Peptides and complexes containing the same

Abstract

The present invention provides a peptide that has high myostatin inhibition activity and that has improved stability in vivo. A peptide represented by formula (1) in the description.

Description

[Technical Field]

[0001] The present invention relates to peptides and conjugates containing the same. [Background technology]

[0002] Muscular dystrophies are genetic diseases characterized by the degeneration and necrosis of skeletal muscle, leading to progressive muscle weakness. Muscle strength development requires a mechanism for transmitting tension generated within myofibrils to the extracellular basement membrane via multiple proteins. Malfunctions in the genes encoding the proteins involved in this process can lead to muscular dystrophies. For example, in the most severe form of Duchenne muscular dystrophy, mutations in the dystrophin gene are thought to be the primary cause of the disease, resulting in the loss or dysfunction of this protein. Therefore, as a means of combating the degeneration and necrosis of skeletal muscle in muscular dystrophies, the development of treatments that increase muscle mass by inhibiting the function of myostatin (growth differentiation factor-8, GDF-8), a factor that negatively regulates skeletal muscle mass, is particularly considered effective.

[0003] Myostatin, a member of the TGF-β family, is a secreted protein abundantly expressed in skeletal muscle. It is synthesized intracellularly as a precursor protein containing an N-terminal prodomain and a C-terminal mature domain. Upon secretion, myostatin undergoes a pro-domain propeptide, called latency-associated protein (LAP), which associates with an active dimer derived from the mature domain, thereby inactivating the active dimer, which negatively regulates skeletal muscle mass. Myostatin is stored in the body in an inactive state, and when needed, the propeptide is enzymatically cleaved to become active. Active myostatin functions as a signaling molecule that negatively regulates skeletal muscle mass through binding to receptors, such as the activin type IIB receptor.

[0004] Therefore, by inhibiting myostatin in vivo using peptides derived from the myostatin propeptide, it is expected that effects such as an increase in skeletal muscle mass and the treatment of muscle atrophy disorders such as muscular dystrophy can be achieved. For example, WO 2018 / 030432 (corresponding to the specification of U.S. Patent No. 20190177370) describes myostatin inhibitory peptides derived from the myostatin propeptide. Summary of the Invention

[0005] The myostatin inhibitory peptides described in WO 2018 / 030432 have high myostatin inhibitory activity. However, in addition to high myostatin inhibitory activity, improved stability in vivo is desired.

[0006] Therefore, an object of the present invention is to provide a peptide that has high myostatin inhibitory activity and improved stability in the body. [Means for solving the problem]

[0007] The present inventors have conducted extensive research to solve the above problems, and as a result have found that the above problems can be solved by a peptide composed entirely of D-amino acids, leading to the completion of the present invention.

[0008] One aspect of the present invention relates to a peptide, a pharmaceutically acceptable salt thereof, or a prodrug thereof, which comprises an amino acid sequence represented by the following formula (1) and has 15 to 17 amino acid residues:

[0009] [ka]

[0010] In the above formula (1), X 0 is an amino acid residue or deletion selected from the group consisting of D-Ala, D-Gly, and D-2-aminoisobutyric acid; X1 is an amino acid residue or deletion selected from the group consisting of D-Leu, D-norleucine, D-Val, D-Ile, D-2-aminobutyric acid, D-norvaline, and D-isovaline; X 2 is an amino acid residue selected from the group consisting of D-Arg, D-ornithine, D-Lys, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 3 is an amino acid residue selected from the group consisting of D-2-cyclohexylglycine, D-norleucine, D-Leu, D-Val, D-Ile, D-2-aminobutyric acid, D-norvaline, and D-isovaline; X 4 is an amino acid residue selected from the group consisting of D-Lys, D-Arg, D-ornithine, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 5 is an amino acid residue selected from the group consisting of D-Ser, D-Arg, D-2-hydroxyglycine, D-homoserine, D-Lys, D-ornithine, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 6 is an amino acid residue selected from the group consisting of D-Trp, D-3-(2-naphthyl)alanine, D-Tyr, D-Phe, and D-3-(1-naphthyl)alanine; X 7 is an amino acid residue selected from the group consisting of D-2-phenylglycine, D-norleucine, D-Ile, D-Leu, D-Val, D-2-aminobutyric acid, D-norvaline, and D-isovaline; X 8 is an amino acid residue selected from the group consisting of D-Gln, D-Arg, D-Asn, D-Lys, D-His, D-2,3-diaminopropionic acid, D-2,4-diaminobutanoic acid, and D-ornithine; X 9 is a D-2-cyclohexylglycine residue; X 10is an amino acid residue selected from the group consisting of D-Lys, D-Arg, D-ornithine, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 11 is an amino acid residue selected from the group consisting of D-2-phenylglycine, D-Ile, D-Leu, D-norleucine, D-Val, D-2-aminobutyric acid, D-norvaline, and D-isovaline; X 12 is an amino acid residue selected from the group consisting of D-Trp, D-homophenylalanine, D-Tyr, and D-Phe; X 13 is an amino acid residue selected from the group consisting of D-Arg, D-ornithine, D-Lys, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 14 is an amino acid residue selected from the group consisting of D-2-phenylglycine, D-Ile, D-Leu, D-norleucine, D-Val, D-2-aminobutyric acid, D-norvaline, and D-isovaline; X 15 is an amino acid residue selected from the group consisting of D-Tyr, D-Trp and D-Phe; and X 16 is an amino acid residue or deletion selected from the group consisting of D-Trp, D-homophenylalanine, D-Tyr and D-Phe. [Brief explanation of the drawings]

[0011] [Figure 1] 1 shows the myostatin inhibitory activity of peptides of Examples and Comparative Examples. [Figure 2] 1 shows the myostatin inhibitory activity of the peptides of the examples. [Figure 3] 1 shows the myostatin inhibitory activity of peptides of Examples and Comparative Examples. [Figure 4] 1 shows the myostatin inhibitory activity of the peptides of the examples. [Figure 5]1 shows the results of stability evaluation of the peptide of the example in a solution of bovine pancreatic trypsin. [Figure 6] 1 shows the results of stability evaluation of the peptide of the example in a solution of bovine pancreatic α-chymotrypsin. [Figure 7] 1 shows the results of the effect (in vivo evaluation) of the peptide of the example on the tibialis anterior muscle of mdx mice, a model of Duchenne muscular dystrophy. [Figure 8] 1 shows the results of myostatin oxygenation evaluation using the peptides of the examples. [Figure 9] 1 shows the results of the effect (in vivo evaluation) of intramuscular administration of the peptide of the example on the grip strength of a cancer cachexia model mouse. [Figure 10] 1 shows the results of the effect (in vivo evaluation) of intramuscular administration of the peptide of an example on the gastrocnemius muscle mass of a cancer cachexia model mouse. [Figure 11] 1 shows the results of the effect (in vivo evaluation) of intramuscular administration of a peptide of an example on muscle fiber area in a cancer cachexia model mouse. DETAILED DESCRIPTION OF THE INVENTION

[0012] An embodiment of the present invention will be described below, but the present invention is not limited to the following embodiment.

[0013] In this specification, the range "X to Y" means "X or more and Y or less." Unless otherwise specified, operations and measurements of physical properties are performed at room temperature (20 to 25°C) and a relative humidity of 40 to 50% RH.

[0014] In this specification, "a peptide containing an amino acid sequence represented by formula (1) and having 15 to 17 amino acid residues, or a pharmaceutically acceptable salt thereof" is also simply referred to as "the peptide of the present invention."

[0015] In the present invention, the term "amino acid residue" refers to a portion of a peptide or protein molecule that corresponds to one unit of an amino acid constituting the peptide or protein. More specifically, it refers to a divalent group derived from an α-amino acid, as represented by the following formula (5):

[0016] [ka]

[0017] However, the above R 0 is the side chain of the amino acid, for example, a hydrogen atom for Gly and a methyl group for Ala.

[0018] The "amino acid residue" is derived from a natural or unnatural α-amino acid. The peptide of the present invention is composed of D-amino acids in order to increase stability in vivo.

[0019] More specifically, examples of "amino acid residues" include Arg, Lys, Asp, Asn, Glu, Gln, His, Pro, Tyr, Trp, Ser, Thr, Gly, Ala, Met, Cys, Phe, Leu, Val, and Ile, and analogs thereof. The analogues may be, for example, derivatives in which the side chains of the 20 amino acid residues are substituted with any substituent, and examples thereof include halogenated derivatives of the 20 amino acid residues (e.g., 3-chloroalanine, 4-fluorophenylalanine, 4-chlorophenylalanine), 2-aminobutyric acid, 4-aminobutyric acid, norleucine, norvaline, isovaline, 2-aminoisobutyric acid, homophenylalanine, 2,3-diaminopropionic acid, 2,4-diaminobutanoic acid, ornithine, 2-hydroxyglycine, homoserine, hydroxylysine, hydroxyproline, 3,4-didehydroproline, homocysteine, homomethionine, aspartic acid esters (e.g., aspartic acid methyl ester, aspartic acid ethyl ester, aspartic acid propyl ester, aspartic acid Examples include, but are not limited to, amino acid residues derived from amino acids such as glutamic acid esters (glutamic acid cyclohexyl ester, glutamic acid ethyl ester, glutamic acid propyl ester, glutamic acid methyl ester, glutamic acid benzyl ester, etc.), glutamic acid esters (glutamic acid cyclohexyl ester, glutamic acid ethyl ester, glutamic acid propyl ester, glutamic acid methyl ester, glutamic acid benzyl ester, etc.), formyltryptophan, 2-cyclopentylglycine, 2-cyclohexylglycine, 2-phenylglycine, β-alanine, 3-cyclopentylalanine, 3-cyclohexylalanine, 3-pyridylalanine, 3-pyrazolylalanine, 3-furanylalanine, 3-thienylalanine, 4-methoxyphenylalanine, and 3-naphthylalanine (3-(1-naphthyl)alanine, 3-(2-naphthyl)alanine). Furthermore, for those having an asymmetric carbon in the side chain, such as Ile and Thr, which exist as diastereomers, the natural form (e.g., (2R * ,3R * )-2-amino-3-methylpentanoic acid, and (2R * ,3S *)-2-amino-3-hydroxybutanoic acid) and unnatural forms (e.g., (2R * ,3S * )-2-amino-3-methylpentanoic acid, and (2R * ,3R * )-2-amino-3-hydroxybutanoic acid) can be used without distinction. That is, "Ile" means (2R * ,3R * )-2-amino-3-methylpentanoic acid and (2R * ,3S * )-2-amino-3-methylpentanoic acid, and "Thr" is used to mean both (2R * ,3S * )-2-amino-3-hydroxybutanoic acid and (2R * ,3R * )-2-amino-3-hydroxybutanoic acid. Preferably, the natural diastereomer (i.e., Ile, (2R * ,3R * )-2-amino-3-methylpentanoic acid, if Thr, (2R * ,3S * )-2-amino-3-hydroxybutanoic acid) is used.

[0020] Unless otherwise specified, the amino acid sequences described herein are conventionally written from the N-terminus (amino terminus) to the C-terminus (carboxyl terminus).

[0021] It is known in the art that each amino acid residue can be substituted with an amino acid residue having similar properties based on differences in its side chain (conservative substitution). For example, the aliphatic hydrophobic amino acids Val, Leu, Ile, 2-aminobutyric acid (Abu), norleucine (Nle), norvaline (Nva), and isovaline (Iva) can be substituted for each other. Gly, Ala, and 2-aminoisobutyric acid (Aib), whose side chains are hydrogen atoms or methyl groups, can be substituted for each other. Phe and homophenylalanine (Hph), whose side chains are phenylalkyl groups, can be substituted for each other. The neutral polar amino acids Asn and Gln can be substituted for each other. The basic amino acids Arg, Lys, His, 2,3-diaminopropionic acid (Dpr), 2,4-diaminobutanoic acid (Dbu), and ornithine (Orn) can be substituted for each other. The acidic amino acids Asp and Glu can be substituted for each other. Ser, 2-hydroxyglycine (Hyg), and homoserine (Hse), whose side chains are hydroxy groups or short-chain hydroxyalkyl groups, can be substituted for each other. Pro and 3,4-didehydroproline (Dhp), whose side chain has a dehydrogenated pyrrolidyl group, can be substituted for each other. Cys and homocysteine ​​(Hcy), whose side chains are short-chain thiolalkyl groups, can be substituted for each other. Met and homomethionine (Hme), whose side chains are short-chain sulfide structures, can be substituted for each other. Trp, Tyr, and Phe, whose side chains are aromatic, can be substituted for each other.

[0022] Additionally, the amino acid residues Gly and Pro, which affect chain orientation, can be substituted for each other.

[0023] As used herein, the term "pharmaceutically acceptable salt" refers to a metal salt, ammonium salt, organic acid salt, inorganic acid salt, or salt with an organic or inorganic base that does not produce undesirable physiological effects after administration to a patient or subject. More specifically, examples include, but are not limited to, sodium salt, potassium salt, calcium salt, magnesium salt, barium salt, aluminum salt, zinc salt, ammonium salt, methylamine salt, ethylamine salt, aniline salt, dimethylamine salt, diethylamine salt, pyrrolidine salt, piperidine salt, morpholine salt, piperazine salt, trimethylamine salt, triethylamine salt, ethanolamine salt, diethanolamine salt, triethanolamine salt, hydrochloride, hydrobromide, nitrate, sulfate, phosphate, formate, acetate, trifluoroacetate, phthalate, fumarate, oxalate, tartrate, maleate, citrate, succinate, malate, methanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0024] A prodrug of the peptide of the present invention (hereinafter, "prodrug of the peptide of the present invention" may also be simply referred to as "prodrug") refers to a peptide derivative that is converted into the peptide of the present invention, i.e., a peptide derivative that is converted into the peptide of the present invention by oxidation, reduction, hydrolysis, etc. with gastric acid, enzymes, etc. These peptide derivatives can be produced from the peptide of the present invention by conventionally known methods described, for example, in Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985.

[0025] When the side chain of the peptide of the present invention has a carboxyl group, examples of prodrugs include ester derivatives obtained by reacting the carboxyl group with an alcohol, or amide derivatives obtained by reacting the carboxyl group with an amine. More specific examples include peptides in which the carboxyl group in the peptide side chain is derivatized with an ester represented by -COOR (R is an alkyl group having 1 to 20 carbon atoms), or an amide group represented by -CONHR or -CONRR' (R and R' are each independently an alkyl group having 1 to 20 carbon atoms).

[0026] When the side chain of the peptide according to the present invention has a hydroxyl group, an example of the prodrug is an acyloxy derivative obtained by acylation by reacting the hydroxyl group with an acid anhydride, etc. More specifically, for example, a peptide in which the hydroxyl group in the peptide side chain is derivatized with an acyloxy group represented by -OCOR (R is an alkyl group having 1 to 20 carbon atoms) can be mentioned.

[0027] When the peptide of the present invention has an amino group in the side chain, examples of the prodrug include derivatives in which the amino group is acylated, N-oxidized, alkylated, or phosphorylated. More specifically, examples include peptides in which the amino group in the side chain is derivatized with an amide group represented by -NHCOR (R is an alkyl group having 1 to 20 carbon atoms) or -NHCOCH(NH2)CH3.

[0028] The N-terminal structure of the peptide according to the present invention is not particularly limited, and may be, for example, a hydrogen atom (i.e., unmodified), or a structure to which a modifying group has been introduced by a conventionally known method. Examples of N-terminal modifying groups include alkyl groups having 1 to 20 carbon atoms, alkenyl groups having 1 to 20 carbon atoms, alkynyl groups having 1 to 20 carbon atoms, aromatic hydrocarbon groups having 6 to 20 carbon atoms, heterocyclic groups, groups represented by the following formula (6), sulfonyl groups, carboxyl groups, glyoxyl groups, formyl groups; polyethylene glycol groups (PEGylated), polyoxyethylene glycol groups, polypropylene glycol groups; tert-butoxycarbonyl groups (Boc groups), benzyloxycarbonyl groups (Z groups), fluorocarbonyl groups, and the like. Examples of protecting groups include cycloalkyloxycarbonyl groups such as cyclopentyloxycarbonyl, cyclohexyloxycarbonyl, adamantyloxycarbonyl, norbornyloxycarbonyl, and isobornyloxycarbonyl; protecting groups derived from amino acids such as pyroglutamic acid and morotanoic acid; carbamate protecting groups; and protecting groups derived from sulfonic acids such as benzenesulfonic acid and phosphoric acid. Among these, from the viewpoint of myostatin inhibitory activity, the N-terminus of the peptide is preferably a hydrogen atom, an alkyl group, an aromatic hydrocarbon group, a heterocyclic group, a group represented by the following formula (6), a sulfonyl group, a carboxyl group, a glyoxyl group, a formyl group, or a polyethylene glycol group, more preferably a hydrogen atom, an acyl group, or a polyethylene glycol group, and even more preferably a hydrogen atom.

[0029] The number of carbon atoms in the alkyl group that may be present at the N-terminus of the peptide is, for example, 1 to 20, and preferably 1 to 10. The alkyl group may have a saturated chain, an unsaturated chain, or a cyclic structure, or may have a branched chain structure. More specific examples of the alkyl group include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an amyl group, an isoamyl group, a tert-amyl group, a hexyl group, a cyclohexyl group, a heptyl group, an octyl group, a 2-ethylhexyl group, a nonyl group, and a decyl group.

[0030] The aromatic hydrocarbon group that may be present at the N-terminus of the peptide has, for example, 6 to 20 carbon atoms, and more specific examples thereof include a phenyl group, a naphthyl group, a tolyl group, and a phenanthryl group. Examples of heterocyclic groups that may be present at the N-terminus of the peptide include substituents having a monocyclic, fused bicyclic, or fused tricyclic structure containing 1 to 3 heteroatoms selected from a nitrogen atom, an oxygen atom, and a sulfur atom in the ring, and more specific examples thereof include a pyrrolidyl group, a pyrrole group, a piperidyl group, a pyridyl group, an imidazolyl group, a pyrazolyl group, an oxazolyl group, a thiazolyl group, a morpholyl group, an indolyl group, a benzimidazolyl group, a quinolyl group, a carbazolyl group, a tetrahydrofuranyl group, a tetrahydrothiophenyl group, a furanyl group, a thiophenyl group, a tetrahydropyranyl group, and a tetrahydrothiopyranyl group. These aromatic hydrocarbon groups and heterocyclic groups may be substituted with a further substituent such as a linear or branched alkyl group having 1 to 6 carbon atoms, a linear or branched alkoxy group having 1 to 6 carbon atoms, an amino group, a carboxyl group, an ester group, a carbamoyl group, an amido group, a nitro group, a sulfo group, a sulfonamido group, and / or a halogen atom.

[0031] The modifying group at the N-terminus may be, for example, a functional group represented by the following formula (6).

[0032] [ka]

[0033] However, in equation (6), X 0Arepresents a single bond, an oxygen atom, or a sulfur atom, or a divalent linking group selected from the group consisting of alkylene groups having 1 to 3 carbon atoms (e.g., methylene, ethylene, trimethylene, and propylene groups), oxyalkylene groups having 1 to 3 carbon atoms (e.g., oxymethylene, oxyethylene, oxytrimethylene, and oxypropylene groups), and alkyleneoxy groups having 1 to 3 carbon atoms (e.g., methyleneoxy, ethyleneoxy, trimethyleneoxy, and propyleneoxy groups), which optionally have a substituent selected from the group consisting of amino groups, acetylamino groups, and propionylamino groups; R 1A represents an alkyl group having 1 to 20 carbon atoms which may have a substituent, and

[0034] [ka]

[0035] is selected from the group consisting of Above R 11 ~R 30 are each independently selected from the group consisting of a hydrogen atom, a halogen atom (e.g., a fluorine atom, a chlorine atom, a bromine atom, an iodine atom), an alkyl group having 1 to 3 carbon atoms (i.e., a methyl group, an ethyl group, a propyl group), an alkoxy group having 1 to 3 carbon atoms (i.e., a methoxy group, an ethoxy group, a propoxy group), a hydroxyl group, and an amino group.

[0036] In the above formula (6), R 1A When R is an alkyl group having 1 to 20 carbon atoms which may have a substituent, the aliphatic chain may have a saturated chain, an unsaturated chain, or a cyclic structure, or may have a branched chain structure. 1A The alkyl group preferably has 2 to 12 carbon atoms.

[0037] R 1AExamples of the substituent include a hydroxy group, an alkoxy group having 1 to 5 carbon atoms (such as a methoxy group or an ethoxy group), an amino group, a carboxyl group, an ester group, a carbamoyl group, an amide group, a nitro group, a sulfo group, and a halogen atom (fluorine, chlorine, bromine, or iodine).

[0038] In the above formula (6), preferably, X 0A is a single bond or a divalent linking group selected from the group consisting of alkylene groups having 1 to 3 carbon atoms and oxyalkylene groups having 1 to 3 carbon atoms, which may have a substituent selected from the group consisting of amino groups and acetylamino groups.

[0039] In one embodiment, the group represented by the formula (6) is an acyl group. Examples of the acyl group include acyl groups derived from various carboxylic acids. More specifically, the acyl group may be an acyl group having an aliphatic chain, an aromatic ring, or a heterocyclic ring, or may be an acyl group derived from a compound selected from the group consisting of an amino acid, a vitamin having an acyl group, and a nucleic acid base having an acyl group.

[0040] R in the above formula (6) 1A

[0039] More specific examples of the acyl group, which is an alkyl group having 1 to 20 carbon atoms and which may have a substituent, include, but are not limited to, an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a valeryl group, an isovaleryl group, a pivaloyl group, a caproyl group, a caprinoyl group, a methylhexanoyl group, a cyclopropanecarbonyl group, an aminocyclopropanecarbonyl group, a cyclohexanecarbonyl group, a cyclohexylacetyl group, a cyclopentylpropionyl group, a cyclohexylpropionyl group, a cyclopentylbutanoyl group, a cyclohexylbutanoyl group, an adamantylacetyl group, a lauroyl group, a myristoyl group, a palmitoyl group, a stearoyl group, an oxalyl group, a malonyl group, a succinyl group, a glutaryl group, an adipoyl group, a glycol group, a lactoyl group, a glyceroyl group, a pyruvoyl group, and an acetoacetyl group.

[0041] Examples of vitamins having an acyl group include nicotinic acid, pantothenic acid, biotin, pteroylglutamic acid (folic acid), orotic acid, fluoroorotic acid, α-lipoic acid, pyridoxic acid, biocytin, pteroic acid, 10-formylpteroic acid, 7,8-dihydrofolic acid, homopteroic acid, pterin-6-carboxylic acid, dihydrolipoic acid, and hydroorotic acid.

[0042] The nucleic acid base derivative having an acyl group refers to a base component constituting a nucleotide and its derivatives, and preferred examples include pyrimidine derivatives, such as 5-carboxymethyluracil and 5-carboxythiouracil.

[0043] Examples of sulfonyl groups that can be present at the N-terminus of a peptide include those having a structure in which the carbonyl structure in the above-mentioned acyl group is converted into a sulfone structure.

[0044] The polyethylene glycol group that can be present at the N-terminus of a peptide has a structure in which polyethylene glycol or its analogues are linked via an ester bond, an amine (-NH-), an acyl group (e.g., an acyl group having 1 to 12 carbon atoms), or a combination thereof. The number of carbon atoms in the polyethylene glycol group can be, for example, 2 to 20 (i.e., -(C2H4O) n -, where n=1 to 10), preferably 4 to 16 (i.e., -(C2H4O) n -, where n = 2 to 8. The end of the polyethylene glycol group opposite to the end linked to the N-terminus of the peptide may be modified with an amino group or a protecting group generally used to protect a hydroxyl group, such as an alkyl group having 1 to 6 carbon atoms (e.g., methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, tert-butyl, amyl, isoamyl, tert-amyl, or hexyl).

[0045] The structure of the C-terminus of the peptide according to the present invention is also not particularly limited, and may be a structure modified with a protecting group commonly used for protecting carboxylic acids. More specifically, the C-terminus of the peptide according to the present invention may be, for example, a carboxyl group (-COOH), a carboxylate (-COO - ), amide (-CONH2), alkylamide (-CONHR 31 , -CONR 31 R 32 ), ester (-COOR 31 ), acyloxyalkyl (-R 33 -OCOR 31 ), a phthalidyl group optionally substituted with an alkyl or alkoxy group having 1 to 4 carbon atoms (for example, a phthalidyl group, a dimethylphthalidyl group, or a dimethoxyphthalidyl group), or a (5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl group. Of these, the C-terminus of the peptide is preferably an amide. In the above alkylamide, ester, and acyloxyalkyl groups, R 31 and R 32 are each independently an alkyl group having 1 to 6 carbon atoms, such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an amyl group, an isoamyl group, a tert-amyl group, a hexyl group, or a cyclohexyl group; an aryl group having 6 to 10 carbon atoms, such as a phenyl group or a naphthyl group; an aralkyl group having 7 to 18 carbon atoms, such as a benzyl group, a phenethyl group, or a benzhydryl group; a sugar, such as glucose; or a polyethylene glycol group optionally modified with an alkyl group having 1 to 6 carbon atoms (e.g., a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an amyl group, an isoamyl group, a tert-amyl group, or a hexyl group). 33 is an alkylene group having 1 to 4 carbon atoms such as a methylene group, an ethylene group, an n-propylene group, an isopropylene group, an n-butylene group, an isobutylene group, an s-butylene group, or a t-butylene group.

[0046] The peptides of the present invention also include peptide derivatives chemically modified by covalent bonding with polymers, lipids, etc., and derivatives in which the α-helix contained within the peptide has been further enhanced. Examples of derivatives in which the α-helix has been further enhanced include derivatives in which a salt bridge is formed at the i, i+4, or other amino acid positions, and derivatives having a cross-linked structure formed by a disulfide bond, a carbon-carbon bond, or the like.

[0047] The number of amino acid residues of the peptide according to the present invention is 15 to 17. Having 17 or fewer amino acid residues is advantageous in terms of both synthesis and bioavailability. If the number of amino acid residues is less than 14, the effects of the present invention cannot be achieved.

[0048] <Peptide> One aspect of the present invention is a peptide, or a pharmaceutically acceptable salt thereof, or a prodrug thereof, which comprises an amino acid sequence represented by the following formula (1) and has 15 to 17 amino acid residues:

[0049] [ka]

[0050] In the above formula (1), X 0 is an amino acid residue or deletion selected from the group consisting of D-Ala, D-Gly, and D-2-aminoisobutyric acid; X 1 is an amino acid residue or deletion selected from the group consisting of D-Leu, D-norleucine, D-Val, D-Ile, D-2-aminobutyric acid, D-norvaline, and D-isovaline; X 2 is an amino acid residue selected from the group consisting of D-Arg, D-ornithine, D-Lys, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 3is an amino acid residue selected from the group consisting of D-2-cyclohexylglycine, D-norleucine, D-Leu, D-Val, D-Ile, D-2-aminobutyric acid, D-norvaline, and D-isovaline; X 4 is an amino acid residue selected from the group consisting of D-Lys, D-Arg, D-ornithine, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 5 is an amino acid residue selected from the group consisting of D-Ser, D-Arg, D-2-hydroxyglycine, D-homoserine, D-Lys, D-ornithine, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 6 is an amino acid residue selected from the group consisting of D-Trp, D-3-(2-naphthyl)alanine, D-Tyr, D-Phe, and D-3-(1-naphthyl)alanine; X 7 is an amino acid residue selected from the group consisting of D-2-phenylglycine, D-norleucine, D-Ile, D-Leu, D-Val, D-2-aminobutyric acid, D-norvaline, and D-isovaline; X 8 is an amino acid residue selected from the group consisting of D-Gln, D-Arg, D-Asn, D-Lys, D-His, D-2,3-diaminopropionic acid, D-2,4-diaminobutanoic acid, and D-ornithine; X 9 is a D-2-cyclohexylglycine residue; X 10 is an amino acid residue selected from the group consisting of D-Lys, D-Arg, D-ornithine, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 11 is an amino acid residue selected from the group consisting of D-2-phenylglycine, D-Ile, D-Leu, D-norleucine, D-Val, D-2-aminobutyric acid, D-norvaline, and D-isovaline; X 12is an amino acid residue selected from the group consisting of D-Trp, D-homophenylalanine, D-Tyr, and D-Phe; X 13 is an amino acid residue selected from the group consisting of D-Arg, D-ornithine, D-Lys, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 14 is an amino acid residue selected from the group consisting of D-2-phenylglycine, D-Ile, D-Leu, D-norleucine, D-Val, D-2-aminobutyric acid, D-norvaline, and D-isovaline; X 15 is an amino acid residue selected from the group consisting of D-Tyr, D-Trp and D-Phe; and X 16 is an amino acid residue or deletion selected from the group consisting of D-Trp, D-homophenylalanine, D-Tyr and D-Phe.

[0051] According to the present invention, it is possible to provide a peptide that has high myostatin inhibitory activity and improved stability in the body.

[0052] More specifically, the peptide of the present invention has high myostatin inhibitory activity, and since it is composed entirely of D-amino acids, it is stable against enzymes in the body and can exhibit sustained, high efficacy when administered to the body.

[0053] In a preferred embodiment, in terms of myostatin inhibitory activity, X 0 is an amino acid residue or deletion selected from the group consisting of D-Ala, D-Gly, and D-2-aminoisobutyric acid; X 1 is an amino acid residue or deletion selected from the group consisting of D-Leu, D-norleucine, D-Val, D-Ile, D-2-aminobutyric acid, D-norvaline, and D-isovaline; X 2is an amino acid residue selected from the group consisting of D-Arg, D-ornithine, D-Lys, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 3 is an amino acid residue selected from the group consisting of D-2-cyclohexylglycine, D-norleucine, D-Leu, D-Val, D-Ile, D-2-aminobutyric acid, D-norvaline, and D-isovaline; X 4 is an amino acid residue selected from the group consisting of D-Lys, D-Arg, D-ornithine, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 5 is an amino acid residue selected from the group consisting of D-Ser, D-Arg, D-2-hydroxyglycine, D-homoserine, D-Lys, D-ornithine, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 6 is an amino acid residue selected from the group consisting of D-Trp, D-3-(2-naphthyl)alanine, D-Tyr, D-Phe and D-3-(1-naphthyl)alanine, more preferably an amino acid residue selected from the group consisting of D-Trp, D-Tyr and D-Phe; X 7 is an amino acid residue selected from the group consisting of D-2-phenylglycine, D-norleucine, D-Ile, D-Leu, D-Val, D-2-aminobutyric acid, D-norvaline, and D-isovaline; X 8 is an amino acid residue selected from the group consisting of D-Gln, D-Arg, D-Asn, D-Lys, D-His, D-2,3-diaminopropionic acid, D-2,4-diaminobutanoic acid, and D-ornithine; X 9 is a D-2-cyclohexylglycine residue; X 10is an amino acid residue selected from the group consisting of D-Lys, D-Arg, D-ornithine, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 11 is a D-2-phenylglycine residue; X 12 is an amino acid residue selected from the group consisting of D-Trp, D-homophenylalanine, D-Tyr, and D-Phe; X 13 is an amino acid residue selected from the group consisting of D-Arg, D-ornithine, D-Lys, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 14 is a D-2-phenylglycine residue; X 15 is an amino acid residue selected from the group consisting of D-Tyr, D-Trp and D-Phe; and X 16 is an amino acid residue or deletion selected from the group consisting of D-Trp, D-homophenylalanine, D-Tyr and D-Phe, more preferably an amino acid residue or deletion selected from the group consisting of D-Trp, D-Tyr and D-Phe.

[0054] In a preferred embodiment, in terms of myostatin inhibitory activity, X 0 is an amino acid residue or deletion selected from the group consisting of D-Ala, D-Gly, and D-2-aminoisobutyric acid; X 1 is an amino acid residue or deletion selected from the group consisting of D-Leu, D-norleucine, D-Val, D-Ile, D-2-aminobutyric acid, D-norvaline, and D-isovaline; X 2 is an amino acid residue selected from the group consisting of D-Arg, D-ornithine, D-Lys, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 3is a D-2-cyclohexylglycine residue; X 4 is an amino acid residue selected from the group consisting of D-Lys, D-Arg, D-ornithine, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 5 is an amino acid residue selected from the group consisting of D-Ser, D-Arg, D-2-hydroxyglycine, D-homoserine, D-Lys, D-ornithine, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 6 is an amino acid residue selected from the group consisting of D-Trp, D-3-(2-naphthyl)alanine, D-Tyr, D-Phe, and D-3-(1-naphthyl)alanine; X 7 is an amino acid residue selected from the group consisting of D-Ile, D-Leu, D-norleucine, D-Val, D-2-aminobutyric acid, D-norvaline, and D-isovaline; X 8 is an amino acid residue selected from the group consisting of D-Gln, D-Arg, D-Asn, D-Lys, D-His, D-2,3-diaminopropionic acid, D-2,4-diaminobutanoic acid, and D-ornithine; X 9 is a D-2-cyclohexylglycine residue; X 10 is an amino acid residue selected from the group consisting of D-Lys, D-Arg, D-ornithine, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 11 is an amino acid residue selected from the group consisting of D-Ile, D-Leu, D-norleucine, D-Val, D-2-aminobutyric acid, D-norvaline, and D-isovaline; X 12 is an amino acid residue selected from the group consisting of D-Trp, D-homophenylalanine, D-Tyr, and D-Phe; X 13is an amino acid residue selected from the group consisting of D-Arg, D-ornithine, D-Lys, D-His, D-2,3-diaminopropionic acid, and D-2,4-diaminobutanoic acid; X 14 is an amino acid residue selected from the group consisting of D-Ile, D-Leu, D-norleucine, D-Val, D-2-aminobutyric acid, D-norvaline, and D-isovaline; X 15 is an amino acid residue selected from the group consisting of D-Tyr, D-Trp and D-Phe; and X 16 is an amino acid residue or deletion selected from the group consisting of D-Trp, D-homophenylalanine, D-Tyr and D-Phe, more preferably an amino acid residue selected from the group consisting of D-Trp, D-homophenylalanine, D-Tyr and D-Phe.

[0055] In a further preferred embodiment, in terms of myostatin inhibitory activity, in the above formula (1), X 0 is a D-Ala residue or deletion; X 1 is a D-Leu residue, a D-norleucine residue, or a deletion; X 2 is a D-Arg residue or a D-ornithine residue; X 3 is a D-2-cyclohexylglycine residue or a D-norleucine residue; X 4 is a D-Lys residue, a D-Arg residue, or a D-ornithine residue; X 5 is a D-Ser or D-Arg residue; X 6 is a D-Trp residue or a D-3-(2-naphthyl)alanine residue; X 7 is a D-2-phenylglycine residue or a D-Ile residue; X 8 is a D-Gln or D-Arg residue; X9 is a D-2-cyclohexylglycine residue; X 10 is an amino acid residue selected from the group consisting of D-Lys, D-Arg, and D-ornithine; X 11 is a D-2-phenylglycine residue or a D-Ile residue; X 12 is a D-Trp residue or a D-homophenylalanine residue; X 13 is a D-Arg residue or a D-ornithine residue; X 14 is a D-2-phenylglycine residue or a D-Ile residue; X 15 is a D-Tyr residue; and X 16 is a D-Trp residue, a D-homophenylalanine residue, or a deletion.

[0056] In the peptide according to the present invention, the peptide represented by the above formula (1) preferably comprises any one of the amino acid sequences represented by SEQ ID NOs: 1 to 25, from the viewpoint of myostatin inhibitory activity.

[0057] [Table 1]

[0058] In the peptides of the present invention, from the viewpoint of myostatin inhibitory activity, the peptide represented by the above formula (1) more preferably comprises any one of the amino acid sequences represented by SEQ ID NOs: 1 to 4, 6, 8 to 23 and 25, and particularly preferably comprises any one of the amino acid sequences represented by SEQ ID NOs: 19 to 23 and 25.

[0059] (Method for producing peptides) The peptides of the present invention can be produced by conventional methods, including chemical synthesis and recombinant technology. To prepare peptides by chemical synthesis, each amino acid can be produced by a method commonly used in peptide chemistry, such as the methods described in "The Peptides," Vol. 1 (Schroder and Luhke, Academic Press, New York, USA, 1966) and "Fundamentals and Experiments of Peptide Synthesis" (Izumiya Nobuo et al., Maruzen Co., Ltd., 1985). Both liquid-phase and solid-phase methods can be used. Furthermore, both column and batch methods can be used.

[0060] The peptides of the present invention may also be produced by recombinant technology using animal cells, insect cells, microorganisms, or the like, for example, by the techniques described in Current Protocols in Molecular Biology, Chapter 16 below. After being produced in cultured cells or microorganisms, the peptides can be purified by conventionally known methods. Methods for purifying and isolating peptides are known to those skilled in the art and can be carried out by the techniques described, for example, in Current Protocols in Molecular Biology, Chapter 16 (Ausubel et al., John Wiley and Sons, 2006).

[0061] Condensation methods for forming peptide bonds include the azide method, acid halide method, acid anhydride method, carbodiimide method, carbodiimide-additive method, active ester method, carbonylimidazole method, oxidation-reduction method, enzymatic method, and methods using Woodward reagent K, HATU reagent, Bop reagent, etc. Of the above-mentioned methods for condensation reactions using solid phase methods, the acid anhydride method, carbodiimide method, and active ester method are the main methods.

[0062] Furthermore, when extending a peptide chain using the solid-phase method, the C-terminal amino acid is bound to a support such as a resin that is insoluble in the organic solvent used. Such resins may include those into which functional groups have been introduced for the purpose of binding the amino acid to the resin, or those into which a spacer has been inserted between the resin and the functional group, depending on the purpose. More specific examples include halomethyl resins such as chloromethyl resin, oxymethyl resin, 4-(oxymethyl)-phenylacetamidomethyl resin, 4-(oxymethyl)-phenoxymethyl resin, and Rink amide resin. Prior to these condensation reactions, carboxyl groups, amino groups, hydroxyl groups, amidino groups, and the like that do not participate in the condensation reaction can be protected by commonly known means. Conversely, carboxyl groups and amino groups that directly participate in the condensation reaction can also be activated.

[0063] The protecting groups used to protect the functional groups not involved in the condensation reaction of each unit can be those commonly used in organic chemistry, such as those described in "Protective Groups in Organic Synthesis" (Greene, John Wiley & Sons, Inc. (1981)). More specifically, protecting groups for carboxyl groups include commonly known protecting groups such as various methyl esters, ethyl esters, benzyl esters, p-nitrobenzyl esters, t-butyl esters, and cyclohexyl esters. Protecting groups for amino groups include benzyloxycarbonyl, t-butoxycarbonyl, isobornyloxycarbonyl, and 9-fluorenylmethoxycarbonyl (Fmoc) groups.

[0064] Examples of activated carboxyl groups include acid anhydrides corresponding to the carboxyl groups, azides, and activated esters with pentafluorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, N-hydroxysuccinimide, N-hydroxy-5-norbornene-2,3-dicarboximide, N-hydroxyphthalimide, 1-hydroxybenzotriazole, etc. Examples of activated amino groups include phosphoric acid amides corresponding to the amino groups.

[0065] Condensation reactions during peptide synthesis are usually carried out in a solvent. Examples of such solvents include chloroform, dichloromethane, ethyl acetate, N,N-dimethylformamide, dimethyl sulfoxide, pyridine, dioxane, tetrahydrofuran, N-methylpyrrolidone, water, methanol, and mixtures thereof. The reaction temperature for the condensation reaction is usually within the range of -30°C to 50°C.

[0066] Furthermore, the type of protecting group elimination reaction in the production process of the peptide of the present invention can be selected depending on the type of protecting group used, as long as it can eliminate the protecting group without affecting the peptide bond. Examples of such reactions include acid treatment with hydrogen chloride, hydrogen bromide, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof; alkali treatment with sodium hydroxide, potassium hydroxide, hydrazine, diethylamine, piperidine, or the like; sodium treatment in liquid ammonia; reduction with palladium on carbon; and silylation treatment with trimethylsilyl triflate, trimethylsilyl bromide, or the like. In the deprotection reaction using the above acid or silylating agent treatment, it is preferable to add a cation scavenger such as anisole, phenol, cresol, thioanisole, or ethanedithiol, from the viewpoint of efficient deprotection.

[0067] The peptide of the present invention synthesized by the solid-phase method can also be cleaved from the solid phase by a commonly known method. For example, the cleavage method can include treatment with the above-mentioned acid or silylating agent. The peptide of the present invention thus produced can be subjected to commonly known separation and purification procedures after completion of the above series of reactions. For example, the peptide of the present invention can be obtained with higher purity by extraction, distribution, reprecipitation, recrystallization, solid-phase extraction, column chromatography, etc.

[0068] The obtained peptides can be analyzed using an automated amino acid analyzer, capillary electrophoresis, reverse-phase high-performance liquid chromatography, mass spectrometry, etc. Peptides may also be selected using their interaction with myostatin as an indicator using various biomolecular interaction analysis techniques, such as phage display, two-hybrid analysis, affinity chromatography, surface plasmon resonance, co-immunoprecipitation, protein chip analysis, three-dimensional structural analysis, far-western blotting, and fluorescence quenching.

[0069] The peptide of the present invention may be isolated or purified. "Isolated or purified" means that a procedure has been performed to remove components other than the target component. The purity of the isolated or purified peptide of the present invention is usually 50% or more (e.g., 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%).

[0070] <Complex> One aspect of the present invention is a complex represented by the following formula (2) or a pharmaceutically acceptable salt thereof:

[0071] [ka]

[0072] In formula (2), Y is expressed by the following formula (3):

[0073] [ka]

[0074] [In formula 3, R 1 and R 2 each independently represents a halogenoalkyl group or a halogen atom, R 3 represents a bromine atom, an iodine atom, or a selenium atom, R 4 and R 5 each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group, R 6 and R 7 each independently represents a hydrogen atom, a halogen atom, an alkoxy group, or a substituted or unsubstituted alkyl group, and R 4 and R 6 or R 5 and R 7 may be taken together to form a substituted or unsubstituted alkylene or alkenylene group, R 8 represents a hydrogen atom or a substituted or unsubstituted alkyl group; R 9 and R 10 each independently represents a hydrogen atom, a halogen atom, an alkoxy group, or a substituted or unsubstituted alkyl group, and R 8 and R 9 or R 10 and may together form a substituted or unsubstituted alkylene or alkenylene group, m and n each independently represent an integer of 1 to 3; * denotes the binding site for L] is a compound represented by the formula: L represents a linker between Y and Z; Z is the peptide according to the present invention.

[0075] In this specification, the "conjugate represented by formula (2) or a pharmaceutically acceptable salt thereof" is also simply referred to as the "conjugate of the present invention."

[0076] The components constituting the complex of the present invention will be explained below, except that the peptide Z of the present invention is the same as above, and therefore will not be explained here.

[0077] (Compound Y represented by formula (3)) In the above formula (2), Y is a compound represented by the above formula (3). The compound represented by formula (3) is an on / off switch catalyst that enables target-selective photooxygenation. By linking the compound represented by formula (3) with the peptide of the present invention to form a complex, the complex can be activated by light with a wavelength of 650 to 800 nm and selectively oxygenate myostatin, thereby exhibiting even greater myostatin inhibitory activity.

[0078] In equation (3), R 1 and R 2 each independently represents a halogenoalkyl group or a halogen atom.

[0079] The halogenoalkyl group is preferably a linear or branched halogenoalkyl group having 1 to 6 carbon atoms, more preferably a linear or branched halogenoalkyl group having 1 to 4 carbon atoms, still more preferably a trifluoromethyl group or a pentafluoroethyl group, and particularly preferably a trifluoromethyl group.

[0080] Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom, and a fluorine atom is preferred.

[0081] In formula (3), preferably R 1 is a halogen atom, and the R 2 is a halogenoalkyl group.

[0082] In equation (3), R 3 represents a bromine atom, an iodine atom, or a selenium atom, and is preferably a bromine atom.

[0083] In equation (3), R 4 and R5 R each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group. 6 and R 7 each independently represents a hydrogen atom, a halogen atom, an alkoxy group, or a substituted or unsubstituted alkyl group. 4 and R 6 or R 5 and R 7 may be taken together to form a substituted or unsubstituted alkylene or alkenylene group.

[0084] In equation (3), R 8 represents a hydrogen atom or a substituted or unsubstituted alkyl group. 9 and R 10 each independently represents a hydrogen atom, a halogen atom, an alkoxy group, or a substituted or unsubstituted alkyl group. 8 and R 9 or R 10 may be taken together to form a substituted or unsubstituted alkylene or alkenylene group.

[0085] In formula (3), preferably R 4 and R 6 , R 5 and R 7 and R 8 and R 10 together form a substituted or unsubstituted alkylene or alkenylene group, wherein the alkylene or alkenylene group has 2 or 3 carbon atoms.

[0086] R 4 ~R 10The alkyl group in the formula (I) is preferably a linear or branched alkyl group having 1 to 6 carbon atoms, more preferably a linear or branched alkyl group having 1 to 4 carbon atoms. Examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, and a tert-butyl group. The groups that can substitute these alkyl groups are preferably 1 to 3 groups selected from a carboxy group, a sulfonic acid group, a hydroxy group, an amino group, -CO-, -CONH-, and a triazole group. From the viewpoint of increasing water solubility, the groups that can substitute the alkyl group are more preferably selected from a carboxy group, a sulfonic acid group, a hydroxy group, and an amino group.

[0087] R 6 , R 7 , R 9 and R 10 The alkoxy group in R is preferably a linear or branched alkoxy group having 1 to 6 carbon atoms, more preferably a linear or branched alkoxy group having 1 to 4 carbon atoms. Specific examples include a methoxy group, an ethoxy group, and a propyloxy group. 6 , R 7 , R 9 and R 10 Examples of the halogen atom in the formula include a chlorine atom, a bromine atom, an iodine atom, and a fluorine atom.

[0088] In a preferred embodiment, R 4 and R 6 , R 5 and R 7 and R 8 and R 10 together form a substituted or unsubstituted alkylene or alkenylene group, wherein the alkylene or alkenylene group has 2 or 3 carbon atoms. Examples of the alkylene group include an ethylene group and a trimethylene group. Examples of the alkenylene group include a vinylene group and a propenylene group.

[0089] Examples of ring structures formed by combining these groups include the following structures.

[0090] [ka]

[0091] In the above structures a-1 to a-9 and b-1 to b-8, R 4 ~R 7 and R 9 ~R 10 represents a group that does not form an alkylene group or an alkenylene group.

[0092] In formula (3), m and n each independently represent an integer of 1 to 3. m and n each independently are preferably 1 or 2, and more preferably 1.

[0093] In formula (3), * is the bonding site to L.

[0094] When the compound represented by formula (3) has an asymmetric carbon atom, optical isomers exist, and the compound represented by formula (3) includes both optical isomers and racemates.

[0095] (Linker L) In formula (2), L represents a linker between the compound Y represented by formula (3) and the peptide Z according to the present invention.

[0096] The binding site of the linker L to the peptide Z of the present invention is not particularly limited, and may be the N-terminus or C-terminus of the peptide Z of the present invention, or may be a side chain of an amino acid residue constituting the peptide Z, as long as the effects of the present invention are achieved. In a preferred embodiment, the linker L is bound to the N-terminus of the peptide Z of the present invention.

[0097] The specific structure of such a linker unit is not particularly limited, and examples thereof include a substituted or unsubstituted alkylene group having 1 to 20 carbon atoms, a substituted or unsubstituted alkenylene group having 2 to 20 carbon atoms, a substituted or unsubstituted alkynylene group having 2 to 20 carbon atoms, a substituted or unsubstituted cycloalkylene group having 3 to 20 carbon atoms, a substituted or unsubstituted cycloalkenylene group having 3 to 20 carbon atoms, a substituted or unsubstituted arylene group having 6 to 20 carbon atoms, a substituted or unsubstituted heteroarylene group having 3 to 20 carbon atoms, -NH-, -O-, -S-, -C(=O)-NH-, -NH-C(=O)-, -O-, -C(=O)-O-, -OC(=O)-, -S-, -C(=O)-, a polyoxyalkylene group, an amino acid residue, a peptide chain, polyethylene glycol, and combinations thereof.

[0098] In one embodiment, L has a structure represented by formula (4):

[0099] [ka]

[0100] Here, V represents -CO-, -CONH-, or a triazole ring. O represents an integer of 0 or 1. Examples of the triazole ring include a 1,2,3-triazole-1,4-diyl group and a 1,2,4-triazole-1,3-diyl group. l and p each independently represent an integer of 1 to 6. l preferably represents an integer of 1 to 6, more preferably an integer of 1 to 4. p preferably represents an integer of 1 to 6, more preferably an integer of 1 to 4. W represents -NH-, -O-, -S-, -C(=O)-NH-, -NH-C(=O)-, -O-, -C(=O)-O-, -OC(=O)-, -S-, or -C(=O)-.

[0101] In formula (4), *1 is the linking site to the compound represented by formula (3), and *2 is the linking site to the peptide of the present invention.

[0102] (Method of manufacturing the composite) There are no particular limitations on the method for producing the complex of the present invention. A person skilled in the art would be able to produce the complex of the present invention based on the description in the Examples section below and taking into consideration the common general technical knowledge at the time of filing this application.

[0103] For example, synthesis can be carried out by appropriately referring to the descriptions in H. Okamoto et al., Chem. Commun., 2019, 55, 9108-9111. and International Publication No. 2017 / 164172.

[0104] Hereinafter, the method for producing the composite according to the present invention will be described using the composite represented by the following formula (2-1) as an example.

[0105] [ka]

[0106] In formula (2-1), R 1 ~R 7 and Z are as defined for formula (2) above.

[0107] The complex represented by formula (2-1) can be produced according to the following reaction scheme.

[0108] [ka]

[0109] Compound a and compound b are condensed to obtain compound c, and then compound c is reacted with compound d to obtain the complex represented by formula (2-1).

[0110] The reaction between compound a and compound b is an aldol condensation reaction.

[0111] The reaction between compound c and compound d is a 1,3-dipolar addition reaction between an alkyne and an azide, which proceeds easily in the presence of a copper catalyst in a polar solvent such as dimethylformamide at room temperature.

[0112] Compounds a to d can be synthesized by appropriately referring to the descriptions in H. Okamoto et al., Chem. Commun., 2019, 55, 9108-9111, WO 2017 / 164172, and the like.

[0113] The resulting complex represented by formula (2-1) can be purified by known means such as chromatography.

[0114] <Myostatin inhibitors, preventive / therapeutic agents, preventive / therapeutic methods> One embodiment of the present invention provides a myostatin inhibitor comprising the peptide of the present invention or a prodrug thereof, or the conjugate of the present invention (hereinafter, a "myostatin inhibitor comprising the peptide of the present invention or a prodrug thereof, or the conjugate of the present invention" will also be referred to simply as a "myostatin inhibitor"). By administering an effective amount of the myostatin inhibitor to a subject, effects such as maintaining, increasing, enhancing, or inhibiting the decline of muscle mass and strength can be achieved. The myostatin inhibitor may consist of one or more peptides of the present invention, one or more prodrugs thereof, one or more conjugates of the present invention, or a mixture thereof, but is typically a pharmaceutical composition comprising one or more peptides of the present invention, their prodrugs, and the conjugate of the present invention, as well as a pharmaceutically acceptable carrier.

[0115] One embodiment of the present invention relates to a method for inhibiting myostatin, comprising administering to a patient an effective amount of a peptide according to the present invention or a prodrug thereof, or a conjugate according to the present invention. One embodiment of the present invention also relates to a peptide according to the present invention or a prodrug thereof, or a conjugate according to the present invention, for use in inhibiting myostatin.

[0116] One embodiment of the present invention provides a preventive and / or therapeutic agent for muscle atrophy disorders, comprising the peptide of the present invention or a prodrug thereof, or the conjugate of the present invention (hereinafter, the "preventive and / or therapeutic agent for muscle atrophy disorders, comprising the peptide of the present invention or a prodrug thereof, or the conjugate of the present invention" will also be simply referred to as the "preventive and / or therapeutic agent for muscle atrophy disorders"). By administering an effective amount of the preventive and / or therapeutic agent for muscle atrophy disorders to a patient, therapeutic effects such as slowing the rate of progression, inhibiting progression, halting progression, ameliorating, curing, and / or preventing muscle atrophy disorders can be achieved. The preventive and / or therapeutic agent for muscle atrophy disorders may consist of one or more peptides of the present invention, one or more prodrugs thereof, one or more conjugates of the present invention, or a mixture thereof. However, it is typically a pharmaceutical composition containing one or more peptides of the present invention, their prodrugs, and the conjugate of the present invention, as well as a pharmaceutically acceptable carrier.

[0117] One embodiment of the present invention relates to a method for preventing and / or treating a muscle wasting disorder, which comprises administering to a patient an effective amount of the peptide according to the present invention or a prodrug thereof, or the conjugate according to the present invention. One embodiment of the present invention also relates to the peptide according to the present invention or a prodrug thereof, or the conjugate according to the present invention, for use in the prevention and / or treatment of a muscle wasting disorder.

[0118] Myostatin inhibitors, preventive and therapeutic agents for muscle atrophy disorders, and the above-mentioned preventive and / or therapeutic methods are also effective for strengthening the tibialis anterior muscle through local administration in elderly people with walking difficulties. Simply strengthening the tibialis anterior muscle facilitates dorsiflexion of the ankle joint, leading to the prevention of falls. Furthermore, continuous local administration during spaceflight could contribute to shortening the rehabilitation period after return, for example.

[0119] Examples of the muscle atrophy disorders include, but are not limited to, muscular dystrophy, distal myopathy, congenital myopathy, inflammatory muscle diseases such as inclusion body myositis, myopathies such as mitochondrial myopathy, disuse muscle atrophy, and sarcopenia. The preventive and therapeutic agent for muscle atrophy disorders is preferably used effectively for muscular dystrophy and sarcopenia. The preventive and therapeutic agent for muscle atrophy disorders is more preferably used effectively for muscular dystrophies such as Duchenne muscular dystrophy, Becker muscular dystrophy, Fukuyama congenital muscular dystrophy, merosin-deficient congenital muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, Emery-Dreifuss muscular dystrophy, Miyoshi muscular dystrophy, and infantile neuroaxonal muscular dystrophy, as well as sarcopenia, and is particularly effective for Duchenne muscular dystrophy.

[0120] Muscle wasting disorders can also result from chronic diseases such as amyotrophic lateral sclerosis, chronic obstructive pulmonary disease (COPD), cancer, AIDS, renal failure, and rheumatoid arthritis. Muscle wasting disorders can also result from metabolic disorders such as diabetes and related disorders. Therefore, the prophylactic and therapeutic agents for muscle wasting disorders of the present invention and the above-mentioned preventive and / or therapeutic methods can be used to improve cachexia associated with muscle wasting. Furthermore, increasing muscle mass through myostatin inhibition can improve bone strength and reduce osteoporosis and other degenerative bone diseases.

[0121] One embodiment of the present invention relates to an agent for preventing and / or treating muscle wasting disorders caused by diabetes, comprising the peptide according to the present invention or a prodrug thereof, or the conjugate according to the present invention. One embodiment of the present invention also relates to a method for preventing and / or treating muscle wasting disorders caused by diabetes, comprising administering to a patient an effective amount of the peptide according to the present invention or a prodrug thereof, or the conjugate according to the present invention.

[0122] One embodiment of the present invention relates to an agent for preventing and / or treating muscle wasting disorders caused by cancer cachexia, comprising the peptide of the present invention or a prodrug thereof, or the conjugate of the present invention. One embodiment of the present invention also relates to a method for preventing and / or treating muscle wasting disorders caused by cancer cachexia, comprising administering to a patient an effective amount of the peptide of the present invention or a prodrug thereof, or the conjugate of the present invention.

[0123] As used herein, a therapeutically "effective amount" is an amount effective for producing some desired therapeutic effect commensurate with a reasonable benefit / risk ratio.

[0124] As used herein, the terms "subject" and "patient" include humans and non-human animals including fish, but are preferably selected from mammals such as humans, dogs, cats, mice, rats, hamsters, guinea pigs, horses (including racehorses), cows, pigs, rabbits, and sheep, and poultry such as chickens, quails, and turkeys, with humans being more preferred.

[0125] Examples of the pharmaceutically acceptable carrier include, but are not limited to, excipients such as lactose, sucrose, mannitol, starch, corn starch, crystalline cellulose, and light anhydrous silicic acid; lubricants such as silica, talc, calcium stearate, and magnesium stearate; binders such as hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, methyl cellulose, polyvinylpyrrolidone, crystalline cellulose, dextrin, and gelatin; antioxidants such as ascorbic acid, sodium sulfite, sodium bisulfite, and tocopherol; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as borate, bicarbonate, Tris-HCl, citrate, phosphate, and other organic acids; water for injection, physiological saline, ethanol, protease inhibitors, and the like. Examples of suitable surfactants include solvents such as alcohol, ethylene glycol, propylene glycol, macrogol, olive oil, and corn oil; surfactants or humectants such as Pluronic®, polyethylene glycol, sorbitan fatty acid esters, polysorbates, Triton®, lecithin, cholesterol, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; isotonicity agents such as sodium chloride, potassium chloride, glycerin, glucose, sorbitol, and mannitol; preservatives such as benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, and chlorhexidine; complexing agents; amino acids; antimicrobial agents; colorants; flavoring agents and diluents; emulsifiers; salt-forming counterions such as sodium; delivery vehicles; and diluents (Remington's Pharmaceutical Sciences, 18th ed., edited by A.R. Gennaro, Mack Publishing Company, 1990).

[0126] The content of the peptide according to the present invention or a prodrug thereof, or the conjugate according to the present invention in the drug may be 0.01 to 100% by weight based on the total weight of the drug.

[0127] The dosage of the peptide or prodrug thereof according to the present invention, or the conjugate according to the present invention varies depending on age, symptoms, administration method, etc., but in the case of oral administration, it is generally about 0.1 to 100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg per day for a human (assuming a body weight of 60 kg). In the case of parenteral administration, the single dose varies depending on age, symptoms, administration method, etc., but for example, in the form of an injection, it is usually convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about 0.1 to 10 mg per day for a human (assuming a body weight of 60 kg). In the case of animals other than humans, the amount converted to per 60 kg body weight can also be administered. [Example]

[0128] The effects of the present invention will be explained using the following examples and comparative examples, although the technical scope of the present invention is not limited to the following examples.

[0129] <Peptide synthesis> (Synthesis Example 1) Synthesis of peptide riDM-4 containing the amino acid sequence of SEQ ID NO: 1 HD-Leu-D-Arg-D-Chg-D-Lys-D-Ser-D-Trp-D-Phg-D-Gln-D-Chg-D-Lys-D-Phg-D-Trp-D-Arg-D-Phg-D-Tyr-D-Trp-NH2 D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-4 was synthesized by the Fmoc solid-phase peptide synthesis method described below.

[0130] 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) was weighed into a reaction vessel for solid-phase synthesis, and the resin was allowed to stand in a dimethylformamide (DMF) solution at room temperature for 1 hour to swell. The resin was then reacted in a 20% (v / v) piperidine / DMF solution (2 mL) at room temperature (25°C) for 20 minutes to remove the protecting group Fmoc (9-fluorenylmethoxycarbonyl) on the resin. The resin was washed 10 times with DMF (2.5 mL) and reacted with Fmoc-D-Trp(Boc)-OH (0.20 mmol, 10 eq.) in the presence of 16 mg (0.10 mmol, 5 eq.) of 1-hydroxybenzotriazole (HOBt) and 0.016 mL (0.10 mmol, 5 eq.) of N,N-diisopropylcarbodiimide (DIPCD) at room temperature for 30 min in DMF (2 mL) to introduce the amino acid onto the resin. To prepare the next amino acid for condensation, the Fmoc group on the resin was removed by reaction in 20% (v / v) piperidine / DMF solution (2.5 mL) for 20 min. The following were prepared in the same manner as for Fmoc-D-Trp(Boc)-OH, from the C-terminus: Fmoc-D-Tyr(tBu)-OH (0.20 mmol, 10 eq.), Fmoc-D-Phg-OH (0.20 mmol, 10 eq.), Fmoc-D-Arg(Pmc)-OH (0.20 mmol, 10 eq.), Fmoc-D-Trp(Boc)-OH (0.20 mmol, 10 eq.), Fmoc-D-Phg-OH (0.20 mmol, 10 eq.), Fmoc-D-Lys(Boc)-OH (0.20 mmol, 10 eq.), Fmoc-D-Chg-OH (0.20 mmol, 10 eq.), Fmoc-D The peptide chain was elongated by introducing Fmoc-Gln(Trt)-OH (0.20 mmol, 10 eq.), Fmoc-D-Phg-OH (0.20 mmol, 10 eq.), Fmoc-D-Trp(Boc)-OH (0.20 mmol, 10 eq.), Fmoc-D-Ser(tBu)-OH (0.20 mmol, 10 eq.), Fmoc-D-Lys(Boc)-OH (0.20 mmol, 10 eq.), Fmoc-D-Chg-OH (0.20 mmol, 10 eq.), Fmoc-D-Arg(Pmc)-OH (0.20 mmol, 10 eq.), and Fmoc-D-Leu-OH (0.20 mmol, 10 eq.).The N-terminal Fmoc group was removed by incubation in 20% (v / v) piperidine / DMF solution (2.5 mL) for 20 minutes. After washing with DMF (2.5 mL, 8 times), methanol (2.5 mL, 5 times), and diethyl ether (2.5 mL, 5 times), the resin was dried. To remove various side chain protecting groups and remove the resin, the resin was incubated in 4.0 mL of trifluoroacetic acid (TFA) in the presence of m-cresol (0.10 mL), thioanisole (0.10 mL), and 1,2-ethanedithiol (0.10 mL) for 2 hours. After removing the resin by filtration using a filter funnel, the TFA was evaporated under nitrogen sparge, and 40 mL of diethyl ether was added to precipitate the crude peptide. The crude peptide was dissolved in a 0.75 M acetic acid-25% (v / v) acetonitrile mixture and purified by gradient elution (purification) using reversed-phase high-performance liquid chromatography with a water-acetonitrile mixture containing 0.1% TFA as the mobile phase to obtain a white solid (6.6 mg, 12% yield). HRMS(ES+)calcd for(M 3+ +3H)1157.1340,found 1157.1326.

[0131] (Synthesis Example 2) Synthesis of peptide riDM-8 containing the amino acid sequence of SEQ ID NO: 2 HD-Ala-D-Leu-D-Arg-D-Chg-D-Lys-D-Ser-D-Trp-D-Phg-D-Gln-D-Chg-D-Lys-D-Phg-D-Trp-D-Arg-D-Phg-D-Tyr-D-Trp-NH2 D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-8 was synthesized and purified (6.2 mg, 11% yield) using 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2384.30,found 2383.69.

[0132] (Synthesis Example 3) Synthesis of peptide riDM-9 containing the amino acid sequence of SEQ ID NO: 3 HD-Arg-D-Chg-D-Lys-D-Ser-D-Trp-D-Phg-D-Gln-D-Chg-D-Lys-D-Phg-D-Trp-D-Arg-D-Phg-D-Tyr-D-Trp-NH2 D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-9 was synthesized and purified (8.4 mg, 16% yield) using 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2200.18,found 2200.30.

[0133] (Synthesis Example 4) Synthesis of peptide containing the amino acid sequence of SEQ ID NO: 4: riDM-10 HD-Leu-D-Arg-D-Chg-D-Lys-D-Ser-D-Trp-D-Phg-D-Gln-D-Chg-D-Lys-D-Phg-D-Trp-D-Arg-D-Phg-D-Tyr-NH2 D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-10 was synthesized and purified (9.5 mg, 19% yield) using 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2127.18,found 2127.69.

[0134] (Synthesis Example 5) Synthesis of peptide: riDM-12 containing the following amino acid sequence (SEQ ID NO: 26: underlined portion) (Comparative Example) H- D-Leu-D-Arg-D-Chg-D-Lys-D-Ser-D-Trp-D-Phg-D-Chg-D-Lys-D-Phg-D-Trp-D-Arg-D-Phg-D-Tyr-D-Trp -NH2 D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-12 was synthesized and purified (4.0 mg, yield 7.6%) using 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2185.20,found 2184.69.

[0135] (Synthesis Example 6) Synthesis of peptide containing the amino acid sequence of SEQ ID NO: 5: riDM-13 HD-Leu-D-Arg-D-Chg-D-Lys-D-Ser-D-Ala(2-Naph)-D-Phg-D-Gln-D-Chg-D-Lys-D-Phg-D-Trp-D-Arg-D-Phg-D-Tyr-D-Trp-NH2 D-Chg: D-cyclohexylglycine D-Ala(2-Naph): D-3-(2-naphthyl)alanine D-Phg: D-phenylglycine riDM-13 was synthesized and purified (2.9 mg, yield 2.6%) using 108 mg (0.040 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2324.26,found 2324.10.

[0136] (Synthesis Example 7) Synthesis of peptide containing the amino acid sequence of SEQ ID NO: 6: riDM-14 HD-Leu-D-Arg-D-Chg-D-Lys-D-Ser-D-Trp-D-Phg-D-Gln-D-Chg-D-Lys-D-Phg-D-homoPhe-D-Arg-D-Phg-D-Tyr-D-Trp-NH2 D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine D-homoPhe: D-homophenylalanine riDM-14 was synthesized and purified (1.7 mg, yield 3.1%) using 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2288.26,found 2288.33.

[0137] (Synthesis Example 8) Synthesis of peptide containing the amino acid sequence of SEQ ID NO: 7: riDM-15 HD-Leu-D-Arg-D-Chg-D-Lys-D-Ser-D-Trp-D-Phg-D-Gln-D-Chg-D-Lys-D-Phg-D-Trp-D-Arg-D-Phg-D-Tyr-D-homoPhe-NH2 D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine D-homoPhe: D-homophenylalanine riDM-15 was synthesized and purified (7.1 mg, 13% yield) using 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2288.26,found 2288.08.

[0138] (Synthesis Example 9) Synthesis of peptide containing the amino acid sequence of SEQ ID NO: 8: riDM-26 HD-Leu-D-Arg-D-Chg-D-Arg-D-Ser-D-Trp-D-Phg-D-Gln-D-Chg-D-Lys-D-Phg-D-Trp-D-Arg-D-Phg-D-Tyr-D-Trp-NH2 D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-26 was synthesized and purified (4.0 mg, yield 7.2%) using 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2341.27,found 2341.11.

[0139] (Synthesis Example 10) Synthesis of peptide containing the amino acid sequence of SEQ ID NO: 9: riDM-27 HD-Leu-D-Arg-D-Chg-D-Lys-D-Arg-D-Trp-D-Phg-D-Gln-D-Chg-D-Lys-D-Phg-D-Trp-D-Arg-D-Phg-D-Tyr-D-Trp-NH2 D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-27 was synthesized and purified (11.4 mg, 19% yield) using 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2382.33,found 2382.04.

[0140] (Synthesis Example 11) Synthesis of peptide containing the amino acid sequence of SEQ ID NO: 10: riDM-28 HD-Leu-D-Arg-D-Chg-D-Lys-D-Ser-D-Trp-D-Phg-D-Arg-D-Chg-D-Lys-D-Phg-D-Trp-D-Arg-D-Phg-D-Tyr-D-Trp-NH2 D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-28 was synthesized and purified (6.2 mg, 11% yield) using 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2341.30,found 2340.74.

[0141] (Synthesis Example 12) Synthesis of riDM-29: a peptide containing the amino acid sequence of SEQ ID NO: 11 HD-Leu-D-Arg-D-Chg-D-Lys-D-Ser-D-Trp-D-Phg-D-Gln-D-Chg-D-Arg-D-Phg-D-Trp-D-Arg-D-Phg-D-Tyr-D-Trp-NH2 D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-29 was synthesized and purified (1.2 mg, yield 2.2%) using 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2341.26,found 2340.85.

[0142] (Synthesis Example 13) Synthesis of peptide containing the amino acid sequence of SEQ ID NO: 12: riDM-18 HD-Nle-D-Arg-D-Chg-D-Lys-D-Ser-D-Trp-D-Phg-D-Gln-D-Chg-D-Lys-D-Phg-D-Trp-D-Arg-D-Phg-D-Tyr-D-Trp-NH2 D-Nle: D-norleucine D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-18 was synthesized and purified (6.3 mg, 11% yield) using 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2313.26,found 2314.13.

[0143] (Synthesis Example 14) Synthesis of riDM-19: a peptide containing the amino acid sequence of SEQ ID NO: 13 HD-Leu-D-Arg-D-Nle-D-Lys-D-Ser-D-Trp-D-Phg-D-Gln-D-Chg-D-Lys-D-Phg-D-Trp-D-Arg-D-Phg-D-Tyr-D-Trp-NH2 D-Nle: D-norleucine D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-19 was synthesized and purified (5.2 mg, yield 9.5%) using 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2287.24,found 2287.13.

[0144] (Synthesis Example 15) Synthesis of peptide containing the amino acid sequence of SEQ ID NO: 14: riDM-20 HD-Leu-D-Arg-D-Chg-D-Lys-D-Ser-D-Trp-D-Nle-D-Gln-D-Chg-D-Lys-D-Phg-D-Trp-D-Arg-D-Phg-D-Tyr-D-Trp-NH2 D-Chg: D-cyclohexylglycine D-Nle: D-norleucine D-Phg: D-phenylglycine riDM-20 was synthesized and purified (2.2 mg, yield 4.0%) using 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2287.24,found 2287.15.

[0145] (Synthesis Example 16) Synthesis of riDM-22: a peptide containing the amino acid sequence of SEQ ID NO: 15 HD-Leu-D-Orn-D-Chg-D-Lys-D-Ser-D-Trp-D-Phg-D-Gln-D-Chg-D-Lys-D-Phg-D-Trp-D-Arg-D-Phg-D-Tyr-D-Trp-NH2 D-Orn: D-ornithine D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-22 was synthesized and purified (7.9 mg, 15% yield) using 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2271.24,found 2271.75.

[0146] (Synthesis Example 17) Synthesis of peptide containing the amino acid sequence of SEQ ID NO: 16: riDM-23 HD-Leu-D-Arg-D-Chg-D-Orn-D-Ser-D-Trp-D-Phg-D-Gln-D-Chg-D-Lys-D-Phg-D-Trp-D-Arg-D-Phg-D-Tyr-D-Trp-NH2 D-Chg: D-cyclohexylglycine D-Orn: D-ornithine D-Phg: D-phenylglycine riDM-23 was synthesized and purified (8.6 mg, 16% yield) using 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2299.24,found 2299.43.

[0147] (Synthesis Example 18) Synthesis of peptide containing the amino acid sequence of SEQ ID NO: 17: riDM-24 HD-Leu-D-Arg-D-Chg-D-Lys-D-Ser-D-Trp-D-Phg-D-Gln-D-Chg-D-Orn-D-Phg-D-Trp-D-Arg-D-Phg-D-Tyr-D-Trp-NH2 D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine D-Orn: D-ornithine riDM-24 was synthesized and purified (5.2 mg, yield 9.4%) using 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2299.24,found 2299.21.

[0148] (Synthesis Example 19) Synthesis of peptide containing the amino acid sequence of SEQ ID NO: 18: riDM-25 HD-Leu-D-Arg-D-Chg-D-Lys-D-Ser-D-Trp-D-Phg-D-Gln-D-Chg-D-Lys-D-Phg-D-Orn-D-Arg-D-Phg-D-Tyr-D-Trp-NH2 D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine D-Orn: D-ornithine riDM-25 was synthesized and purified (5.7 mg, 10% yield) using 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2271.24,found 2271.11.

[0149] (Synthesis Example 20) Synthesis of peptide n-66ri containing the amino acid sequence of SEQ ID NO: 19 HD-Leu-D-Arg-D-Chg-D-Lys-D-Ser-D-Trp-D-Ile-D-Gln-D-Chg-D-Lys-D-Ile-D-Trp-D-Arg-D-Ile-D-Tyr-D-Trp-NH2 D-Chg: D-cyclohexylglycine n-66ri was synthesized and purified (5.4 mg, 10% yield) in the same manner as in Synthesis Example 1 using 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.). HRMS(ES+)calcd for(M 3+ +3H)751.7899,found 751.7890.

[0150] (Synthesis Example 21) Synthesis of peptide containing the amino acid sequence of SEQ ID NO: 20: riDM-33 HD-Leu-D-Arg-D-Chg-D-Lys-D-Arg-D-Trp-D-Ile-D-Gln-D-Chg-D-Lys-D-Ile-D-Trp-D-Arg-D-Ile-D-Tyr-D-Trp-NH2 D-Chg: D-cyclohexylglycine riDM-33 was synthesized and purified (26.1 mg, yield 23%) using 108 mg (0.040 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M ++H)2323.41,found 2322.53.

[0151] (Synthesis Example 22) Synthesis of peptide containing the amino acid sequence of SEQ ID NO: 21: riDM-34 HD-Leu-D-Arg-D-Chg-D-Lys-D-Ser-D-Trp-D-Ile-D-Arg-D-Chg-D-Lys-D-Ile-D-Trp-D-Arg-D-Ile-D-Tyr-D-Trp-NH2 D-Chg: D-cyclohexylglycine riDM-34 was synthesized and purified (21.3 mg, 19% yield) using 108 mg (0.040 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2281.40,found 2282.18.

[0152] (Synthesis Example 23) Synthesis of peptide containing the amino acid sequence of SEQ ID NO: 22: riDM-35 HD-Leu-D-Arg-D-Chg-D-Lys-D-Arg-D-Trp-D-Ile-D-Arg-D-Chg-D-Lys-D-Ile-D-Trp-D-Arg-D-Ile-D-Tyr-D-Trp-NH2 D-Chg: D-cyclohexylglycine riDM-35 was synthesized and purified (26.2 mg, yield 22%) using 108 mg (0.040 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. HRMS(ES+)calcd for(M + +H)2350.4657,found 2350.4646.

[0153] (Synthesis Example 24) Synthesis of peptide: riDM-42 containing the following amino acid sequence (SEQ ID NO: 27: underlined portion) (Comparative Example) H- D-Arg-D-Chg-D-Lys-D-Arg-D-Trp-D-Ile-D-Arg-D-Chg-D-Lys-D-Ile-D-Trp-D-Arg-D-Ile-D-Tyr -NH2 D-Chg: D-cyclohexylglycine riDM-42 was synthesized and purified (30.2 mg, yield 28%) using 108 mg (0.040 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2052.63 (average MS),found 2053.26.

[0154] (Synthesis Example 25) Synthesis of peptide containing the amino acid sequence of SEQ ID NO: 23: riDM-36 HD-Arg-D-Chg-D-Lys-D-Arg-D-Trp-D-Ile-D-Arg-D-Chg-D-Lys-D-Ile-D-Trp-D-Arg-D-Ile-D-Tyr-D-Trp-NH2 D-Chg: D-cyclohexylglycine riDM-36 was synthesized and purified (16.1 mg, 28% yield) using 108 mg (0.040 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. HRMS(ES+)calcd for(M 3+ +3H)746.4645,found 746.7000.

[0155] (Synthesis Example 26) Synthesis of peptide containing the amino acid sequence of SEQ ID NO: 24: riDM-39 HD-Leu-D-Arg-D-Chg-D-Lys-D-Arg-D-Trp-D-Ile-D-Arg-D-Chg-D-Lys-D-Ile-D-Trp-D-Arg-D-Ile-D-Tyr-NH2 D-Chg: D-cyclohexylglycine riDM-39 was synthesized and purified (19.0 mg, yield 33%) using 108 mg (0.040 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. HRMS(ES+)calcd for(M 3+ +3H)722.1345,found 722.4000.

[0156] (Synthesis Example 27) Synthesis of peptide containing the amino acid sequence of SEQ ID NO: 25: riDM-46 HD-Leu-D-Arg-D-Chg-D-Lys-D-Arg-D-Ala(2-Naph)-D-Ile-D-Arg-D-Chg-D-Lys-D-Ile-D-homoPhe-D-Arg-D-Ile-D-Tyr-D-homoPhe-NH2 D-Chg: D-cyclohexylglycine D-Ala(2-Naph): D-3-(2-naphthyl)alanine D-homoPhe: D-homophenylalanine riDM-46 was synthesized and purified (44 mg, yield 37%) using 108 mg (0.040 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2311.48,found 2311.45.

[0157] (Synthesis Example 28) Synthesis of complex 16PC-N X 1 -D-Leu-D-Arg-D-Chg-D-Lys-D-Arg-D-Ala(2-Naph)-D-Ile-D-Arg-D-Chg-D-Lys-D-Ile-D-homoPhe-D-Arg-D-Ile-D-Tyr-D-homoPhe-NH2 D-Chg: D-cyclohexylglycine D-Ala(2-Naph): D-3-(2-naphthyl)alanine D-homoPhe: D-homophenylalanine

[0158] [ka]

[0159] [ka]

[0160] Peptide 1 was synthesized and purified (8.2 mg, 24%) using 30 mg (0.011 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in the same manner as in Synthesis Example 1. LRMS (MALDI+) calculation for (M + +H)2392.03,found 2391.04.

[0161] Compound 2 was synthesized using a method similar to that previously reported (H. Okamoto et al., Chem. Commun., 2019, 55, 9108-9111.).

[0162] Peptide 1 (0.60 μmol, 1 eq.) and compound 2 (0.66 μmol, 1.1 eq.) were stirred in N,N-dimethylformamide (DMF) / methanol (MeOH) (1:1) in the presence of tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, 2.4 μmol, 4 eq.), L-ascorbic acid (14 μmol, 24 eq.), and tetrakis(acetonitrile)copper(I) hexafluorophosphate (12 μmol, 20 eq.) at room temperature (25°C) for 1 hour, and the resulting mixture was purified by high-performance liquid chromatography to give a dark blue solid (0.45 mg, 20% yield). HRMS(ESI+)calcd for(M + +H)3062.6752,found 3062.6809.

[0163] (Test Example 1) Evaluation of myostatin inhibitory activity of peptide derivatives by in vitro reporter assay The myostatin inhibitory activity of each peptide derivative (0.3 μM) was evaluated by the following method. The results for the derivatives synthesized in Synthesis Examples 1 to 12 are shown in Figure 1, the results for the derivatives synthesized in Synthesis Examples 13 to 19 in Figure 2, the results for the derivatives synthesized in Synthesis Examples 20 to 24 in Figure 3, and the results for the derivatives synthesized in Synthesis Examples 25 to 27 in Figure 4. With the exception of the peptide derivatives of Synthesis Examples 5 and 24, which were synthesized as comparative examples, the peptide derivatives of the present invention showed significant myostatin inhibitory activity at a concentration of 0.3 μM.

[0164] (1)Cell culture Human embryonic kidney cells, HEK293, were cultured in a 10% FBS DMEM (Nacalai Tesque, Inc.) medium supplemented with non-essential amino acids (Fujifilm Wako Pure Chemical Industries, Ltd.) in a 37°C, 5% CO 2 incubator.

[0165] (2) In vitro reporter assay HEK293 cells were plated at 2.0 x 10 per well in a D-Lys-coated 96-well clear plate (Thermo Fisher Scientific). 4 The cells (100 μL DMEM + 10% (v / v) FBS) were seeded and cultured for 24 hours.

[0166] The next day, 100 ng of pGL4.48[luc2P / SBE / Hygro] (Promega) and 10 ng of pGL4[hRluc / TK] (Promega) as an internal control were mixed with FuGENE HD (Promega) in OPTI-MEM to a final concentration of 41.25 μg / mL per well. The cells were cultured at 37°C for 24 hours, after which the cell culture medium was replaced with serum-free DMEM and cultured for 8 hours.

[0167] The test peptides were suspended in DMSO to a stock concentration of 10 mM and stored at -30°C. One hour before addition to the culture medium, the peptides were suspended in serum-free DMEM medium and allowed to stand at room temperature (25°C) for 20 minutes. Next, the peptides were added to the culture medium at a final concentration of 0.3 μM and myostatin (Merck Millipore) was added at 8 ng / mL, followed by incubation for 4 hours.

[0168] The propeptide protein (RSD) used as a positive control was suspended in PBS containing 0.1% (v / v) BSA to a stock concentration of 10 μM and stored at -30°C. One hour before addition to the culture medium, it was suspended in serum-free DMEM medium and allowed to stand at room temperature (25°C) for 20 minutes. Next, the propeptide protein and myostatin (Merck Millipore) were added to the culture medium to a final concentration of 10 nM, and then cultured for 4 hours.

[0169] After 4 hours of incubation, the culture medium was removed using an aspirator, and the cells were washed with 1x PBS. Then, 50 μL of Passive Lysis buffer (Promega) was added per well to lyse the cells. The lysate was centrifuged at 4500 rpm for 6 minutes at 4°C. After centrifugation, 20 μL of the supernatant was transferred to a white 96-well plate (Costar), and 50 μL of Luciferase Assay Reagent (Promega) was added. Firefly luciferase activity was measured by detecting luminescence using a Luminoskan Ascent (Thermo Fisher Scientific). Furthermore, 50 μL of Stop & Glo Buffer was added, and Renilla luciferase activity was measured using a Luminoskan Ascent for luminescence detection, serving as an internal control.

[0170] (Test Example 2) Evaluation of peptide stability in bovine pancreatic trypsin solution The stability of riDM-35 synthesized in Synthesis Example 23 in a solution of bovine pancreatic trypsin was evaluated by the following method.

[0171] riDM-35 was dissolved in 50 mM Tris-HCl buffer (pH 7.5, 0.15 M NaCl, 10 mM CaCl, 0.05% (w / v) Brij-35) to a final concentration of 50 μM. TPCK-treated bovine pancreatic trypsin (Sigma-Aldrich) was added to a final concentration of 1 μg / mL (total volume 100 μL). The mixture was incubated at 37°C for 400 minutes. 20 μL aliquots were taken at 0 minutes and 400 minutes after the start of incubation. The aliquots were diluted with 80 μL of 25% aqueous acetonitrile and then injected into a high-performance liquid chromatograph (HPLC, Hitachi High-Tech Corporation, Chromaster®). The stability of the riDM-35 was analyzed based on the residual peak area. The column used was a COSMOSIL 5C18-AR-II 4.6 × 150 mm (Nacalai Tesque, Inc.), and a linear gradient of acetonitrile (25–40%, 30 min) in a water-acetonitrile system containing 0.1% trifluoroacetic acid was applied at a flow rate of 1 mL / min, and peptide peaks were detected with a UV detector (220 nm).

[0172] The results are shown in Figure 5. No degradation products derived from riDM-35 were identified by mass spectrometry using an LCMS-2020 (Shimadzu Corporation). riDM-35 exhibited extremely high stability in a solution of bovine pancreatic trypsin.

[0173] (Test Example 3) Evaluation of the stability of riDM-35 in bovine pancreatic α-chymotrypsin solution The stability of riDM-35 synthesized in Synthesis Example 23 in a solution of bovine pancreatic α-chymotrypsin was evaluated by the following method.

[0174] riDM-35 was dissolved in 100 mM Tris-HCl buffer (pH 7.8, containing 10 mM CaCl) to a final concentration of 50 μM, and TLCK-treated bovine pancreatic α-chymotrypsin (Sigma-Aldrich) was added to a final concentration of 2 μg / mL (total volume 100 μL). The mixture was incubated at 37°C for 400 minutes. 20 μL aliquots were taken at 0 minutes and 400 minutes after the start of incubation, diluted with 80 μL of 25% aqueous acetonitrile, and 20 μL was injected into a high-performance liquid chromatograph (HPLC, Hitachi High-Tech Corporation, Chromaster®). Stability was analyzed based on the residual percentage of riDM-35, as determined by the peak area. The column used was a COSMOSIL 5C18-AR-II 4.6 × 150 mm (Nacalai Tesque, Inc.), and a linear gradient of acetonitrile (25–40%, 30 min) in a water-acetonitrile system containing 0.1% trifluoroacetic acid was applied at a flow rate of 1 mL / min, and peptide peaks were detected with a UV detector (220 nm).

[0175] The results are shown in Figure 6. No degradation products derived from riDM-35 were identified by mass spectrometry using an LCMS-2020 (Shimadzu Corporation). riDM-35 exhibited extremely high stability in a solution of bovine pancreatic α-chymotrypsin.

[0176] (Test Example 4) Effect of riDM-4 on the tibialis anterior muscle of mdx mice, a model of Duchenne muscular dystrophy (in vivo evaluation) To verify the grip strength-increasing effect in vivo of riDM-4 synthesized in Synthesis Example 1, evaluation was carried out by the following method.

[0177] riDM-4 was dissolved in saline to a concentration of 0.75 mM. 40 μL of the solution was administered intramuscularly to eight sites on both hind limbs of anesthetized 5-week-old male mdx mice (purchased from CLEA Japan) (40 μL of saline was administered to the control group). Two weeks later, the same amount of riDM-4 (saline was administered to the control group) was administered again, and after another 4 weeks, the mice's grip strength was measured (Saito Mouse Grip Strength Measurement Apparatus MK-380M, Muromachi Kikai Co., Ltd.).

[0178] The results are shown in Figure 7. riDM-4 significantly increased the hindlimb grip strength of mdx mice by approximately 60% based on its myostatin inhibitory activity. This was a greater improvement than the existing MIPE-1686.

[0179] (Test Example 5) Evaluation of myostatin oxygenation using 16PC-N Myostatin oxygenation of 16PC-N synthesized in Synthesis Example 28 was carried out by the following method.

[0180] 16PC-N (3 μM) and myostatin (1 μM) in phosphate buffer (10 mM, pH 7.4) were irradiated with light (730 nm, 14 mW) at 37°C for 30 minutes. After reduction with dithiothreitol, digestion with lysyl endopeptidase, and desalting with ZipTip C18, the mixture was analyzed by MALDI-TOF MS.

[0181] The results are shown in Figure 8. Oxygenated myostatin was observed in the sample after the reaction, suggesting that myostatin was photooxygenated by 16PC-N.

[0182] (Test Example 5) Verification of the effect of intramuscular administration of riDM-35 on improving muscle wasting in cancer cachexia model mice (in vivo evaluation) The ri-DM35 synthesized in Synthesis Example 23 was dissolved in PBS (phosphate buffer solution; 10 mM, pH 7.4) to a concentration of 1 mM. Lewis lung adenocarcinoma cells (LLC) were subcutaneously injected into the dorsal skin of anesthetized C57BL6 / J male mice (purchased from Oriental Yeast Co., Ltd.) at a concentration of 5.0 × 10 6Cancer cachexia model mice were created by LLC transplantation. 4, 11, and 18 days after transplantation, 30 μL of ri-DM35 solution was intramuscularly administered to the gastrocnemius muscles of both legs (control groups received 30 μL of PBS). 22 days after LLC transplantation, the grip strength of the mice's limbs was measured using an Imada digital force gauge.

[0183] The results are shown in Figure 9. The ri-DM35 administration group improved the grip strength of the cancer cachexia model mice by 41.3%.

[0184] In addition, 22 days after LLC transplantation, the mice were autopsied, and the gastrocnemius muscles of both legs were excised and the weight and muscle fiber area were measured.

[0185] The gastrocnemius muscle weight per body weight of the cancer cachexia model mice is shown in Figure 10. The gastrocnemius muscle weight per body weight of the cancer cachexia model mice in the ri-DM35 administration group increased by 19.6%.

[0186] The frequency distribution table of muscle fiber area in the cancer cachexia model mice is shown in Figure 11. The ri-DM35 administration group enlarged the muscle fiber area in the cancer cachexia model mice.

[0187] This application is based on Japanese Patent Application No. 2020-116583 filed on July 6, 2020, the disclosure of which is incorporated herein by reference in its entirety.

Claims

1. A peptide or a pharmaceutically acceptable salt thereof, which comprises an amino acid sequence represented by the following formula (1), has 15 to 17 amino acid residues, and has myostatin inhibitory activity: 【Chemical 1】 In the above formula (1), X ０ is a D-Ala residue or deletion; X １ is a D-Leu residue, a D-norleucine residue, or a deletion; X ２ is a D-Arg residue or a D-ornithine residue; X ３ is a D-2-cyclohexylglycine residue or a D-norleucine residue; X ４ is a D-Lys residue, a D-Arg residue, or a D-ornithine residue; X ５ is a D-Ser or D-Arg residue; X ６ is a D-Trp residue or a D-3-(2-naphthyl)alanine residue; X ７ is a D-2-phenylglycine residue, a D-norleucine residue, or a D-Ile residue; X ８ is a D-Gln or D-Arg residue; X ９ is a D-2-cyclohexylglycine residue; X １０ is an amino acid residue selected from the group consisting of D-Lys, D-Arg, and D-ornithine; X １１ is a D-2-phenylglycine residue or a D-Ile residue; X １２ is a D-Trp residue, a D-ornithine residue, or a D-homophenylalanine residue; X １３ is a D-Arg residue; X １４ is a D-2-phenylglycine residue or a D-Ile residue; X １５ is a D-Tyr residue; and X １６ is a D-Trp residue, a D-homophenylalanine residue, or a deletion.

2. The peptide according to claim 1, or a pharmaceutically acceptable salt thereof, comprising any one of the amino acid sequences represented by SEQ ID NOs: 1 to 25. 【Table 1】

3. The peptide according to claim 2, comprising any one of the amino acid sequences represented by SEQ ID NOs: 1 to 4, 6, 8 to 18, 20 to 23 and 25, or a pharmaceutically acceptable salt thereof.

4. 4. The peptide according to claim 2 or 3, comprising any one of the amino acid sequences represented by SEQ ID NOs: 20 to 23 and 25, or a pharmaceutically acceptable salt thereof.

5. A complex represented by the following formula (2) or a pharmaceutically acceptable salt thereof: 【Chemistry 2】 In formula (2), Y is represented by the following formula (3): 【Chemistry 3】 [In formula 3, R １ and R ２ each independently represents a halogenoalkyl group or a halogen atom, R ３ represents a bromine atom, an iodine atom, or a selenium atom, R ４ and R ５ each independently represents a hydrogen atom or a substituted or unsubstituted alkyl group, R ６ and R ７ each independently represents a hydrogen atom, a halogen atom, an alkoxy group, or a substituted or unsubstituted alkyl group, and R ４ and R ６ or R ５ and R ７ may be taken together to form a substituted or unsubstituted alkylene or alkenylene group, R ８ represents a hydrogen atom or a substituted or unsubstituted alkyl group; R ９ and R １０ each independently represents a hydrogen atom, a halogen atom, an alkoxy group, or a substituted or unsubstituted alkyl group, and R ８ and R ９ or R １０ and may together form a substituted or unsubstituted alkylene or alkenylene group, m and n represent integers of 1 to 3; * is the binding site to L. A compound represented by the formula: L represents a linker between Y and Z; Z is a peptide according to any one of claims 1 to 4.

6. The R １ is a halogen atom, and the R ２ The complex of claim 5 , wherein is a halogenoalkyl group.

7. The complex according to claim 5 or 6, wherein m and n are 1.

8. R ４ and R ６ , R ５ and R ７ and R ８ and R １０ and (b) are bonded together to form a substituted or unsubstituted alkylene or alkenylene group, wherein the alkylene or alkenylene group has 2 or 3 carbon atoms.

9. A myostatin inhibitor comprising the peptide according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, or the complex according to any one of claims 5 to 8 or a pharmaceutically acceptable salt thereof.

10. A preventive and / or therapeutic agent for muscle atrophy disorders, comprising the peptide according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, or the complex according to any one of claims 5 to 8 or a pharmaceutically acceptable salt thereof.

11. The preventive and / or therapeutic agent for muscle wasting disorders according to claim 10, wherein the muscle wasting disorder is muscular dystrophy or sarcopenia.

12. The preventive and / or therapeutic agent for muscle atrophy disorder according to claim 10, wherein the muscle atrophy disorder is a muscle atrophy disorder caused by diabetes or cancer cachexia.

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