 minutes. The 13th residue was introduced by two reactions at 75°C for 30 minutes. The 14th residue was introduced by one reaction at 75°C for 30 minutes. The 15th residue was introduced by one reaction at 20 minutes. The 16th residue was introduced by one reaction at 75°C for 10 minutes. Furthermore, Fmoc removal was carried out by reacting with a 10% pyrrolidine solution in DMF at 75°C for 3 minutes. However, the 12th, 13th, and 16th residues were introduced by reacting at 25°C for 1 minute and then for 1 minute. The introduction of the 11th residue was carried out by using Fmoc-E(allyl)-OH / HATU / DIEA (5 equivalents / 5 equivalents / 10 equivalents) relative to 1 equivalent of the resin, and the reaction was carried out once at room temperature for 60 minutes. The obtained solid phase resin was suspended in DCM, and 0.2 equivalents of tetrakis(triphenylphosphine)palladium(0) and 10 equivalents of phenylsilane were added relative to 1 equivalent of the resin, shaken for 1.5 hours, and washed with DCM and DMF. The obtained solid phase resin was suspended in DMF and reacted with Boc-apa-H / PyAOP / DIEA (10 equivalents / 10 equivalents / 10 equivalents) at 25°C for 120 minutes relative to 1 equivalent of the solid phase resin, and washed with DMF. Next, the introduction of each residue was carried out by using Fmoc-AA / DIPCI / Oxyma Pure (5.3 equivalents / 10 equivalents / 5 equivalents) relative to 1 equivalent of the obtained solid phase resin, and the reaction was carried out once in DMF at 90°C for 10 minutes, thereby continuously performing the elongation reaction. However, the first, sixth, and eighth residues were reacted once at 90°C for 3 minutes. The second and fourth residues were reacted twice at 90°C for 10 minutes. In addition, Fmoc removal was carried out by reacting with a 10% pyrrolidine solution in DMF at 90°C for 1 minute. However, Fmoc removal of the second and fourth residues was performed by reacting at 25°C for 1 minute and then for 1 minute.Chloroacetyl groups were introduced by reacting the solid-phase resin holding the Fmoc-protected peptide obtained in the previous step with a 10% pyrrolidine solution in DMF at 25°C twice for 1 minute to remove the α-amino Fmoc group. After washing with DMF, the resin was shaken with a solution of N-(chloroacetoxy)succinimide (approximately 10 equivalents) in DMF for 60 minutes. To deprotect the side chain and cleave it from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with chloromethane, followed by washing with diethyl ether and drying under reduced pressure. Next, reactant mixture A (4 mL, a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5 / 2.5 / 2.5 / 2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 60 minutes. The reaction solution was recovered by filtration through the frit. The solid-phase resin remaining in the reaction vessel is shaken again with the excision mixture, and the solution components are recovered from the frit and mixed with the filtrate. This filtrate is added to an excess of a 1 / 1 diisopropyl ether / hexane mixture cooled to 0°C, resulting in a cloudy precipitate. The mixture is centrifuged (9000 rpm, 2 minutes), and the solution is decanted. The resulting solid is washed again with a small amount of diethyl ether cooled to 0°C and then dried under reduced pressure. The resulting solid is used in the subsequent cyclization reaction. The peptide cyclization reaction is performed by dissolving the solid-phase resin in DMSO / water / acetonitrile (2 / 1 / 1) at a final peptide concentration of 5 mM based on the molar ratio. Then, 10 equivalents of triethylamine are added and stirred at room temperature for 1 hour. The reaction solution is quenched with acetic acid and concentrated under reduced pressure using a Genevac EZ-II elite.

[0761] The crude product was purified using the following conditions (column: Waters Xbridge (registered trademark) C18 5μm 50x150mm; mobile phase: A = 0.1% TFA in water, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (%B): 5% in 2 minutes, 5-28% in 1 minute, 28-33% in 8 minutes, 33-60% in 1 minute; flow rate: 20 mL / min in 1 minute, then 20-120 mL / min in 1 minute, then 120 mL / min).

[0762] The purity of the target product was calculated from the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under analytical conditions and was 90.9%.

[0763] Analysis conditions: retention time = 3.51 minutes; column: Kinetex EVO C18 2.6 μm 2.1x150 mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% Bconc): 20-60% in 7.15 minutes, then 60-95% in 0.3 minutes, then 95-95% in 1.55 minutes; Flow rate: 0.5 mL / min

[0764] ESI-MS (+) observed value m / z = 807.44 (M+3H) 3+

[0765] [Example 4-16]

[0766] Synthesis of 894_0646 (SEQ ID NO: 178)

[0767] [Chemistry 16]

[0768]

[0769] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.60 mmol / g, 0.21 g) according to the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue solid-phase synthesizer was used for synthesis according to the manufacturer's manual. For the introduction of each residue, Fmoc-AA / DIPCI / Oxyma Pure (4.2 equiv. / 8 equiv. / 4 equiv.) was used per equivalent of resin in DMF at 90°C for a single reaction of 3 minutes. However, for the second and fourth residues, two reactions were performed at 75°C for 30 minutes. For the 12th residue, one reaction was performed at 75°C for 10 minutes. For the 13th residue, two reactions were performed at 90°C for 10 minutes. For the 14th residue, Fmoc-MeF4COO(allyl)-OH was used for a single reaction at 90°C for 3 minutes. The 15th residue was reacted once at 50°C for 15 minutes. Fmoc removal was performed by reacting with a 10% pyrrolidine solution in DMF at 90°C for 1 minute. However, Fmoc removal of the 4th and 13th residues was performed by reacting at room temperature for 1 minute and then for 1 minute. Fmoc removal of the 1st residue was performed after the MeF4COO side chain reacted with H-PEG4c-OtBu. The solid-phase resin removed from the solid-phase synthesis base was suspended in DCM / acetic acid (99 / 1). 0.2 equivalents of tetrakis(triphenylphosphine)palladium(0) and 10 equivalents of phenylsilane were added per equivalent of the resin. The mixture was shaken for 1 hour and washed with DCM and DMF. The resin was suspended in DMF and reacted with H-PEG4c(tBu) / HATU / DIEA (4.2 equivalents / 4 equivalents / 8 equivalents) per equivalent of the resin at room temperature for 10 minutes. Chloroacetyl groups were introduced by reacting the solid-phase resin holding the Fmoc-protected peptide obtained in the previous step with a 10% pyrrolidine solution in DMF at room temperature for 5 minutes. After reacting for 5 minutes to remove the Fmoc group from the α-amino group, the resin was washed with DMF and then shaken for 30 minutes with a solution of chloroacetic acid / HATU / DIEA (5 equivalents / 5 equivalents / 10 equivalents) in DMF. To deprotect the side chain and cleave it from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with chloromethane, followed by washing with diethyl ether and drying under reduced pressure. Next, reactant mixture A (5 mL, a mixture of TFA / H₂O / TIS / DODT in a volume ratio of 92.5 / 2.5 / 2.5 / 2.5) was added to the reaction vessel containing the solid-phase resin and shaken at room temperature for 90 minutes. The reaction solution was recovered by filtration through the frit. The solid phase resin remaining in the reaction container is shaken again with the excision mixture, and the solution component is recovered from the frit and mixed with the aforementioned filtrate.When the filtrate is added to an excess of a diethyl ether / hexane (1 / 1) mixed solvent cooled to 0°C, a white precipitate is formed. The mixture is centrifuged (9000 rpm, 0°C, 2 min), and the solution is decanted. The resulting solid is washed again with a small amount of diethyl ether cooled to 0°C and then dried under reduced pressure. The resulting solid is used in the subsequent cyclization reaction. The peptide cyclization reaction is performed by dissolving the peptide in DMSO / water (95 / 5) to a final concentration of 5 mM based on the molar number of the solid phase resin, adding 10 equivalents of triethylamine, and stirring at room temperature for 1 hour. The reaction solution is quenched with acetic acid and concentrated under reduced pressure using a Genevac EZ-II elite.

[0770] The crude product was purified using the following conditions (column: Waters XBridge (registered trademark) C18 5μm 50x150mm; mobile phase: A = 0.1% TFA in water, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (% B): 6% in 2 minutes, 6-31% in 1 minute, 31-36% in 8 minutes, and 36-60% in 1 minute; flow rate: 20 mL / min in 1 minute, 20-120 mL / min in 1 minute, and then 120 mL / min).

[0771] The purity of the target product was calculated from the area ratio of the LC / MS chromatogram (UV wavelength 225 nm) under analytical conditions and was 91.8%.

[0772] Analysis conditions: retention time = 3.95 minutes; column: Kinetex EVO C18 2.6 μm 2.1x150 mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% Bconc): 20-60% in 7.15 minutes, then 60-95% in 0.3 minutes, then 95-95% in 1.55 minutes; Flow rate: 0.5 mL / min

[0773] ESI-MS (+) observed value m / z = 909.45 (M+3H) 3+

[0774] Industrial applicability

[0775] This invention can be used in the pharmaceutical industry.

Claims

1. A peptide or a pharmaceutically acceptable salt thereof, which consists of the following amino acid sequence: The amino acid sequence of Ala-Val-MeF3C-Val-W7N-Asn-3Py6NH2-F4OMe-Ile-Ile-Arg-Arg-4Py-MeTyr-Cys (SEQ ID NO: 1), or An amino acid sequence having one or more substitutions selected from the following group in the amino acid sequence described in SEQ ID NO: 1: (I) the second valine residue in SEQ ID NO: 1 is replaced by a polar amino acid; (II) the MeF3C residue at position 3 of SEQ ID NO: 1 is substituted with an N-methyl amino acid having an aromatic ring or a heterocyclic ring in its side chain; (III) the W7N residue in the fifth position of SEQ ID NO: 1 is replaced by tryptophan or N-methyltryptophan, which may have a substituent and may have the carbon atom on the indole ring replaced by an N atom; (IV) substitution of the aspartic acid residue at position 6 of SEQ ID NO: 1 into Nmm; (V) The 3Py6NH2 residue at position 7 of SEQ ID NO: 1 is substituted with an amino acid having an aromatic ring or a heterocyclic ring which may have a substituent in its side chain; (VI) the F4OMe residue at position 8 of SEQ ID NO: 1 is substituted with an amino acid having an aromatic ring or a heterocyclic ring which may have a substituent in its side chain; (VII) substitution of the 9th isoleucine residue in SEQ ID NO: 1 with Eva; (VIII) the isoleucine residue at position 10 of SEQ ID NO: 1 is replaced by any amino acid; (IX) the 11th arginine residue in SEQ ID NO: 1 is substituted with lysine which may have a substituent on its side chain or glutamine which may have a substituent on its side chain; (X) the arginine residue at position 12 of SEQ ID NO: 1 is substituted with lysine, histidine, or glutamine, which may have a substituent on the side chain; (XI) the 4Py residue at position 13 of SEQ ID NO: 1 is substituted with an amino acid having an aromatic ring or a heterocyclic ring which may have a substituent on its side chain, or with glutamic acid which may have a substituent on its side chain; and (XII) The 14th MeTyr residue in SEQ ID NO: 1 is substituted with an N-methyl amino acid having an optionally substituted aromatic or heterocyclic ring in its side chain.

2. The peptide or pharmaceutically acceptable salt thereof according to claim 1, which consists of the amino acid sequence described in SEQ ID NO: 1, or consists of an amino acid sequence having one or more substitutions selected from the following group in the amino acid sequence described in SEQ ID NO: 1: (I) the second valine residue in SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of T and Kmor; (II) the third MeF3C residue in SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of MeF and Me3Py; (III) the W7N residue in the fifth position of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of W1aa, W, W1EtOH, W1Me, 3Imp, and MeW; (IV) the 3Py6NH2 residue at position 7 of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of Y, Y26Df, 3Py6NHaa, Y3Me, and R; (V) the F4OMe residue at position 8 of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of F4aao, F4aao(Glucamine), F4aao(pipzaa), YaeCOpipzaa, 3Py6Ome, 3Py6NHaa, 3Imp, Y, and F4aao(PEG8Me); (VI) the isoleucine residue at position 10 of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of Har, Kmor, T, Mor, Hpr, and S; (VII) the arginine residue at position 11 of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of Kmor, KCOpipzaa, K, E (glucamine), Q, Q which may have a linker on its side chain, E which may have a linker on its side chain, and E (apa); (VIII) the arginine residue at position 12 of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of Kmor, KCOpipzaa, Q, and H; (IX) the 4Py residue at position 13 of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of F3COO, F, F3CONPEG4c, Y3Me, 3Py6Me, 3Py6Ome, 3Py6NH2, 4Py2Me, 4Py2Ome, 4Py2NH2, 3Py, Y, F3COO which may have a linker, and E which may have a linker on its side chain; and (X) The MeTyr residue at position 14 of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of MeF4COO, MeF3COO, MeF4C, MeF4Ome, MeF3C, MeF3Ome, Me4Py, Me3Py, and MeF4COO(PEG4c).

3. The peptide or pharmaceutically acceptable salt thereof according to claim 1 or 2, comprising an amino acid sequence that satisfies at least one of the following conditions: (I) the first position of SEQ ID NO: 1 is an alanine residue; (II) the fourth position of SEQ ID NO: 1 is a valine residue; (III) the sixth residue of SEQ ID NO: 1 is an aspartic acid residue; (IV) the 7th position of SEQ ID NO: 1 is a tyrosine residue or a 3Py6NH2 residue; (V) The ninth position of SEQ ID NO: 1 is an isoleucine residue.

4. The peptide or pharmaceutically acceptable salt thereof according to claim 3, which is composed of an amino acid sequence that satisfies at least one of the following A or B: A: An amino acid sequence that satisfies at least two of the following: (a) the fifth residue of SEQ ID NO: 1 is a W7N residue or a 3Imp residue; (b) The 7th residue of SEQ ID NO: 1 is 3Py6NH2 residue; (c) The eighth residue of SEQ ID NO: 1 is F4aao (pipzaa); (d) The 13th residue of SEQ ID NO: 1 is a 4Py residue, B: (i) The 8th amino acid sequence of SEQ ID NO: 1 is a F4aao (pipzaa) residue, a F4aao (Glucamine) residue, a F4aao (PEG8Me) residue or a YaeCOpipzaa residue.

5. The peptide or pharmaceutically acceptable salt thereof according to claim 1, which consists of the first to fifteenth amino acid sequences of the amino acid sequence described in any one of SEQ ID NOs: 1 to 202 or a combination of the amino acid sequence described in any one of SEQ ID NOs: 1 to 202 and a linker, and wherein the amino acid sequence portion has a cyclic structure. The peptide or a pharmaceutically acceptable salt thereof according to claim 1 , which is a cyclic peptide. The peptide or a pharmaceutically acceptable salt thereof according to claim 3, which has hTfR-binding activity. The peptide or a pharmaceutically acceptable salt thereof according to claim 4, which has hTfR-binding activity in a pH-dependent manner.

9. The peptide or pharmaceutically acceptable salt thereof according to claim 1, wherein A linker is bound to the 8th, 11th, 13th or 15th amino acid of SEQ ID NO:

1. 10 . The peptide or a pharmaceutically acceptable salt thereof according to claim 9 , which is a peptide represented by any one of SEQ ID NOs: 1 to 167 or a pharmaceutically acceptable salt thereof.

11. The peptide or pharmaceutically acceptable salt thereof according to claim 9, wherein The linker is a polyethylene glycol (PEG) linker or a GKN3 linker. 12 . A complex comprising the peptide according to claim 9 or a pharmaceutically acceptable salt thereof and a substance bound to the linker.

13. The composite according to claim 12, wherein The linker is a polyethylene glycol (PEG) linker or a GKN3 linker.

14. A composition comprising the peptide according to claim 1 or a pharmaceutically acceptable salt thereof.

15. A pharmaceutical or diagnostic composition comprising the peptide according to claim 1 or a pharmaceutically acceptable salt thereof.

16. A composition comprising the complex according to claim 12 or 13.

17. A pharmaceutical or diagnostic composition comprising the complex according to claim 12 or 13.

18. A method for producing a pharmaceutical or diagnostic composition, comprising the step of obtaining the complex according to claim 12 or 13.

19. The method according to claim 18, wherein The linker is a polyethylene glycol (PEG) linker or a GKN3 linker.

20. A method for testing a peptide or a pharmaceutically acceptable salt thereof a) Solubility in solvent b) Binding ability to hTfR c) Toxicity to cells and / or tissues d) a test method for the toxicity of at least one peptide or a pharmaceutically acceptable salt thereof to experimental animals, The peptide or its pharmaceutically acceptable salt is a peptide or its pharmaceutically acceptable salt having an amino acid sequence in which 1 to 3 amino acid residues are deleted, substituted, inserted and / or added to the amino acid sequence of the peptide or its pharmaceutically acceptable salt according to claim 1.

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