WO2022009698A1 - Peptide and complex including same - Google Patents

Peptide and complex including same Download PDF

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Publication number
WO2022009698A1
WO2022009698A1 PCT/JP2021/024125 JP2021024125W WO2022009698A1 WO 2022009698 A1 WO2022009698 A1 WO 2022009698A1 JP 2021024125 W JP2021024125 W JP 2021024125W WO 2022009698 A1 WO2022009698 A1 WO 2022009698A1
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group
residue
amino acid
arg
peptide
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PCT/JP2021/024125
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French (fr)
Japanese (ja)
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良雄 林
健太郎 高山
敦彦 谷口
洋一 根岸
史子 伊東
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学校法人東京薬科大学
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Publication of WO2022009698A1 publication Critical patent/WO2022009698A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/69—Boron compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the present invention relates to peptides and complexes containing them.
  • Muscular dystrophy is a hereditary disease in which degeneration and necrosis of skeletal muscle are the main lesions and muscle weakness progresses.
  • the expression of muscle strength requires a mechanism to transmit the tension generated in the intracellular myofibrils to the extracellular basement membrane via multiple proteins. Defects in the genes that encode the proteins involved in this cause the development of muscular dystrophy. For example, in the most severe Duchenne-type muscular dystrophy, the dystrophin gene is mutated, and it is said that the main cause is deletion or dysfunction of the protein.
  • muscle mass is inhibited by inhibiting the function of myostatin (proliferative differentiation factor-8, GDF-8), which is a factor that negatively controls skeletal muscle mass.
  • GDF-8 proliferation differentiation factor-8
  • Myostatin is a secretory protein that belongs to the TGF- ⁇ family and is highly expressed in skeletal muscle, and is synthesized intracellularly as a precursor protein containing a prodomain on the N-terminal side and a mature domain on the C-terminal side.
  • Myostatin secreted from cells is a propeptide derived from a prodomain called latency assisted protein (LAP), which associates with an active dimer derived from a mature domain and does not eliminate the active dimer, which is the main component that negatively controls skeletal muscle mass.
  • LAP latency assisted protein
  • myostatin stocked in vivo in an inactivated state in this way becomes an active substance by decomposing a propeptide by an enzyme when necessary.
  • the activated myostatin functions as a signal molecule that negatively regulates skeletal muscle mass through binding to a receptor typified by the activin type IIB receptor.
  • WO 2018/030432 (corresponding to US Pat. No. 20130177370) describes a myostatin-inhibiting peptide derived from a myostatin propeptide.
  • the myostatin-inhibiting peptide described in International Publication No. 2018/030432 has high myostatin-inhibiting activity.
  • improvement of stability in vivo is desired.
  • an object of the present invention is to provide a peptide having high myostatin inhibitory activity and improved in vivo stability.
  • the present inventors have conducted diligent research in order to solve the above problems. As a result, it was found that the above-mentioned problem can be solved by a peptide composed of all D-form amino acids, and the present invention has been completed.
  • One embodiment 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. ..
  • X 0 is an amino acid residue or defect selected from the group consisting of D-Ala, D-Gly and D-2-aminoisobutyric acid
  • X 1 is an amino acid residue or defect 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, D-Lys, D-Arg , D- ornithine, D-His, with an amino acid residue selected from the group consisting of D-2,3-diaminopropionic acid and D-2,4-diaminobutane acid can be;
  • X 5 contains D-Ser, D-Arg, D-2-hydroxyglycine, D-homoserine, D-Lys, D-ornithine, D-His, D-2,3-diaminopropionic acid and D-2,4.
  • 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, D-Gln, D-Arg , D-Asn, D-Lys, D-His, D-2,3- diaminopropionic acid, the group consisting of D-2,4-diaminobutanoic acid and D- ornithine Amino acid residues selected from;
  • 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. Is the basis;
  • 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. Is the basis;
  • X 15 is an amino acid residue selected from the group consisting of D-Tyr, D-Trp and D-Phe; and
  • X 16 is from D-Trp, D-homophenylalanine, D-Tyr and D-Phe. Amino acid residues or defects selected from the group consisting of.
  • the peptides of Examples and Comparative Examples show myostatin inhibitory activity.
  • the peptide of the example shows the myostatin inhibitory activity.
  • the peptides of Examples and Comparative Examples show myostatin inhibitory activity.
  • the peptide of the example shows the myostatin inhibitory activity.
  • the results of the stability evaluation of the peptide of the example in a bovine pancreas-derived trypsin solution are shown.
  • the results of the stability evaluation of the peptide of the example in a bovine pancreas-derived ⁇ -chymotrypsin solution are shown.
  • the results of the effect (in vivo evaluation) of the peptide of the example on the Duchenne muscular dystrophy model mdx mouse tibialis anterior muscle are shown.
  • the result of myostatin oxygenation evaluation using the peptide of Example is shown.
  • the results of the effect (in vivo evaluation) on the grip strength of the cancer cachexia model mouse by intramuscular administration of the peptide of the example are shown.
  • the results of the effect (in vivo evaluation) on the gastrocnemius muscle mass of the cancer cachexia model mouse by intramuscular administration of the peptide of the example are shown.
  • the results of the effect (in vivo evaluation) on the muscle fiber area of the cancer cachexia model mouse by intramuscular administration of the peptide of the example are shown.
  • XY indicating a range means "X or more and Y or less”. Unless otherwise specified, the operation and physical properties are measured under the conditions of room temperature (20 to 25 ° C.) / relative humidity of 40 to 50% RH.
  • a peptide containing an amino acid sequence represented by the formula (1) and having 15 to 17 amino acid residues, or a pharmaceutically acceptable salt thereof is simply referred to as “the present invention.” Also called “peptide”.
  • amino acid residue in the present invention means a portion corresponding to one unit of the amino acid constituting the peptide or protein on the peptide or protein molecule. More specifically, it means a divalent group derived from an ⁇ -amino acid, which is represented by the following formula (5):
  • R 0 is a side chain of an amino acid, for example, Gly is a hydrogen atom and Ala is a methyl group.
  • amino acid residues are derived from natural or unnatural ⁇ -amino acids.
  • the peptide according to the present invention is composed of D-form amino acids from the viewpoint of enhancing stability in a living body.
  • amino acid residue is Arg, Lys, Asp, Asn, Glu, Gln, His, Pro, Tyr, Trp, Ser, Thr, Gly, Ala, Met, Cys, Phe, Leu, Val. , And Ile, and their analogs can be exemplified.
  • the analog may be, for example, a derivative in which the side chain of the 20 kinds of amino acid residues is substituted with an arbitrary substituent, and for example, a halogenated derivative of the above 20 kinds of amino acid residues (for example, , 3-Chloroalanine, 4-Fluorophenylalanine, 4-Chlorophenylalanine), 2-aminobutyric acid, 4-aminobutyric acid, norleucine, norvaline, isovalin, 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 ester (eg, aspartic acid-methyl ester, aspartic acid) -Ethyl ester, aspart
  • Natural diastereomers ie, (2R * , 3R * ) for Ile, -2-amino-3-methylpentanoic acid, (2R * , 3S * ) for Thr, -2-amino-3-hydroxybutane Acid
  • amino acid sequence described in the present specification is described in the direction from the N-terminal (amino-terminal) side to the C-terminal (carboxyl-terminal) side according to the convention.
  • each amino acid residue can be replaced with an amino acid residue having similar properties based on the difference in its side chain (conservative substitution).
  • the aliphatic hydrophobic amino acids Val, Leu, Ile, 2-aminobutyric acid (Abu), norleucine (Nle), norvaline (Nva), and isovaline (Iva) can be substituted with each other.
  • Gly, Ala and 2-aminoisobutyric acid (Aib), whose side chains are hydrogen atoms or methyl groups, can be replaced with each other.
  • Phe and homophenylalanine (Hph) whose side chains are phenylalkyl groups, can be substituted with each other.
  • the neutral polar amino acids Asn and Gln can be replaced with each other.
  • the basic amino acids Arg, Lys, His, 2,3-diaminopropionic acid (Dpr), 2,4-diaminobutanoic acid (Dbu), and ornithine (Orn) can be replaced with each other.
  • the acidic amino acids Asp and Glu can be replaced with each other.
  • Ser, 2-hydroxyglycine (Hyg) and homoserine (Hse), whose side chains are hydroxy groups or short hydroxyalkyl groups, can be substituted with each other.
  • Pro and 3,4-didehydroproline (Dhp) having a side chain in which the pyrrolidyl group of the side chain is dehydrogenated can be mutually substituted.
  • Cys and homocysteine (Hcy) whose side chains are short thiolalkyl groups, can be replaced with each other.
  • Met and homomethionine (Hme), whose side chains are short-chain sulfide structures, can be replaced with each other.
  • Trp, Tyr and Phe whose side chains are aromatic, can be replaced with each other.
  • Gly and Pro which are amino acid residues that affect the orientation of the chain, can be replaced with each other.
  • a "pharmaceutically acceptable salt” is a metal salt, ammonium salt, organic acid salt, inorganic acid salt, or organic salt that does not produce an undesired physiological effect after being administered to a patient or subject. It is a salt with a base or an inorganic base.
  • the peptide prodrug according to the present invention is a peptide derivative that converts the peptide according to the present invention, that is, gastric acid or enzyme. It refers to a peptide derivative that undergoes oxidation, reduction, hydrolysis, etc. to change into the peptide according to the present invention.
  • These peptide derivatives are, for example, Bundgard, H. et al. , Design of Prodrugs, pp. It can be produced from the peptide according to the present invention by a conventionally known method described in 7-9, 21-24, Elsevier, Amsterdam 1985 and the like.
  • an ester derivative obtained by reacting the carboxyl group with an alcohol, or by reacting the carboxyl group with an amine can be exemplified. More specifically, for example, an ester in which the carboxyl group of the peptide side chain is represented by -COOR (R is an alkyl group having 1 to 20 carbon atoms), or -CONHR or -CONRR'(R and R'is Independently, a peptide derivatized into an amide group represented by an alkyl group having 1 to 20 carbon atoms can be mentioned.
  • the side chain of the peptide according to the present invention has a hydroxyl group
  • an acyloxy derivative obtained by reacting the hydroxyl group with an acid anhydride or the like to acylate it can be exemplified. More specifically, for example, a peptide obtained by derivatizing the hydroxyl group of the peptide side chain into an acyloxy group represented by ⁇ OCOR (R is an alkyl group having 1 to 20 carbon atoms) can be mentioned.
  • prodrug examples include derivatives in which the amino group is acylated, N-oxidized, alkylated, or phosphorylated when the side chain of the peptide according to the present invention has an amino group. More specifically, for example, a peptide in which the amino group of the side chain is derivatized into an amide group represented by -NHCOR (R is an alkyl group having 1 to 20 carbon atoms) or -NHCOCH (NH 2 ) CH 3. Can be mentioned.
  • -NHCOR R is an alkyl group having 1 to 20 carbon atoms
  • -NHCOCH NH 2
  • the structure of the N-terminal of the peptide according to the present invention is not particularly limited, and may be, for example, a hydrogen atom (that is, unmodified) or a structure in which a modifying group is introduced by a conventionally known method.
  • the N-terminal modifying group includes an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 1 to 20 carbon atoms, an alkynyl group having 1 to 20 carbon atoms, an aromatic hydrocarbon group having 6 to 20 carbon atoms, and a heterocyclic group.
  • a group represented by the following formula (6) a sulfonyl group, a carboxyl group, a glyoxyl group, a formyl group; a polyethylene glycol group (PEGylated), a polyoxyethylene glycol group, a polypropylene glycol group; a tert-butoxycarbonyl group (Boc group).
  • benzyloxycarbonyl group Z group
  • protective group such as fluorenylmethoxycarbonyl group (Fmoc group); cyclopentyloxycarbonyl group, cyclohexyloxycarbonyl group, adamantyloxycarbonyl group, norbornyloxycarbonyl group, iso Cycloalkyloxycarbonyl groups such as Bornyloxycarbonyl groups; Protective groups derived from amino acids such as pyroglutamic acid and morothanic acid; Carbamate-based protective groups; sulfonic acid such as benzenesulfonic acid and protective groups derived from phosphoric acid, etc. , Can be exemplified.
  • the peptide has a hydrogen atom, an alkyl group, an aromatic hydrocarbon group, a heterocyclic group at the N-terminal, a group represented by the following formula (6), a sulfonyl group, a carboxyl group, and the like. It is preferably 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.
  • the number of carbon atoms of the alkyl group that can be present at the N-terminal of the peptide is, for example, 1 to 20, preferably 1 to 10.
  • the alkyl group may have a saturated chain, an unsaturated chain, or a cyclic structure, and may have a branched chain structure. More specifically, the alkyl group includes 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, and a tert.
  • the carbon number of the aromatic hydrocarbon group that can exist at the N-terminal of the peptide is, for example, 6 to 20, and more specifically, a phenyl group, a naphthyl group, a tolyl group, a phenanthryl group and the like can be exemplified.
  • Heterocyclic groups that can be present at the N-terminal of the peptide include monocyclic, fused bicyclic or fused tricyclic groups containing 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur atoms in the ring.
  • Examples of the substituent of the structure can be exemplified, and more specifically, 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 morphoryl group, an indolyl group, a benzoimidazolyl group, a quinolyl group, Examples thereof include a carbazolyl group, a tetrahydrofuranyl group, a tetrahydrothiophenyl group, a furanyl group, a thiophenyl group, a tetrahydropyranyl group, a tetrahydrothiopyranyl group and the like.
  • aromatic hydrocarbon groups and heterocyclic groups are linear or branched alkyl groups having 1 to 6 carbon atoms, linear or branched alkoxy groups having 1 to 6 carbon atoms, amino groups, carboxyl groups and esters. It may be substituted with a group, a carbamoyl group, an amide group, a nitro group, a sulfo group, a sulfonamide group, and / or a further substituent such as a halogen.
  • the N-terminal modifying group may be, for example, a functional group represented by the following formula (6).
  • X 0A is a single bond, an oxygen atom or a sulfur atom, or may have a substituent selected from the group consisting of an amino group, an acetylamino group and a propionylamino group, and is an alkylene having 1 to 3 carbon atoms.
  • Group eg, methylene group, ethylene group, trimethylene group and propylene group
  • oxyalkylene group having 1 to 3 carbon atoms eg, oxymethylene group, oxyethylene group, oxytrimethylene group and oxypropylene group
  • a divalent linking group selected from the group consisting of ⁇ 3 alkyleneoxy groups (eg, methyleneoxy group, ethyleneoxy group, trimethyleneoxy group and propyleneoxy group);
  • R 1A is an alkyl group having 1 to 20 carbon atoms which may have a substituent, as well as an alkyl group having 1 to 20 carbon atoms.
  • R 11 to R 30 are independently hydrogen atom, halogen atom (for example, fluorine atom, chlorine atom, bromine atom, iodine atom) and alkyl group having 1 to 3 carbon atoms (that is, methyl group, ethyl group, propyl group). ), An alkoxy group having 1 to 3 carbon atoms (that is, a methoxy group, an ethoxy group, a propoxy group), a hydroxyl group, and an amino group.
  • halogen atom for example, fluorine atom, chlorine atom, bromine atom, iodine atom
  • alkyl group having 1 to 3 carbon atoms that is, methyl group, ethyl group, propyl group.
  • An alkoxy group having 1 to 3 carbon atoms that is, a methoxy group, an ethoxy group, a propoxy group
  • hydroxyl group and an amino group.
  • R 1A when R 1A is an alkyl group having 1 to 20 carbon atoms which may have a substituent, the fatty chain may have a saturated chain, an unsaturated chain, or a cyclic structure. , May have a branched chain structure.
  • the alkyl group of R 1A preferably has 2 to 12 carbon atoms.
  • the substituent of R 1A includes a hydroxy group, an alkoxy group having 1 to 5 or less carbon atoms (for example, a methoxy group, an ethoxy group, etc.), an amino group, a carboxyl group, an ester group, a carbamoyl group, an amide group, a nitro group, and a sulfo group.
  • Halogen fluorine, chlorine, bromine, iodine
  • R 1A includes a hydroxy group, an alkoxy group having 1 to 5 or less carbon atoms (for example, a methoxy group, an ethoxy group, etc.), an amino group, a carboxyl group, an ester group, a carbamoyl group, an amide group, a nitro group, and a sulfo group.
  • Halogen fluorine, chlorine, bromine, iodine
  • X 0A is a single bond or may have a substituent selected from the group consisting of an amino group and an acetylamino group, and has 1 to 3 carbon atoms. It is a divalent linking group selected from the group consisting of an alkylene group and an oxyalkylene group having 1 to 3 carbon atoms.
  • the group represented by the above formula (6) is an acyl group.
  • Acyl groups include acyl groups derived from various carboxylic acids. More specifically, it may be an acyl group having a fat chain, an aromatic ring or a heterocycle, or 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. It may be an acyl group to be derived.
  • the acyl group which is an alkyl group having 1 to 20 carbon atoms in which R 1A in the above formula (6) may have a substituent, is an acetyl group, a propionyl group, a butyryl group, or an isobutyryl group.
  • Valeryl group isobarrel group, pivaloyl group, caproyl group, caprinoyle group, methylhexanoyl group, cyclopropanecarbonyl group, aminocyclopropanecarbonyl group, cyclohexanecarbonyl group, cyclohexylacetyl group, cyclopentylpropionyl group, cyclohexylpropionyl group, cyclopentylbuta Noyl group, cyclohexylbutanoyl group, adamantylacetyl group, lauroyl group, myritoyl group, palmitoyl group, stearoyl group, oxalyl group, malonyl group, succinyl group, glutalyl group, adipoyl group, glycol group, lactoyl group, glyceroyl group, pyruboyl group , Acetacetyl group and the like can be exemp
  • vitamins having an acyl group examples include nicotinic acid, pantothenic acid, biotin, pteroylglutamic acid (folic acid), orotic acid, fluoroorotic acid, ⁇ -lipoic acid, pyridoxic acid, biocithin, pteroic acid, and 10-formylpteroin. Acids, 7,8-dihydrofolic acid, homopteroic acid, pterin-6-carboxylic acid, dihydrolipoic acid, hydroorothic acid and the like can be mentioned.
  • the nucleic acid base derivative having an acyl group refers to a base component constituting a nucleotide and a derivative thereof, and preferably a pyrimidine derivative or the like, for example, 5-carboxymethyl uracil, 5-carboxythiouracil and the like can be exemplified.
  • Examples of the sulfonyl group that can exist at the N-terminal of the peptide include those having a structure obtained by converting the carbonyl structure of the above-mentioned acyl group into a sulfone structure.
  • the polyethylene glycol group that may be present at the N-terminal of the peptide is polyethylene glycol or via an ester bond, an amine (-NH-), an acyl group (for example, an acyl group having 1 to 12 carbon atoms), or a combination thereof. It is a structure in which the analogs are linked.
  • the terminal of the polyethylene glycol group opposite to the side connected to the N-terminal of the peptide is an alkyl group having 1 to 6 carbon atoms (for example, a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, or butyl). Modified with a protective group or amino group commonly used to protect hydroxyl groups, such as group, isobutyl group, sec-butyl group, tert-butyl group, amyl group, isoamyl group, tert-amyl group, hexyl group). It may have been done.
  • the structure of the C-terminal of the peptide according to the present invention is also not particularly limited, and may be a structure modified with a protecting group generally used for protection of a carboxylic acid. More specifically, the structure of the C-terminus of the peptide according to the present invention, for example, a carboxyl group (-COOH), a carboxylate (-COO -), amide (-CONH 2), alkylamide (-CONHR 31, - It may be substituted with a CONR 31 R 32 ), an ester (-COOR 31 ), an acyloxyalkyl (-R 33- OCOR 31 ) such as a pivaloyloxymethyl group, an alkyl group having 1 to 4 carbon atoms or an alkoxy group.
  • a carboxyl group -COOH
  • a carboxylate a carboxylate
  • amide -CONH 2
  • alkylamide -CONHR 31, - It may be substituted with a CONR 31 R 32 ), an
  • a phthalidyl group eg, a phthalidyl group, a dimethylphthalidyl group, a dimethoxyphthalidyl group
  • the C-terminal of the peptide is preferably an amide.
  • R 31 and R 32 in the above alkylamides, esters, and acyloxyalkyls are independently methyl group, ethyl group, propyl group, isopropyl group, cyclopropyl group, butyl group, isobutyl group, sec-butyl group, tert.
  • -Alkyl group having 1 to 6 carbon atoms such as butyl group, amyl group, isoamyl group, tert-amyl group, hexyl group and cyclohexyl group; aryl group having 6 to 10 carbon atoms such as phenyl group and naphthyl; benzyl group and phenethyl An aralkyl group having 7 to 18 carbon atoms such as a group and a benzhydryl group; a sugar such as glucose; an alkyl group having 1 to 6 carbon atoms (for example, a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, etc.
  • R 33 in the acyloxyalkyl 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 and a t-butylene group. Is.
  • the peptide according to the present invention also includes peptide derivatives chemically modified by covalent bonds with polymers, lipids and the like, and derivatives having further enhanced ⁇ -helix properties contained in the peptides.
  • the derivative having further enhanced ⁇ -helix property include a derivative having a salt bridge formed at the i, i + 4-position and the like as an amino acid arrangement, and a derivative having a crosslinked structure by a disulfide bond, a carbon-carbon bond, and the like.
  • the number of amino acid residues of the peptide according to the present invention is 15 to 17. When the number of amino acid residues is 17 or less, it is advantageous in terms of both synthesis and utilization in vivo. If the number of amino acid residues is less than 14, the effect of the present invention cannot be exerted.
  • One embodiment of the present invention comprises 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.
  • X 0 is an amino acid residue or defect selected from the group consisting of D-Ala, D-Gly and D-2-aminoisobutyric acid
  • X 1 is an amino acid residue or defect 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, D-Lys, D-Arg , D- ornithine, D-His, with an amino acid residue selected from the group consisting of D-2,3-diaminopropionic acid and D-2,4-diaminobutane acid can be;
  • X 5 contains D-Ser, D-Arg, D-2-hydroxyglycine, D-homoserine, D-Lys, D-ornithine, D-His, D-2,3-diaminopropionic acid and D-2,4.
  • 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, D-Gln, D-Arg , D-Asn, D-Lys, D-His, D-2,3- diaminopropionic acid, the group consisting of D-2,4-diaminobutanoic acid and D- ornithine Amino acid residues selected from;
  • 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. Is the basis;
  • 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. Is the basis;
  • X 15 is an amino acid residue selected from the group consisting of D-Tyr, D-Trp and D-Phe; and
  • X 16 is from D-Trp, D-homophenylalanine, D-Tyr and D-Phe. Amino acid residues or defects selected from the group consisting of.
  • the peptide according to the present invention has high myostatin inhibitory activity, and in addition, since it is composed of all D-form amino acids, it has stability against in vivo enzymes and is effective for living organisms. It can show a long-lasting high effect at the time of administration.
  • X 0 is an amino acid residue or defect selected from the group consisting of D-Ala, D-Gly and D-2-aminoisobutyric acid
  • X 1 is an amino acid residue or defect 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, D-Lys, D-Arg , D- ornithine, D-His, with an amino acid residue selected from the group consisting of D-2,3-diaminopropionic acid and D-2,4-diaminobutane acid can be;
  • X 5 contains D-Ser, D-Arg, D-2-hydroxyglycine, D-homoserine, D-Lys, D-ornithine, D-His, D-2,3-diaminopropionic acid and D-2,4.
  • -Amino acid residues selected from the group consisting of 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, D-Gln, D-Arg , D-Asn, D-Lys, D-His, D-2,3- diaminopropionic acid, the group consisting of D-2,4-diaminobutanoic acid and D- ornithine Amino acid residues selected from;
  • 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 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
  • X 16 is from D-Trp, D-homophenylalanine, D-Tyr and D-Phe.
  • X 0 is an amino acid residue or defect selected from the group consisting of D-Ala, D-Gly and D-2-aminoisobutyric acid
  • X 1 is an amino acid residue or defect 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 a D-2-cyclohexylglycine residue
  • X 4 is, D-Lys, D-Arg , D- ornithine, D-His, with an amino acid residue selected from the group consisting of D-2,3-diaminopropionic acid and D-2,4-diaminobutane acid can be
  • X 5 contains D-Ser, D-Arg, D-2-hydroxyglycine, D-homoserine, D-Lys, D-ornithine, D-His, D-2,3-diaminopropionic acid and D-2,4.
  • 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, D-Gln, D-Arg , D-Asn, D-Lys, D-His, D-2,3- diaminopropionic acid, the group consisting of D-2,4-diaminobutanoic acid and D- ornithine Amino acid residues selected from;
  • X 9 is a D-2-cyclohexylgly
  • 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 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-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
  • X 16 is from D-Trp, D-homophenylalanine, D-Tyr and D-Phe.
  • Amino acid residues or defects selected from the group consisting of, more preferably amino acid residues selected from the group consisting of D-Trp, D-homophenylalanine, D-Tyr and D-Phe.
  • X 0 is a D-Ala residue or defect
  • X 1 is a D-Leu residue, a D-norleucine residue or a defect
  • 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, D-Lys residues, be a D-Arg residue, or D- ornithine residues
  • X 5 is a D-Ser residue or a 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 an D-Gln residue or D-Arg residues
  • X 9 is a D-Gln residue or D-Arg residues
  • the peptide represented by the above formula (1) preferably contains any one of the amino acid sequences represented by SEQ ID NOs: 1 to 25.
  • the peptide represented by the above formula (1) is more preferably the amino acid sequence represented by SEQ ID NOs: 1 to 4, 6, 8 to 23 and 25. It comprises any one, and particularly preferably any one of the amino acid sequences represented by SEQ ID NOs: 19-23 and 25.
  • the peptide according to the present invention can be produced by a conventionally known method including a chemical synthesis method and a recombination technique.
  • a chemical synthesis method and a recombination technique.
  • each amino acid is commonly used in peptide chemistry, eg, "The Peptides,” Volume 1 [Schroder and Luhke, Academic Press, New York, U.S.A. S. A. (1966)], "Basics and Experiments of Peptide Synthesis” (Nobuo Izumiya et al., Maruzen Co., Ltd., 1985), etc.
  • the peptide according to the present invention may also be produced by a recombination technique using animal cells, insect cells, microorganisms or the like by a method as described in, for example, Current Protocols in Molecular Biology, Chapter 16 below.
  • the peptide can be purified by a conventionally known method after being produced by cultured cells or microorganisms. Methods for purifying and isolating peptides are known to engineers in the art, and can be carried out by the methods described in, for example, Current Protocols in Molecular Biology, Chapter 16 (Ausube et al., John Wiley and Sons, 2006).
  • Condensation methods for forming peptide bonds include azide method, acid halide method, acid anhydride method, carbodiimide method, carbodiimide-additive method, active ester method, carbonyl imidazole method, oxidation-reduction method, enzyme method, Woodward reagent K. , HATU reagent, method using Bop reagent and the like can be exemplified.
  • the acid anhydride method, the carbodiimide method, and the active ester method are mainly mentioned as the condensation reaction in 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.
  • a resin a resin having a functional group introduced for the purpose of binding an amino acid to the resin, a resin having a spacer inserted between the resin and the functional group, and the like can also be used depending on the purpose. More specifically, for example, halomethyl resin such as chloromethyl resin, oxymethyl resin, 4- (oxymethyl) -phenylacetamidemethyl resin, 4- (oxymethyl) -phenoxymethyl resin, Rinkamide resin and the like can be mentioned. Can be done.
  • protective means such as a carboxyl group, an amino group, a hydroxyl group, and an amidino group that are not involved in the condensation reaction can be applied by a commonly known means. On the contrary, it is also possible to activate the carboxyl group or amino group directly involved in the condensation reaction.
  • Protecting groups commonly used in organic chemistry as protecting groups for functional groups that are not involved in the condensation reaction of each unit include, for example, "Protective Groups in Organic Synthesis (by Greene, John Wiley & Sons, Inc.). It can be protected by the protecting group described in 1981)) and the like. More specifically, as the protecting group for the carboxyl group, for example, various methyl esters, ethyl esters, benzyl esters, p- Examples of commonly known protecting groups such as nitrobenzyl ester, t-butyl ester and cyclohexyl ester can be mentioned.
  • Examples of the protecting group for the amino group include a benzyloxycarbonyl group, a t-butoxycarbonyl group and an isobornyloxycarbonyl.
  • Groups, 9-fluorenylmethoxycarbonyl group (Fmoc group) and the like can be mentioned.
  • Examples of the activated carboxyl group include acid anhydrides corresponding to the carboxyl group; azide; pentafluorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, and N-hydroxysuccinic acid. Examples thereof include active esters with imide, N-hydroxy-5-norbornen-2,3-dicarboxymid, N-hydroxyphthalimide, 1-hydroxybenzotriazole and the like. Examples of the activated amino group include phosphoric acid amides corresponding to the amino group.
  • the condensation reaction during peptide synthesis is usually carried out in a solvent.
  • the solvent include chloroform, dichloromethane, ethyl acetate, N, N-dimethylformamide, dimethyl sulfoxide, pyridine, dioxane, tetrahydrofuran, N-methylpyrrolidone, water, methanol and the like, or a mixture thereof. ..
  • the reaction temperature of the condensation reaction can be carried out in the range of ⁇ 30 ° C. to 50 ° C., as in the usual case.
  • the type of the protecting group elimination reaction in the peptide production process of the present invention is selected according to the type of protecting group to be used as long as the protecting group can be eliminated without affecting the peptide bond.
  • Examples thereof include alkali treatment with, sodium treatment in liquid ammonia, reduction with palladium carbon, and silylation treatment with trimethylsilyl triflate, trimethylsilyl bromide and the like.
  • the deprotecting group reaction is efficiently carried out by adding a cation scavenger such as anisole, phenol, cresol, thioanisole and ethanedithiol. It is preferable from the viewpoint.
  • the method for cleaving the peptide of the present invention synthesized by the solid phase method from the solid phase also follows a generally known method.
  • the above-mentioned treatment with an acid or a silylating agent can be mentioned as the cutting method.
  • generally known separation and purification means can be fully utilized after the completion of the above series of reactions.
  • the peptide of the present invention can be obtained with higher purity by extraction, partitioning, reprecipitation, recrystallization, solid phase extraction, column chromatography and the like.
  • the obtained peptide can be analyzed by an automatic amino acid analyzer, capillary electrophoresis, reverse phase high performance liquid chromatography, mass spectrometry, etc.
  • various biomolecular interaction analysis methods such as phage display method, two-hybrid method, affinity chromatography, surface plasmon resonance method, co-immunoprecipitation method, protein chip method, three-dimensional structure analysis, far western method, and fluorescence quenching method are used.
  • peptides may be selected using the interaction with myostatin as an index.
  • the peptide according to the present invention may be isolated or purified. "Isolation or purification” means that an operation has been performed to remove components other than the target component.
  • the purity of the isolated or purified peptide according to the present invention is usually 50% or more (for example, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, 100%). be.
  • One form of the present invention is a complex represented by the following formula (2) or a pharmaceutically acceptable salt thereof.
  • R 1 and R 2 independently represent a halogenoalkyl group or a halogen atom, respectively.
  • R 3 represents a bromine atom, an iodine atom or a selenium atom.
  • R 4 and R 5 each independently represent a hydrogen atom or a substituted or unsubstituted alkyl group.
  • R 6 and R 7 each independently represent a hydrogen atom, a halogen atom, an alkoxy group or a substituted or unsubstituted alkyl group, wherein R 4 and R 6 or R 5 and R 7 are combined.
  • Substituted or unsubstituted alkylene groups or alkenylene groups may be formed.
  • R 8 represents a hydrogen atom or a substituted or unsubstituted alkyl group.
  • R 9 and R 10 each independently represent a hydrogen atom, a halogen atom, an alkoxy group or a substituted or unsubstituted alkyl group, wherein R 8 and R 9 or R 10 are substituted or substituted together. It may form an unsubstituted alkylene group or an alkenylene group.
  • m and n independently represent integers of 1 to 3, respectively. * Is the binding site with L] It is a compound represented by; L represents a linker between Y and Z; Z is the peptide according to the present invention.
  • Y is a compound represented by the above formula (3).
  • the compound represented by the formula (3) is an on / off switch catalyst that enables target-selective photooxygenation.
  • the complex is activated by light having a wavelength of 650 to 800 nm, and myostatin is selectively oxygenated. By making it, it is possible to exert even higher myostatin inhibitory activity.
  • R 1 and R 2 independently represent a halogenoalkyl group or a halogen atom, respectively.
  • 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, and even more preferably a tri. It is a fluoromethyl group or a pentafluoroethyl group, and particularly preferably a trifluoromethyl group.
  • halogen atom examples include a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, and a fluorine atom is preferable.
  • R 1 is preferably a halogen atom and R 2 is a halogenoalkyl group.
  • R 3 represents a bromine atom, an iodine atom or a selenium atom, and is preferably a bromine atom.
  • R 4 and R 5 each independently represent a hydrogen atom or a substituted or unsubstituted alkyl group.
  • R 6 and R 7 each independently represent a hydrogen atom, a halogen atom, an alkoxy group or a substituted or unsubstituted alkyl group. At this time, R 4 and R 6 or R 5 and R 7 may be combined to form a substituted or unsubstituted alkylene group or alkenylene group.
  • R 8 represents a hydrogen atom or a substituted or unsubstituted alkyl group.
  • R 9 and R 10 each independently represent a hydrogen atom, a halogen atom, an alkoxy group or a substituted or unsubstituted alkyl group. At this time, R 8 and R 9 or R 10 may be combined to form a substituted or unsubstituted alkylene group or alkenylene group.
  • R 4 and R 6, R 5 and R 7 and R 8 and R 10 form a substituted or unsubstituted alkylene or alkenylene group, this time the alkylene group
  • the alkaneylene group has 2 or 3 carbon atoms.
  • the alkyl group in R 4 to R 10 is preferably an alkyl group having 1 to 6 carbon atoms in a straight chain or a branched chain, and more preferably an alkyl group having 1 to 4 carbon atoms in a straight chain or a branched chain.
  • 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, a tert-butyl group and the like.
  • the group capable of substituting these alkyl groups is 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.
  • the group capable of substituting the alkyl group is more preferably selected from a carboxy group, a sulfonic acid group, a hydroxy group and an amino group from the viewpoint of increasing water solubility.
  • the alkoxy group in R 6 , R 7 , R 9 and R 10 is preferably an alkoxy group having 1 to 6 carbon atoms in a straight chain or a branched chain, and an alkoxy group having 1 to 4 carbon atoms in a more linear or branched chain. It is the basis. Specific examples thereof include a methoxy group, an ethoxy group, and a propyloxy group.
  • Examples of the halogen atom in R 6 , R 7 , R 9 and R 10 include a chlorine atom, a bromine atom, an iodine atom and a fluorine atom.
  • 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 said alkylene or alkenylene group.
  • the alkylene group include an ethylene group and a trimethylene group.
  • the alkenylene group include a vinylene group and a propenylene group.
  • Examples of the ring structure formed by these groups together include the following structures.
  • R 4 ⁇ R 7 and R 9 ⁇ R 10 represents a group that does not form an alkylene group or alkenylene group.
  • m and n each independently represent an integer of 1 to 3.
  • m and n are independently, preferably 1 or 2, and more preferably 1.
  • * is the binding site with L.
  • Linker L represents a linker between compound Y represented by formula (3) and peptide Z according to the present invention.
  • the binding site of the linker L to the peptide Z according to the present invention is not particularly limited and may be the N-terminal or the C-terminal of the peptide Z according to the present invention. It may be a side chain of the group.
  • the linker L is attached to the N-terminus of peptide Z according to the present invention.
  • linker unit is not particularly limited, but for example, 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 non-substituted alkenyl group.
  • L has a structure represented by the following formula (4).
  • V represents a -CO-, -CONH- or a triazole ring.
  • O represents an integer of 0 or 1.
  • the triazole ring include 1,2,3-triazole-1,4-diyl group and 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, and more preferably represents an integer of 1 to 4.
  • p preferably represents an integer of 1 to 6, and more preferably represents an integer of 1 to 4.
  • * 1 is a linking site with the compound represented by the formula (3)
  • * 2 is a linking site with the peptide according to the present invention.
  • R 1 to R 7 and Z are as defined for the above formula (2).
  • the complex represented by the formula (2-1) can be produced according to the following reaction formula.
  • the compound represented by the formula (2-1) can be obtained by condensing the compound a and the compound b to obtain the compound c, and then reacting the compound c with the compound d.
  • the reaction between compound a and compound b is an aldol condensation reaction.
  • reaction between compound c and compound d is a 1,3-dipole addition reaction between alkyne and azide, and easily proceeds at room temperature in a polar solvent such as dimethylformamide in the presence of a copper catalyst.
  • the compounds a to d are the above-mentioned H. Okamoto et al. , Chem. Commun. , 2019, 55, 9108-9111. , International Publication No. 2017/164172 and the like can be appropriately referred to and synthesized.
  • the obtained complex represented by the formula (2-1) can be purified by a known means such as chromatography.
  • a myostatin inhibitor comprising the peptide according to the invention or a prodrug thereof or a complex according to the present invention (hereinafter, "the peptide according to the present invention or a prodrug thereof or the present invention”.
  • a "myostatin inhibitor” containing the complex according to the invention is also simply referred to as a "myostatin inhibitor").
  • the myostatin inhibitor may be composed of one or more peptides according to the present invention, one or more prodrugs thereof, one or more complexes according to the present invention, or a mixture thereof, but usually.
  • a pharmaceutical composition comprising a peptide according to the present invention and a prodrug thereof, one or more selected from the complex according to the present invention, and a pharmaceutically acceptable carrier.
  • One embodiment of the present invention relates to a method for inhibiting myostatin, which comprises administering to a patient an effective amount of a peptide according to the present invention or a prodrug thereof or a complex according to the present invention.
  • One embodiment of the invention also relates to a peptide according to the invention or a prodrug thereof or a complex according to the invention for use in inhibiting myostatin.
  • a prophylactic and / or therapeutic agent for muscular atrophy disorder which comprises the peptide according to the present invention or a prodrug thereof or a complex according to the present invention (hereinafter, "the peptide according to the present invention”).
  • a prodrug thereof or a prophylactic and / or therapeutic agent for muscular atrophy disorder containing the complex according to the present invention is also simply referred to as a "preventive / therapeutic agent for muscular atrophy disorder").
  • the prophylactic / therapeutic agent for muscle atrophy disorder may be composed of one or more peptides according to the present invention, one or more prodrugs thereof, one or more complexes according to the present invention, or a mixture thereof. However, it is usually a pharmaceutical composition comprising a peptide according to the present invention and a prodrug thereof, one or more selected from the complex according to the present invention, and a pharmaceutically acceptable carrier.
  • One embodiment of the present invention relates to a method for preventing and / or treating a muscular atrophy disorder, which comprises administering to a patient an effective amount of a peptide according to the present invention or a prodrug thereof or a complex according to the present invention.
  • One embodiment of the invention also relates to a peptide according to the invention or a prodrug thereof or a complex according to the invention for use in the prevention and / or treatment of muscular atrophy disorder.
  • Myostatin inhibitors, prophylactic / therapeutic agents for muscular atrophy disorders, and the above prophylactic and / or therapeutic methods are also effective for strengthening the tibialis anterior muscle by local administration for elderly people with normal walking disorders. Just strengthening the tibialis anterior muscle facilitates dorsiflexion of the ankle joint, which helps prevent falls. Further, for example, if continuous local administration during staying in space can be carried out, it can contribute to shortening the rehabilitation period after returning.
  • the above-mentioned muscular atrophy disorder is not particularly limited, and is, for example, muscular dystrophy, distal myopathy, congenital myopathy, inflammatory myopathy such as inclusion body myositis, myopathy such as mitochondrial myopathy; and disused muscular atrophy. ; Sarcopenia etc. can be exemplified.
  • the prophylactic / therapeutic agent for muscular atrophy disorder is preferably effectively used for muscular dystrophy and sarcopenia.
  • the preventive / therapeutic agents for muscular dystrophy are more preferably Duchenne muscular dystrophy, Becker muscular dystrophy, Fukuyama muscular dystrophy, melosine deficient muscular dystrophy, limb muscular dystrophy, facial muscular dystrophy, emery muscular dystrophy, and emery muscular dystrophy. , Miyoshi muscular dystrophy, and muscular dystrophy such as infant muscular dystrophy as well as sarcopenia, and is particularly effective against Duchenne muscular dystrophy.
  • Muscle atrophy 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 atrophy disorders can also result from metabolic disorders such as diabetes and related disorders. Therefore, the preventive / therapeutic agent for muscular atrophy disorder of the present invention, and the above-mentioned preventive and / or therapeutic method can be used for improving cachexia associated with muscular atrophy. In addition, inhibition of myostatin can increase muscle mass, improve bone strength, and reduce osteoporosis and other degenerative bone diseases.
  • COPD chronic obstructive pulmonary disease
  • One embodiment of the present invention relates to a prophylactic and / or therapeutic agent for a muscular atrophy disorder caused by diabetes, which comprises a peptide according to the present invention or a prodrug thereof or a complex according to the present invention.
  • One embodiment of the invention also prevents and / or prevents diabetes-induced muscular atrophy disorders, comprising administering to a patient an effective amount of a peptide according to the invention or a prodrug thereof or a complex according to the invention.
  • the treatment method Regarding the treatment method.
  • One embodiment of the present invention relates to a prophylactic and / or therapeutic agent for muscular atrophy disorder caused by a vicious cancer solution, which comprises a peptide according to the present invention or a prodrug thereof or a complex according to the present invention.
  • One embodiment of the present invention also comprises administering to a patient an effective amount of a peptide according to the invention or a prodrug thereof or a complex according to the present invention to prevent muscular atrophy disorder caused by a vicious cancer solution. And / or regarding treatment methods.
  • a therapeutically “effective amount” is an amount that is effective in producing some desired therapeutic effect commensurate with a reasonable benefit / risk ratio.
  • the "subject” and “patient” include non-human animals including humans and fish, but preferably humans, dogs, cats, mice, rats, hamsters, guinea pigs, and horses (race horses). Included), mammals such as cows, pigs, rabbits, and sheep, and poultry such as chickens, quails, and turkeys, more preferably humans.
  • the above-mentioned pharmaceutically acceptable carrier is not particularly limited, but is an excipient such as lactose, sucrose, mannitol, starch, corn starch, crystalline cellulose, and light anhydrous silicic acid; silica, talc, calcium stearate, stearic acid, etc.
  • Lubricants such as magnesium; binders such as hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, methyl cellulose, polyvinylpyrrolidone, crystalline cellulose, dextrin, gelatin; ascorbic acid, sodium sulfite, sodium hydrogen sulfite, tocopherol, etc.
  • Chelating agents such as ethylenediamine tetraacetic acid (EDTA); Buffers such as borates, bicarbonates, Tris-HCl, citrates, phosphates, other organic acids; water for injection, physiological saline Solponics such as water, ethanol, propanol, ethylene glycol, propylene glycol, macrogol, olive oil, corn oil; Pluronic®, polyethylene glycol, sorbitan fatty acid ester, polysorbate, Triton®, lecithin, cholesterol, benza chloride
  • Surface active agents or wetting agents such as luconium, benzethonium chloride, glycerin monostearate; isotonic agents such as sodium chloride, potassium chloride, glycerin, glucose, sorbitol, mannitol; benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben
  • Preservatives such as, propylparaben
  • the content of the peptide according to the present invention or the prodrug thereof or the complex according to the present invention in the drug can be 0.01 to 100% by weight based on the whole drug.
  • the dose of the peptide according to the present invention or the prodrug thereof or the complex according to the present invention varies depending on the age, symptoms, administration method, etc., but in the case of oral administration, it is generally given to humans (assuming a body weight of 60 kg). On the other hand, it is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg per day.
  • the single dose varies depending on age, symptoms, administration method, etc., but for example, in the form of an injection, it is usually about about a day for a human (assuming a body weight of 60 kg). It is 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. In the case of animals other than humans, the amount converted per 60 kg of body weight can also be administered.
  • Trifluoroacetic acid in the presence of m-cresol (0.10 mL), thioanisole (0.10 mL) and 1,2-ethanedithiol (0.10 mL) for removal of various side chain protecting groups and removal of resin.
  • the reaction was carried out in 4.0 mL for 2 hours. After removing the resin by filtration using a funnel with a filter, TFA was distilled off by nitrogen spraying, and 40 mL of diethyl ether was added to precipitate a crude peptide.
  • Peptide 1 was synthesized and purified by the same method as in Synthesis Example 1 using Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 30 mg (0.011 mmol) (8.2 mg, 24%).
  • LRMS (MALDI +) calcd for (M + + H) 2392.03, found 2391.04.
  • the peptide as the test material was suspended in DMSO so as to be 10 mM as a stock solution, and stored at -30 ° C. 1 hour before addition to the medium, the cells were suspended in serum-free DMEM medium and allowed to stand at room temperature (25 ° C.) for 20 minutes. Then, the peptide was added to a medium having a final concentration of 0.3 ⁇ M and 8 ng / mL myostatin (Merck Millipore), and then cultured for 4 hours.
  • the propeptide protein (RSD) used as a positive control was suspended using PBS containing 0.1% (v / v) BSA so as to be 10 ⁇ M as a stock solution, and stored at -30 ° C. 1 hour before addition to the medium, the cells were suspended in serum-free DMEM medium and allowed to stand at room temperature (25 ° C.) for 20 minutes. Then, a final concentration of 10 nM of the propeptide protein and 8 ng / mL of myostatin (Merck Millipore) were added to the medium, and then the cells were cultured for 4 hours.
  • the culture medium was removed with an aspirator, and the cells were washed with 1 ⁇ PBS. Then, 50 ⁇ L of Passive Lysis buffer (Promega) was added per well to lyse the cells. The lysate was centrifuged at 4500 rpm for 6 minutes under 4 ° C. conditions. After transferring 20 ⁇ L of the supernatant after centrifugation to a white 96-well plate (Coster), 50 ⁇ L of Luciferase Assay Reagent (Promega) was added, and the luminescence was detected by Luminoscan Ascent (Thermo Fisher Scientific). Firefly luciferase activity was measured. Further, after adding 50 ⁇ L of Stop & Glo Buffer, luminescence was detected by Luminoscan Ascent and the luciferase activity was measured to obtain internal control.
  • Passive Lysis buffer Promega
  • RiDM-35 was dissolved in a 50 mM Tris-HCl buffer (pH 7.5, 0.15 M NaCl, 10 mM CaCl 2 , 0.05% (w / v) Brij-35 containing) solution to a final concentration of 50 ⁇ M and TPCK.
  • Treated bovine pancreatic trypsin Sigma-Aldrich was added to a final concentration of 1 ⁇ g / mL (total volume 100 ⁇ L) and incubated at 37 ° C. for 400 minutes.
  • the column used was COSMOSIL 5C18-AR-II 4.6 ⁇ 150 mm (Nacalai Tesque, Inc.), and a linear gradient of acetonitrile in a water-acetonitrile system containing 0.1% trifluoroacetic acid (25 to 25 to min) at a flow rate of 1 mL / min. By applying 40%, 30 minutes), the peak of the peptide was detected with a UV detector (220 nm).
  • riDM-35 The decomposition product derived from riDM-35 was not identified by mass spectrometry using LCMS-2020 (Shimadzu Corporation). riDM-35 showed extremely high stability in bovine pancreas-derived trypsin solution.
  • riDM-35 Dissolve riDM-35 in 100 mM Tris-HCl buffer (pH 7.8, containing 10 mM CaCl 2 ) solution to a final concentration of 50 ⁇ M, and add TLCK-treated bovine pancreas-derived ⁇ -chymotrypsin (Sigma-Aldrich) to a final concentration of 2 ⁇ g. It was added to / mL (total volume 100 ⁇ L) and incubated at 37 ° C. for 400 minutes.
  • Tris-HCl buffer pH 7.8, containing 10 mM CaCl 2
  • TLCK-treated bovine pancreas-derived ⁇ -chymotrypsin Sigma-Aldrich
  • the column used was COSMOSIL 5C18-AR-II 4.6 ⁇ 150 mm (Nacalai Tesque, Inc.), and a linear gradient of acetonitrile in a water-acetonitrile system containing 0.1% trifluoroacetic acid (25 to 25 to min) at a flow rate of 1 mL / min. By applying 40%, 30 minutes), the peak of the peptide was detected with a UV detector (220 nm).
  • Test Example 4 Effect of riDM-4 on Duchenne muscular dystrophy model mdx mouse tibialis anterior muscle (in vivo evaluation) In order to verify the in vivo grip strength increasing effect of riDM-4 synthesized in Synthesis Example 1, evaluation was performed by the following method.
  • RiDM-4 was dissolved in physiological saline to a concentration of 0.75 mM. 40 ⁇ L intramuscularly was intramuscularly administered to a total of 8 muscles of both hind limbs of a 5-week-old mdx male mouse (purchased from Claire Japan) under anesthesia (40 ⁇ L of saline was similarly administered to the control group). Two weeks later, the same amount of riDM-4 (physiological saline in the control group) was administered again, and four weeks later, the mouse grip strength was measured (Saito-type mouse grip strength measuring device MK-380M, Muromachi Kikai Co., Ltd.). Was done.
  • riDM-4 significantly increased the grip strength of the hind limbs of mdx mice by about 60% based on its myostatin inhibitory activity. The improvement effect was greater than that of the existing MIPE-1686.
  • 16PC-N (3 ⁇ M) and myostatin (1 ⁇ M) 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 lysylendopeptide dase, and desalting with ZipTip C18, MALDI-TOF MS analysis was performed.
  • Test Example 5 Verification of the effect of intramuscular administration of riDM-35 on improving muscle wasting in a cancer cachexia model mouse (in vivo evaluation)
  • the ri-DM35 synthesized in Synthesis Example 23 was dissolved in PBS (phosphate buffer; 10 mM, pH 7.4) to 1 mM.
  • PBS phosphate buffer; 10 mM, pH 7.4
  • a 5.0 ⁇ 10 6 Lewis lung adenocarcinoma cell (LLC) was transplanted subcutaneously into the back of an anesthetized C57BL6 / J male mouse (purchased from Oriental yeast) to prepare a cancer cachexia model mouse.
  • LLC Lewis lung adenocarcinoma cell
  • mice were performed on the 22nd day of LLC transplantation, and the gastrocnemius muscles of both feet were excised to measure the weight and muscle fiber area.
  • Figure 10 shows the weight of the gastrocnemius muscle per body weight of the cancer cachexia model mouse.
  • the gastrocnemius muscle weight per body weight of the cancer cachexia model mice increased by 19.6%.
  • Figure 11 shows the frequency distribution table of the muscle fiber area of the cancer cachexia model mouse.
  • the ri-DM35-administered group enlarged the muscle fiber area of the cancer cachexia model mice.

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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

ペプチドおよびそれを含む複合体Peptides and complexes containing them
　本発明は、ペプチドおよびそれを含む複合体に関する。 The present invention relates to peptides and complexes containing them.
　筋ジストロフィーは、骨格筋の変性・壊死を主病変とし、筋力低下が進行する遺伝性疾患である。筋力の発現には、細胞内の筋原繊維で生じた張力を複数のタンパク質を介して細胞外の基底膜まで伝達する仕組みが必要である。これに携わるタンパク質群をコードする遺伝子の不具合は、筋ジストロフィー発症の原因となる。例えば、最も重篤なデュシェンヌ型の筋ジストロフィーでは、ジストロフィン遺伝子が変異し、当該タンパク質の欠失や機能不全が主因といわれている。そこで、筋ジストロフィーでの骨格筋の変性壊死に対抗する一手段として、骨格筋量を負に制御する因子であるマイオスタチン（増殖分化因子－８、ＧＤＦ－８）の機能を阻害することで筋肉量を増加させる治療法の開発が、特に有用と考えられている。 Muscular dystrophy is a hereditary disease in which degeneration and necrosis of skeletal muscle are the main lesions and muscle weakness progresses. The expression of muscle strength requires a mechanism to transmit the tension generated in the intracellular myofibrils to the extracellular basement membrane via multiple proteins. Defects in the genes that encode the proteins involved in this cause the development of muscular dystrophy. For example, in the most severe Duchenne-type muscular dystrophy, the dystrophin gene is mutated, and it is said that the main cause is deletion or dysfunction of the protein. Therefore, as a means to counter degenerative necrosis of skeletal muscle in muscular dystrophy, muscle mass is inhibited by inhibiting the function of myostatin (proliferative differentiation factor-8, GDF-8), which is a factor that negatively controls skeletal muscle mass. The development of treatments that increase the number of patients is considered to be particularly useful.
　マイオスタチンは、ＴＧＦ－βファミリーに属し、骨格筋に多く発現する分泌性タンパク質であり、Ｎ末端側のプロドメインとＣ末端側の成熟ドメインとを含む前駆体タンパク質として細胞内で合成される。細胞から分泌されたマイオスタチンは、ｌａｔｅｎｃｙ　ａｓｓｏｃｉａｔｅｄ　ｐｒｏｔｅｉｎ（ＬＡＰ）と呼ばれるプロドメイン由来のプロペプチドが、成熟ドメイン由来の活性ダイマーと会合し、骨格筋量を負に制御する主体である活性ダイマーを不活性化する。このように不活化状態で生体内にストックされたマイオスタチンは、必要時に、プロペプチドが酵素によって分解され、活性体となると考えられている。活性体となったマイオスタチンは、アクチビンタイプＩＩＢ受容体に代表される受容体との結合を介して、骨格筋量を負に制御するシグナル分子としての機能を果たす。 Myostatin is a secretory protein that belongs to the TGF-β family and is highly expressed in skeletal muscle, and is synthesized intracellularly as a precursor protein containing a prodomain on the N-terminal side and a mature domain on the C-terminal side. Myostatin secreted from cells is a propeptide derived from a prodomain called latency assisted protein (LAP), which associates with an active dimer derived from a mature domain and does not eliminate the active dimer, which is the main component that negatively controls skeletal muscle mass. Activate. It is considered that myostatin stocked in vivo in an inactivated state in this way becomes an active substance by decomposing a propeptide by an enzyme when necessary. The activated myostatin functions as a signal molecule that negatively regulates skeletal muscle mass through binding to a receptor typified by the activin type IIB receptor.
　したがって、マイオスタチンプロペプチドに由来するペプチドを用いて生体内のマイオスタチンを阻害することで、骨格筋量の増大や、筋ジストロフィーに代表されるような筋萎縮性障害の治療といった効果が期待できる。例えば、国際公開第２０１８／０３０４３２号（米国特許第２０１９０１７７３７０号明細書に対応）には、マイオスタチンプロペプチドに由来する、マイオスタチン阻害ペプチドに関して記載されている。 Therefore, by inhibiting myostatin in vivo using a peptide derived from myostatin propeptide, it can be expected to have effects such as increase in skeletal muscle mass and treatment of muscular atrophic disorders such as muscular dystrophy. For example, WO 2018/030432 (corresponding to US Pat. No. 20130177370) describes a myostatin-inhibiting peptide derived from a myostatin propeptide.
　国際公開第２０１８／０３０４３２号に記載のマイオスタチン阻害ペプチドは、高いマイオスタチン阻害活性を有している。一方で、高いマイオスタチン阻害活性に加えて、生体中での安定性の向上が望まれている。 The myostatin-inhibiting peptide described in International Publication No. 2018/030432 has high myostatin-inhibiting activity. On the other hand, in addition to high myostatin inhibitory activity, improvement of stability in vivo is desired.
　そこで本発明は、高いマイオスタチン阻害活性を有し、かつ改善した生体中での安定性を有するペプチドを提供することを目的とする。 Therefore, an object of the present invention is to provide a peptide having high myostatin inhibitory activity and improved in vivo stability.
　本発明者らは、上記の問題を解決すべく、鋭意研究を行った。その結果、すべてＤ体のアミノ酸で構成されるペプチドによって上記課題が解決されることを見出し、本発明の完成に至った。 The present inventors have conducted diligent research in order to solve the above problems. As a result, it was found that the above-mentioned problem can be solved by a peptide composed of all D-form amino acids, and the present invention has been completed.
　本発明の一形態は、下記式（１）で表されるアミノ酸配列を含み、アミノ酸残基数が１５～１７である、ペプチド、もしくはその薬学的に許容される塩、またはそれらのプロドラッグに関する。 One embodiment 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. ..
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000005
　上記式（１）において、
　Ｘ０は、Ｄ－Ａｌａ、Ｄ－ＧｌｙおよびＤ－２－アミノイソ酪酸からなる群から選択されるアミノ酸残基または欠損であり；
　Ｘ１は、Ｄ－Ｌｅｕ、Ｄ－ノルロイシン、Ｄ－Ｖａｌ、Ｄ－Ｉｌｅ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基または欠損であり；
　Ｘ２は、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｌｙｓ、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ３は、Ｄ－２－シクロヘキシルグリシン、Ｄ－ノルロイシン、Ｄ－Ｌｅｕ、Ｄ－Ｖａｌ、Ｄ－Ｉｌｅ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基であり；
　Ｘ４は、Ｄ－Ｌｙｓ、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ５は、Ｄ－Ｓｅｒ、Ｄ－Ａｒｇ、Ｄ－２－ヒドロキシグリシン、Ｄ－ホモセリン、Ｄ－Ｌｙｓ、Ｄ－オルニチン、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ６は、Ｄ－Ｔｒｐ、Ｄ－３－（２－ナフチル）アラニン、Ｄ－Ｔｙｒ、Ｄ－ＰｈｅおよびＤ－３－（１－ナフチル）アラニンからなる群から選択されるアミノ酸残基であり；
　Ｘ７は、Ｄ－２－フェニルグリシン、Ｄ－ノルロイシン、Ｄ－Ｉｌｅ、Ｄ－Ｌｅｕ、Ｄ－Ｖａｌ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基であり；
　Ｘ８は、Ｄ－Ｇｌｎ、Ｄ－Ａｒｇ、Ｄ－Ａｓｎ、Ｄ－Ｌｙｓ、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸、Ｄ－２，４－ジアミノブタン酸およびＤ－オルニチンからなる群から選択されるアミノ酸残基であり；
　Ｘ９は、Ｄ－２－シクロヘキシルグリシン残基であり；
　Ｘ１０は、Ｄ－Ｌｙｓ、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ１１は、Ｄ－２－フェニルグリシン、Ｄ－Ｉｌｅ、Ｄ－Ｌｅｕ、Ｄ－ノルロイシン、Ｄ－Ｖａｌ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基であり；
　Ｘ１２は、Ｄ－Ｔｒｐ、Ｄ－ホモフェニルアラニン、Ｄ－ＴｙｒおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基であり；
　Ｘ１３は、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｌｙｓ、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ１４は、Ｄ－２－フェニルグリシン、Ｄ－Ｉｌｅ、Ｄ－Ｌｅｕ、Ｄ－ノルロイシン、Ｄ－Ｖａｌ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基であり；
　Ｘ１５は、Ｄ－Ｔｙｒ、Ｄ－ＴｒｐおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基であり；ならびに
　Ｘ１６は、Ｄ－Ｔｒｐ、Ｄ－ホモフェニルアラニン、Ｄ－ＴｙｒおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基または欠損である。 In the above formula (1)
X 0 is an amino acid residue or defect selected from the group consisting of D-Ala, D-Gly and D-2-aminoisobutyric acid;
X 1 is an amino acid residue or defect 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. can be;
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. Is the basis;
X 4 is, D-Lys, D-Arg , D- ornithine, D-His, with an amino acid residue selected from the group consisting of D-2,3-diaminopropionic acid and D-2,4-diaminobutane acid can be;
X 5 contains D-Ser, D-Arg, D-2-hydroxyglycine, D-homoserine, D-Lys, D-ornithine, D-His, D-2,3-diaminopropionic acid and D-2,4. -Amino acid residues selected from the group consisting of 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. Is the basis;
X 8 is, D-Gln, D-Arg , D-Asn, D-Lys, D-His, D-2,3- diaminopropionic acid, the group consisting of D-2,4-diaminobutanoic acid and D- ornithine Amino acid residues selected from;
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. can be;
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. Is the basis;
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. can be;
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. Is the basis;
X 15 is an amino acid residue selected from the group consisting of D-Tyr, D-Trp and D-Phe; and X 16 is from D-Trp, D-homophenylalanine, D-Tyr and D-Phe. Amino acid residues or defects selected from the group consisting of.
実施例および比較例のペプチドによる、マイオスタチン阻害活性を示す。The peptides of Examples and Comparative Examples show myostatin inhibitory activity. 実施例のペプチドによる、マイオスタチン阻害活性を示す。The peptide of the example shows the myostatin inhibitory activity. 実施例および比較例のペプチドによる、マイオスタチン阻害活性を示す。The peptides of Examples and Comparative Examples show myostatin inhibitory activity. 実施例のペプチドによる、マイオスタチン阻害活性を示す。The peptide of the example shows the myostatin inhibitory activity. 実施例のペプチドのウシ膵臓由来トリプシン溶液中での安定性評価の結果を示す。The results of the stability evaluation of the peptide of the example in a bovine pancreas-derived trypsin solution are shown. 実施例のペプチドのウシ膵臓由来α－キモトリプシン溶液中での安定性評価の結果を示す。The results of the stability evaluation of the peptide of the example in a bovine pancreas-derived α-chymotrypsin solution are shown. 実施例のペプチドのデュシェンヌ型筋ジストロフィーモデルｍｄｘマウス前脛骨筋に及ぼす影響（ｉｎ　ｖｉｖｏ評価）の結果を示す。The results of the effect (in vivo evaluation) of the peptide of the example on the Duchenne muscular dystrophy model mdx mouse tibialis anterior muscle are shown. 実施例のペプチドを用いたマイオスタチン酸素化評価の結果を示す。The result of myostatin oxygenation evaluation using the peptide of Example is shown. 実施例のペプチドの筋肉内投与によるがん悪液質モデルマウスの握力に及ぼす影響（ｉｎ　ｖｉｖｏ評価）の結果を示す。The results of the effect (in vivo evaluation) on the grip strength of the cancer cachexia model mouse by intramuscular administration of the peptide of the example are shown. 実施例のペプチドの筋肉内投与によるがん悪液質モデルマウスの腓腹筋量に及ぼす影響（ｉｎ　ｖｉｖｏ評価）の結果を示す。The results of the effect (in vivo evaluation) on the gastrocnemius muscle mass of the cancer cachexia model mouse by intramuscular administration of the peptide of the example are shown. 実施例のペプチドの筋肉内投与によるがん悪液質モデルマウスの筋線維面積に及ぼす影響（ｉｎ　ｖｉｖｏ評価）の結果を示す。The results of the effect (in vivo evaluation) on the muscle fiber area of the cancer cachexia model mouse by intramuscular administration of the peptide of the example are shown.
　以下、本発明の一形態に係る実施の形態を説明する。本発明は、以下の実施の形態のみには限定されない。 Hereinafter, embodiments according to one embodiment of the present invention will be described. The present invention is not limited to the following embodiments.
　本明細書において、範囲を示す「Ｘ～Ｙ」は「Ｘ以上Ｙ以下」を意味する。また、特記しない限り、操作および物性等の測定は室温（２０～２５℃）／相対湿度４０～５０％ＲＨの条件で測定する。 In the present specification, "XY" indicating a range means "X or more and Y or less". Unless otherwise specified, the operation and physical properties are measured under the conditions of room temperature (20 to 25 ° C.) / relative humidity of 40 to 50% RH.
　本明細書において、「式（１）で表されるアミノ酸配列を含み、アミノ酸残基数が１５～１７である、ペプチド、もしくはその薬学的に許容される塩」を、単に「本発明に係るペプチド」とも称する。 In the present specification, "a peptide containing an amino acid sequence represented by the formula (1) and having 15 to 17 amino acid residues, or a pharmaceutically acceptable salt thereof" is simply referred to as "the present invention." Also called "peptide".
　本発明における「アミノ酸残基」とは、ペプチドまたはタンパク質分子上で、ペプチドまたはタンパク質を構成しているアミノ酸の一単位に当たる部分を意味する。より具体的には、以下の式（５）のように表される、α－アミノ酸から誘導される２価の基を意味する： The "amino acid residue" in the present invention means a portion corresponding to one unit of the amino acid constituting the peptide or protein on the peptide or protein molecule. More specifically, it means a divalent group derived from an α-amino acid, which is represented by the following formula (5):
Figure JPOXMLDOC01-appb-C000006
Figure JPOXMLDOC01-appb-C000006
ただし、上記Ｒ０はアミノ酸の側鎖であり、例えばＧｌｙであれば水素原子、Ａｌａであればメチル基である。 However, R 0 is a side chain of an amino acid, for example, Gly is a hydrogen atom and Ala is a methyl group.
　「アミノ酸残基」は、天然もしくは非天然のα－アミノ酸に由来する。本発明に係るペプチドは、生体中での安定性を高めるとの観点から、Ｄ体のアミノ酸で構成される。 "Amino acid residues" are derived from natural or unnatural α-amino acids. The peptide according to the present invention is composed of D-form amino acids from the viewpoint of enhancing stability in a living body.
　より具体的には、「アミノ酸残基」は、Ａｒｇ、Ｌｙｓ、Ａｓｐ、Ａｓｎ、Ｇｌｕ、Ｇｌｎ、Ｈｉｓ、Ｐｒｏ、Ｔｙｒ、Ｔｒｐ、Ｓｅｒ、Ｔｈｒ、Ｇｌｙ、Ａｌａ、Ｍｅｔ、Ｃｙｓ、Ｐｈｅ、Ｌｅｕ、Ｖａｌ、およびＩｌｅ、ならびにこれらの類縁体が例示できる。上記の類縁体としては、例えば上記２０種のアミノ酸残基の側鎖が任意の置換基で置換された誘導体等であってもよく、例えば、上記２０種のアミノ酸残基のハロゲン化誘導体（例えば、３－クロロアラニン、４－フルオロフェニルアラニン、４－クロロフェニルアラニン）、２－アミノ酪酸、４－アミノ酪酸、ノルロイシン、ノルバリン、イソバリン、２－アミノイソ酪酸、ホモフェニルアラニン、２，３－ジアミノプロピオン酸、２，４－ジアミノブタン酸、オルニチン、２－ヒドロキシグリシン、ホモセリン、ヒドロキシリジン、ヒドロキシプロリン、３，４－ジデヒドロプロリン、ホモシステイン、ホモメチオニン、アスパラギン酸エステル（例えば、アスパラギン酸－メチルエステル、アスパラギン酸－エチルエステル、アスパラギン酸－プロピルエステル、アスパラギン酸－シクロヘキシルエステル、アスパラギン酸－ベンジルエステルなど）、グルタミン酸エステル（グルタミン酸－シクロヘキシルエステル、グルタミン酸－エチルエステル、グルタミン酸－プロピルエステル、グルタミン酸－メチルエステル、グルタミン酸－ベンジルエステルなど）、ホルミルトリプトファン、２－シクロペンチルグリシン、２－シクロヘキシルグリシン、２－フェニルグリシン、β－アラニン、３－シクロペンチルアラニン、３－シクロヘキシルアラニン、３－ピリジルアラニン、３－ピラゾリルアラニン、３－フラニルアラニン、３－チエニルアラニン、４－メトキシフェニルアラニン、および３－ナフチルアラニン（３－（１－ナフチル）アラニン、３－（２－ナフチル）アラニン）等のアミノ酸に由来するアミノ酸残基が例示できるが、これらに制限されない。また、ＩｌｅやＴｈｒのように、側鎖に不斉炭素を有するジアステレオマーが存在するものについては、天然型（例えば、（２Ｒ＊，３Ｒ＊）－２－アミノ－３－メチルペンタン酸、および（２Ｒ＊，３Ｓ＊）－２－アミノ－３－ヒドロキシブタン酸）および非天然型（例えば、（２Ｒ＊，３Ｓ＊）－２－アミノ－３－メチルペンタン酸、および（２Ｒ＊，３Ｒ＊）－２－アミノ－３－ヒドロキシブタン酸）が特に区別なく使用され得る。すなわち、「Ｉｌｅ」は（２Ｒ＊，３Ｒ＊）－２－アミノ－３－メチルペンタン酸および（２Ｒ＊，３Ｓ＊）－２－アミノ－３－メチルペンタン酸の両方を含む意味として使用され、「Ｔｈｒ」は（２Ｒ＊，３Ｓ＊）－２－アミノ－３－ヒドロキシブタン酸および（２Ｒ＊，３Ｒ＊）－２－アミノ－３－ヒドロキシブタン酸の両方を含む意味として使用される。好ましくは。天然型ジアステレオマー（すなわち、Ｉｌｅであれば（２Ｒ＊，３Ｒ＊）－２－アミノ－３－メチルペンタン酸、Ｔｈｒであれば（２Ｒ＊，３Ｓ＊）－２－アミノ－３－ヒドロキシブタン酸）が使用される。 More specifically, the "amino acid residue" is Arg, Lys, Asp, Asn, Glu, Gln, His, Pro, Tyr, Trp, Ser, Thr, Gly, Ala, Met, Cys, Phe, Leu, Val. , And Ile, and their analogs can be exemplified. The analog may be, for example, a derivative in which the side chain of the 20 kinds of amino acid residues is substituted with an arbitrary substituent, and for example, a halogenated derivative of the above 20 kinds of amino acid residues (for example, , 3-Chloroalanine, 4-Fluorophenylalanine, 4-Chlorophenylalanine), 2-aminobutyric acid, 4-aminobutyric acid, norleucine, norvaline, isovalin, 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 ester (eg, aspartic acid-methyl ester, aspartic acid) -Ethyl ester, aspartic acid-propyl ester, aspartic acid-cyclohexyl ester, aspartic acid-benzyl ester, etc.), Glutamic acid ester (glutamic acid-cyclohexyl ester, glutamic acid-ethyl ester, glutamic acid-propyl ester, glutamic acid-methyl ester, glutamate-benzyl Esters, etc.), formyltryptophan, 2-cyclopentylglycine, 2-cyclohexylglycine, 2-phenylglycine, β-alanine, 3-cyclopentylalanine, 3-cyclohexylalanine, 3-pyridylalanine, 3-pyrazolylalanine, 3-furanyl Amino acid residues derived from amino acids such as alanine, 3-thienylalanine, 4-methoxyphenylalanine, and 3-naphthylalanine (3- (1-naphthyl) alanine, 3- (2-naphthyl) alanine) can be exemplified. Not limited to these. In addition, for those having a diastereomer having an asymmetric carbon in the side chain, such as Ile and Thr, the natural type (for example, (2R * , 3R * ) -2-amino-3-methylpentanoic acid, And (2R * , 3S * ) -2-amino-3-hydroxybutanoic acid) and non-natural forms (eg, (2R * , 3S * ) -2-amino-3-methylpentanoic acid, and (2R * , 3R). * ) -2-Amino-3-hydroxybutanoic acid) can be used without particular distinction. That is, "Ile" is used to mean both (2R * , 3R * ) -2-amino-3-methylpentanoic acid and (2R * , 3S * ) -2-amino-3-methylpentanoic acid. "Thr" is used to mean both (2R * , 3S * ) -2-amino-3-hydroxybutanoic acid and (2R * , 3R * ) -2-amino-3-hydroxybutanoic acid. Preferably. Natural diastereomers (ie, (2R * , 3R * ) for Ile, -2-amino-3-methylpentanoic acid, (2R * , 3S * ) for Thr, -2-amino-3-hydroxybutane Acid) is used.
　本明細書に記載のアミノ酸配列は、特に言及がない限り、慣例に従ってＮ末端（アミノ末端）側からＣ末端（カルボキシル末端）側への方向に表記される。 Unless otherwise specified, the amino acid sequence described in the present specification is described in the direction from the N-terminal (amino-terminal) side to the C-terminal (carboxyl-terminal) side according to the convention.
　各アミノ酸残基は、その側鎖の相違に基づいて、類似の性質を有するアミノ酸残基と置換し得ることが本技術分野において知られている（保存的置換）。例えば、脂肪族疎水性アミノ酸であるＶａｌ、Ｌｅｕ、Ｉｌｅ、２－アミノ酪酸（Ａｂｕ）、ノルロイシン（Ｎｌｅ）、ノルバリン（Ｎｖａ）、およびイソバリン（Ｉｖａ）は相互に置換し得る。側鎖が水素原子またはメチル基であるＧｌｙ、Ａｌａおよび２－アミノイソ酪酸（Ａｉｂ）は相互に置換し得る。側鎖がフェニルアルキル基であるＰｈｅおよびホモフェニルアラニン（Ｈｐｈ）は相互に置換し得る。中性極性アミノ酸であるＡｓｎおよびＧｌｎは相互に置換し得る。塩基性アミノ酸であるＡｒｇ、Ｌｙｓ、Ｈｉｓ、２，３－ジアミノプロピオン酸（Ｄｐｒ）、２，４－ジアミノブタン酸（Ｄｂｕ）、およびオルニチン（Ｏｒｎ）は相互に置換し得る。酸性アミノ酸であるＡｓｐおよびＧｌｕは相互に置換し得る。側鎖がヒドロキシ基または短鎖のヒドロキシアルキル基であるＳｅｒ、２－ヒドロキシグリシン（Ｈｙｇ）およびホモセリン（Ｈｓｅ）は相互に置換し得る。Ｐｒｏと、その側鎖のピロリジル基が脱水素した構造の側鎖を有する３，４－ジデヒドロプロリン（Ｄｈｐ）とは、相互に置換し得る。側鎖が短鎖のチオールアルキル基であるＣｙｓおよびホモシステイン（Ｈｃｙ）は相互に置換し得る。側鎖が短鎖のスルフィド構造であるＭｅｔおよびホモメチオニン（Ｈｍｅ）は相互に置換し得る。側鎖が芳香族性であるＴｒｐ、ＴｙｒおよびＰｈｅは相互に置換し得る。 It is known in the art that each amino acid residue can be replaced with an amino acid residue having similar properties based on the difference 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 with each other. Gly, Ala and 2-aminoisobutyric acid (Aib), whose side chains are hydrogen atoms or methyl groups, can be replaced with each other. Phe and homophenylalanine (Hph), whose side chains are phenylalkyl groups, can be substituted with each other. The neutral polar amino acids Asn and Gln can be replaced with each other. The basic amino acids Arg, Lys, His, 2,3-diaminopropionic acid (Dpr), 2,4-diaminobutanoic acid (Dbu), and ornithine (Orn) can be replaced with each other. The acidic amino acids Asp and Glu can be replaced with each other. Ser, 2-hydroxyglycine (Hyg) and homoserine (Hse), whose side chains are hydroxy groups or short hydroxyalkyl groups, can be substituted with each other. Pro and 3,4-didehydroproline (Dhp) having a side chain in which the pyrrolidyl group of the side chain is dehydrogenated can be mutually substituted. Cys and homocysteine (Hcy), whose side chains are short thiolalkyl groups, can be replaced with each other. Met and homomethionine (Hme), whose side chains are short-chain sulfide structures, can be replaced with each other. Trp, Tyr and Phe, whose side chains are aromatic, can be replaced with each other.
　さらに、鎖の配向に影響するアミノ酸残基であるＧｌｙおよびＰｒｏは相互に置換し得る。 Furthermore, Gly and Pro, which are amino acid residues that affect the orientation of the chain, can be replaced with each other.
　本明細書において「薬学的に許容される塩」は、患者や被験体へ投与された後、望ましくない生理学的効果を生じさせない、金属塩、アンモニウム塩、有機酸塩、無機酸塩、または有機塩基もしくは無機塩基との塩である。より具体的には、ナトリウム塩、カリウム塩、カルシウム塩、マグネシウム塩、バリウム塩、アルミニウム塩、亜鉛塩、アンモニウム塩、メチルアミン塩、エチルアミン塩、アニリン塩、ジメチルアミン塩、ジエチルアミン塩、ピロリジン塩、ピペリジン塩、モルホリン塩、ピペラジン塩、トリメチルアミン塩、トリエチルアミン塩、エタノールアミン塩、ジエタノールアミン塩、トリエタノールアミン塩、塩酸塩、臭化水素酸塩、硝酸塩、硫酸塩、リン酸塩、ギ酸塩、酢酸塩、トリフルオロ酢酸塩、フタル酸塩、フマル酸塩、シュウ酸塩、酒石酸塩、マレイン酸塩、クエン酸塩、コハク酸塩、リンゴ酸塩、メタンスルホン酸塩、ベンゼンスルホン酸塩、およびｐ－トルエンスルホン酸塩等が例示できるが、これらに限定されない。 As used herein, a "pharmaceutically acceptable salt" is a metal salt, ammonium salt, organic acid salt, inorganic acid salt, or organic salt that does not produce an undesired physiological effect after being administered to a patient or subject. It is a salt with a base or an inorganic base. More specifically, 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- Examples thereof include, but are not limited to, toluene sulfonates and the like.
　本発明に係るペプチドのプロドラッグ（以下、「本発明に係るペプチドのプロドラッグ」を、単に「プロドラッグ」とも称する。）は、本発明に係るペプチドに変換するペプチド誘導体、すなわち、胃酸や酵素等により酸化、還元、加水分解等を起こして本発明に係るペプチドに変化するペプチド誘導体を指す。これらのペプチド誘導体は、例えばＢｕｎｄｇａｒｄ，　Ｈ．，　Ｄｅｓｉｇｎ　ｏｆ　Ｐｒｏｄｒｕｇｓ，　ｐｐ．　７－９，　２１－２４，　Ｅｌｓｅｖｉｅｒ，　Ａｍｓｔｅｒｄａｍ　１９８５等に記載の従来公知の方法によって、本発明に係るペプチドから製造することができる。 The peptide prodrug according to the present invention (hereinafter, "prodrug of the peptide according to the present invention" is also simply referred to as "prodrug") is a peptide derivative that converts the peptide according to the present invention, that is, gastric acid or enzyme. It refers to a peptide derivative that undergoes oxidation, reduction, hydrolysis, etc. to change into the peptide according to the present invention. These peptide derivatives are, for example, Bundgard, H. et al. , Design of Prodrugs, pp. It can be produced from the peptide according to the present invention by a conventionally known method described in 7-9, 21-24, Elsevier, Amsterdam 1985 and the like.
　プロドラッグとしては、本発明に係るペプチドの側鎖がカルボキシル基を有する場合には、当該カルボキシル基とアルコールとを反応させることによって得られるエステル誘導体、または当該カルボキシル基とアミンとを反応させることによって得られるアミド誘導体が例示できる。より具体的には、例えば、ペプチド側鎖のカルボキシル基を－ＣＯＯＲ（Ｒは、炭素数１～２０のアルキル基）で表されるエステル、または－ＣＯＮＨＲもしくは－ＣＯＮＲＲ’（ＲおよびＲ’は、それぞれ独立に、炭素数１～２０のアルキル基）で表されるアミド基に誘導体化したペプチドが挙げられる。 As a prodrug, when the side chain of the peptide according to the present invention has a carboxyl group, an ester derivative obtained by reacting the carboxyl group with an alcohol, or by reacting the carboxyl group with an amine The obtained amide derivative can be exemplified. More specifically, for example, an ester in which the carboxyl group of the peptide side chain is represented by -COOR (R is an alkyl group having 1 to 20 carbon atoms), or -CONHR or -CONRR'(R and R'is Independently, a peptide derivatized into an amide group represented by an alkyl group having 1 to 20 carbon atoms can be mentioned.
　プロドラッグとしては、本発明に係るペプチドの側鎖が水酸基を有する場合には、当該水酸基と酸無水物等を反応させてアシル化させたアシルオキシ誘導体が例示できる。より具体的には、例えば、ペプチド側鎖の水酸基を－ＯＣＯＲ（Ｒは、炭素数１～２０のアルキル基）で表されるアシルオキシ基に誘導体化したペプチドが挙げられる。 As the prodrug, when the side chain of the peptide according to the present invention has a hydroxyl group, an acyloxy derivative obtained by reacting the hydroxyl group with an acid anhydride or the like to acylate it can be exemplified. More specifically, for example, a peptide obtained by derivatizing the hydroxyl group of the peptide side chain into an acyloxy group represented by −OCOR (R is an alkyl group having 1 to 20 carbon atoms) can be mentioned.
　プロドラッグとしては、本発明に係るペプチドの側鎖がアミノ基を有する場合には、当該アミノ基がアシル化、Ｎ－オキシド化、アルキル化、またはリン酸化された誘導体が例示できる。より具体的には、例えば、側鎖のアミノ基を－ＮＨＣＯＲ（Ｒは、炭素数１～２０のアルキル基）や－ＮＨＣＯＣＨ（ＮＨ２）ＣＨ３で表されるアミド基に誘導体化したペプチドが挙げられる。 Examples of the prodrug include derivatives in which the amino group is acylated, N-oxidized, alkylated, or phosphorylated when the side chain of the peptide according to the present invention has an amino group. More specifically, for example, a peptide in which the amino group of the side chain is derivatized into an amide group represented by -NHCOR (R is an alkyl group having 1 to 20 carbon atoms) or -NHCOCH (NH 2 ) CH 3. Can be mentioned.
　本発明に係るペプチドのＮ末端の構造は特に制限されず、例えば、水素原子（すなわち、未修飾）、または従来公知の手法により修飾基を導入した構造であってもよい。Ｎ末端の修飾基としては、炭素数１～２０のアルキル基、炭素数１～２０のアルケニル基、炭素数１～２０のアルキニル基、炭素数６～２０の芳香族炭化水素基、複素環基、下記式（６）で表される基、スルホニル基、カルボキシル基、グリオキシル基、ホルミル基；ポリエチレングリコール基（ＰＥＧ化）、ポリオキシエチレングリコール基、ポリプロピレングリコール基；ｔｅｒｔ－ブトキシカルボニル基（Ｂｏｃ基）、ベンジルオキシカルボニル基（Ｚ基）、フルオレニルメトキシカルボニル基（Ｆｍｏｃ基）のような保護基；シクロペンチルオキシカルボニル基、シクロヘキシルオキシカルボニル基、アダマンチルオキシカルボニル基、ノルボルニルオキシカルボニル基、イソボルニルオキシカルボニル基等のシクロアルキルオキシカルボニル基；ピログルタミン酸やモロタン酸などのアミノ酸から誘導される保護基；カルバメート系保護基；ベンゼンスルホン酸などのスルホン酸やリン酸から誘導される保護基等、が例示できる。このうち、マイオスタチン阻害活性の観点から、ペプチドは、Ｎ末端が水素原子、アルキル基、芳香族炭化水素基、複素環基、下記式（６）で表される基、スルホニル基、カルボキシル基、グリオキシル基、ホルミル基またはポリエチレングリコール基であることが好ましく、水素原子、アシル基またはポリエチレングリコール基であることがより好ましく、水素原子であることがさらに好ましい。 The structure of the N-terminal of the peptide according to the present invention is not particularly limited, and may be, for example, a hydrogen atom (that is, unmodified) or a structure in which a modifying group is introduced by a conventionally known method. The N-terminal modifying group includes an alkyl group having 1 to 20 carbon atoms, an alkenyl group having 1 to 20 carbon atoms, an alkynyl group having 1 to 20 carbon atoms, an aromatic hydrocarbon group having 6 to 20 carbon atoms, and a heterocyclic group. , A group represented by the following formula (6), a sulfonyl group, a carboxyl group, a glyoxyl group, a formyl group; a polyethylene glycol group (PEGylated), a polyoxyethylene glycol group, a polypropylene glycol group; a tert-butoxycarbonyl group (Boc group). ), benzyloxycarbonyl group (Z group), protective group such as fluorenylmethoxycarbonyl group (Fmoc group); cyclopentyloxycarbonyl group, cyclohexyloxycarbonyl group, adamantyloxycarbonyl group, norbornyloxycarbonyl group, iso Cycloalkyloxycarbonyl groups such as Bornyloxycarbonyl groups; Protective groups derived from amino acids such as pyroglutamic acid and morothanic acid; Carbamate-based protective groups; sulfonic acid such as benzenesulfonic acid and protective groups derived from phosphoric acid, etc. , Can be exemplified. Of these, from the viewpoint of myostatin inhibitory activity, the peptide has a hydrogen atom, an alkyl group, an aromatic hydrocarbon group, a heterocyclic group at the N-terminal, a group represented by the following formula (6), a sulfonyl group, a carboxyl group, and the like. It is preferably 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.
　ペプチドのＮ末端に存在し得るアルキル基の炭素数は、例えば１～２０であり、好ましくは１～１０である。アルキル基は飽和鎖、不飽和鎖、または環状の構造であって良く、分岐鎖構造を取っていても良い。より具体的には、アルキル基としては、メチル基、エチル基、プロピル基、イソプロピル基、シクロプロピル基、ブチル基、イソブチル基、ｓｅｃ－ブチル基、ｔｅｒｔ－ブチル基、アミル基、イソアミル基、ｔｅｒｔ－アミル基、ヘキシル基、シクロヘキシル基、ヘプチル基、オクチル基、２－エチルヘキシル基、ノニル基、デシル基等が例示できる。 The number of carbon atoms of the alkyl group that can be present at the N-terminal of the peptide is, for example, 1 to 20, preferably 1 to 10. The alkyl group may have a saturated chain, an unsaturated chain, or a cyclic structure, and may have a branched chain structure. More specifically, the alkyl group includes 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, and a tert. -Amil group, hexyl group, cyclohexyl group, heptyl group, octyl group, 2-ethylhexyl group, nonyl group, decyl group and the like can be exemplified.
　ペプチドのＮ末端に存在し得る芳香族炭化水素基の炭素数は、例えば６～２０であり、より具体的には、フェニル基、ナフチル基、トリル基、およびフェナントリル基等が例示できる。ペプチドのＮ末端に存在し得る複素環基としては、環内に窒素原子、酸素原子および硫黄原子から選択されるヘテロ原子を１～３個含む、単環、縮合二環式または縮合三環式構造の置換基が例示でき、より具体的には、ピロリジル基、ピロール基、ピペリジル基、ピリジル基、イミダゾリル基、ピラゾリル基、オキサゾリル基、チアゾリル基、モルホリル基、インドリル基、ベンゾイミダゾリル基、キノリル基、カルバゾリル基、テトラヒドロフラニル基、テトラヒドロチオフェニル基、フラニル基、チオフェニル基、テトラヒドロピラニル基、およびテトラヒドロチオピラニル基等が例示できる。これらの芳香族炭化水素基や複素環基は、炭素数１～６の直鎖もしくは分岐鎖のアルキル基、炭素数１～６の直鎖もしくは分岐鎖のアルコキシ基、アミノ基、カルボキシル基、エステル基、カルバモイル基、アミド基、ニトロ基、スルホ基、スルホンアミド基、および／またはハロゲン等のさらなる置換基によって置換されていてもよい。 The carbon number of the aromatic hydrocarbon group that can exist at the N-terminal of the peptide is, for example, 6 to 20, and more specifically, a phenyl group, a naphthyl group, a tolyl group, a phenanthryl group and the like can be exemplified. Heterocyclic groups that can be present at the N-terminal of the peptide include monocyclic, fused bicyclic or fused tricyclic groups containing 1 to 3 heteroatoms selected from nitrogen, oxygen and sulfur atoms in the ring. Examples of the substituent of the structure can be exemplified, and more specifically, 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 morphoryl group, an indolyl group, a benzoimidazolyl group, a quinolyl group, Examples thereof include a carbazolyl group, a tetrahydrofuranyl group, a tetrahydrothiophenyl group, a furanyl group, a thiophenyl group, a tetrahydropyranyl group, a tetrahydrothiopyranyl group and the like. These aromatic hydrocarbon groups and heterocyclic groups are linear or branched alkyl groups having 1 to 6 carbon atoms, linear or branched alkoxy groups having 1 to 6 carbon atoms, amino groups, carboxyl groups and esters. It may be substituted with a group, a carbamoyl group, an amide group, a nitro group, a sulfo group, a sulfonamide group, and / or a further substituent such as a halogen.
　Ｎ末端の修飾基は、例えば下記式（６）で表される官能基であってもよい。 The N-terminal modifying group may be, for example, a functional group represented by the following formula (6).
Figure JPOXMLDOC01-appb-C000007
Figure JPOXMLDOC01-appb-C000007
　ただし、式（６）において、
Ｘ０Ａは単結合、酸素原子もしくは硫黄原子であるか、または、アミノ基、アセチルアミノ基およびプロピオニルアミノ基からなる群から選択される置換基を有してもよい、炭素数１～３のアルキレン基（例えば、メチレン基、エチレン基、トリメチレン基およびプロピレン基）、炭素数１～３のオキシアルキレン基（例えば、オキシメチレン基、オキシエチレン基、オキシトリメチレン基およびオキシプロピレン基）および炭素数１～３のアルキレンオキシ基（例えば、メチレンオキシ基、エチレンオキシ基、トリメチレンオキシ基およびプロピレンオキシ基）からなる群から選択される２価の連結基であり；
Ｒ１Ａは、置換基を有していてもよい炭素数１～２０のアルキル基、ならびに However, in equation (6),
X 0A is a single bond, an oxygen atom or a sulfur atom, or may have a substituent selected from the group consisting of an amino group, an acetylamino group and a propionylamino group, and is an alkylene having 1 to 3 carbon atoms. Group (eg, methylene group, ethylene group, trimethylene group and propylene group), oxyalkylene group having 1 to 3 carbon atoms (eg, oxymethylene group, oxyethylene group, oxytrimethylene group and oxypropylene group) and 1 carbon number. A divalent linking group selected from the group consisting of ~ 3 alkyleneoxy groups (eg, methyleneoxy group, ethyleneoxy group, trimethyleneoxy group and propyleneoxy group);
R 1A is an alkyl group having 1 to 20 carbon atoms which may have a substituent, as well as an alkyl group having 1 to 20 carbon atoms.
Figure JPOXMLDOC01-appb-C000008
Figure JPOXMLDOC01-appb-C000008
からなる群から選択され、
上記Ｒ１１～Ｒ３０はそれぞれ独立に水素原子、ハロゲン原子（例えば、フッ素原子、塩素原子、臭素原子、ヨウ素原子）、炭素数１～３のアルキル基（すなわち、メチル基、エチル基、プロピル基）、炭素数１～３のアルコキシ基（すなわち、メトキシ基、エトキシ基、プロポキシ基）、水酸基、およびアミノ基からなる群から選択される。 Selected from a group of
The above R 11 to R 30 are independently hydrogen atom, halogen atom (for example, fluorine atom, chlorine atom, bromine atom, iodine atom) and alkyl group having 1 to 3 carbon atoms (that is, methyl group, ethyl group, propyl group). ), An alkoxy group having 1 to 3 carbon atoms (that is, a methoxy group, an ethoxy group, a propoxy group), a hydroxyl group, and an amino group.
　上記式（６）において、Ｒ１Ａが置換基を有していてもよい炭素数１～２０のアルキル基である場合、その脂肪鎖は飽和鎖、不飽和鎖、または環状の構造であって良く、分岐鎖構造を取っていても良い。Ｒ１Ａのアルキル基の炭素数は、２～１２であることが好ましい。 In the above formula (6), when R 1A is an alkyl group having 1 to 20 carbon atoms which may have a substituent, the fatty chain may have a saturated chain, an unsaturated chain, or a cyclic structure. , May have a branched chain structure. The alkyl group of R 1A preferably has 2 to 12 carbon atoms.
　Ｒ１Ａの置換基としては、ヒドロキシ基、炭素数１～５以下のアルコキシ基（例えばメトキシ基、エトキシ基など）、アミノ基、カルボキシル基、エステル基、カルバモイル基、アミド基、ニトロ基、スルホ基、ハロゲン（フッ素、塩素、臭素、ヨウ素）などが例示できる。 The substituent of R 1A includes a hydroxy group, an alkoxy group having 1 to 5 or less carbon atoms (for example, a methoxy group, an ethoxy group, etc.), an amino group, a carboxyl group, an ester group, a carbamoyl group, an amide group, a nitro group, and a sulfo group. , Halogen (fluorine, chlorine, bromine, iodine) and the like can be exemplified.
　上記式（６）において、好ましくは、Ｘ０Ａは単結合であるか、または、アミノ基およびアセチルアミノ基からなる群から選択される置換基を有していてもよい、炭素数１～３のアルキレン基および炭素数１～３のオキシアルキレン基からなる群から選択される２価の連結基である。 In the above formula (6), preferably, X 0A is a single bond or may have a substituent selected from the group consisting of an amino group and an acetylamino group, and has 1 to 3 carbon atoms. It is a divalent linking group selected from the group consisting of an alkylene group and an oxyalkylene group having 1 to 3 carbon atoms.
　一実施形態では、上記式（６）で表される基は、アシル基である。アシル基としては、種々のカルボン酸から誘導されるアシル基が含まれる。より具体的には、脂肪鎖、芳香族環または複素環を有するアシル基であってもよく、あるいは、アミノ酸、アシル基を有するビタミン、アシル基を有する核酸塩基よりなる群から選択される化合物から誘導されるアシル基であってもよい。 In one embodiment, the group represented by the above formula (6) is an acyl group. Acyl groups include acyl groups derived from various carboxylic acids. More specifically, it may be an acyl group having a fat chain, an aromatic ring or a heterocycle, or 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. It may be an acyl group to be derived.
　上記式（６）におけるＲ１Ａが置換基を有していてもよい炭素数１～２０のアルキル基であるアシル基としては、より具体的には、アセチル基、プロピオニル基、ブチリル基、イソブチリル基、バレリル基、イソバレル基、ピバロイル基、カプロイル基、カプリノイル基、メチルヘキサノイル基、シクロプロパンカルボニル基、アミノシクロプロパンカルボニル基、シクロヘキサンカルボニル基、シクロヘキシルアセチル基、シクロペンチルプロピオニル基、シクロヘキシルプロピオニル基、シクロペンチルブタノイル基、シクロヘキシルブタノイル基、アダマンチルアセチル基、ラウロイル基、ミリストイル基、パルミトイル基、ステアロイル基、オキサリル基、マロニル基、スクシニル基、グルタリル基、アジポイル基、グリコール基、ラクトイル基、グリセロイル基、ピルボイル基、アセトアセチル基等が例示できるが、これらに限定されない。 More specifically, the acyl group, which is an alkyl group having 1 to 20 carbon atoms in which R 1A in the above formula (6) may have a substituent, is an acetyl group, a propionyl group, a butyryl group, or an isobutyryl group. , Valeryl group, isobarrel group, pivaloyl group, caproyl group, caprinoyle group, methylhexanoyl group, cyclopropanecarbonyl group, aminocyclopropanecarbonyl group, cyclohexanecarbonyl group, cyclohexylacetyl group, cyclopentylpropionyl group, cyclohexylpropionyl group, cyclopentylbuta Noyl group, cyclohexylbutanoyl group, adamantylacetyl group, lauroyl group, myritoyl group, palmitoyl group, stearoyl group, oxalyl group, malonyl group, succinyl group, glutalyl group, adipoyl group, glycol group, lactoyl group, glyceroyl group, pyruboyl group , Acetacetyl group and the like can be exemplified, but the present invention is not limited thereto.
　アシル基を有するビタミンとしては、例えば、ニコチン酸、パントテン酸、ビオチン、プテロイルグルタミン酸（葉酸）、オロチン酸、フルオロオロチン酸、α－リポ酸、ピリドキシン酸、ビオシチン、プテロイン酸、１０－ホルミルプテロイン酸、７，８－ジヒドロ葉酸、ホモプテロイン酸、プテリン－６－カルボン酸、ジヒドロリポ酸、ハイドロオロチン酸などが挙げられる。 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, biocithin, pteroic acid, and 10-formylpteroin. Acids, 7,8-dihydrofolic acid, homopteroic acid, pterin-6-carboxylic acid, dihydrolipoic acid, hydroorothic acid and the like can be mentioned.
　アシル基を有する核酸塩基誘導体は、ヌクレオチドを構成する塩基成分およびその誘導体を指し、好ましくはピリミジン誘導体等、例えば５－カルボキシメチルウラシル、５－カルボキシチオウラシル等を例示することができる。 The nucleic acid base derivative having an acyl group refers to a base component constituting a nucleotide and a derivative thereof, and preferably a pyrimidine derivative or the like, for example, 5-carboxymethyl uracil, 5-carboxythiouracil and the like can be exemplified.
　ペプチドのＮ末端に存在し得るスルホニル基としては、例えば、上記したアシル基におけるカルボニル構造をスルホン構造に変換した構造を有するものが例示できる。 Examples of the sulfonyl group that can exist at the N-terminal of the peptide include those having a structure obtained by converting the carbonyl structure of the above-mentioned acyl group into a sulfone structure.
　ペプチドのＮ末端に存在し得るポリエチレングリコール基は、エステル結合、アミン（－ＮＨ－）、アシル基（例えば、炭素数１～１２のアシル基）等や、これらの組み合わせを介して、ポリエチレングリコールまたはその類似体が連結された構造である。ポリエチレングリコール基の炭素数は、例えば２～２０（すなわち、－（Ｃ２Ｈ４Ｏ）ｎ－で表される構造単位を有し、ｎ＝１～１０である。）、好ましくは４～１６（すなわち、－（Ｃ２Ｈ４Ｏ）ｎ－で表される構造単位を有し、ｎ＝２～８である。）である。ポリエチレングリコール基におけるペプチドのＮ末端と連結している側とは反対側の末端は、炭素数１～６のアルキル基（例えば、メチル基、エチル基、プロピル基、イソプロピル基、シクロプロピル基、ブチル基、イソブチル基、ｓｅｃ－ブチル基、ｔｅｒｔ－ブチル基、アミル基、イソアミル基、ｔｅｒｔ－アミル基、ヘキシル基）のような、一般的に水酸基の保護に使用される保護基やアミノ基で修飾されていてもよい。 The polyethylene glycol group that may be present at the N-terminal of the peptide is polyethylene glycol or via an ester bond, an amine (-NH-), an acyl group (for example, an acyl group having 1 to 12 carbon atoms), or a combination thereof. It is a structure in which the analogs are linked. The number of carbon atoms of the polyethylene glycol group is, for example, 2 to 20 (that is, it has a structural unit represented by − (C 2 H 4 O) n −, and n = 1 to 10), preferably 4 to 16. (That is, it has a structural unit represented by − (C 2 H 4 O) n −, and n = 2 to 8). The terminal of the polyethylene glycol group opposite to the side connected to the N-terminal of the peptide is an alkyl group having 1 to 6 carbon atoms (for example, a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, or butyl). Modified with a protective group or amino group commonly used to protect hydroxyl groups, such as group, isobutyl group, sec-butyl group, tert-butyl group, amyl group, isoamyl group, tert-amyl group, hexyl group). It may have been done.
　本発明に係るペプチドのＣ末端の構造もまた特に制限されず、カルボン酸の保護に一般的に使用される保護基で修飾された構造であってもよい。より具体的には、本発明に係るペプチドのＣ末端の構造は、例えば、カルボキシル基（－ＣＯＯＨ）、カルボキシレート（－ＣＯＯ－）、アミド（－ＣＯＮＨ２）、アルキルアミド（－ＣＯＮＨＲ３１、－ＣＯＮＲ３１Ｒ３２）、エステル（－ＣＯＯＲ３１）、ピバロイルオキシメチル基のようなアシルオキシアルキル（－Ｒ３３－ＯＣＯＲ３１）、炭素数１～４のアルキル基もしくはアルコキシ基で置換されてもよいフタリジル基（例えば、フタリジル基、ジメチルフタリジル基、ジメトキシフタリジル基）、または（５－メチル－２－オキソ－１，３－ジオキソレン－４－イル）メチル基であり得る。このうち、ペプチドのＣ末端はアミドであることが好ましい。上記のアルキルアミド、エステル、およびアシルオキシアルキルにおけるＲ３１およびＲ３２は、それぞれ独立に、メチル基、エチル基、プロピル基、イソプロピル基、シクロプロピル基、ブチル基、イソブチル基、ｓｅｃ－ブチル基、ｔｅｒｔ－ブチル基、アミル基、イソアミル基、ｔｅｒｔ－アミル基、ヘキシル基、シクロヘキシル基等の炭素数１～６のアルキル基；フェニル基、ナフチル等の炭素数６～１０のアリール基；ベンジル基、フェネチル基、ベンズヒドリル基等の炭素数７～１８のアラルキル基；グルコース等の糖；炭素数１～６のアルキル基（例えば、メチル基、エチル基、プロピル基、イソプロピル基、シクロプロピル基、ブチル基、イソブチル基、ｓｅｃ－ブチル基、ｔｅｒｔ－ブチル基、アミル基、イソアミル基、ｔｅｒｔ－アミル基、ヘキシル基）で修飾されていてもよいポリエチレングリコール基などが挙げられる。アシルオキシアルキルにおけるＲ３３は、メチレン基、エチレン基、ｎ－プロピレン基、イソプロピレン基、ｎ－ブチレン基、イソブチレン基、ｓ－ブチレン基、ｔ－ブチレン基のような炭素数１～４のアルキレン基である。 The structure of the C-terminal of the peptide according to the present invention is also not particularly limited, and may be a structure modified with a protecting group generally used for protection of a carboxylic acid. More specifically, the structure of the C-terminus of the peptide according to the present invention, for example, a carboxyl group (-COOH), a carboxylate (-COO -), amide (-CONH 2), alkylamide (-CONHR 31, - It may be substituted with a CONR 31 R 32 ), an ester (-COOR 31 ), an acyloxyalkyl (-R 33- OCOR 31 ) such as a pivaloyloxymethyl group, an alkyl group having 1 to 4 carbon atoms or an alkoxy group. It can be a phthalidyl group (eg, a phthalidyl group, a dimethylphthalidyl group, a dimethoxyphthalidyl group), or a (5-methyl-2-oxo-1,3-dioxolen-4-yl) methyl group. Of these, the C-terminal of the peptide is preferably an amide. R 31 and R 32 in the above alkylamides, esters, and acyloxyalkyls are independently methyl group, ethyl group, propyl group, isopropyl group, cyclopropyl group, butyl group, isobutyl group, sec-butyl group, tert. -Alkyl group having 1 to 6 carbon atoms such as butyl group, amyl group, isoamyl group, tert-amyl group, hexyl group and cyclohexyl group; aryl group having 6 to 10 carbon atoms such as phenyl group and naphthyl; benzyl group and phenethyl An aralkyl group having 7 to 18 carbon atoms such as a group and a benzhydryl group; a sugar such as glucose; an alkyl group having 1 to 6 carbon atoms (for example, a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, etc. Examples thereof include polyethylene glycol groups that may be modified with an isobutyl group, sec-butyl group, tert-butyl group, amyl group, isoamyl group, tert-amyl group, hexyl group). R 33 in the acyloxyalkyl 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 and a t-butylene group. Is.
　本発明に係るペプチドには、ポリマー、脂質等との共有結合によって化学的に修飾されたペプチド誘導体や、ペプチド内に含まれるα－へリックス性をさらに強化した誘導体も包含される。α－へリックス性をさらに強化した誘導体としては、アミノ酸配置としてｉ，ｉ＋４位等に塩橋を形成した誘導体や、ジスルフィド結合、炭素－炭素結合などによる架橋構造を有する誘導体が例示できる。 The peptide according to the present invention also includes peptide derivatives chemically modified by covalent bonds with polymers, lipids and the like, and derivatives having further enhanced α-helix properties contained in the peptides. Examples of the derivative having further enhanced α-helix property include a derivative having a salt bridge formed at the i, i + 4-position and the like as an amino acid arrangement, and a derivative having a crosslinked structure by a disulfide bond, a carbon-carbon bond, and the like.
　本発明に係るペプチドのアミノ酸残基数は１５～１７である。アミノ酸残基数が１７以下であることにより、合成および生体内での利用率の両面から有利となる。アミノ酸残基数が１４未満であると、本発明の効果を発揮することができない。 The number of amino acid residues of the peptide according to the present invention is 15 to 17. When the number of amino acid residues is 17 or less, it is advantageous in terms of both synthesis and utilization in vivo. If the number of amino acid residues is less than 14, the effect of the present invention cannot be exerted.
　＜ペプチド＞
　本発明の一形態は、下記式（１）で表されるアミノ酸配列を含み、アミノ酸残基数が１５～１７である、ペプチド、もしくはその薬学的に許容される塩、またはそれらのプロドラッグである： <Peptide>
One embodiment of the present invention comprises 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. be:
Figure JPOXMLDOC01-appb-C000009
Figure JPOXMLDOC01-appb-C000009
　上記式（１）において、
　Ｘ０は、Ｄ－Ａｌａ、Ｄ－ＧｌｙおよびＤ－２－アミノイソ酪酸からなる群から選択されるアミノ酸残基または欠損であり；
　Ｘ１は、Ｄ－Ｌｅｕ、Ｄ－ノルロイシン、Ｄ－Ｖａｌ、Ｄ－Ｉｌｅ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基または欠損であり；
　Ｘ２は、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｌｙｓ、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ３は、Ｄ－２－シクロヘキシルグリシン、Ｄ－ノルロイシン、Ｄ－Ｌｅｕ、Ｄ－Ｖａｌ、Ｄ－Ｉｌｅ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基であり；
　Ｘ４は、Ｄ－Ｌｙｓ、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ５は、Ｄ－Ｓｅｒ、Ｄ－Ａｒｇ、Ｄ－２－ヒドロキシグリシン、Ｄ－ホモセリン、Ｄ－Ｌｙｓ、Ｄ－オルニチン、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ６は、Ｄ－Ｔｒｐ、Ｄ－３－（２－ナフチル）アラニン、Ｄ－Ｔｙｒ、Ｄ－ＰｈｅおよびＤ－３－（１－ナフチル）アラニンからなる群から選択されるアミノ酸残基であり；
　Ｘ７は、Ｄ－２－フェニルグリシン、Ｄ－ノルロイシン、Ｄ－Ｉｌｅ、Ｄ－Ｌｅｕ、Ｄ－Ｖａｌ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基であり；
　Ｘ８は、Ｄ－Ｇｌｎ、Ｄ－Ａｒｇ、Ｄ－Ａｓｎ、Ｄ－Ｌｙｓ、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸、Ｄ－２，４－ジアミノブタン酸およびＤ－オルニチンからなる群から選択されるアミノ酸残基であり；
　Ｘ９は、Ｄ－２－シクロヘキシルグリシン残基であり；
　Ｘ１０は、Ｄ－Ｌｙｓ、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ１１は、Ｄ－２－フェニルグリシン、Ｄ－Ｉｌｅ、Ｄ－Ｌｅｕ、Ｄ－ノルロイシン、Ｄ－Ｖａｌ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基であり；
　Ｘ１２は、Ｄ－Ｔｒｐ、Ｄ－ホモフェニルアラニン、Ｄ－ＴｙｒおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基であり；
　Ｘ１３は、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｌｙｓ、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ１４は、Ｄ－２－フェニルグリシン、Ｄ－Ｉｌｅ、Ｄ－Ｌｅｕ、Ｄ－ノルロイシン、Ｄ－Ｖａｌ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基であり；
　Ｘ１５は、Ｄ－Ｔｙｒ、Ｄ－ＴｒｐおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基であり；ならびに
　Ｘ１６は、Ｄ－Ｔｒｐ、Ｄ－ホモフェニルアラニン、Ｄ－ＴｙｒおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基または欠損である。 In the above formula (1)
X 0 is an amino acid residue or defect selected from the group consisting of D-Ala, D-Gly and D-2-aminoisobutyric acid;
X 1 is an amino acid residue or defect 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. can be;
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. Is the basis;
X 4 is, D-Lys, D-Arg , D- ornithine, D-His, with an amino acid residue selected from the group consisting of D-2,3-diaminopropionic acid and D-2,4-diaminobutane acid can be;
X 5 contains D-Ser, D-Arg, D-2-hydroxyglycine, D-homoserine, D-Lys, D-ornithine, D-His, D-2,3-diaminopropionic acid and D-2,4. -Amino acid residues selected from the group consisting of 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. Is the basis;
X 8 is, D-Gln, D-Arg , D-Asn, D-Lys, D-His, D-2,3- diaminopropionic acid, the group consisting of D-2,4-diaminobutanoic acid and D- ornithine Amino acid residues selected from;
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. can be;
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. Is the basis;
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. can be;
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. Is the basis;
X 15 is an amino acid residue selected from the group consisting of D-Tyr, D-Trp and D-Phe; and X 16 is from D-Trp, D-homophenylalanine, D-Tyr and D-Phe. Amino acid residues or defects selected from the group consisting of.
　本発明によれば、高いマイオスタチン阻害活性を有し、かつ改善した生体中での安定性を有するペプチドを提供することができる。 According to the present invention, it is possible to provide a peptide having high myostatin inhibitory activity and improved in vivo stability.
　より具体的には、本発明に係るペプチドは、高いマイオスタチン阻害活性を有し、加えてすべてＤ体のアミノ酸で構成されるため、生体内酵素に対する安定性を有しており、生体への投与時において持続的な高い効果を示すことができる。 More specifically, the peptide according to the present invention has high myostatin inhibitory activity, and in addition, since it is composed of all D-form amino acids, it has stability against in vivo enzymes and is effective for living organisms. It can show a long-lasting high effect at the time of administration.
　好ましい実施形態では、マイオスタチン阻害活性の観点から、上記式（１）において、
　Ｘ０は、Ｄ－Ａｌａ、Ｄ－ＧｌｙおよびＤ－２－アミノイソ酪酸からなる群から選択されるアミノ酸残基または欠損であり；
　Ｘ１は、Ｄ－Ｌｅｕ、Ｄ－ノルロイシン、Ｄ－Ｖａｌ、Ｄ－Ｉｌｅ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基または欠損であり；
　Ｘ２は、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｌｙｓ、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ３は、Ｄ－２－シクロヘキシルグリシン、Ｄ－ノルロイシン、Ｄ－Ｌｅｕ、Ｄ－Ｖａｌ、Ｄ－Ｉｌｅ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基であり；
　Ｘ４は、Ｄ－Ｌｙｓ、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ５は、Ｄ－Ｓｅｒ、Ｄ－Ａｒｇ、Ｄ－２－ヒドロキシグリシン、Ｄ－ホモセリン、Ｄ－Ｌｙｓ、Ｄ－オルニチン、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ６は、Ｄ－Ｔｒｐ、Ｄ－３－（２－ナフチル）アラニン、Ｄ－Ｔｙｒ、Ｄ－ＰｈｅおよびＤ－３－（１－ナフチル）アラニンからなる群から選択されるアミノ酸残基であり、より好ましくはＤ－Ｔｒｐ、Ｄ－ＴｙｒおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基であり；
　Ｘ７は、Ｄ－２－フェニルグリシン、Ｄ－ノルロイシン、Ｄ－Ｉｌｅ、Ｄ－Ｌｅｕ、Ｄ－Ｖａｌ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基であり；
　Ｘ８は、Ｄ－Ｇｌｎ、Ｄ－Ａｒｇ、Ｄ－Ａｓｎ、Ｄ－Ｌｙｓ、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸、Ｄ－２，４－ジアミノブタン酸およびＤ－オルニチンからなる群から選択されるアミノ酸残基であり；
　Ｘ９は、Ｄ－２－シクロヘキシルグリシン残基であり；
　Ｘ１０は、Ｄ－Ｌｙｓ、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ１１は、Ｄ－２－フェニルグリシン残基であり；
　Ｘ１２は、Ｄ－Ｔｒｐ、Ｄ－ホモフェニルアラニン、Ｄ－ＴｙｒおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基であり；
　Ｘ１３は、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｌｙｓ、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ１４は、Ｄ－２－フェニルグリシン残基であり；
　Ｘ１５は、Ｄ－Ｔｙｒ、Ｄ－ＴｒｐおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基であり；ならびに
　Ｘ１６は、Ｄ－Ｔｒｐ、Ｄ－ホモフェニルアラニン、Ｄ－ＴｙｒおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基または欠損であり、より好ましくはＤ－Ｔｒｐ、Ｄ－ＴｙｒおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基または欠損である。 In a preferred embodiment, in the above formula (1), from the viewpoint of myostatin inhibitory activity,
X 0 is an amino acid residue or defect selected from the group consisting of D-Ala, D-Gly and D-2-aminoisobutyric acid;
X 1 is an amino acid residue or defect 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. can be;
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. Is the basis;
X 4 is, D-Lys, D-Arg , D- ornithine, D-His, with an amino acid residue selected from the group consisting of D-2,3-diaminopropionic acid and D-2,4-diaminobutane acid can be;
X 5 contains D-Ser, D-Arg, D-2-hydroxyglycine, D-homoserine, D-Lys, D-ornithine, D-His, D-2,3-diaminopropionic acid and D-2,4. -Amino acid residues selected from the group consisting of 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, it is 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. Is the basis;
X 8 is, D-Gln, D-Arg , D-Asn, D-Lys, D-His, D-2,3- diaminopropionic acid, the group consisting of D-2,4-diaminobutanoic acid and D- ornithine Amino acid residues selected from;
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. can be;
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. can be;
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 from D-Trp, D-homophenylalanine, D-Tyr and D-Phe. Amino acid residues or deficiencies selected from the group consisting of, more preferably amino acid residues or deficiencies selected from the group consisting of D-Trp, D-Tyr and D-Phe.
　好ましい実施形態では、マイオスタチン阻害活性の観点から、上記式（１）において、
　Ｘ０は、Ｄ－Ａｌａ、Ｄ－ＧｌｙおよびＤ－２－アミノイソ酪酸からなる群から選択されるアミノ酸残基または欠損であり；
　Ｘ１は、Ｄ－Ｌｅｕ、Ｄ－ノルロイシン、Ｄ－Ｖａｌ、Ｄ－Ｉｌｅ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基または欠損であり；
　Ｘ２は、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｌｙｓ、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ３は、Ｄ－２－シクロヘキシルグリシン残基であり；
　Ｘ４は、Ｄ－Ｌｙｓ、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ５は、Ｄ－Ｓｅｒ、Ｄ－Ａｒｇ、Ｄ－２－ヒドロキシグリシン、Ｄ－ホモセリン、Ｄ－Ｌｙｓ、Ｄ－オルニチン、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ６は、Ｄ－Ｔｒｐ、Ｄ－３－（２－ナフチル）アラニン、Ｄ－Ｔｙｒ、Ｄ－ＰｈｅおよびＤ－３－（１－ナフチル）アラニンからなる群から選択されるアミノ酸残基であり；
　Ｘ７は、Ｄ－Ｉｌｅ、Ｄ－Ｌｅｕ、Ｄ－ノルロイシン、Ｄ－Ｖａｌ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基であり；
　Ｘ８は、Ｄ－Ｇｌｎ、Ｄ－Ａｒｇ、Ｄ－Ａｓｎ、Ｄ－Ｌｙｓ、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸、Ｄ－２，４－ジアミノブタン酸およびＤ－オルニチンからなる群から選択されるアミノ酸残基であり；
　Ｘ９は、Ｄ－２－シクロヘキシルグリシン残基であり；
　Ｘ１０は、Ｄ－Ｌｙｓ、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ１１は、Ｄ－Ｉｌｅ、Ｄ－Ｌｅｕ、Ｄ－ノルロイシン、Ｄ－Ｖａｌ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基であり；
　Ｘ１２は、Ｄ－Ｔｒｐ、Ｄ－ホモフェニルアラニン、Ｄ－ＴｙｒおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基であり；
　Ｘ１３は、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｌｙｓ、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
　Ｘ１４は、Ｄ－Ｉｌｅ、Ｄ－Ｌｅｕ、Ｄ－ノルロイシン、Ｄ－Ｖａｌ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基であり；
　Ｘ１５は、Ｄ－Ｔｙｒ、Ｄ－ＴｒｐおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基であり；ならびに
　Ｘ１６は、Ｄ－Ｔｒｐ、Ｄ－ホモフェニルアラニン、Ｄ－ＴｙｒおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基または欠損であり、より好ましくはＤ－Ｔｒｐ、Ｄ－ホモフェニルアラニン、Ｄ－ＴｙｒおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基である。 In a preferred embodiment, in the above formula (1), from the viewpoint of myostatin inhibitory activity,
X 0 is an amino acid residue or defect selected from the group consisting of D-Ala, D-Gly and D-2-aminoisobutyric acid;
X 1 is an amino acid residue or defect 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. can be;
X 3 is a D-2-cyclohexylglycine residue;
X 4 is, D-Lys, D-Arg , D- ornithine, D-His, with an amino acid residue selected from the group consisting of D-2,3-diaminopropionic acid and D-2,4-diaminobutane acid can be;
X 5 contains D-Ser, D-Arg, D-2-hydroxyglycine, D-homoserine, D-Lys, D-ornithine, D-His, D-2,3-diaminopropionic acid and D-2,4. -Amino acid residues selected from the group consisting of 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, D-Gln, D-Arg , D-Asn, D-Lys, D-His, D-2,3- diaminopropionic acid, the group consisting of D-2,4-diaminobutanoic acid and D- ornithine Amino acid residues selected from;
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. can be;
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 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. can be;
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 from D-Trp, D-homophenylalanine, D-Tyr and D-Phe. Amino acid residues or defects selected from the group consisting of, more preferably amino acid residues selected from the group consisting of D-Trp, D-homophenylalanine, D-Tyr and D-Phe.
　さらに好ましい実施形態では、マイオスタチン阻害活性の観点から、上記式（１）において、
　Ｘ０は、Ｄ－Ａｌａ残基または欠損であり；
　Ｘ１は、Ｄ－Ｌｅｕ残基、Ｄ－ノルロイシン残基または欠損であり；
　Ｘ２は、Ｄ－Ａｒｇ残基またはＤ－オルニチン残基であり；
　Ｘ３は、Ｄ－２－シクロヘキシルグリシン残基またはＤ－ノルロイシン残基であり；
　Ｘ４は、Ｄ－Ｌｙｓ残基、Ｄ－Ａｒｇ残基またはＤ－オルニチン残基であり；
　Ｘ５は、Ｄ－Ｓｅｒ残基またはＤ－Ａｒｇ残基であり；
　Ｘ６は、Ｄ－Ｔｒｐ残基またはＤ－３－（２－ナフチル）アラニン残基であり；
　Ｘ７は、Ｄ－２－フェニルグリシン残基またはＤ－Ｉｌｅ残基であり；
　Ｘ８は、Ｄ－Ｇｌｎ残基またはＤ－Ａｒｇ残基であり；
　Ｘ９は、Ｄ－２－シクロヘキシルグリシン残基であり；
　Ｘ１０は、Ｄ－Ｌｙｓ、Ｄ－ＡｒｇおよびＤ－オルニチンからなる群から選択されるアミノ酸残基であり；
　Ｘ１１は、Ｄ－２－フェニルグリシン残基またはＤ－Ｉｌｅ残基であり；
　Ｘ１２は、Ｄ－Ｔｒｐ残基またはＤ－ホモフェニルアラニン残基であり；
　Ｘ１３は、Ｄ－Ａｒｇ残基またはＤ－オルニチン残基であり；
　Ｘ１４は、Ｄ－２－フェニルグリシン残基またはＤ－Ｉｌｅ残基であり；
　Ｘ１５は、Ｄ－Ｔｙｒ残基であり；ならびに
　Ｘ１６は、Ｄ－Ｔｒｐ残基、Ｄ－ホモフェニルアラニン残基または欠損である。 In a more preferred embodiment, in the above formula (1), from the viewpoint of myostatin inhibitory activity,
X 0 is a D-Ala residue or defect;
X 1 is a D-Leu residue, a D-norleucine residue or a defect;
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, D-Lys residues, be a D-Arg residue, or D- ornithine residues;
X 5 is a D-Ser residue or a 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 an D-Gln residue or D-Arg residues;
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 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, D-homophenylalanine residue or defect.
　本発明に係るペプチドにおいて、マイオスタチン阻害活性の観点から、上記式（１）で表されるペプチドは、好ましくは配列番号１～２５で表されるアミノ酸配列のいずれか１つを含む。 In the peptide according to the present invention, from the viewpoint of myostatin inhibitory activity, the peptide represented by the above formula (1) preferably contains any one of the amino acid sequences represented by SEQ ID NOs: 1 to 25.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
　本発明に係るペプチドにおいて、マイオスタチン阻害活性の観点から、上記式（１）で表されるペプチドは、さらに好ましくは配列番号１～４、６、８～２３および２５で表されるアミノ酸配列のいずれか１つを含み、特に好ましくは配列番号１９～２３および２５で表されるアミノ酸配列のいずれか１つを含む。 In the peptide according to the present invention, from the viewpoint of myostatin inhibitory activity, the peptide represented by the above formula (1) is more preferably the amino acid sequence represented by SEQ ID NOs: 1 to 4, 6, 8 to 23 and 25. It comprises any one, and particularly preferably any one of the amino acid sequences represented by SEQ ID NOs: 19-23 and 25.
　（ペプチドの製造方法）
　本発明に係るペプチドは、化学的合成法や組換え技術を含む従来公知の手法によって製造することができる。ペプチドを化学合成により調製するには、各アミノ酸をペプチド化学において通常用いられる方法、例えば、「ザ　ペプチド（Ｔｈｅ　Ｐｅｐｔｉｄｅｓ）」第１巻〔Ｓｃｈｒｏｄｅｒ　ａｎｄ　Ｌｕｈｋｅ著，　Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓ，　Ｎｅｗ　Ｙｏｒｋ，　Ｕ．Ｓ．Ａ．（１９６６年）〕、「ペプチド合成の基礎と実験」（泉屋信夫ら著丸善株式会社、１９８５年）等に記載されている方法によって製造することが可能であり、液相法および固相法のいずれによっても製造できる。さらに、カラム法、バッチ法のいずれの方法も用いることができる。 (Peptide production method)
The peptide according to the present invention can be produced by a conventionally known method including a chemical synthesis method and a recombination technique. To prepare peptides by chemical synthesis, each amino acid is commonly used in peptide chemistry, eg, "The Peptides," Volume 1 [Schroder and Luhke, Academic Press, New York, U.S.A. S. A. (1966)], "Basics and Experiments of Peptide Synthesis" (Nobuo Izumiya et al., Maruzen Co., Ltd., 1985), etc., can be produced by the methods described in the liquid phase method and the solid phase method. It can be manufactured by either. Further, either the column method or the batch method can be used.
　本発明に係るペプチドはまた、例えば下記のＣｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ、Ｃｈａｐｔｅｒ　１６に記載されるような手法により、動物細胞、昆虫細胞、または微生物等を利用した組み換え技術により製造してもよい。ペプチドは、培養細胞や微生物によって生成された後、従来公知の方法によって精製し得る。ペプチドの精製および単離法は当分野の技術者に公知であり、例えばＣｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ、Ｃｈａｐｔｅｒ　１６（Ａｕｓｕｂｅｌら、Ｊｏｈｎ　Ｗｉｌｅｙ　ａｎｄ　Ｓｏｎｓ、２００６年）等に記載の手法により行うことができる。 The peptide according to the present invention may also be produced by a recombination technique using animal cells, insect cells, microorganisms or the like by a method as described in, for example, Current Protocols in Molecular Biology, Chapter 16 below. The peptide can be purified by a conventionally known method after being produced by cultured cells or microorganisms. Methods for purifying and isolating peptides are known to engineers in the art, and can be carried out by the methods described in, for example, Current Protocols in Molecular Biology, Chapter 16 (Ausube et al., John Wiley and Sons, 2006).
　ペプチド結合を形成するための縮合方法として、アジド法、酸ハライド法、酸無水物法、カルボジイミド法、カルボジイミド－アディティブ法、活性エステル法、カルボニルイミダゾール法、酸化還元法、酵素法、ウッドワード試薬Ｋ、ＨＡＴＵ試薬、Ｂｏｐ試薬を用いる方法等を例示することができる。なお、固相法での縮合反応は上記した方法のうち、酸無水物法、カルボジイミド法、および活性エステル法が主な方法として挙げられる。 Condensation methods for forming peptide bonds include azide method, acid halide method, acid anhydride method, carbodiimide method, carbodiimide-additive method, active ester method, carbonyl imidazole method, oxidation-reduction method, enzyme method, Woodward reagent K. , HATU reagent, method using Bop reagent and the like can be exemplified. Among the above-mentioned methods, the acid anhydride method, the carbodiimide method, and the active ester method are mainly mentioned as the condensation reaction in the solid phase method.
　さらに、固相法でペプチド鎖を延長するときは、用いる有機溶媒に対して不溶な樹脂等の支持体に、Ｃ末端アミノ酸を結合する。かような樹脂としては、アミノ酸を樹脂に結合させる目的で官能基を導入した樹脂や、樹脂と官能基の間にスペーサーを挿入したもの等を目的に応じて用いることもできる。より具体的には、例えば、クロロメチル樹脂などのハロメチル樹脂、オキシメチル樹脂、４－（オキシメチル）－フェニルアセトアミドメチル樹脂、４－（オキシメチル）－フェノキシメチル樹脂、Ｒｉｎｋアミド樹脂などを挙げることができる。なお、これらの縮合反応を行う前に、通常公知の手段によって当該縮合反応に関与しないカルボキシル基やアミノ基や水酸基やアミジノ基等の保護手段を施すことができる。また逆に当該縮合反応に直接関与するカルボキシル基やアミノ基を活性化することもできる。 Furthermore, when extending the peptide chain by 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. As such a resin, a resin having a functional group introduced for the purpose of binding an amino acid to the resin, a resin having a spacer inserted between the resin and the functional group, and the like can also be used depending on the purpose. More specifically, for example, halomethyl resin such as chloromethyl resin, oxymethyl resin, 4- (oxymethyl) -phenylacetamidemethyl resin, 4- (oxymethyl) -phenoxymethyl resin, Rinkamide resin and the like can be mentioned. Can be done. Before performing these condensation reactions, protective means such as a carboxyl group, an amino group, a hydroxyl group, and an amidino group that are not involved in the condensation reaction can be applied by a commonly known means. On the contrary, it is also possible to activate the carboxyl group or amino group directly involved in the condensation reaction.
　各ユニットの縮合反応に関与しない官能基の保護手段に用いる保護基としては有機化学において通常用いられている保護基、例えば、「Ｐｒｏｔｅｃｔｉｖｅ　Ｇｒｏｕｐｓ　ｉｎ　Ｏｒｇａｎｉｃ　Ｓｙｎｔｈｅｓｉｓ（Ｇｒｅｅｎｅ著、Ｊｏｈｎ　Ｗｉｌｅｙ　＆　Ｓｏｎｓ，　Ｉｎｃ．（１９８１年））等に記載されている保護基によって保護することが可能である。より具体的には、カルボキシル基の保護基としては、例えば、各種のメチルエステル、エチルエステル、ベンジルエステル、ｐ－ニトロベンジルエステル、ｔ－ブチルエステル、シクロヘキシルエステル等の通常公知の保護基を挙げることができる。アミノ基の保護基としては、例えば、ベンジルオキシカルボニル基、ｔ－ブトキシカルボニル基、イソボルニルオキシカルボニル基、９－フルオレニルメトキシカルボニル基（Ｆｍｏｃ基）等を挙げることができる。 Protecting groups commonly used in organic chemistry as protecting groups for functional groups that are not involved in the condensation reaction of each unit include, for example, "Protective Groups in Organic Synthesis (by Greene, John Wiley & Sons, Inc.). It can be protected by the protecting group described in 1981)) and the like. More specifically, as the protecting group for the carboxyl group, for example, various methyl esters, ethyl esters, benzyl esters, p- Examples of commonly known protecting groups such as nitrobenzyl ester, t-butyl ester and cyclohexyl ester can be mentioned. Examples of the protecting group for the amino group include a benzyloxycarbonyl group, a t-butoxycarbonyl group and an isobornyloxycarbonyl. Groups, 9-fluorenylmethoxycarbonyl group (Fmoc group) and the like can be mentioned.
　カルボキシル基の活性化されたものとしては、例えば、当該カルボキシル基に対応する酸無水物；アジド；ペンタフルオロフェノール、２，４－ジニトロフェノール、シアノメチルアルコール、ｐ－ニトロフェノール、Ｎ－ヒドロキシコハク酸イミド、Ｎ－ヒドロキシ－５－ノルボルネン－２，３－ジカルボキシミド、Ｎ－ヒドロキシフタルイミド、１－ヒドロキシベンゾトリアゾール等との活性エステル等が挙げられる。アミノ基の活性化されたものとしては、当該アミノ基に対応する燐酸アミド等を挙げることができる。 Examples of the activated carboxyl group include acid anhydrides corresponding to the carboxyl group; azide; pentafluorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, and N-hydroxysuccinic acid. Examples thereof include active esters with imide, N-hydroxy-5-norbornen-2,3-dicarboxymid, N-hydroxyphthalimide, 1-hydroxybenzotriazole and the like. Examples of the activated amino group include phosphoric acid amides corresponding to the amino group.
　ペプチド合成の際の縮合反応は、通常溶媒中で行われる。当該溶媒としては、例えば、クロロホルム、ジクロロメタン、酢酸エチル、Ｎ，Ｎ－ジメチルホルムアミド、ジメチルスルホキシド、ピリジン、ジオキサン、テトラヒドロフラン、Ｎ－メチルピロリドン、水、メタノール等、または、これらの混合物を挙げることができる。また、当該縮合反応の反応温度は、通常の場合と同様に、－３０℃～５０℃の範囲で行なうことができる。 The condensation reaction during peptide synthesis is usually carried out in a solvent. Examples of the solvent include chloroform, dichloromethane, ethyl acetate, N, N-dimethylformamide, dimethyl sulfoxide, pyridine, dioxane, tetrahydrofuran, N-methylpyrrolidone, water, methanol and the like, or a mixture thereof. .. Further, the reaction temperature of the condensation reaction can be carried out in the range of −30 ° C. to 50 ° C., as in the usual case.
　さらに、本発明のペプチドの製造工程における保護基の脱離反応の種類は、ペプチド結合に影響を与えずに保護基を脱離させることができる限りにおいて、用いる保護基の種類に応じて選択することができる。例えば、塩化水素、臭化水素、無水フッ化水素、メタンスルホン酸、トリフルオロメタンスルホン酸、トリフルオロ酢酸、またはこれらの混合物等による酸処理、水酸化ナトリウム、水酸化カリウム、ヒドラジン、ジエチルアミン、ピペリジン等によるアルカリ処理、液体アンモニア中におけるナトリウム処理やパラジウム炭素による還元および、トリメチルシリルトリフラート、トリメチルシリルブロマイド等のシリル化処理等が挙げられる。なお、上記の酸またはシリル化剤処理による脱保護基反応においては、アニソール、フェノール、クレゾール、チオアニソール、エタンジチオール等のカチオン捕捉剤を添加するのが、脱保護基反応が効率的に行われるという観点から好ましい。 Further, the type of the protecting group elimination reaction in the peptide production process of the present invention is selected according to the type of protecting group to be used as long as the protecting group can be eliminated without affecting the peptide bond. be able to. For example, acid treatment with hydrogen chloride, hydrogen bromide, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof, sodium hydroxide, potassium hydroxide, hydrazine, diethylamine, piperidine, etc. Examples thereof include alkali treatment with, sodium treatment in liquid ammonia, reduction with palladium carbon, and silylation treatment with trimethylsilyl triflate, trimethylsilyl bromide and the like. In the deprotecting group reaction by the above acid or silylating agent treatment, the deprotecting group reaction is efficiently carried out by adding a cation scavenger such as anisole, phenol, cresol, thioanisole and ethanedithiol. It is preferable from the viewpoint.
　なお、固相法で合成した本発明のペプチドの固相からの切断方法も通常公知の方法に従う。例えば、上記の酸またはシリル化剤による処理等を当該切断方法として挙げることができる。このようにして製造された本発明のペプチドに対しては、上記の一連の反応の終了後に通常公知の分離、精製手段を駆使することができる。例えば、抽出、分配、再沈澱、再結晶、固相抽出、カラムクロマトグラフィー等によって、より高純度で本発明のペプチドを収得することができる。 The method for cleaving the peptide of the present invention synthesized by the solid phase method from the solid phase also follows a generally known method. For example, the above-mentioned treatment with an acid or a silylating agent can be mentioned as the cutting method. For the peptide of the present invention produced in this manner, generally known separation and purification means can be fully utilized after the completion of the above series of reactions. For example, the peptide of the present invention can be obtained with higher purity by extraction, partitioning, reprecipitation, recrystallization, solid phase extraction, column chromatography and the like.
　得られたペプチドは、アミノ酸自動分析機、キャピラリー電気泳動、逆相高速液体クロマトグラフィー、質量分析法等によって解析できる。また、ファージディスプレイ法、ツーハイブリッド法、アフィニティークロマトグラフィー、表面プラズモン共鳴法、共免疫沈降法、プロテインチップ法、立体構造解析、ファーウェスタン法、蛍光消光法等の各種生体分子相互作用解析手法を利用し、マイオスタチンとの相互作用を指標としてペプチドを選別してもよい。 The obtained peptide can be analyzed by an automatic amino acid analyzer, capillary electrophoresis, reverse phase high performance liquid chromatography, mass spectrometry, etc. In addition, various biomolecular interaction analysis methods such as phage display method, two-hybrid method, affinity chromatography, surface plasmon resonance method, co-immunoprecipitation method, protein chip method, three-dimensional structure analysis, far western method, and fluorescence quenching method are used. However, peptides may be selected using the interaction with myostatin as an index.
　本発明に係るペプチドは、単離または精製されていてもよい。「単離または精製」とは、目的とする成分以外の成分を除去する操作が施されていることを意味する。単離または精製された本発明に係るペプチドの純度は、通常５０％以上（例えば、７０％以上、８０％以上、９０％以上、９５％以上、９８％以上、９９％以上、１００％）である。 The peptide according to the present invention may be isolated or purified. "Isolation or purification" means that an operation has been performed to remove components other than the target component. The purity of the isolated or purified peptide according to the present invention is usually 50% or more (for example, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, 100%). be.
　＜複合体＞
　本発明の一形態は、下記式（２）で表される複合体またはその薬学的に許容される塩である。 <Complex>
One form of the present invention is a complex represented by the following formula (2) or a pharmaceutically acceptable salt thereof.
Figure JPOXMLDOC01-appb-C000011
Figure JPOXMLDOC01-appb-C000011
　式（２）中、
　Ｙは、下記式（３）： In equation (2),
Y is the following formula (3):
Figure JPOXMLDOC01-appb-C000012
Figure JPOXMLDOC01-appb-C000012
［式３中、
　Ｒ１およびＲ２は、それぞれ独立して、ハロゲノアルキル基またはハロゲン原子を表し、
　Ｒ３は、臭素原子、ヨウ素原子またはセレン原子を表し、
　Ｒ４およびＲ５は、それぞれ独立して、水素原子または置換もしくは非置換のアルキル基を表し、
　Ｒ６およびＲ７は、それぞれ独立して、水素原子、ハロゲン原子、アルコキシ基または置換もしくは非置換のアルキル基を表し、この際Ｒ４およびＲ６またはＲ５およびＲ７が一緒になって、置換または非置換のアルキレン基またはアルケニレン基を形成してもよく、
　Ｒ８は、水素原子または置換もしくは非置換のアルキル基を表し、
　Ｒ９およびＲ１０は、それぞれ独立して、水素原子、ハロゲン原子、アルコキシ基または置換もしくは非置換のアルキル基を表し、この際Ｒ８とＲ９またはＲ１０とが一緒になって、置換または非置換のアルキレン基またはアルケニレン基を形成してもよく、
　ｍおよびｎは、それぞれ独立して、１～３の整数を表し、
　＊は、Ｌとの結合部位である］
で表される化合物であり；
　Ｌは、ＹおよびＺ間のリンカーを表し；
　Ｚは、上記本発明に係るペプチドである。 [In Equation 3,
R 1 and R 2 independently represent a halogenoalkyl group or a halogen atom, respectively.
R 3 represents a bromine atom, an iodine atom or a selenium atom.
R 4 and R 5 each independently represent a hydrogen atom or a substituted or unsubstituted alkyl group.
R 6 and R 7 each independently represent a hydrogen atom, a halogen atom, an alkoxy group or a substituted or unsubstituted alkyl group, wherein R 4 and R 6 or R 5 and R 7 are combined. Substituted or unsubstituted alkylene groups or alkenylene groups may be formed.
R 8 represents a hydrogen atom or a substituted or unsubstituted alkyl group.
R 9 and R 10 each independently represent a hydrogen atom, a halogen atom, an alkoxy group or a substituted or unsubstituted alkyl group, wherein R 8 and R 9 or R 10 are substituted or substituted together. It may form an unsubstituted alkylene group or an alkenylene group.
m and n independently represent integers of 1 to 3, respectively.
* Is the binding site with L]
It is a compound represented by;
L represents a linker between Y and Z;
Z is the peptide according to the present invention.
　本明細書において、「式（２）で表される複合体またはその薬学的に許容される塩」を、単に「本発明に係る複合体」とも称する。 In the present specification, the "complex represented by the formula (2) or a pharmaceutically acceptable salt thereof" is also simply referred to as "complex according to the present invention".
　以下、本発明に係る複合体を構成する要素について説明する。ただし、本発明に係るペプチドＺについては、上記と同様であるため、ここでは説明を省略する。 Hereinafter, the elements constituting the complex according to the present invention will be described. However, since the peptide Z according to the present invention is the same as above, the description thereof is omitted here.
　（式（３）で表される化合物Ｙ）
　上記式（２）において、Ｙは、上記式（３）で表される化合物である。式（３）で表される化合物は、ターゲット選択的光酸素化を可能とするｏｎ／ｏｆｆスイッチ触媒である。式（３）で表される化合物と本発明に係るペプチドとを連結して複合体を形成することにより、当該複合体は、波長６５０～８００ｎｍの光により活性化し、マイオスタチンを選択的に酸素化することで、さらに高いマイオスタチン阻害活性を発揮することができる。 (Compound Y represented by the formula (3))
In the above formula (2), Y is a compound represented by the above formula (3). The compound represented by the formula (3) is an on / off switch catalyst that enables target-selective photooxygenation. By linking the compound represented by the formula (3) and the peptide according to the present invention to form a complex, the complex is activated by light having a wavelength of 650 to 800 nm, and myostatin is selectively oxygenated. By making it, it is possible to exert even higher myostatin inhibitory activity.
　式（３）において、Ｒ１およびＲ２は、それぞれ独立して、ハロゲノアルキル基またはハロゲン原子を表す。 In formula (3), R 1 and R 2 independently represent a halogenoalkyl group or a halogen atom, respectively.
　ハロゲノアルキル基は、好ましくは直鎖または分岐鎖の炭素数１～６のハロゲノアルキル基であり、より好ましくは直鎖または分岐鎖の炭素数１～４のハロゲノアルキル基であり、さらに好ましくはトリフルオロメチル基またはペンタフルオロエチル基であり、特に好ましくはトリフルオロメチル基である。 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, and even more preferably a tri. It is a fluoromethyl group or a pentafluoroethyl group, and particularly preferably a trifluoromethyl group.
　ハロゲン原子としては、フッ素原子、塩素原子、臭素原子またはヨウ素原子が挙げられ、好ましくはフッ素原子である。 Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, and a fluorine atom is preferable.
　式（３）において、好ましくはＲ１がハロゲン原子であり、前記Ｒ２がハロゲノアルキル基である。 In the formula (3), R 1 is preferably a halogen atom and R 2 is a halogenoalkyl group.
　式（３）において、Ｒ３は、臭素原子、ヨウ素原子またはセレン原子を表し、好ましくは臭素原子である。 In formula (3), R 3 represents a bromine atom, an iodine atom or a selenium atom, and is preferably a bromine atom.
　式（３）において、Ｒ４およびＲ５は、それぞれ独立して、水素原子または置換もしくは非置換のアルキル基を表す。Ｒ６およびＲ７は、それぞれ独立して、水素原子、ハロゲン原子、アルコキシ基または置換もしくは非置換のアルキル基を表す。この際Ｒ４およびＲ６またはＲ５およびＲ７が一緒になって、置換または非置換のアルキレン基またはアルケニレン基を形成してもよい。 In formula (3), R 4 and R 5 each independently represent a hydrogen atom or a substituted or unsubstituted alkyl group. R 6 and R 7 each independently represent a hydrogen atom, a halogen atom, an alkoxy group or a substituted or unsubstituted alkyl group. At this time, R 4 and R 6 or R 5 and R 7 may be combined to form a substituted or unsubstituted alkylene group or alkenylene group.
　式（３）において、Ｒ８は、水素原子または置換もしくは非置換のアルキル基を表す。Ｒ９およびＲ１０は、それぞれ独立して、水素原子、ハロゲン原子、アルコキシ基または置換もしくは非置換のアルキル基を表す。この際Ｒ８とＲ９またはＲ１０とが一緒になって、置換または非置換のアルキレン基またはアルケニレン基を形成してもよい。 In formula (3), R 8 represents a hydrogen atom or a substituted or unsubstituted alkyl group. R 9 and R 10 each independently represent a hydrogen atom, a halogen atom, an alkoxy group or a substituted or unsubstituted alkyl group. At this time, R 8 and R 9 or R 10 may be combined to form a substituted or unsubstituted alkylene group or alkenylene group.
　式（３）において、好ましくはＲ４およびＲ６、Ｒ５およびＲ７ならびにＲ８およびＲ１０が一緒になって、置換または非置換のアルキレン基またはアルケニレン基を形成し、この際前記アルキレン基またはアルケニレン基の炭素数が２または３である。 In the formula (3), preferably together R 4 and R 6, R 5 and R 7 and R 8 and R 10, form a substituted or unsubstituted alkylene or alkenylene group, this time the alkylene group Alternatively, the alkaneylene group has 2 or 3 carbon atoms.
　Ｒ４～Ｒ１０におけるアルキル基としては、好ましくは直鎖または分岐鎖の炭素数１～６のアルキル基であり、より好ましくは直鎖または分岐鎖の炭素数１～４のアルキル基である。アルキル基の例としては、メチル基、エチル基、ｎ－プロピル基、イソプロピル基、ｎ－ブチル基、イソブチル基、ｔｅｒｔ－ブチル基などが挙げられる。これらのアルキル基を置換し得る基としては、好ましくはカルボキシ基、スルホン酸基、ヒドロキシ基、アミノ基、－ＣＯ－、－ＣＯＮＨ－およびトリアゾール基から選択される１～３個の基である。アルキル基を置換し得る基は、水溶性を高めるとの観点から、より好ましくはカルボキシ基、スルホン酸基、ヒドロキシ基およびアミノ基から選択される。 The alkyl group in R 4 to R 10 is preferably an alkyl group having 1 to 6 carbon atoms in a straight chain or a branched chain, and more preferably an alkyl group having 1 to 4 carbon atoms in a straight chain or a branched chain. 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, a tert-butyl group and the like. The group capable of substituting these alkyl groups is 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. The group capable of substituting the alkyl group is more preferably selected from a carboxy group, a sulfonic acid group, a hydroxy group and an amino group from the viewpoint of increasing water solubility.
　Ｒ６、Ｒ７、Ｒ９およびＲ１０におけるアルコキシ基としては、好ましくは直鎖または分岐鎖の炭素数１～６のアルコキシ基であり、より直鎖または分岐鎖の炭素数１～４のアルコキシ基である。具体的には、メトキシ基、エトキシ基、プロピルオキシ基などが挙げられる。また、Ｒ６、Ｒ７、Ｒ９およびＲ１０におけるハロゲン原子としては、塩素原子、臭素原子、ヨウ素原子、フッ素原子などが挙げられる。 The alkoxy group in R 6 , R 7 , R 9 and R 10 is preferably an alkoxy group having 1 to 6 carbon atoms in a straight chain or a branched chain, and an alkoxy group having 1 to 4 carbon atoms in a more linear or branched chain. It is the basis. Specific examples thereof include a methoxy group, an ethoxy group, and a propyloxy group. Examples of the halogen atom in R 6 , R 7 , R 9 and R 10 include a chlorine atom, a bromine atom, an iodine atom and a fluorine atom.
　好ましい実施形態では、Ｒ４およびＲ６、Ｒ５およびＲ７ならびにＲ８およびＲ１０が一緒になって、置換または非置換のアルキレン基またはアルケニレン基を形成し、この際前記アルキレン基またはアルケニレン基の炭素数が２または３である。アルキレン基としては、エチレン基、トリメチレン基などが挙げられる。アルケニレン基としては、ビニレン基、プロペニレン基などが挙げられる。 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 said 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.
　これらの基が一緒になって形成する環構造としては、以下の構造が挙げられる。 Examples of the ring structure formed by these groups together include the following structures.
Figure JPOXMLDOC01-appb-C000013
Figure JPOXMLDOC01-appb-C000013
　上記ａ－１～ａ－９およびｂ－１～ｂ－８の構造中、Ｒ４～Ｒ７およびＲ９～Ｒ１０は、アルキレン基またはアルケニレン基を形成しない基を示す。 Structure of the a-1 ~ a-9 and b-1 ~ b-8, R 4 ~ R 7 and R 9 ~ R 10 represents a group that does not form an alkylene group or alkenylene group.
　式（３）において、ｍおよびｎは、それぞれ独立して、１～３の整数を表す。ｍおよびｎは、それぞれ独立して、好ましくは１または２であり、より好ましくは１である。 In equation (3), m and n each independently represent an integer of 1 to 3. m and n are independently, preferably 1 or 2, and more preferably 1.
　式（３）において、＊は、Ｌとの結合部位である。 In formula (3), * is the binding site with L.
　式（３）で表される化合物が不斉炭素原子を有する場合、光学異性体が存在するが、式（３）で表される化合物は、光学異性体およびラセミ体のいずれも含む。 When the compound represented by the formula (3) has an asymmetric carbon atom, an optical isomer exists, but the compound represented by the formula (3) includes both an optical isomer and a racemate.
　（リンカーＬ）
　式（２）において、Ｌは、式（３）で表される化合物Ｙおよび本発明に係るペプチドＺ間のリンカーを表す。 (Linker L)
In formula (2), L represents a linker between compound Y represented by formula (3) and peptide Z according to the present invention.
　本発明に係るペプチドＺへのリンカーＬの結合部位は、特に制限されず、本発明に係るペプチドＺのＮ末端またはＣ末端でもよく、本発明の効果を奏する限り、ペプチドＺを構成するアミノ酸残基の側鎖でもよい。好ましい実施形態では、リンカーＬは、本発明に係るペプチドＺのＮ末端に結合している。 The binding site of the linker L to the peptide Z according to the present invention is not particularly limited and may be the N-terminal or the C-terminal of the peptide Z according to the present invention. It may be a side chain of the group. In a preferred embodiment, the linker L is attached to the N-terminus of peptide Z according to the present invention.
　このようなリンカーユニットの具体的な構造について特に制限はないが、例えば、置換または非置換の炭素数１～２０のアルキレン基、置換または非置換の炭素数２～２０のアルケニレン基、置換または非置換の炭素数２～２０のアルキニレン基、置換または非置換の炭素数３～２０のシクロアルキレン基、置換または非置換の炭素数３～２０のシクロアルケニレン基、置換または非置換の炭素数６～２０のアリーレン基、置換または非置換の炭素数３～２０のヘテロアリーレン基、－ＮＨ－、－Ｏ－、－Ｓ－、－Ｃ（＝Ｏ）－ＮＨ－、－ＮＨ－Ｃ（＝Ｏ）－、－Ｏ－、－Ｃ（＝Ｏ）－Ｏ－、－Ｏ－Ｃ（＝Ｏ）－、－Ｓ－、－Ｃ（＝Ｏ）－、ポリオキシアルキレン基、アミノ酸残基、ペプチド鎖、ポリエチレングリコールおよびこれらの組み合わせが挙げられる。 The specific structure of such a linker unit is not particularly limited, but for example, 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 non-substituted alkenyl group. Substituent alkynylene group with 2 to 20 carbon atoms, substituted or unsubstituted cycloalkylene group with 3 to 20 carbon atoms, substituted or unsubstituted cycloalkenylene group with 3 to 20 carbon atoms, substituted or unsubstituted carbon number 6 to 20 20 arylene groups, substituted or unsubstituted heteroarylene groups having 3 to 20 carbon atoms, -NH-, -O-, -S-, -C (= O) -NH-, -NH-C (= O) -, -O-, -C (= O) -O-, -OC (= O)-, -S-, -C (= O)-, polyoxyalkylene group, amino acid residue, peptide chain, Examples include polyethylene glycol and combinations thereof.
　一実施形態では、Ｌは、下記式（４）で表される構造を有する。 In one embodiment, L has a structure represented by the following formula (4).
Figure JPOXMLDOC01-appb-C000014
Figure JPOXMLDOC01-appb-C000014
　ここで、Ｖは、－ＣＯ－、－ＣＯＮＨ－またはトリアゾール環を表す。また、Ｏは、０または１の整数を表す。トリアゾール環としては、１，２，３－トリアゾール－１，４－ジイル基、１，２，４－トリアゾール－１，３－ジイル基などが挙げられる。ｌおよびｐは、それぞれ独立して、１～６の整数を表す。ｌは、好ましくは１～６の整数を表し、より好ましくは１～４の整数を表す。ｐは、好ましくは１～６の整数を表し、より好ましくは１～４の整数を表す。Ｗは、－ＮＨ－、－Ｏ－、－Ｓ－、－Ｃ（＝Ｏ）－ＮＨ－、－ＮＨ－Ｃ（＝Ｏ）－、－Ｏ－、－Ｃ（＝Ｏ）－Ｏ－、－Ｏ－Ｃ（＝Ｏ）－、－Ｓ－または－Ｃ（＝Ｏ）－を表す。 Here, V represents a -CO-, -CONH- or a triazole ring. Further, O represents an integer of 0 or 1. Examples of the triazole ring include 1,2,3-triazole-1,4-diyl group and 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, and more preferably represents an integer of 1 to 4. p preferably represents an integer of 1 to 6, and more preferably represents an integer of 1 to 4. W is -NH-, -O-, -S-, -C (= O) -NH-, -NH-C (= O)-, -O-, -C (= O) -O-,- Represents OC (= O)-, -S- or -C (= O)-.
　式（４）において、＊１は、式（３）で表される化合物との連結部位であり、＊２は、本発明に係るペプチドとの連結部位である。 In the formula (4), * 1 is a linking site with the compound represented by the formula (3), and * 2 is a linking site with the peptide according to the present invention.
　（複合体の製造方法）
　本発明に係る複合体の製造方法について特に制限はない。当業者であれば、後述する実施例の欄の記載に基づき、本願の出願時における技術常識を参酌して、本発明に係る複合体を製造することが可能である。 (Manufacturing method of complex)
There are no particular restrictions on the method for producing the complex according to the present invention. A person skilled in the art can produce a complex according to the present invention based on the description in the column of Examples described later, taking into consideration the common general knowledge as of the filing of the present application.
　例えば、Ｈ．Ｏｋａｍｏｔｏ　ｅｔ　ａｌ．，Ｃｈｅｍ．Ｃｏｍｍｕｎ．，２０１９，５５，９１０８－９１１１．、国際公開第２０１７／１６４１７２号などの記載を適宜参照して合成することができる。 For example, H. Okamoto et al. , Chem. Commun. , 2019, 55, 9108-9111. , International Publication No. 2017/164172 and the like can be appropriately referred to and synthesized.
　以下では、本発明に係る複合体のうち、以下の式（２－１）で表される複合体を例に挙げて、その製造方法を説明する。 In the following, among the complexes according to the present invention, a complex represented by the following formula (2-1) will be taken as an example, and a method for producing the complex will be described.
Figure JPOXMLDOC01-appb-C000015
Figure JPOXMLDOC01-appb-C000015
　式（２－１）において、Ｒ１～Ｒ７およびＺは、上記式（２）について定義した通りである。 In the formula (2-1), R 1 to R 7 and Z are as defined for the above formula (2).
　式（２－１）で表される複合体は、以下の反応式に従って製造することができる。 The complex represented by the formula (2-1) can be produced according to the following reaction formula.
Figure JPOXMLDOC01-appb-C000016
Figure JPOXMLDOC01-appb-C000016
　化合物ａと化合物ｂとを縮合させて、化合物ｃを得、次いで化合物ｃに化合物ｄを反応させることにより式（２－１）で表される複合体を得ることができる。 The compound represented by the formula (2-1) can be obtained by condensing the compound a and the compound b to obtain the compound c, and then reacting the compound c with the compound d.
　化合物ａと化合物ｂとの反応は、アルドール縮合反応である。 The reaction between compound a and compound b is an aldol condensation reaction.
　化合物ｃと化合物ｄとの反応は、アルキンとアジドとの１，３－双極子付加反応であり、銅触媒の存在下、ジメチルホルムアミドなどの極性溶媒中、室温で容易に進行する。 The reaction between compound c and compound d is a 1,3-dipole addition reaction between alkyne and azide, and easily proceeds at room temperature in a polar solvent such as dimethylformamide in the presence of a copper catalyst.
　化合物ａ～ｄは、上述のＨ．Ｏｋａｍｏｔｏ　ｅｔ　ａｌ．，Ｃｈｅｍ．Ｃｏｍｍｕｎ．，２０１９，５５，９１０８－９１１１．、国際公開第２０１７／１６４１７２号などの記載を適宜参照して合成することができる。 The compounds a to d are the above-mentioned H. Okamoto et al. , Chem. Commun. , 2019, 55, 9108-9111. , International Publication No. 2017/164172 and the like can be appropriately referred to and synthesized.
　得られた式（２－１）で表される複合体は、クロマトグラフィーなどの公知の手段により、精製することができる。 The obtained complex represented by the formula (2-1) can be purified by a known means such as chromatography.
　＜マイオスタチン阻害剤、予防剤／治療剤、予防方法／治療方法＞
　本発明の一実施形態では、本発明に係るペプチドもしくはそのプロドラッグまたは本発明に係る複合体を含む、マイオスタチン阻害剤が提供される（以下、「本発明に係るペプチドもしくはそのプロドラッグまたは本発明に係る複合体を含むマイオスタチン阻害剤」を、単に「マイオスタチン阻害剤」とも称する。）。上記マイオスタチン阻害剤の有効量を対象に投与することにより、筋肉量や筋力の維持、増加、増強、低下の抑制等の効果を達成し得る。マイオスタチン阻害剤は、本発明に係るペプチドの１種以上、そのプロドラッグの１種以上、もしくは本発明に係る複合体の１種以上、またはこれらの混合物から構成されてもよいが、通常は、本発明に係るペプチドおよびそのプロドラッグならびに本発明に係る複合体から選択される１種以上、ならびに薬学的に許容される担体を含む医薬組成物である。 <Myostatin inhibitor, preventive / therapeutic agent, preventive / therapeutic method>
In one embodiment of the invention, a myostatin inhibitor comprising the peptide according to the invention or a prodrug thereof or a complex according to the present invention is provided (hereinafter, "the peptide according to the present invention or a prodrug thereof or the present invention". A "myostatin inhibitor" containing the complex according to the invention is also simply referred to as a "myostatin inhibitor"). By administering an effective amount of the above-mentioned myostatin inhibitor to a subject, effects such as maintenance, increase, enhancement, and suppression of decrease in muscle mass and strength can be achieved. The myostatin inhibitor may be composed of one or more peptides according to the present invention, one or more prodrugs thereof, one or more complexes according to the present invention, or a mixture thereof, but usually. , A pharmaceutical composition comprising a peptide according to the present invention and a prodrug thereof, one or more selected from the complex according to the present invention, and a pharmaceutically acceptable carrier.
　本発明の一実施形態は、本発明に係るペプチドもしくはそのプロドラッグまたは本発明に係る複合体の有効量を患者に投与することを含む、マイオスタチンの阻害方法に関する。本発明の一実施形態は、また、マイオスタチンの阻害に使用するための、本発明に係るペプチドもしくはそのプロドラッグまたは本発明に係る複合体に関する。 One embodiment of the present invention relates to a method for inhibiting myostatin, which comprises administering to a patient an effective amount of a peptide according to the present invention or a prodrug thereof or a complex according to the present invention. One embodiment of the invention also relates to a peptide according to the invention or a prodrug thereof or a complex according to the invention for use in inhibiting myostatin.
　本発明の一実施形態では、本発明に係るペプチドもしくはそのプロドラッグまたは本発明に係る複合体を含む、筋萎縮障害の予防および／または治療剤が提供される（以下、「本発明に係るペプチドもしくはそのプロドラッグまたは本発明に係る複合体を含む筋萎縮障害の予防および／または治療剤」を、単に「筋萎縮障害の予防・治療剤」とも称する）。上記筋萎縮障害の予防・治療剤の有効量を患者に投与することにより、筋萎縮障害の進行速度の低下、進行の阻害、進行の停止、改善、治癒、および／または予防等の治療的効果を達成し得る。筋萎縮障害の予防・治療剤は、本発明に係るペプチドの１種以上、そのプロドラッグの１種以上、もしくは本発明に係る複合体の１種以上、またはこれらの混合物から構成されてもよいが、通常は、本発明に係るペプチドおよびそのプロドラッグならびに本発明に係る複合体から選択される１種以上、ならびに薬学的に許容される担体を含む医薬組成物である。 In one embodiment of the present invention, there is provided a prophylactic and / or therapeutic agent for muscular atrophy disorder, which comprises the peptide according to the present invention or a prodrug thereof or a complex according to the present invention (hereinafter, "the peptide according to the present invention"). Alternatively, a prodrug thereof or a prophylactic and / or therapeutic agent for muscular atrophy disorder containing the complex according to the present invention is also simply referred to as a "preventive / therapeutic agent for muscular atrophy disorder"). By administering an effective amount of the above-mentioned prophylactic / therapeutic agent for muscular atrophy disorder to a patient, therapeutic effects such as slowing down the progression rate of the muscular atrophy disorder, inhibiting the progression, stopping the progression, improving, healing, and / or preventing the muscular atrophy disorder. Can be achieved. The prophylactic / therapeutic agent for muscle atrophy disorder may be composed of one or more peptides according to the present invention, one or more prodrugs thereof, one or more complexes according to the present invention, or a mixture thereof. However, it is usually a pharmaceutical composition comprising a peptide according to the present invention and a prodrug thereof, one or more selected from the complex according to the present invention, and a pharmaceutically acceptable carrier.
　本発明の一実施形態は、本発明に係るペプチドもしくはそのプロドラッグまたは本発明に係る複合体の有効量を患者に投与することを含む、筋萎縮障害の予防および／または治療方法に関する。本発明の一実施形態は、また、筋萎縮障害の予防および／または治療に使用するための、本発明に係るペプチドもしくはそのプロドラッグまたは本発明に係る複合体に関する。 One embodiment of the present invention relates to a method for preventing and / or treating a muscular atrophy disorder, which comprises administering to a patient an effective amount of a peptide according to the present invention or a prodrug thereof or a complex according to the present invention. One embodiment of the invention also relates to a peptide according to the invention or a prodrug thereof or a complex according to the invention for use in the prevention and / or treatment of muscular atrophy disorder.
　マイオスタチン阻害剤、筋萎縮障害の予防・治療剤、ならびに上記予防および／または治療方法は、通常歩行に障害をもつ老人を対象とした局所投与による前脛骨筋の強化にも有効である。前脛骨筋を強化するのみでも足関節の背屈が容易になり、転倒防止に繋がる。また、たとえば、宇宙滞在時の継続的な局所投与を実施できれば、帰還後のリハビリ期間短縮等に貢献できる。 Myostatin inhibitors, prophylactic / therapeutic agents for muscular atrophy disorders, and the above prophylactic and / or therapeutic methods are also effective for strengthening the tibialis anterior muscle by local administration for elderly people with normal walking disorders. Just strengthening the tibialis anterior muscle facilitates dorsiflexion of the ankle joint, which helps prevent falls. Further, for example, if continuous local administration during staying in space can be carried out, it can contribute to shortening the rehabilitation period after returning.
　上記の筋萎縮障害としては、特に限定するものではないが、例えば、筋ジストロフィー、遠位型ミオパチー、先天性ミオパチー、封入体筋炎等の炎症性筋疾患、ミトコンドリアミオパチー等のミオパチー；廃用性筋萎縮；サルコペニアなどが例示できる。筋萎縮障害の予防・治療剤は、好ましくは筋ジストロフィーおよびサルコペニアに有効に用いられる。筋萎縮障害の予防・治療剤は、より好ましくはデュシェンヌ型筋ジストロフィー、ベッカー型筋ジストロフィー、福山型先天性筋ジストロフィー、メロシン欠損型先天性筋ジストロフィー、肢体型筋ジストロフィー、顔面肩甲上腕型筋ジストロフィー、エメリー・ドレフュス型筋ジストロフィー、三好型筋ジストロフィー、および乳児型神経軸索筋ジストロフィーなどの筋ジストロフィーならびにサルコペニアに有効に用いられ、この中でもデュシェンヌ型筋ジストロフィーに対して特に有効である。 The above-mentioned muscular atrophy disorder is not particularly limited, and is, for example, muscular dystrophy, distal myopathy, congenital myopathy, inflammatory myopathy such as inclusion body myositis, myopathy such as mitochondrial myopathy; and disused muscular atrophy. ; Sarcopenia etc. can be exemplified. The prophylactic / therapeutic agent for muscular atrophy disorder is preferably effectively used for muscular dystrophy and sarcopenia. The preventive / therapeutic agents for muscular dystrophy are more preferably Duchenne muscular dystrophy, Becker muscular dystrophy, Fukuyama muscular dystrophy, melosine deficient muscular dystrophy, limb muscular dystrophy, facial muscular dystrophy, emery muscular dystrophy, and emery muscular dystrophy. , Miyoshi muscular dystrophy, and muscular dystrophy such as infant muscular dystrophy as well as sarcopenia, and is particularly effective against Duchenne muscular dystrophy.
　筋萎縮障害は、筋萎縮性側索硬化症、慢性閉塞性肺疾患（ＣＯＰＤ）、癌、ＡＩＤＳ、腎不全、および関節リウマチなどの慢性疾患からも生じ得る。また、筋萎縮障害は、糖尿病および関連障害などの代謝障害からも生じうる。したがって、本発明の筋萎縮障害の予防・治療剤、ならびに上記予防および／または治療方法は、筋肉の萎縮を伴う悪液質の改善に利用できる。さらに、マイオスタチンの阻害によって、筋量を増加させると、骨強度が改善され、そして骨粗しょう症および他の変性骨疾患を減少させることも可能である。 Muscle atrophy 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 atrophy disorders can also result from metabolic disorders such as diabetes and related disorders. Therefore, the preventive / therapeutic agent for muscular atrophy disorder of the present invention, and the above-mentioned preventive and / or therapeutic method can be used for improving cachexia associated with muscular atrophy. In addition, inhibition of myostatin can increase muscle mass, improve bone strength, and reduce osteoporosis and other degenerative bone diseases.
　本発明の一実施形態は、本発明に係るペプチドもしくはそのプロドラッグまたは本発明に係る複合体を含む、糖尿病に起因する筋萎縮障害の予防および／または治療剤に関する。本発明の一実施形態は、また、本発明に係るペプチドもしくはそのプロドラッグまたは本発明に係る複合体の有効量を患者に投与することを含む、糖尿病に起因する筋萎縮障害の予防および／または治療方法に関する。 One embodiment of the present invention relates to a prophylactic and / or therapeutic agent for a muscular atrophy disorder caused by diabetes, which comprises a peptide according to the present invention or a prodrug thereof or a complex according to the present invention. One embodiment of the invention also prevents and / or prevents diabetes-induced muscular atrophy disorders, comprising administering to a patient an effective amount of a peptide according to the invention or a prodrug thereof or a complex according to the invention. Regarding the treatment method.
　本発明の一実施形態は、本発明に係るペプチドもしくはそのプロドラッグまたは本発明に係る複合体を含む、がん悪質液に起因する筋萎縮障害の予防および／または治療剤に関する。本発明の一実施形態は、また、本発明に係るペプチドもしくはそのプロドラッグまたは本発明に係る複合体の有効量を患者に投与することを含む、がん悪質液に起因する筋萎縮障害の予防および／または治療方法に関する。 One embodiment of the present invention relates to a prophylactic and / or therapeutic agent for muscular atrophy disorder caused by a vicious cancer solution, which comprises a peptide according to the present invention or a prodrug thereof or a complex according to the present invention. One embodiment of the present invention also comprises administering to a patient an effective amount of a peptide according to the invention or a prodrug thereof or a complex according to the present invention to prevent muscular atrophy disorder caused by a vicious cancer solution. And / or regarding treatment methods.
　本明細書において、治療上の「有効量」とは、合理的な利益／リスク比に見合う、なんらかの望ましい治療効果を生じさせるのに有効な量である。 As used herein, a therapeutically "effective amount" is an amount that is effective in producing some desired therapeutic effect commensurate with a reasonable benefit / risk ratio.
　本明細書において、「対象」および「患者」とは、ヒトおよび魚類を含む非ヒト動物が挙げられるが、好ましくは、ヒト、イヌ、ネコ、マウス、ラット、ハムスター、モルモット、ウマ（競走馬を含む）、ウシ、ブタ、ウサギ、およびヒツジ等の哺乳動物、ならびにニワトリ、ウズラ、および七面鳥等の家禽から選択され、ヒトであることがより好ましい。 As used herein, the "subject" and "patient" include non-human animals including humans and fish, but preferably humans, dogs, cats, mice, rats, hamsters, guinea pigs, and horses (race horses). Included), mammals such as cows, pigs, rabbits, and sheep, and poultry such as chickens, quails, and turkeys, more preferably humans.
　上記の薬学的に許容される担体としては、特に限定されないが、乳糖、ショ糖、マンニトール、デンプン、コーンスターチ、結晶セルロース、軽質無水ケイ酸等の賦形剤；シリカ、タルク、ステアリン酸カルシウム、ステアリン酸マグネシウム等の滑沢剤；ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、カルボキシメチルセルロース、カルボキシメチルセルロースナトリウム、メチルセルロース、ポリビニルピロリドン、結晶セルロース、デキストリン、ゼラチン等の結合剤；アスコルビン酸、亜硫酸ナトリウム、亜硫酸水素ナトリウム、トコフェロール等の酸化防止剤；エチレンジアミン四酢酸（ＥＤＴＡ）等のキレート剤；ホウ酸塩、重炭酸塩、Ｔｒｉｓ－ＨＣｌ、クエン酸塩、リン酸塩、他の有機酸等の緩衝剤；注射用水、生理食塩水、エタノール、プロパノール、エチレングリコール、プロピレングリコール、マクロゴール、オリーブ油、トウモロコシ油等の溶媒；プルロニック（登録商標）、ポリエチレングリコール、ソルビタン脂肪酸エステル、ポリソルベート、トリトン（登録商標）、レシチン、コレステロール、塩化ベンザルコニウム、塩化ベンゼトニウム、モノステアリン酸グリセリン等の界面活性剤または湿潤剤；塩化ナトリウム、塩化カリウム、グリセリン、ブドウ糖、ソルビトール、マンニトール等の等張化剤；安息香酸、サリチル酸、チメロサール、フェネチルアルコール、メチルパラベン、プロピルパラベン、クロルヘキシジン等の保存剤；錯化剤；アミノ酸；抗菌剤；着色剤；フレーバー剤および希釈剤；乳化剤；ナトリウム等の塩形成対イオン；搬送ビヒクル；希釈剤などが挙げられる（Ｒｅｍｉｎｇｔｏｎ’ｓ　Ｐｈａｒｍａｃｅｕｔｉｃａｌ　Ｓｃｉｅｎｃｅｓ，　第１８版，　Ａ．Ｒ．　Ｇｅｎｎａｒｏ監修，　Ｍａｃｋ　Ｐｕｂｌｉｓｈｉｎｇ　Ｃｏｍｐａｎｙ，　１９９０）。 The above-mentioned pharmaceutically acceptable carrier is not particularly limited, but is an excipient such as lactose, sucrose, mannitol, starch, corn starch, crystalline cellulose, and light anhydrous silicic acid; silica, talc, calcium stearate, stearic acid, etc. Lubricants such as magnesium; binders such as hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, methyl cellulose, polyvinylpyrrolidone, crystalline cellulose, dextrin, gelatin; ascorbic acid, sodium sulfite, sodium hydrogen sulfite, tocopherol, etc. Antioxidants; Chelating agents such as ethylenediamine tetraacetic acid (EDTA); Buffers such as borates, bicarbonates, Tris-HCl, citrates, phosphates, other organic acids; water for injection, physiological saline Solponics such as water, ethanol, propanol, ethylene glycol, propylene glycol, macrogol, olive oil, corn oil; Pluronic®, polyethylene glycol, sorbitan fatty acid ester, polysorbate, Triton®, lecithin, cholesterol, benza chloride Surface active agents or wetting agents such as luconium, benzethonium chloride, glycerin monostearate; isotonic agents such as sodium chloride, potassium chloride, glycerin, glucose, sorbitol, mannitol; benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben Preservatives such as, propylparaben, chlorhexidine; complexing agents; amino acids; antibacterial agents; colorants; flavoring agents and diluents; emulsifiers; salt-forming counterions such as sodium; transport vehicles; diluents and the like (Remington' s Pharmaceutical Sciences, 18th Edition, supervised by AR Gennaro, Mack Publishing Company, 1990).
　本発明に係るペプチドもしくはそのプロドラッグまたは本発明に係る複合体の薬剤中の含有量は、薬剤全体に対して０．０１～１００重量％であり得る。 The content of the peptide according to the present invention or the prodrug thereof or the complex according to the present invention in the drug can be 0.01 to 100% by weight based on the whole drug.
　本発明に係るペプチドもしくはそのプロドラッグまたは本発明に係る複合体の投与量は、年齢、症状、投与方法などにより差異はあるが、経口投与の場合、一般的に、ヒト（体重６０ｋｇとして）に対して、一日につき約０．１～１００ｍｇ、好ましくは約１．０～５０ｍｇ、より好ましくは約１．０～２０ｍｇである。非経口的に投与する場合は、その１回投与量は年齢、症状、投与方法などによっても異なるが、例えば、注射剤の形では通常、ヒト（体重６０ｋｇとして）に対して、一日につき約０．０１～３０ｍｇ程度、好ましくは約０．１～２０ｍｇ程度、より好ましくは約０．１～１０ｍｇ程度を投与するのが好都合である。ヒト以外の動物の場合も、体重６０ｋｇ当たりに換算した量を投与することができる。 The dose of the peptide according to the present invention or the prodrug thereof or the complex according to the present invention varies depending on the age, symptoms, administration method, etc., but in the case of oral administration, it is generally given to humans (assuming a body weight of 60 kg). On the other hand, it is about 0.1 to 100 mg, preferably about 1.0 to 50 mg, and more preferably about 1.0 to 20 mg per day. When administered parenterally, the single dose varies depending on age, symptoms, administration method, etc., but for example, in the form of an injection, it is usually about about a day for a human (assuming a body weight of 60 kg). It is 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. In the case of animals other than humans, the amount converted per 60 kg of body weight can also be administered.
　本発明の効果を、以下の実施例および比較例を用いて説明する。ただし、本発明の技術的範囲が以下の実施例のみに制限されるわけではない。 The effects of the present invention will be described with reference to the following examples and comparative examples. However, the technical scope of the present invention is not limited to the following examples.
　＜ペプチドの合成＞
　（合成例１）
　配列番号１のアミノ酸配列を含むペプチド：ｒｉＤＭ－４の合成
　Ｈ－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－シクロヘキシルグリシン
　Ｄ－Ｐｈｇ：Ｄ－フェニルグリシン
　ｒｉＤＭ－４は以下に示すＦｍｏｃ固相ペプチド合成法により合成した。 <Peptide synthesis>
(Synthesis Example 1)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 1: Synthesis of riDM-4 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-NH 2
D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-4 was synthesized by the Fmoc solid phase peptide synthesis method shown below.
　Ｒｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）５４ｍｇ（０．０２０ｍｍｏｌ）を固相合成用反応容器に量りとり、ジメチルホルムアミド（ＤＭＦ）溶液中室温で１時間静置し樹脂を膨潤させた後、２０％（ｖ／ｖ）ピペリジン／ＤＭＦ溶液（２ｍＬ）中室温（２５℃）で２０分間反応させることで樹脂上の保護基Ｆｍｏｃ（９－フルオレニルメトキシカルボニル）基を除去した。ＤＭＦ（２．５ｍＬ）で１０回樹脂を洗浄し、１－ヒドロキシベンゾトリアゾール（ＨＯＢｔ）１６ｍｇ（０．１０ｍｍｏｌ，５ｅｑ．）、Ｎ，Ｎ－ジイソプロピルカルボジイミド（ＤＩＰＣＤ）０．０１６ｍＬ（０．１０ｍｍｏｌ，５ｅｑ．）存在下で、Ｆｍｏｃ－Ｄ－Ｔｒｐ（Ｂｏｃ）－ＯＨ（０．２０ｍｍｏｌ，１０ｅｑ．）を室温で３０分間ＤＭＦ（２　ｍＬ）中にて反応させ、樹脂上にアミノ酸を導入した。次のアミノ酸を縮合させるため、２０％（ｖ／ｖ）ピペリジン／ＤＭＦ溶液（２．５ｍＬ）中にて２０分間反応させることで樹脂上のＦｍｏｃ基を除去した。以下、Ｆｍｏｃ－Ｄ－Ｔｒｐ（Ｂｏｃ）－ＯＨの場合と同様にして順次Ｃ末端側からＦｍｏｃ－Ｄ－Ｔｙｒ（ｔＢｕ）－ＯＨ（０．２０ｍｍｏｌ，１０ｅｑ．）、Ｆｍｏｃ－Ｄ－Ｐｈｇ－ＯＨ（０．２０ｍｍｏｌ，１０ｅｑ．）、Ｆｍｏｃ－Ｄ－Ａｒｇ（Ｐｍｃ）－ＯＨ（０．２０ｍｍｏｌ，１０ｅｑ．）、Ｆｍｏｃ－Ｄ－Ｔｒｐ（Ｂｏｃ）－ＯＨ（０．２０ｍｍｏｌ，１０ｅｑ．）、Ｆｍｏｃ－Ｄ－Ｐｈｇ－ＯＨ（０．２０ｍｍｏｌ，１０ｅｑ．）、Ｆｍｏｃ－Ｄ－Ｌｙｓ（Ｂｏｃ）－ＯＨ（０．２０ｍｍｏｌ，１０ｅｑ．）、Ｆｍｏｃ－Ｄ－Ｃｈｇ－ＯＨ（０．２０ｍｍｏｌ，１０ｅｑ．）、Ｆｍｏｃ－Ｄ－Ｇｌｎ（Ｔｒｔ）－ＯＨ（０．２０ｍｍｏｌ，１０ｅｑ．）、Ｆｍｏｃ－Ｄ－Ｐｈｇ－ＯＨ（０．２０ｍｍｏｌ，１０ｅｑ．）、Ｆｍｏｃ－Ｄ－Ｔｒｐ（Ｂｏｃ）－ＯＨ（０．２０ｍｍｏｌ，１０ｅｑ．）、Ｆｍｏｃ－Ｄ－Ｓｅｒ（ｔＢｕ）－ＯＨ（０．２０ｍｍｏｌ，１０ｅｑ．）、Ｆｍｏｃ－Ｄ－Ｌｙｓ（Ｂｏｃ）－ＯＨ（０．２０ｍｍｏｌ，１０ｅｑ．）、Ｆｍｏｃ－Ｄ－Ｃｈｇ－ＯＨ（０．２０ｍｍｏｌ，１０ｅｑ．）、Ｆｍｏｃ－Ｄ－Ａｒｇ（Ｐｍｃ）－ＯＨ（０．２０ｍｍｏｌ，１０ｅｑ．）、Ｆｍｏｃ－Ｄ－Ｌｅｕ－ＯＨ（０．２０ｍｍｏｌ，１０ｅｑ．）を導入しペプチド鎖を伸長させた。２０％（ｖ／ｖ）ピペリジン／ＤＭＦ溶液（２．５ｍＬ）中にて２０分間反応させることでＮ末端のＦｍｏｃ基を除去し、ＤＭＦ（２．５ｍＬ，８回）、メタノール（２．５ｍＬ，５回）およびジエチルエーテル（２．５ｍＬ，５回）洗浄後、樹脂を乾燥させた。各種側鎖保護基の除去および脱樹脂のため、ｍ－クレゾール（０．１０ｍＬ）、チオアニソール（０．１０ｍＬ）および１，２－エタンジチオール（０．１０ｍＬ）存在下でトリフルオロ酢酸（ＴＦＡ）４．０ｍＬ中にて２時間反応させた。フィルター付きロートを用いてろ過することで樹脂を除去した後、窒素噴霧によりＴＦＡを留去し、ジエチルエーテル４０ｍＬを加えて粗精製ペプチドを析出させた。粗精製ペプチドを０．７５Ｍ酢酸－２５％（ｖ／ｖ）アセトニトリル混液に溶解し、０．１％ＴＦＡを含有する水－アセトニトリル混合溶媒を移動相として用いる逆相高速液体クロマトグラフィーによりグラジエント溶離（精製）することで、白色固体を得た（６．６ｍｇ，収率１２％）。
ＨＲＭＳ（ＥＳ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ３＋＋３Ｈ）１１５７．１３４０，ｆｏｕｎｄ　１１５７．１３２６。 Weigh 54 mg (0.020 mmol) of Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) in a reaction vessel for solid-phase synthesis, and allow it to stand in a dimethylformamide (DMF) solution at room temperature for 1 hour to swell the resin. After that, the protecting group Fmoc (9-fluorenylmethoxycarbonyl) group on the resin was removed by reacting in a 20% (v / v) piperidine / DMF solution (2 mL) at room temperature (25 ° C.) for 20 minutes. .. Wash the resin 10 times with DMF (2.5 mL), 16 mg (0.10 mmol, 5 eq.) Of 1-hydroxybenzotriazole (HOBt), 0.016 mL (0.10 mmol, 5 eq) of N, N-diisopropylcarbodiimide (DIPCD). In the presence of Fmoc-D-Trp (Boc) -OH (0.20 mmol, 10 eq.) Was reacted in DMF (2 mL) for 30 minutes at room temperature to introduce amino acids onto the resin. To condense the following amino acids, the Fmoc group on the resin was removed by reaction in a 20% (v / v) piperidine / DMF solution (2.5 mL) for 20 minutes. Hereinafter, in the same manner as in the case of Fmoc-D-Trp (Boc) -OH, 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 -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.), Fmoc-D-Leu-OH (0.20 mmol, 10 eq.) To extend the peptide chain. The N-terminal Fmoc group was removed by reacting in 20% (v / v) piperidine / DMF solution (2.5 mL) for 20 minutes, and DMF (2.5 mL, 8 times), methanol (2.5 mL, After washing 5 times) and diethyl ether (2.5 mL, 5 times), the resin was dried. Trifluoroacetic acid (TFA) in the presence of m-cresol (0.10 mL), thioanisole (0.10 mL) and 1,2-ethanedithiol (0.10 mL) for removal of various side chain protecting groups and removal of resin. The reaction was carried out in 4.0 mL for 2 hours. After removing the resin by filtration using a funnel with a filter, TFA was distilled off by nitrogen spraying, and 40 mL of diethyl ether was added to precipitate a crude peptide. The crude peptide is dissolved in a 0.75 M acetate-25% (v / v) acetonitrile mixture and gradient elution by reverse phase high performance liquid chromatography using a water-acetonitrile mixed solvent containing 0.1% TFA as the mobile phase. By purification), a white solid was obtained (6.6 mg, yield 12%).
HRMS (ES +) calcd for (M 3+ + 3H) 1157.1340, found 1157.1326.
　（合成例２）
　配列番号２のアミノ酸配列を含むペプチド：ｒｉＤＭ－８の合成
　Ｈ－Ｄ－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－シクロヘキシルグリシン
　Ｄ－Ｐｈｇ：Ｄ－フェニルグリシン
　ｒｉＤＭ－８はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）５４ｍｇ（０．０２０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（６．２ｍｇ，収率１１％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２３８４．３０，ｆｏｕｎｄ　２３８３．６９。 (Synthesis Example 2)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 2: Synthesis of riDM-8 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-NH 2
D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-8 is the same as in Synthesis Example 1 using Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 54 mg (0.020 mmol). (6.2 mg, yield 11%).
LRMS (MALDI +) calcd for (M + + H) 2384.30, found 2383.69.
　（合成例３）
　配列番号３のアミノ酸配列を含むペプチド：ｒｉＤＭ－９の合成
　Ｈ－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－シクロヘキシルグリシン
　Ｄ－Ｐｈｇ：Ｄ－フェニルグリシン
　ｒｉＤＭ－９はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）５４ｍｇ（０．０２０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（８．４ｍｇ，収率１６％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２２００．１８，ｆｏｕｎｄ　２２００．３０。 (Synthesis Example 3)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 3: Synthesis of riDM-9 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-NH 2
D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-9 is the same as in Synthesis Example 1 using Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 54 mg (0.020 mmol). (8.4 mg, 16% yield).
LRMS (MALDI +) calcd for (M + + H) 2200.18, found 2200.30.
　（合成例４）
　配列番号４のアミノ酸配列を含むペプチド：ｒｉＤＭ－１０の合成
　Ｈ－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－ＮＨ２
　Ｄ－Ｃｈｇ：Ｄ－シクロヘキシルグリシン
　Ｄ－Ｐｈｇ：Ｄ－フェニルグリシン
　ｒｉＤＭ－１０はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）５４ｍｇ（０．０２０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（９．５ｍｇ，収率１９％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２１２７．１８，ｆｏｕｎｄ　２１２７．６９。 (Synthesis Example 4)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 4: Synthesis of 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-NH 2
D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-10 is the same as in Synthesis Example 1 using Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 54 mg (0.020 mmol). (9.5 mg, yield 19%).
LRMS (MALDI +) calcd for (M + + H) 2127.18, found 2127.69.
　（合成例５）
　以下のアミノ酸配列（配列番号２６：下線部）を含むペプチド：ｒｉＤＭ－１２の合成（比較例）
　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－シクロヘキシルグリシン
　Ｄ－Ｐｈｇ：Ｄ－フェニルグリシン
　ｒｉＤＭ－１２はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）５４ｍｇ（０．０２０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（４．０ｍｇ，収率７．６％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２１８５．２０，ｆｏｕｎｄ　２１８４．６９。 (Synthesis Example 5)
Synthesis of peptide: riDM-12 containing the following amino acid sequence (SEQ ID NO: 26: underlined part) (comparative example)
HD- 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- NH 2
D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-12 is the same as in Synthesis Example 1 using Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 54 mg (0.020 mmol). Synthesized and purified by the method of (4.0 mg, yield 7.6%).
LRMS (MALDI +) calcd for (M + + H) 2185.20, found 2184.69.
　（合成例６）
　配列番号５のアミノ酸配列を含むペプチド：ｒｉＤＭ－１３の合成
　Ｈ－Ｄ－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－シクロヘキシルグリシン
　Ｄ－Ａｌａ（２－Ｎａｐｈ）：Ｄ－３－（２－ナフチル）アラニン
　Ｄ－Ｐｈｇ：Ｄ－フェニルグリシン
　ｒｉＤＭ－１３はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）１０８ｍｇ（０．０４０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（２．９ｍｇ，収率２．６％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２３２４．２６，ｆｏｕｎｄ　２３２４．１０。 (Synthesis Example 6)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 5: Synthesis of 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-NH 2
D-Chg: D-cyclohexylglycine D-Ala (2-Naph): D-3- (2-naphthyl) alanine D-Phg: D-phenylglycine riDM-13 is Link Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) Synthesized and purified using 108 mg (0.040 mmol) by the same method as in Synthesis Example 1 (2.9 mg, yield 2.6%).
LRMS (MALDI +) calcd for (M + + H) 2324.26, found 2324.10.
　（合成例７）
　配列番号６のアミノ酸配列を含むペプチド：ｒｉＤＭ－１４の合成
　Ｈ－Ｄ－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－シクロヘキシルグリシン
　Ｄ－Ｐｈｇ：Ｄ－フェニルグリシン
　Ｄ－ｈｏｍｏＰｈｅ：Ｄ－ホモフェニルアラニン
　ｒｉＤＭ－１４はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）５４ｍｇ（０．０２０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（１．７ｍｇ，収率３．１％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２２８８．２６，ｆｏｕｎｄ　２２８８．３３。 (Synthesis Example 7)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 6: Synthesis of 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-NH 2
D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine D-homophhe: D-homophenylalanine riDM-14 is Link Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 54 mg (0.020 mmol) Was synthesized and purified by the same method as in Synthesis Example 1 (1.7 mg, yield 3.1%).
LRMS (MALDI +) calcd for (M + + H) 2288.26, found 2288.33.
　（合成例８）
　配列番号７のアミノ酸配列を含むペプチド：ｒｉＤＭ－１５の合成
　Ｈ－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－ｈｏｍｏＰｈｅ－ＮＨ２
　Ｄ－Ｃｈｇ：Ｄ－シクロヘキシルグリシン
　Ｄ－Ｐｈｇ：Ｄ－フェニルグリシン
　Ｄ－ｈｏｍｏＰｈｅ：Ｄ－ホモフェニルアラニン
　ｒｉＤＭ－１５はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）５４ｍｇ（０．０２０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（７．１ｍｇ，収率１３％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２２８８．２６，ｆｏｕｎｄ　２２８８．０８。 (Synthesis Example 8)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 7: Synthesis of 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-NH 2
D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine D-homophhe: D-homophenylalanine riDM-15 is Link Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 54 mg (0.020 mmol) Was synthesized and purified by the same method as in Synthesis Example 1 (7.1 mg, yield 13%).
LRMS (MALDI +) calcd for (M + + H) 2288.26, found 2288.08.
　（合成例９）
　配列番号８のアミノ酸配列を含むペプチド：ｒｉＤＭ－２６の合成
　Ｈ－Ｄ－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－シクロヘキシルグリシン
　Ｄ－Ｐｈｇ：Ｄ－フェニルグリシン
　ｒｉＤＭ－２６はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）５４ｍｇ（０．０２０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（４．０ｍｇ，収率７．２％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２３４１．２７，ｆｏｕｎｄ　２３４１．１１。 (Synthesis Example 9)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 8: Synthesis of 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-NH 2
D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-26 is the same as in Synthesis Example 1 using Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 54 mg (0.020 mmol). (4.0 mg, yield 7.2%).
LRMS (MALDI +) calcd for (M + + H) 2341.27, found 2341.11.
　（合成例１０）
　配列番号９のアミノ酸配列を含むペプチド：ｒｉＤＭ－２７の合成
　Ｈ－Ｄ－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－シクロヘキシルグリシン
　Ｄ－Ｐｈｇ：Ｄ－フェニルグリシン
　ｒｉＤＭ－２７はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）５４ｍｇ（０．０２０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（１１．４ｍｇ，収率１９％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２３８２．３３，ｆｏｕｎｄ　２３８２．０４。 (Synthesis Example 10)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 9: Synthesis of 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-NH 2
D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-27 is the same as in Synthesis Example 1 using Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 54 mg (0.020 mmol). (11.4 mg, yield 19%).
LRMS (MALDI +) calcd for (M + + H) 2382.33, found 382.04.
　（合成例１１）
　配列番号１０のアミノ酸配列を含むペプチド：ｒｉＤＭ－２８の合成
　Ｈ－Ｄ－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－シクロヘキシルグリシン
　Ｄ－Ｐｈｇ：Ｄ－フェニルグリシン
　ｒｉＤＭ－２８はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）５４ｍｇ（０．０２０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（６．２ｍｇ，収率１１％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２３４１．３０，ｆｏｕｎｄ　２３４０．７４。 (Synthesis Example 11)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 10: Synthesis of 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-NH 2
D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-28 is the same as in Synthesis Example 1 using Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 54 mg (0.020 mmol). (6.2 mg, yield 11%).
LRMS (MALDI +) calcd for (M + + H) 2341.30, found 2340.74.
　（合成例１２）
　配列番号１１のアミノ酸配列を含むペプチド：ｒｉＤＭ－２９の合成
　Ｈ－Ｄ－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－シクロヘキシルグリシン
　Ｄ－Ｐｈｇ：Ｄ－フェニルグリシン
　ｒｉＤＭ－２９はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）５４ｍｇ（０．０２０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（１．２ｍｇ，収率２．２％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２３４１．２６，ｆｏｕｎｄ　２３４０．８５。 (Synthesis Example 12)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 11: Synthesis of riDM-29 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-NH 2
D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-29 is the same as in Synthesis Example 1 using Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 54 mg (0.020 mmol). (1.2 mg, yield 2.2%).
LRMS (MALDI +) calcd for (M + + H) 2341.26, found 2340.85.
　（合成例１３）
　配列番号１２のアミノ酸配列を含むペプチド：ｒｉＤＭ－１８の合成
　Ｈ－Ｄ－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－ノルロイシン
　Ｄ－Ｃｈｇ：Ｄ－シクロヘキシルグリシン
　Ｄ－Ｐｈｇ：Ｄ－フェニルグリシン
　ｒｉＤＭ－１８はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）５４ｍｇ（０．０２０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（６．３ｍｇ，収率１１％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２３１３．２６，ｆｏｕｎｄ　２３１４．１３。 (Synthesis Example 13)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 12: Synthesis of 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-NH 2
D-Nle: D-norleucine D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-18 is Link Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 54 mg (0.020 mmol). It was synthesized and purified by the same method as in Synthesis Example 1 (6.3 mg, yield 11%).
LRMS (MALDI +) calcd for (M + + H) 2313.26, found 2314.13.
　（合成例１４）
　配列番号１３のアミノ酸配列を含むペプチド：ｒｉＤＭ－１９の合成
　Ｈ－Ｄ－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－ノルロイシン
　Ｄ－Ｃｈｇ：Ｄ－シクロヘキシルグリシン
　Ｄ－Ｐｈｇ：Ｄ－フェニルグリシン
　ｒｉＤＭ－１９はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）５４ｍｇ（０．０２０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（５．２ｍｇ，収率９．５％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２２８７．２４，ｆｏｕｎｄ　２２８７．１３。 (Synthesis Example 14)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 13: Synthesis of riDM-19 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-NH 2
D-Nle: D-norleucine D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-19 contains Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 54 mg (0.020 mmol). It was synthesized and purified by the same method as in Synthesis Example 1 (5.2 mg, yield 9.5%).
LRMS (MALDI +) calcd for (M + + H) 2287.24, found 2287.13.
　（合成例１５）
　配列番号１４のアミノ酸配列を含むペプチド：ｒｉＤＭ－２０の合成
　Ｈ－Ｄ－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－シクロヘキシルグリシン
　Ｄ－Ｎｌｅ：Ｄ－ノルロイシン
　Ｄ－Ｐｈｇ：Ｄ－フェニルグリシン
　ｒｉＤＭ－２０はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）５４ｍｇ（０．０２０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（２．２ｍｇ，収率４．０％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２２８７．２４，ｆｏｕｎｄ　２２８７．１５。 (Synthesis Example 15)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 14: Synthesis of 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-NH 2
D-Chg: D-cyclohexylglycine D-Nle: D-norleucine D-Phg: D-phenylglycine riDM-20 contains Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 54 mg (0.020 mmol). It was synthesized and purified by the same method as in Synthesis Example 1 (2.2 mg, yield 4.0%).
LRMS (MALDI +) calcd for (M + + H) 2287.24, found 2287.15.
　（合成例１６）
　配列番号１５のアミノ酸配列を含むペプチド：ｒｉＤＭ－２２の合成
　Ｈ－Ｄ－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－オルニチン
　Ｄ－Ｃｈｇ：Ｄ－シクロヘキシルグリシン
　Ｄ－Ｐｈｇ：Ｄ－フェニルグリシン
　ｒｉＤＭ－２２はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）５４ｍｇ（０．０２０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（７．９ｍｇ，収率１５％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２２７１．２４，ｆｏｕｎｄ　２２７１．７５。 (Synthesis Example 16)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 15: Synthesis of riDM-22 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-NH 2
D-Orn: D-ornithine D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine riDM-22 contains Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 54 mg (0.020 mmol). It was synthesized and purified by the same method as in Synthesis Example 1 (7.9 mg, yield 15%).
LRMS (MALDI +) calcd for (M + + H) 2271.24, found 2271.75.
　（合成例１７）
　配列番号１６のアミノ酸配列を含むペプチド：ｒｉＤＭ－２３の合成
　Ｈ－Ｄ－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－シクロヘキシルグリシン
　Ｄ－Ｏｒｎ：Ｄ－オルニチン
　Ｄ－Ｐｈｇ：Ｄ－フェニルグリシン
　ｒｉＤＭ－２３はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）５４ｍｇ（０．０２０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（８．６ｍｇ，収率１６％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２２９９．２４，ｆｏｕｎｄ　２２９９．４３。 (Synthesis Example 17)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 16: Synthesis of 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-NH 2
D-Chg: D-cyclohexylglycine D-Orn: D-ornithine D-Phg: D-phenylglycine riDM-23 is Link Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 54 mg (0.020 mmol). It was synthesized and purified by the same method as in Synthesis Example 1 (8.6 mg, yield 16%).
LRMS (MALDI +) calcd for (M + + H) 2299.24, found 2299.43.
　（合成例１８）
　配列番号１７のアミノ酸配列を含むペプチド：ｒｉＤＭ－２４の合成
　Ｈ－Ｄ－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－シクロヘキシルグリシン
　Ｄ－Ｐｈｇ：Ｄ－フェニルグリシン
　Ｄ－Ｏｒｎ：Ｄ－オルニチン
　ｒｉＤＭ－２４はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）５４ｍｇ（０．０２０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（５．２ｍｇ，収率９．４％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２２９９．２４，ｆｏｕｎｄ　２２９９．２１。 (Synthesis Example 18)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 17: Synthesis of 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-NH 2
D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine D-Orn: D-ornithine riDM-24 contains Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 54 mg (0.020 mmol). It was synthesized and purified by the same method as in Synthesis Example 1 (5.2 mg, yield 9.4%).
LRMS (MALDI +) calcd for (M + + H) 2299.24, found 2299.21.
　（合成例１９）
　配列番号１８のアミノ酸配列を含むペプチド：ｒｉＤＭ－２５の合成
　Ｈ－Ｄ－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－シクロヘキシルグリシン
　Ｄ－Ｐｈｇ：Ｄ－フェニルグリシン
　Ｄ－Ｏｒｎ：Ｄ－オルニチン
　ｒｉＤＭ－２５はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）５４ｍｇ（０．０２０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（５．７ｍｇ，収率１０％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２２７１．２４，ｆｏｕｎｄ　２２７１．１１。 (Synthesis Example 19)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 18: Synthesis of 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-NH 2
D-Chg: D-cyclohexylglycine D-Phg: D-phenylglycine D-Orn: D-ornithine riDM-25 contains Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 54 mg (0.020 mmol). It was synthesized and purified by the same method as in Synthesis Example 1 (5.7 mg, yield 10%).
LRMS (MALDI +) calcd for (M + + H) 2271.24, found 2271.11.
　（合成例２０）
　配列番号１９のアミノ酸配列を含むペプチド：ｎ－６６ｒｉの合成
　Ｈ－Ｄ－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－シクロヘキシルグリシン
　ｎ－６６ｒｉはＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）５４ｍｇ（０．０２０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（５．４ｍｇ，収率１０％）。
ＨＲＭＳ（ＥＳ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ３＋＋３Ｈ）７５１．７８９９，ｆｏｕｎｄ　７５１．７８９０。 (Synthesis Example 20)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 19: Synthesis of n-66ri 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-NH 2
D-Chg: D-cyclohexylglycine n-66ri was synthesized and purified by the same method as in Synthesis Example 1 using Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 54 mg (0.020 mmol). 5.4 mg, yield 10%).
HRMS (ES +) calcd for (M 3+ + 3H) 751.7899, found 751.7890.
　（合成例２１）
　配列番号２０のアミノ酸配列を含むペプチド：ｒｉＤＭ－３３の合成
　Ｈ－Ｄ－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－シクロヘキシルグリシン
　ｒｉＤＭ－３３はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）１０８ｍｇ（０．０４０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（２６．１ｍｇ，収率２３％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２３２３．４１，ｆｏｕｎｄ　２３２２．５３。 (Synthesis Example 21)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 20: Synthesis of 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-NH 2
D-Chg: D-cyclohexylglycine riDM-33 was synthesized and purified by the same method as in Synthesis Example 1 using Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 108 mg (0.040 mmol). 26.1 mg, yield 23%).
LRMS (MALDI +) calcd for (M + + H) 2323.41, found 3222.53.
　（合成例２２）
　配列番号２１のアミノ酸配列を含むペプチド：ｒｉＤＭ－３４の合成
　Ｈ－Ｄ－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－シクロヘキシルグリシン
　ｒｉＤＭ－３４はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）１０８ｍｇ（０．０４０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（２１．３ｍｇ，収率１９％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２２８１．４０，ｆｏｕｎｄ　２２８２．１８。 (Synthesis Example 22)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 21: Synthesis of 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-NH 2
D-Chg: D-cyclohexylglycine riDM-34 was synthesized and purified by the same method as in Synthesis Example 1 using Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 108 mg (0.040 mmol). 21.3 mg, yield 19%).
LRMS (MALDI +) calcd for (M + + H) 2281.40, found 2282.18.
　（合成例２３）
　配列番号２２のアミノ酸配列を含むペプチド：ｒｉＤＭ－３５の合成
　Ｈ－Ｄ－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－シクロヘキシルグリシン
　ｒｉＤＭ－３５はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）１０８ｍｇ（０．０４０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（２６．２ｍｇ，収率２２％）。
ＨＲＭＳ（ＥＳ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２３５０．４６５７，ｆｏｕｎｄ　２３５０．４６４６。 (Synthesis Example 23)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 22: Synthesis of 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-NH 2
D-Chg: D-cyclohexylglycine riDM-35 was synthesized and purified by the same method as in Synthesis Example 1 using Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 108 mg (0.040 mmol). 26.2 mg, yield 22%).
HRMS (ES +) calcd for (M + + H) 2350.4657, found 2350.4646.
　（合成例２４）
　以下のアミノ酸配列（配列番号２７：下線部）を含むペプチド：ｒｉＤＭ－４２の合成（比較例）
　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－シクロヘキシルグリシン
　ｒｉＤＭ－４２はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）１０８ｍｇ（０．０４０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（３０．２ｍｇ，収率２８％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２０５２．６３　（ａｖｅｒａｇｅ　ＭＳ），ｆｏｕｎｄ　２０５３．２６。 (Synthesis Example 24)
Synthesis of peptide: riDM-42 containing the following amino acid sequence (SEQ ID NO: 27: underlined part) (comparative example)
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- NH 2
D-Chg: D-cyclohexylglycine riDM-42 was synthesized and purified using Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 108 mg (0.040 mmol) in the same manner as in Synthesis Example 1 (Synthesis Example 1). 30.2 mg, yield 28%).
LRMS (MALDI +) calcd for (M + + H) 2052.63 (average MS), found 2053.26.
　（合成例２５）
　配列番号２３のアミノ酸配列を含むペプチド：ｒｉＤＭ－３６の合成
　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－Ｄ－Ｔｒｐ－ＮＨ２
　Ｄ－Ｃｈｇ：Ｄ－シクロヘキシルグリシン
　ｒｉＤＭ－３６はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）１０８ｍｇ（０．０４０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（１６．１ｍｇ，収率２８％）。
ＨＲＭＳ（ＥＳ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ３＋＋３Ｈ）７４６．４６４５，ｆｏｕｎｄ　７４６．７０００。 (Synthesis Example 25)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 23: Synthesis of 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-NH 2
D-Chg: D-cyclohexylglycine riDM-36 was synthesized and purified by the same method as in Synthesis Example 1 using Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 108 mg (0.040 mmol). 16.1 mg, yield 28%).
HRMS (ES +) calcd for (M 3+ + 3H) 746.4645, found 746.7000.
　（合成例２６）
　配列番号２４のアミノ酸配列を含むペプチド：ｒｉＤＭ－３９の合成
　Ｈ－Ｄ－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－シクロヘキシルグリシン
　ｒｉＤＭ－３９はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）１０８ｍｇ（０．０４０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（１９．０ｍｇ，収率３３％）。
ＨＲＭＳ（ＥＳ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ３＋＋３Ｈ）７２２．１３４５，ｆｏｕｎｄ　７２２．４０００。 (Synthesis Example 26)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 24: Synthesis of 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-NH 2
D-Chg: D-cyclohexylglycine riDM-39 was synthesized and purified by the same method as in Synthesis Example 1 using Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 108 mg (0.040 mmol). 19.0 mg, yield 33%).
HRMS (ES +) calcd for (M 3+ + 3H) 722.1345, found 722.4000.
　（合成例２７）
　配列番号２５のアミノ酸配列を含むペプチド：ｒｉＤＭ－４６の合成
　Ｈ－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－シクロヘキシルグリシン
　Ｄ－Ａｌａ（２－Ｎａｐｈ）：Ｄ－３－（２－ナフチル）アラニン
　Ｄ－ｈｏｍｏＰｈｅ：Ｄ－ホモフェニルアラニン
　ｒｉＤＭ－４６はＲｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）１０８ｍｇ（０．０４０ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（４４ｍｇ，収率３７％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２３１１．４８，ｆｏｕｎｄ　２３１１．４５。 (Synthesis Example 27)
Peptide Containing the Amino Acid Sequence of SEQ ID NO: 25: Synthesis of 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-NH 2
D-Chg: D-cyclohexylglycine D-Ala (2-Naph): D-3- (2-naphthyl) alanine D-homoPhe: D-homophenylalanine riDM-46 is Link Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) Synthesized and purified using 108 mg (0.040 mmol) by the same method as in Synthesis Example 1 (44 mg, yield 37%).
LRMS (MALDI +) calcd for (M + + H) 2311.48, found 2311.45.
　（合成例２８）
　複合体１６ＰＣ－Ｎの合成
　Ｘ１－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－シクロヘキシルグリシン
　Ｄ－Ａｌａ（２－Ｎａｐｈ）：Ｄ－３－（２－ナフチル）アラニン
　Ｄ－ｈｏｍｏＰｈｅ：Ｄ－ホモフェニルアラニン (Synthesis Example 28)
Synthesis of the 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-NH 2
D-Chg: D-cyclohexylglycine D-Ala (2-Naph): D-3- (2-naphthyl) alanine D-homoPhe: D-homophenylalanine
Figure JPOXMLDOC01-appb-C000017
Figure JPOXMLDOC01-appb-C000017
Figure JPOXMLDOC01-appb-C000018
Figure JPOXMLDOC01-appb-C000018
　ペプチド１は、Ｒｉｎｋ　Ａｍｉｄｅ　ｒｅｓｉｎ（０．３７ｍｍｏｌ／ｇ，渡辺化学工業株式会社）３０ｍｇ（０．０１１ｍｍｏｌ）を用いて合成例１と同様の手法により合成および精製した（８．２ｍｇ，２４％）。
ＬＲＭＳ（ＭＡＬＤＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）２３９２．０３，ｆｏｕｎｄ　２３９１．０４。 Peptide 1 was synthesized and purified by the same method as in Synthesis Example 1 using Rink Amide resin (0.37 mmol / g, Watanabe Chemical Industry Co., Ltd.) 30 mg (0.011 mmol) (8.2 mg, 24%).
LRMS (MALDI +) calcd for (M + + H) 2392.03, found 2391.04.
　化合物２は、以前の報告と同様の手法により合成した（Ｈ．Ｏｋａｍｏｔｏ　ｅｔ　ａｌ．，Ｃｈｅｍ．Ｃｏｍｍｕｎ．，２０１９，５５，９１０８－９１１１．）。 Compound 2 was synthesized by the same method as previously reported (H. Okamoto et al., Chem.Commun., 2019, 55, 9108-9111.).
　Ｎ，Ｎ－ジメチルホルムアミド（ＤＭＦ）／メタノール（ＭｅＯＨ）（１：１）中、ペプチド１（０．６０μｍｏｌ，１ｅｑ．）および化合物２（０．６６μｍｏｌ，１．１ｅｑ．）を、トリス［（１－ベンジル－１Ｈ－１，２，３－トリアゾール－４－イル）メチル］アミン（ＴＢＴＡ，２．４μｍｏｌ，４ｅｑ．）、Ｌ－アスコルビン酸（１４μｍｏｌ，２４ｅｑ．）およびテトラキス（アセトニトリル）銅（Ｉ）ヘキサフルオロホスファート（１２μｍｏｌ，２０ｅｑ．）存在下、室温（２５℃）で１時間攪拌し、高速液体クロマトグラフィーを用いて精製することにより、濃紺色固体を得た（０．４５ｍｇ，収率２０％）。
ＨＲＭＳ（ＥＳＩ＋）ｃａｌｃｄ　ｆｏｒ（Ｍ＋＋Ｈ）３０６２．６７５２，ｆｏｕｎｄ　３０６２．６８０９。 Peptide 1 (0.60 μmol, 1 eq.) And compound 2 (0.66 μmol, 1.1 eq.) In N, N-dimethylformamide (DMF) / methanol (MeOH) (1: 1) were added to Tris [(1). -Benzyl-1H-1,2,3-triazole-4-yl) methyl] amine (TBTA, 2.4 μmol, 4 eq.), L-ascorbic acid (14 μmol, 24 eq.) And tetrakis (nitrile) copper (I). In the presence of hexafluorophosphate (12 μmol, 20 eq.), The mixture was stirred at room temperature (25 ° C.) for 1 hour and purified using high-speed liquid chromatography to obtain a dark blue solid (0.45 mg, yield 20). %).
HRMS (ESI +) calcd for (M + + H) 3062.6752, found 3062.6809.
　（試験例１）ｉｎ　ｖｉｔｒｏ　レポーターアッセイによるペプチド誘導体のマイオスタチン阻害活性評価
　各ペプチド誘導体（０．３μＭ）のマイオスタチン阻害活性評価は、以下の手法により実施した。合成例１～１２にて合成した誘導体の結果を図１に、合成例１３～１９にて合成した誘導体の結果を図２に、合成例２０～２４にて合成した誘導体の結果を図３に、合成例２５～２７にて合成した誘導体の結果を図４に示す。比較例として合成した合成例５および２４のペプチド誘導体を除き、本発明に係るペプチド誘導体は０．３μＭの濃度において顕著なマイオスタチン阻害活性を示した。 (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 of the derivatives synthesized in Synthesis Examples 1 to 12, the results of the derivatives synthesized in Synthesis Examples 13 to 19 are shown in FIG. 2, and the results of the derivatives synthesized in Synthesis Examples 20 to 24 are shown in FIG. The results of the derivatives synthesized in Synthesis Examples 25 to 27 are shown in FIG. Except for the peptide derivatives of Synthesis Examples 5 and 24 synthesized as comparative examples, the peptide derivatives according to the present invention showed remarkable myostatin inhibitory activity at a concentration of 0.3 μM.
　（１）細胞培養
　ヒト胎児腎細胞ＨＥＫ２９３細胞は、非必須アミノ酸（富士フイルム和光純薬株式会社）が添加された１０％ＦＢＳ　ＤＭＥＭ（ナカライテスク株式会社）培地を用いて、３７℃、５％　ＣＯ２インキュベータで培養した。 (1) Cell culture Human fetal bovine kidney cells HEK293 cells were prepared at 37 ° C. and 5% CO using 10% FBS DMEM (Nakalitesk Co., Ltd.) medium supplemented with non-essential amino acids (Fujifilm Wako Pure Chemical Industries, Ltd.). 2 Incubator cultured.
　（２）ｉｎ　ｖｉｔｒｏレポーターアッセイ
　Ｄ－Ｌｙｓ－ｃｏａｔｅｄ　９６ウェル透明プレート（サーモフィッシャーサイエンティフィック社）にＨＥＫ２９３細胞を１ウェルたり２．０ｘ１０４　ｃｅｌｌｓ（１００μＬ　ＤＭＥＭ＋１０％（ｖ／ｖ）ＦＢＳ）播種し、２４時間培養した。 (2) In vitro reporter assay 1 well of HEK293 cells or 2.0x10 4 cells (100 μL DMEM + 10% (v / v) FBS) was seeded on a D-Lys-coated 96-well transparent plate (Thermo Fisher Scientific). It was cultured for 24 hours.
　その翌日、１ウェルあたり１００ｎｇのｐＧＬ４．４８［ｌｕｃ２Ｐ／ＳＢＥ／Ｈｙｇｒｏ］（プロメガ社）、およびインターナルコントロールとして１０ｎｇのｐＧＬ４［ｈＲｌｕｃ／ＴＫ］（プロメガ社）をＦｕＧＥＮＥ　ＨＤ（プロメガ社）の最終濃度が４１．２５μｇ／ｍＬになるようＯＰＴＩ－ＭＥＭを用いて混合し、ウェル中の培地に添加した。この細胞を３７℃、２４時間培養したのち、細胞培養液を無血清ＤＭＥＭに交換して８時間培養した。 The next day, 100 ng of pGL4.48 [luc2P / SBE / Hygro] (Promega) per well and 10 ng of pGL4 [hRluc / TK] (Promega) as an internal control at the final concentration of FuGENE HD (Promega). Was mixed using OPTI-MEM to a concentration of 41.25 μg / mL and added to the medium in the wells. After culturing these cells at 37 ° C. for 24 hours, the cell culture medium was replaced with serum-free DMEM and cultured for 8 hours.
　試験資料であるペプチドはストック溶液として１０ｍＭになるようＤＭＳＯを用いて懸濁させ、－３０℃で保存した。培地に添加する１時間前に、無血清ＤＭＥＭ培地で懸濁して、室温（２５℃）で２０分間静置した。次いで、０．３μＭの最終濃度のペプチドと、８ｎｇ／ｍＬのマイオスタチン（メルクミリポア社）とになるように培地に添加し、その後４時間培養を行った。 The peptide as the test material was suspended in DMSO so as to be 10 mM as a stock solution, and stored at -30 ° C. 1 hour before addition to the medium, the cells were suspended in serum-free DMEM medium and allowed to stand at room temperature (25 ° C.) for 20 minutes. Then, the peptide was added to a medium having a final concentration of 0.3 μM and 8 ng / mL myostatin (Merck Millipore), and then cultured for 4 hours.
　ポジティブコントロールとして用いたプロペプチドタンパク質（ＲＳＤ社）は、ストック溶液として１０μＭになるように０．１％（ｖ／ｖ）ＢＳＡ含有ＰＢＳを用いて懸濁し、－３０℃で保存した。培地に添加する１時間前に、無血清ＤＭＥＭ培地で懸濁して室温（２５℃）で２０分間静置した。次いで、１０ｎＭの最終濃度のプロペプチドタンパク質と、８ｎｇ／ｍＬのマイオスタチン（メルクミリポア社）とになるように培地に添加し、その後４時間培養を行った。 The propeptide protein (RSD) used as a positive control was suspended using PBS containing 0.1% (v / v) BSA so as to be 10 μM as a stock solution, and stored at -30 ° C. 1 hour before addition to the medium, the cells were suspended in serum-free DMEM medium and allowed to stand at room temperature (25 ° C.) for 20 minutes. Then, a final concentration of 10 nM of the propeptide protein and 8 ng / mL of myostatin (Merck Millipore) were added to the medium, and then the cells were cultured for 4 hours.
　４時間の培養後、アスピレーターで培養液を取り除き、細胞を１×ＰＢＳで洗浄した。その後、１ウェルあたり５０μＬのＰａｓｓｉｖｅ　Ｌｙｓｉｓ　ｂｕｆｆｅｒ（プロメガ社）を加えて細胞を溶解した。溶解液を４℃条件下、４５００ｒｐｍで６分間遠心分離した。遠心分離後の上清２０μＬを白色９６ウェルプレート（コスター社）へ移した後、５０μＬのＬｕｃｉｆｅｒａｓｅ　Ａｓｓａｙ　Ｒｅａｇｅｎｔ（プロメガ社）を添加し、発光をＬｕｍｉｎｏｓｋａｎ　Ａｓｃｅｎｔ（サーモフィッシャーサイエンティフィック社）で検出してホタルルシフェラーゼ活性を測定した。さらに、５０μＬのＳｔｏｐ　＆　Ｇｌｏ　Ｂｕｆｆｅｒを添加した後、発光をＬｕｍｉｎｏｓｋａｎ　Ａｓｃｅｎｔで検出してウミシイタケルシフェラーゼ活性を測定することで、インターナルコントロールとした。 After culturing for 4 hours, the culture medium was removed with an aspirator, and the cells were washed with 1 × 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 under 4 ° C. conditions. After transferring 20 μL of the supernatant after centrifugation to a white 96-well plate (Coster), 50 μL of Luciferase Assay Reagent (Promega) was added, and the luminescence was detected by Luminoscan Ascent (Thermo Fisher Scientific). Firefly luciferase activity was measured. Further, after adding 50 μL of Stop & Glo Buffer, luminescence was detected by Luminoscan Ascent and the luciferase activity was measured to obtain internal control.
　（試験例２）ペプチドのウシ膵臓由来トリプシン溶液中での安定性評価
　合成例２３で合成したｒｉＤＭ－３５のウシ膵臓由来トリプシン溶液中での安定性を検証するため以下の手法により評価した。 (Test Example 2) Evaluation of Peptide Stability in Bovine Pancreas-Derived Trypsin Solution The stability of riDM-35 synthesized in Synthesis Example 23 in bovine pancreas-derived trypsin solution was evaluated by the following method.
　ｒｉＤＭ－３５を最終濃度５０μＭになるように５０ｍＭ　Ｔｒｉｓ－ＨＣｌバッファー（ｐＨ７．５、０．１５Ｍ　ＮａＣｌ、１０ｍＭ　ＣａＣｌ２、０．０５％（ｗ／ｖ）Ｂｒｉｊ－３５含有）溶液に溶解し、ＴＰＣＫ処理済みウシ膵臓由来トリプシン（シグマアルドリッチ社）を最終濃度１μｇ／ｍＬになるように添加し（総容量１００μＬ）、３７℃で４００分間インキュベートした。インキュベート開始０分およびインキュベート開始後４００分に２０μＬをサンプリングし、２５％アセトニトリル水溶液８０μＬで希釈後、２０μＬを高速液体クロマトグラフィー（ＨＰＬＣ、株式会社日立ハイテク、Ｃｈｒｏｍａｓｔｅｒ（登録商標））に注入し、得られたｒｉＤＭ－３５のピーク面積に基づく残存率から安定性を解析した。カラムはＣＯＳＭＯＳＩＬ　５Ｃ１８－ＡＲ－ＩＩ　４．６×１５０ｍｍ（ナカライテスク株式会社）を用い、１ｍＬ／ｍｉｎの流速にて、０．１％トリフルオロ酢酸含有水－アセトニトリル系におけるアセトニトリルの直線グラジエント（２５～４０％，３０分）をかけることで、ＵＶ検出器（２２０ｎｍ）でペプチドのピークを検出した。 RiDM-35 was dissolved in a 50 mM Tris-HCl buffer (pH 7.5, 0.15 M NaCl, 10 mM CaCl 2 , 0.05% (w / v) Brij-35 containing) solution to a final concentration of 50 μM and TPCK. Treated bovine pancreatic trypsin (Sigma-Aldrich) was added to a final concentration of 1 μg / mL (total volume 100 μL) and incubated at 37 ° C. for 400 minutes. 20 μL was sampled 0 minutes after the start of the incubation and 400 minutes after the start of the incubation, diluted with 80 μL of a 25% acetonitrile aqueous solution, and then 20 μL was injected into high performance liquid chromatography (HPLC, Hitachi High-Tech Co., Ltd., Chromaster®) to obtain the obtained product. Stability was analyzed from the residual rate based on the peak area of riDM-35. The column used was COSMOSIL 5C18-AR-II 4.6 × 150 mm (Nacalai Tesque, Inc.), and a linear gradient of acetonitrile in a water-acetonitrile system containing 0.1% trifluoroacetic acid (25 to 25 to min) at a flow rate of 1 mL / min. By applying 40%, 30 minutes), the peak of the peptide was detected with a UV detector (220 nm).
　この結果を図５に示す。ｒｉＤＭ－３５に由来する分解物は、ＬＣＭＳ－２０２０（株式会社島津製作所）を用いた質量分析にて同定されなかった。ｒｉＤＭ－３５はウシ膵臓由来トリプシン溶液中で極めて高い安定性を示した。 This result is shown in Fig. 5. The decomposition product derived from riDM-35 was not identified by mass spectrometry using LCMS-2020 (Shimadzu Corporation). riDM-35 showed extremely high stability in bovine pancreas-derived trypsin solution.
　（試験例３）ｒｉＤＭ－３５のウシ膵臓由来α－キモトリプシン溶液中での安定性評価
　合成例２３で合成したｒｉＤＭ－３５のウシ膵臓由来α－キモトリプシン溶液中での安定性を検証するため以下の手法により評価した。 (Test Example 3) Evaluation of stability of riDM-35 in bovine pancreas-derived α-chymotrypsin solution In order to verify the stability of riDM-35 synthesized in Synthesis Example 23 in bovine pancreas-derived α-chymotrypsin solution, the following It was evaluated by the method.
　ｒｉＤＭ－３５を最終濃度５０μＭになるように１００ｍＭ　Ｔｒｉｓ－ＨＣｌバッファー（ｐＨ７．８、１０ｍＭ　ＣａＣｌ２含有）溶液に溶解し、ＴＬＣＫ処理済みウシ膵臓由来α－キモトリプシン（シグマアルドリッチ社）を最終濃度２　μｇ／ｍＬになるように添加して（総容量１００μＬ）、３７℃で４００分間インキュベートした。インキュベート開始０分およびインキュベート開始後４００分に２０μＬをサンプリングし、２５％アセトニトリル水溶液８０μＬで希釈後、２０μＬを高速液体クロマトグラフィー（ＨＰＬＣ、株式会社日立ハイテク、Ｃｈｒｏｍａｓｔｅｒ（登録商標））に注入し、得られたｒｉＤＭ－３５のピーク面積に基づく残存率から安定性を解析した。カラムはＣＯＳＭＯＳＩＬ　５Ｃ１８－ＡＲ－ＩＩ　４．６×１５０ｍｍ（ナカライテスク株式会社）を用い、１ｍＬ／ｍｉｎの流速にて、０．１％トリフルオロ酢酸含有水－アセトニトリル系におけるアセトニトリルの直線グラジエント（２５～４０％，３０分）をかけることで、ＵＶ検出器（２２０ｎｍ）でペプチドのピークを検出した。 Dissolve riDM-35 in 100 mM Tris-HCl buffer (pH 7.8, containing 10 mM CaCl 2 ) solution to a final concentration of 50 μM, and add TLCK-treated bovine pancreas-derived α-chymotrypsin (Sigma-Aldrich) to a final concentration of 2 μg. It was added to / mL (total volume 100 μL) and incubated at 37 ° C. for 400 minutes. 20 μL was sampled 0 minutes after the start of the incubation and 400 minutes after the start of the incubation, diluted with 80 μL of a 25% acetonitrile aqueous solution, and then 20 μL was injected into high performance liquid chromatography (HPLC, Hitachi High-Tech Co., Ltd., Chromaster®) to obtain the obtained product. Stability was analyzed from the residual rate based on the peak area of riDM-35. The column used was COSMOSIL 5C18-AR-II 4.6 × 150 mm (Nacalai Tesque, Inc.), and a linear gradient of acetonitrile in a water-acetonitrile system containing 0.1% trifluoroacetic acid (25 to 25 to min) at a flow rate of 1 mL / min. By applying 40%, 30 minutes), the peak of the peptide was detected with a UV detector (220 nm).
　この結果を図６に示す。ｒｉＤＭ－３５に由来する分解物は、ＬＣＭＳ－２０２０（株式会社島津製作所）を用いた質量分析にて同定されなかった。ｒｉＤＭ－３５はウシ膵臓由来α－キモトリプシン溶液中で極めて高い安定性を示した。 This result is shown in FIG. The decomposition product derived from riDM-35 was not identified by mass spectrometry using LCMS-2020 (Shimadzu Corporation). riDM-35 showed extremely high stability in a bovine pancreas-derived α-chymotrypsin solution.
　（試験例４）ｒｉＤＭ－４のデュシェンヌ型筋ジストロフィーモデルｍｄｘマウス前脛骨筋に及ぼす影響（ｉｎ　ｖｉｖｏ評価）
　合成例１で合成したｒｉＤＭ－４のｉｎ　ｖｉｖｏにおける握力増大効果を検証するため以下の手法により評価を行った。 (Test Example 4) Effect of riDM-4 on Duchenne muscular dystrophy model mdx mouse tibialis anterior muscle (in vivo evaluation)
In order to verify the in vivo grip strength increasing effect of riDM-4 synthesized in Synthesis Example 1, evaluation was performed by the following method.
　ｒｉＤＭ－４を生理食塩水に０．７５ｍＭになるよう溶解した。麻酔下にある５週齢のｍｄｘ雄マウス（日本クレア社より購入）の両後肢計８箇所の筋肉内に４０μＬ筋肉内投与した（対照群には生理食塩水を同様に４０μＬ投与した）。２週間後、再度同じ量のｒｉＤＭ－４（対照群には生理食塩水）を投与し、さらに４週間後、マウスの握力測定（齋藤式マウス用握力測定装置ＭＫ－３８０Ｍ、室町機械株式会社）を行った。 RiDM-4 was dissolved in physiological saline to a concentration of 0.75 mM. 40 μL intramuscularly was intramuscularly administered to a total of 8 muscles of both hind limbs of a 5-week-old mdx male mouse (purchased from Claire Japan) under anesthesia (40 μL of saline was similarly administered to the control group). Two weeks later, the same amount of riDM-4 (physiological saline in the control group) was administered again, and four weeks later, the mouse grip strength was measured (Saito-type mouse grip strength measuring device MK-380M, Muromachi Kikai Co., Ltd.). Was done.
　この結果を図７に示す。ｒｉＤＭ－４はそのマイオスタチン阻害活性に基づいて６０％程度ｍｄｘマウスの後肢の握力を有意に増大させた。既存のＭＩＰＥ－１６８６よりも改善効果が大きかった。 This result is shown in Fig. 7. riDM-4 significantly increased the grip strength of the hind limbs of mdx mice by about 60% based on its myostatin inhibitory activity. The improvement effect was greater than that of the existing MIPE-1686.
　（試験例５）１６ＰＣ－Ｎを用いたマイオスタチン酸素化評価
　合成例２８で合成した１６ＰＣ－Ｎのマイオスタチン酸素化は、以下の手法により実施した。 (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.
　リン酸緩衝液（１０ｍＭ，ｐＨ７．４）中の１６ＰＣ－Ｎ（３μＭ）とマイオスタチン（１μＭ）とに、３７℃下で３０分間光照射（７３０ｎｍ，１４ｍＷ）を行った。ジチオスレイトールによる還元、リシルエンドペプチドダーゼによる消化、ＺｉｐＴｉｐ　Ｃ１８による脱塩処理を経て、ＭＡＬＤＩ－ＴＯＦ　ＭＳ分析を行った。 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 lysylendopeptide dase, and desalting with ZipTip C18, MALDI-TOF MS analysis was performed.
　この結果を図８に示す。反応後のサンプルでマイオスタチン酸素化体が観測され、１６ＰＣ－Ｎによってマイオスタチンが光酸素化されたことが示唆された。 This result is shown in FIG. An oxygenated myostatin was observed in the post-reaction sample, suggesting that myostatin was photooxygenated by 16PC-N.
　（試験例５）ｒｉＤＭ－３５の筋肉内投与によるがん悪液質モデルマウスの筋肉消耗改善効果の検証（ｉｎ　ｖｉｖｏ評価）
　合成例２３で合成したｒｉ－ＤＭ３５をＰＢＳ（リン酸緩衝液；１０ｍＭ，ｐＨ７．４）に１ｍＭになるよう溶解した。麻酔下にあるＣ５７ＢＬ６／Ｊ雄マウス（オリエンタル酵母より購入）の背部皮下に、ルイス肺腺癌細胞（ＬＬＣ）を５．０×１０６移植してがん悪液質モデルマウスを作製した。移植後４、１１、１８日にｒｉ－ＤＭ３５溶液を両足腓腹筋に３０μＬずつ筋肉内投与した（対象群にはＰＢＳを同様に３０μＬ投与した）。ＬＬＣ移植２２日目にマウスの四肢握力をイマダデジタルフォースゲージにより測定した。 (Test Example 5) Verification of the effect of intramuscular administration of riDM-35 on improving muscle wasting in a cancer cachexia model mouse (in vivo evaluation)
The ri-DM35 synthesized in Synthesis Example 23 was dissolved in PBS (phosphate buffer; 10 mM, pH 7.4) to 1 mM. A 5.0 × 10 6 Lewis lung adenocarcinoma cell (LLC) was transplanted subcutaneously into the back of an anesthetized C57BL6 / J male mouse (purchased from Oriental yeast) to prepare a cancer cachexia model mouse. On 4, 11 and 18 days after transplantation, 30 μL of ri-DM35 solution was intramuscularly administered to both gastrocnemius muscles (30 μL of PBS was also administered to the target group). On the 22nd day of LLC transplantation, the grip strength of the extremities of the mice was measured by the Imada Digital Force Gauge.
　この結果を図９に示す。ｒｉ－ＤＭ３５投与群はがん悪液質モデルマウスの握力を４１．３％改善した。 This result is shown in Fig. 9. The ri-DM35-administered group improved the grip strength of the cancer cachexia model mice by 41.3%.
　また、ＬＬＣ移植２２日目にマウスの剖検を行い、両足腓腹筋を摘出して重量および筋線維面積を測定した。 In addition, an autopsy of the mice was performed on the 22nd day of LLC transplantation, and the gastrocnemius muscles of both feet were excised to measure the weight and muscle fiber area.
　がん悪液質モデルマウスの体重当たりの腓腹筋重量を図１０に示す。ｒｉ－ＤＭ３５投与群はがん悪液質モデルマウスの体重当たりの腓腹筋重量が１９．６％増加した。 Figure 10 shows the weight of the gastrocnemius muscle per body weight of the cancer cachexia model mouse. In the ri-DM35-administered group, the gastrocnemius muscle weight per body weight of the cancer cachexia model mice increased by 19.6%.
　がん悪液質モデルマウスの筋線維面積の度数分布表を図１１に示す。ｒｉ－ＤＭ３５投与群はがん悪液質モデルマウスの筋線維面積を肥大した。 Figure 11 shows the frequency distribution table of the muscle fiber area of the cancer cachexia model mouse. The ri-DM35-administered group enlarged the muscle fiber area of the cancer cachexia model mice.
　本出願は、２０２０年７月６日に出願された日本国特許出願第２０２０－１１６５８３号に基づいており、その開示内容は、参照により全体として引用されている。 This application is based on Japanese Patent Application No. 2020-116583 filed on July 6, 2020, and the disclosure content is cited as a whole by reference.

Claims (20)

  1. 　下記式（１）で表されるアミノ酸配列を含み、アミノ酸残基数が１５～１７である、ペプチド、もしくはその薬学的に許容される塩、またはそれらのプロドラッグ：
    Figure JPOXMLDOC01-appb-C000001
    　上記式（１）において、
    　Ｘ０は、Ｄ－Ａｌａ、Ｄ－ＧｌｙおよびＤ－２－アミノイソ酪酸からなる群から選択されるアミノ酸残基または欠損であり；
    　Ｘ１は、Ｄ－Ｌｅｕ、Ｄ－ノルロイシン、Ｄ－Ｖａｌ、Ｄ－Ｉｌｅ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基または欠損であり；
    　Ｘ２は、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｌｙｓ、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
    　Ｘ３は、Ｄ－２－シクロヘキシルグリシン、Ｄ－ノルロイシン、Ｄ－Ｌｅｕ、Ｄ－Ｖａｌ、Ｄ－Ｉｌｅ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基であり；
    　Ｘ４は、Ｄ－Ｌｙｓ、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
    　Ｘ５は、Ｄ－Ｓｅｒ、Ｄ－Ａｒｇ、Ｄ－２－ヒドロキシグリシン、Ｄ－ホモセリン、Ｄ－Ｌｙｓ、Ｄ－オルニチン、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
    　Ｘ６は、Ｄ－Ｔｒｐ、Ｄ－３－（２－ナフチル）アラニン、Ｄ－Ｔｙｒ、Ｄ－ＰｈｅおよびＤ－３－（１－ナフチル）アラニンからなる群から選択されるアミノ酸残基であり；　Ｘ７は、Ｄ－２－フェニルグリシン、Ｄ－ノルロイシン、Ｄ－Ｉｌｅ、Ｄ－Ｌｅｕ、Ｄ－Ｖａｌ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基であり；
    　Ｘ８は、Ｄ－Ｇｌｎ、Ｄ－Ａｒｇ、Ｄ－Ａｓｎ、Ｄ－Ｌｙｓ、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸、Ｄ－２，４－ジアミノブタン酸およびＤ－オルニチンからなる群から選択されるアミノ酸残基であり；
    　Ｘ９は、Ｄ－２－シクロヘキシルグリシン残基であり；
    　Ｘ１０は、Ｄ－Ｌｙｓ、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
    　Ｘ１１は、Ｄ－２－フェニルグリシン、Ｄ－Ｉｌｅ、Ｄ－Ｌｅｕ、Ｄ－ノルロイシン、Ｄ－Ｖａｌ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基であり；
    　Ｘ１２は、Ｄ－Ｔｒｐ、Ｄ－ホモフェニルアラニン、Ｄ－ＴｙｒおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基であり；
    　Ｘ１３は、Ｄ－Ａｒｇ、Ｄ－オルニチン、Ｄ－Ｌｙｓ、Ｄ－Ｈｉｓ、Ｄ－２，３－ジアミノプロピオン酸およびＤ－２，４－ジアミノブタン酸からなる群から選択されるアミノ酸残基であり；
    　Ｘ１４は、Ｄ－２－フェニルグリシン、Ｄ－Ｉｌｅ、Ｄ－Ｌｅｕ、Ｄ－ノルロイシン、Ｄ－Ｖａｌ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基であり；
    　Ｘ１５は、Ｄ－Ｔｙｒ、Ｄ－ＴｒｐおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基であり；ならびに
    　Ｘ１６は、Ｄ－Ｔｒｐ、Ｄ－ホモフェニルアラニン、Ｄ－ＴｙｒおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基または欠損である。     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.
    Figure JPOXMLDOC01-appb-C000001
    In the above formula (1)
    X 0 is an amino acid residue or defect selected from the group consisting of D-Ala, D-Gly and D-2-aminoisobutyric acid;
    X 1 is an amino acid residue or defect 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. can be;
    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. Is the basis;
    X 4 is, D-Lys, D-Arg , D- ornithine, D-His, with an amino acid residue selected from the group consisting of D-2,3-diaminopropionic acid and D-2,4-diaminobutane acid can be;
    X 5 contains D-Ser, D-Arg, D-2-hydroxyglycine, D-homoserine, D-Lys, D-ornithine, D-His, D-2,3-diaminopropionic acid and D-2,4. -Amino acid residues selected from the group consisting of 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. Is the basis;
    X 8 is, D-Gln, D-Arg , D-Asn, D-Lys, D-His, D-2,3- diaminopropionic acid, the group consisting of D-2,4-diaminobutanoic acid and D- ornithine Amino acid residues selected from;
    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. can be;
    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. Is the basis;
    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. can be;
    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. Is the basis;
    X 15 is an amino acid residue selected from the group consisting of D-Tyr, D-Trp and D-Phe; and X 16 is from D-Trp, D-homophenylalanine, D-Tyr and D-Phe. Amino acid residues or defects selected from the group consisting of.
  2. 　Ｘ１１は、Ｄ－２－フェニルグリシン残基であり；および
    　Ｘ１４は、Ｄ－２－フェニルグリシン残基である、請求項１に記載のペプチド、もしくはその薬学的に許容される塩、またはそれらのプロドラッグ。     X 11 is a D-2-phenylglycine residue; and X 14 is a D-2-phenylglycine residue, the peptide of claim 1, or a pharmaceutically acceptable salt thereof, or. Those prodrugs.
  3. 　Ｘ６は、Ｄ－Ｔｒｐ、Ｄ－ＴｙｒおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基であり；ならびに
    　Ｘ１６は、Ｄ－Ｔｒｐ、Ｄ－ＴｙｒおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基または欠損である、請求項２に記載のペプチド、もしくはその薬学的に許容される塩、またはそれらのプロドラッグ。     X 6 is an amino acid residue selected from the group consisting of D-Trp, D-Tyr and D-Phe; and X 16 is selected from the group consisting of D-Trp, D-Tyr and D-Phe. The peptide according to claim 2, or a pharmaceutically acceptable salt thereof, or a prodrug thereof, which is an amino acid residue or a defect thereof.
  4. 　Ｘ３は、Ｄ－２－シクロヘキシルグリシン残基であり；
    　Ｘ７は、Ｄ－Ｉｌｅ、Ｄ－Ｌｅｕ、Ｄ－ノルロイシン、Ｄ－Ｖａｌ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基であり；
    　Ｘ１１は、Ｄ－Ｉｌｅ、Ｄ－Ｌｅｕ、Ｄ－ノルロイシン、Ｄ－Ｖａｌ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基であり；ならびに
    　Ｘ１４は、Ｄ－Ｉｌｅ、Ｄ－Ｌｅｕ、Ｄ－ノルロイシン、Ｄ－Ｖａｌ、Ｄ－２－アミノ酪酸、Ｄ－ノルバリンおよびＤ－イソバリンからなる群から選択されるアミノ酸残基である、請求項１に記載のペプチド、もしくはその薬学的に許容される塩、またはそれらのプロドラッグ。     X 3 is a D-2-cyclohexylglycine residue;
    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 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; and X 14 1 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, according to claim 1. Peptides, or pharmaceutically acceptable salts thereof, or prodrugs thereof.
  5. 　Ｘ１６は、Ｄ－Ｔｒｐ、Ｄ－ホモフェニルアラニン、Ｄ－ＴｙｒおよびＤ－Ｐｈｅからなる群から選択されるアミノ酸残基である、請求項４に記載のペプチド、もしくはその薬学的に許容される塩、またはそれらのプロドラッグ。 X 16 is the peptide according to claim 4, or a pharmaceutically acceptable salt thereof, which is an amino acid residue selected from the group consisting of D-Trp, D-homophenylalanine, D-Tyr and D-Phe. , Or their prodrugs.
  6. 　Ｘ０は、Ｄ－Ａｌａ残基または欠損であり；
    　Ｘ１は、Ｄ－Ｌｅｕ残基、Ｄ－ノルロイシン残基または欠損であり；
    　Ｘ２は、Ｄ－Ａｒｇ残基またはＤ－オルニチン残基であり；
    　Ｘ３は、Ｄ－２－シクロヘキシルグリシン残基またはＤ－ノルロイシン残基であり；
    　Ｘ４は、Ｄ－Ｌｙｓ残基、Ｄ－Ａｒｇ残基またはＤ－オルニチン残基であり；
    　Ｘ５は、Ｄ－Ｓｅｒ残基またはＤ－Ａｒｇ残基であり；
    　Ｘ６は、Ｄ－Ｔｒｐ残基またはＤ－３－（２－ナフチル）アラニン残基であり；
    　Ｘ７は、Ｄ－２－フェニルグリシン残基またはＤ－Ｉｌｅ残基であり；
    　Ｘ８は、Ｄ－Ｇｌｎ残基またはＤ－Ａｒｇ残基であり；
    　Ｘ９は、Ｄ－２－シクロヘキシルグリシン残基であり；
    　Ｘ１０は、Ｄ－Ｌｙｓ、Ｄ－ＡｒｇおよびＤ－オルニチンからなる群から選択されるアミノ酸残基であり；
    　Ｘ１１は、Ｄ－２－フェニルグリシン残基またはＤ－Ｉｌｅ残基であり；
    　Ｘ１２は、Ｄ－Ｔｒｐ残基またはＤ－ホモフェニルアラニン残基であり；
    　Ｘ１３は、Ｄ－Ａｒｇ残基またはＤ－オルニチン残基であり；
    　Ｘ１４は、Ｄ－２－フェニルグリシン残基またはＤ－Ｉｌｅ残基であり；
    　Ｘ１５は、Ｄ－Ｔｙｒ残基であり；ならびに
    　Ｘ１６は、Ｄ－Ｔｒｐ残基、Ｄ－ホモフェニルアラニン残基または欠損である、請求項１に記載のペプチド、もしくはその薬学的に許容される塩、またはそれらのプロドラッグ。     X 0 is a D-Ala residue or defect;
    X 1 is a D-Leu residue, a D-norleucine residue or a defect;
    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, D-Lys residues, be a D-Arg residue, or D- ornithine residues;
    X 5 is a D-Ser residue or a 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 an D-Gln residue or D-Arg residues;
    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 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 defect, the peptide of claim 1 or pharmaceutically acceptable thereof. Salt, or their prodrugs.
  7. 　配列番号１～２５で表されるアミノ酸配列のいずれか１つを含む、請求項６に記載のペプチドもしくはその薬学的に許容される塩、またはそれらのプロドラッグ。
    Figure JPOXMLDOC01-appb-T000002
         The peptide according to claim 6, a pharmaceutically acceptable salt thereof, or a prodrug thereof, which comprises any one of the amino acid sequences represented by SEQ ID NOs: 1 to 25.
    Figure JPOXMLDOC01-appb-T000002
  8. 　配列番号１～４、６、８～２３および２５で表されるアミノ酸配列のいずれか１つを含む、請求項６または７に記載のペプチドもしくはその薬学的に許容される塩、またはそれらのプロドラッグ。 The peptide according to claim 6 or 7, a pharmaceutically acceptable salt thereof, or a prodrug thereof, comprising any one of the amino acid sequences represented by SEQ ID NOs: 1 to 4, 6, 8 to 23 and 25. drag.
  9. 　配列番号１９～２３および２５で表されるアミノ酸配列のいずれか１つを含む、請求項６～８のいずれか１項に記載のペプチドもしくはその薬学的に許容される塩、またはそれらのプロドラッグ。 The peptide according to any one of claims 6 to 8, which comprises any one of the amino acid sequences represented by SEQ ID NOs: 19 to 23 and 25, or a pharmaceutically acceptable salt thereof, or a prodrug thereof. ..
  10. 　下記式（２）で表される複合体またはその薬学的に許容される塩：
    Figure JPOXMLDOC01-appb-C000003
    　式（２）中、
    　Ｙは、下記式（３）：
    Figure JPOXMLDOC01-appb-C000004
    ［式３中、
    　Ｒ１およびＲ２は、それぞれ独立して、ハロゲノアルキル基またはハロゲン原子を表し、
    　Ｒ３は、臭素原子、ヨウ素原子またはセレン原子を表し、
    　Ｒ４およびＲ５は、それぞれ独立して、水素原子または置換もしくは非置換のアルキル基を表し、
    　Ｒ６およびＲ７は、それぞれ独立して、水素原子、ハロゲン原子、アルコキシ基または置換もしくは非置換のアルキル基を表し、この際Ｒ４およびＲ６またはＲ５およびＲ７が一緒になって、置換または非置換のアルキレン基またはアルケニレン基を形成してもよく、
    　Ｒ８は、水素原子または置換もしくは非置換のアルキル基を表し、
    　Ｒ９およびＲ１０は、それぞれ独立して、水素原子、ハロゲン原子、アルコキシ基または置換もしくは非置換のアルキル基を表し、この際Ｒ８とＲ９またはＲ１０とが一緒になって、置換または非置換のアルキレン基またはアルケニレン基を形成してもよく、
    　ｍおよびｎは、１～３の整数を表し、
    　＊は、Ｌとの結合部位である］
    で表される化合物であり；
    　Ｌは、ＹおよびＺ間のリンカーを表し；
    　Ｚは、請求項１～９のいずれか１項に記載のペプチドである。     A complex represented by the following formula (2) or a pharmaceutically acceptable salt thereof:
    Figure JPOXMLDOC01-appb-C000003
    In equation (2),
    Y is the following formula (3):
    Figure JPOXMLDOC01-appb-C000004
    [In Equation 3,
    R 1 and R 2 independently represent a halogenoalkyl group or a halogen atom, respectively.
    R 3 represents a bromine atom, an iodine atom or a selenium atom.
    R 4 and R 5 each independently represent a hydrogen atom or a substituted or unsubstituted alkyl group.
    R 6 and R 7 each independently represent a hydrogen atom, a halogen atom, an alkoxy group or a substituted or unsubstituted alkyl group, wherein R 4 and R 6 or R 5 and R 7 are combined. Substituted or unsubstituted alkylene groups or alkenylene groups may be formed.
    R 8 represents a hydrogen atom or a substituted or unsubstituted alkyl group.
    R 9 and R 10 each independently represent a hydrogen atom, a halogen atom, an alkoxy group or a substituted or unsubstituted alkyl group, wherein R 8 and R 9 or R 10 are substituted or substituted together. It may form an unsubstituted alkylene group or an alkenylene group.
    m and n represent integers of 1 to 3 and represent
    * Is the binding site with L]
    It is a compound represented by;
    L represents a linker between Y and Z;
    Z is the peptide according to any one of claims 1 to 9.
  11. 　前記Ｒ１がハロゲン原子であり、前記Ｒ２がハロゲノアルキル基である、請求項１０に記載の複合体。 The complex according to claim 10, wherein R 1 is a halogen atom and R 2 is a halogenoalkyl group.
  12. 　前記ｍおよびｎが１である、請求項１０または１１に記載の複合体。 The complex according to claim 10 or 11, wherein m and n are 1.
  13. 　Ｒ４およびＲ６、Ｒ５およびＲ７ならびにＲ８およびＲ１０が一緒になって、置換または非置換のアルキレン基またはアルケニレン基を形成し、この際前記アルキレン基またはアルケニレン基の炭素数が２または３である、請求項１０～１２のいずれか１項に記載の複合体。 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 carbon atoms. Or 3, the complex according to any one of claims 10 to 12.
  14. 　請求項１～９のいずれか１項に記載のペプチドもしくはその薬学的に許容される塩、または請求項１０～１３のいずれか１項に記載の複合体もしくはその薬学的に許容される塩を含む、マイオスタチン阻害剤。 The peptide according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, or the complex according to any one of claims 10 to 13 or a pharmaceutically acceptable salt thereof. Including myostatin inhibitors.
  15. 　請求項１～９のいずれか１項に記載のペプチドもしくはその薬学的に許容される塩、または請求項１０～１３のいずれか１項に記載の複合体もしくはその薬学的に許容される塩を含む、筋萎縮障害の予防および／または治療剤。 The peptide according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, or the complex according to any one of claims 10 to 13 or a pharmaceutically acceptable salt thereof. A prophylactic and / or therapeutic agent for muscular atrophy disorders, including.
  16. 　前記筋萎縮障害が、筋ジストロフィーまたはサルコペニアである、請求項１５に記載の筋萎縮障害の予防および／または治療剤。 The prophylactic and / or therapeutic agent for a muscular atrophy disorder according to claim 15, wherein the muscular atrophy disorder is muscular dystrophy or sarcopenia.
  17. 　前記筋萎縮障害が、糖尿病またはがん悪質液に起因する筋萎縮障害である、請求項１５に記載の筋萎縮障害の予防および／または治療剤。 The preventive and / or therapeutic agent for a muscular atrophy disorder according to claim 15, wherein the muscular atrophy disorder is a muscular atrophy disorder caused by diabetes or a vicious cancer solution.
  18. 　請求項１～９のいずれか１項に記載のペプチドもしくはその薬学的に許容される塩、または請求項１０～１３のいずれか１項に記載の複合体もしくはその薬学的に許容される塩の有効量を患者に投与することを含む、筋萎縮障害の予防および／または治療方法。 The peptide according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof, or the complex according to any one of claims 10 to 13 or a pharmaceutically acceptable salt thereof. A method for preventing and / or treating a muscular atrophy disorder, which comprises administering an effective amount to a patient.
  19. 　前記筋萎縮障害が、筋ジストロフィーまたはサルコペニアである、請求項１８に記載の筋萎縮障害の予防および／または治療方法。 The method for preventing and / or treating a muscular atrophy disorder according to claim 18, wherein the muscular atrophy disorder is muscular dystrophy or sarcopenia.
  20. 　前記筋萎縮障害が、糖尿病またはがん悪質液に起因する筋萎縮障害である、請求項１８に記載の筋萎縮障害の予防および／または治療方法。 The method for preventing and / or treating a muscular atrophy disorder according to claim 18, wherein the muscular atrophy disorder is a muscular atrophy disorder caused by diabetes or a vicious cancer solution.
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