peptide

Abstract

The present invention pertains to a peptide and a composition including the peptide. A peptide according to the present invention includes or comprises an amino acid sequence disclosed in any one of SEQ ID NOs: 3 and 110-140.

Description

peptide

[0001] The present invention relates to peptides, compositions containing said peptides, and uses of said peptides.

[0002] Myostatin, also known as growth differentiation factor 8 (GDF-8), is a secreted protein that is a member of the transforming growth factor beta (TGF-β) superfamily. Like other members of the TGF-β family, myostatin is synthesized as a large precursor protein containing an N-terminal propeptide domain and a C-terminal domain, which is the active molecule. Two molecules of the myostatin precursor are covalently linked via a single disulfide bond present in the C-terminal growth factor domain. Active mature myostatin (a disulfide-linked homodimer consisting of the C-terminal growth factor domain) is released from the myostatin precursor through multiple steps of proteolytic processing.

[0003] In the first step of the myostatin activation pathway, the peptide bond Arg266-Asp267 between the N-terminal propeptide domain and the C-terminal growth factor domain is cleaved in both chains of the homodimeric precursor by a furin-type proprotein convertase. However, the resulting three peptides (two propeptides and one mature myostatin (i.e., a disulfide-linked homodimer consisting of the growth factor domain)) remain associated, forming a noncovalent, inactive complex called "latent myostatin." Mature myostatin can then be released from latent myostatin via degradation of the propeptide. Members of the bone morphogenetic protein 1 (BMP1) family of metalloproteinases cleave a single peptide bond Arg98-Asp99 within the propeptide, concomitantly releasing the homodimeric, active mature myostatin. Furthermore, latent myostatin can also be activated in vitro by dissociating the complex with either acid or heat treatment (Non-Patent Documents 2 and 3).

[0004] Myostatin is produced in striated muscle and is a factor that negatively regulates the hypertrophy of striated muscle fibers. Significant skeletal muscle hypertrophy has been reported in myostatin knockout mice (Non-Patent Document 1). Negative regulation of muscle hypertrophy is not only achieved by myostatin, but also by GDF-11 (Non-Patent Document 4) and activin A (Non-Patent Document 5), both of which belong to the TGF-β superfamily. Myostatin exerts its effects via the transmembrane serine / threonine kinase heterotetramer receptor family. Its activation is achieved by enhancing transphosphorylation of the receptor and its serine / threonine kinase activity. Activated forms of myostatin, GDF-11, and activin A all bind with high affinity to the activin type IIB receptor (ActRIIB). Subsequently, low-affinity receptors such as activin-like kinase 4 (ALK4) or activin-like kinase 5 (ALK5) are recruited, activating their transphosphorylation. Subsequently, the proteins Smad2 and Smad3 are phosphorylated in the cytoplasm, and a complex is formed between these and Smad4, which then translocates into the nucleus and induces target gene expression (Non-Patent Document 6).

[0005] However, it is known that GDF-11 acts as a rejuvenating factor to improve muscle function (Non-Patent Document 7), and activin acts on the pituitary gland to promote the production of follicle-stimulating hormone (FSH) (Non-Patent Document 8). Hypertrophy of striated muscle fibers is controlled by multiple factors with multiple functions. Therefore, it is unclear which factors control muscle strength, which is the original function of striated muscle. The target molecules intended to improve muscle weakness in muscular dystrophy, spinal muscular atrophy (SMA), sarcopenia, frailty, cachexia, stroke, etc., were also unknown.

[0006] In particular, there are reports that blood levels of GDF-11 increase with aging (Non-Patent Document 9) and reports that blood levels decrease with aging (Non-Patent Document 7). Therefore, the role of GDF-11 in regulating muscle strength has not been clear.

[0007] However, it has now been shown that an anti-latent myostatin antibody that specifically inhibits myostatin signaling exhibits stronger muscle strength enhancement in Duchenne muscular dystrophy model mice than antibodies or proteins that also inhibit GDF-11 or activin, and that GDF-11 administered to muscle atrophy model mice improved muscle strength. These experimental results suggest that specific inhibition of myostatin by an anti-latent myostatin antibody in situations where muscle strength is reduced is a useful therapeutic strategy (Non-Patent Document 10). Similarly, an anti-pro-myostatin antibody that specifically inhibits myostatin signaling exhibited an additive effect on muscle strength improvement induced by a small molecule splicing modifier of the SMN2 gene in SMA model mice. This suggests that selective inhibition of myostatin by an anti-pro-myostatin antibody is useful for increasing muscle strength in SMA (Non-Patent Document 11).

[0008] Based on these findings, selective myostatin signal inhibitors using antibodies such as those described above have been developed. However, it is known that antibodies, including IgG, have very low distribution in muscle interstitial fluid (Non-Patent Documents 12 and 13). Myostatin acts in vivo through autocrine, paracrine, and endocrine pathways, but it is believed that most of its action is through autocrine and paracrine pathways in muscle tissue (Non-Patent Document 16). It has also been reported that clathrin-dependent early endosomes are involved in signaling by Smads of the TGF-β superfamily (Non-Patent Documents 14 and 15).

[0009] However, no pharmaceutical composition has been developed that inhibits myostatin signaling not only extracellularly but also intracellularly.

[0010] McPherron et al., Nature 1997; 387(6628):83-90.Szlama et al., FEBS J 280(16):3822-3839(2013)Lee, PloS One 3(2):e1628(2008)Hammers et al., EMBO Mol Med. 2017;9(4):531-44.Latres et al., Nat Commun.2017;8:15153.Lee, J Clin Invest.2021;131(9):e148372.Sinha et al., Science 2014;344(6184):649-52.Bloise et al., Physiol Rev.2019;99(1):739-80.Egerman et al., Cell Metab.2015;22(1):164-74.Muramatsu et al., Sci Rep.2021;11(1):2160.Long et al., Hum Mol Genet.2019;28(7):1076-89.Jadhav et al., J Pharm Sci.2017;106(9):2853-59.Wiig et al., Am J Physiol Heart Circ Physiol.2001;280(4):H1505-12.Derynck et al., Sci Signal.2019;12(570):1-58.Chen et al., Cell Res.2009;19:58-70.Lee et al., J Appl Physiol(1985).2016;120(6):592-8.Bulfield et al., Proc Natl Acad Sci U S A.1984;81:1189-92.Fukada et al., Am J Pathol.2010;176(5):2414-24.van Putten et al., FASEB J. 2019;33(7):8110-24.

[0011] If myostatin signaling can be selectively inhibited not only extracellularly but also intracellularly, for example, both in the interstitial fluid and cytoplasm of muscle, a stronger inhibitory effect on myostatin signaling than antibodies can be expected. If a compound with such an effect can be obtained, it is expected to have a stronger muscle strength-enhancing effect than previously discovered compounds and antibodies, and will be a promising therapeutic agent for diseases or symptoms accompanied by muscle dysfunction, such as muscular dystrophy and spinal muscular atrophy (SMA).

[0012] The present inventors have intensively studied compounds with myostatin inhibitory activity and have arrived at the present invention as a result. The present invention provides novel peptides with myostatin inhibitory activity, compositions containing said peptides, and uses of said peptides.

[0013] This application includes, but is not limited to, the following inventions. [1] A peptide comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3, 110-140, or a pharmaceutically acceptable salt thereof. [2] The peptide according to [1], wherein the peptide is a cyclic peptide, or a pharmaceutically acceptable salt thereof. [3] The peptide according to [1], wherein a cyclic structure is formed by bonding a chloroacetylated amino acid to a cysteine ​​residue contained in the peptide, or a pharmaceutically acceptable salt thereof. [4] The peptide according to [1], wherein a cyclic structure is formed by bonding the amino group of the first amino acid residue and the carboxy group of the 15th amino acid residue contained in the peptide, or a pharmaceutically acceptable salt thereof. [5] The peptide according to [1], wherein the peptide further comprises an additional amino acid residue, or a pharmaceutically acceptable salt thereof. [6] The peptide according to [1], wherein the peptide comprises a linker at the C-terminus, or a pharmaceutically acceptable salt thereof. [7] A pharmaceutical composition comprising the peptide according to any one of [1] to [6], or a pharmaceutically acceptable salt thereof. [8] The pharmaceutical composition according to [7], which has myostatin inhibitory activity. [9] The pharmaceutical composition according to [7], for preventing or treating a myostatin-related disease or a disease or symptom accompanied by muscle decline.

[10] A method for inhibiting myostatin signaling using the peptide according to any one of [1] to [6], or a pharmaceutically acceptable salt thereof.

[11] A diagnostic composition for diagnosing a myostatin-related disease or a disease or symptom accompanied by muscle decline, comprising the peptide according to any one of [1] to [6], or a pharmaceutically acceptable salt thereof.

[12] A method for testing a peptide or a pharmaceutically acceptable salt thereof, which tests at least one of the following for a peptide or a pharmaceutically acceptable salt thereof: a) solubility in a solvent; b) myostatin inhibitory activity; c) toxicity to cells and / or tissues; or d) toxicity to laboratory animals, wherein the peptide or pharmaceutically acceptable salt thereof is the peptide according to any one of [1] to [6], or a pharmaceutically acceptable salt thereof.

[0014] The peptide of the present invention has myostatin inhibitory activity and is therefore useful as a pharmaceutical composition, diagnostic composition, research composition, etc. for the prevention or treatment of diseases or symptoms accompanied by decreased muscle function.

[0015] FIG. 1 is a graph showing the progression of weight change in wild-type mice upon administration of a peptide of the present invention. In A, the black squares represent the results of administration of 1 mg / kg of Myostatin_99_Variant_07 (SEQ ID NO: 3), the black triangles represent the same peptide at 3 mg / kg, and the gray circles represent PBS as a control. The horizontal axis represents the number of days after administration, and the vertical axis represents the change in mouse weight (grams) after administration. FIG. 2 is a graph showing the improvement in grip strength and the progression of weight change in B10 mdx mice upon administration of a peptide of the present invention. The black squares represent the results of administration of 10 mg / kg of Myostatin_99_Variant_07 (SEQ ID NO: 3), and the white circles represent the results of administration of PBS as a control. The horizontal axis represents the number of days after administration, and the vertical axis represents the change in grip strength. FIG. 3 is a graph showing the progression of weight change in B10 mdx mice upon administration of a peptide of the present invention. The black squares indicate the results of administering 10 mg / kg of Myostatin_99_Variant_07 (SEQ ID NO: 3), and the white circles indicate the results of administering PBS as a control. The horizontal axis indicates the number of days after administration, and the vertical axis indicates the change in body weight.

[0016] The present invention includes, but is not limited to, the following embodiments. Unless otherwise specified herein, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The substances, materials, and examples disclosed herein are merely illustrative and are not intended to be limiting. When referring to "in one embodiment" in this specification, it means that the embodiment is not limited, i.e., is not limited.

[0017] 1. Abbreviations In this specification, the following abbreviations are used with the following meanings unless otherwise specified.

[0018] Abbreviations (general) Å: Angstrom (unit); ClAc: chloroacetyl; Cy5SAlk: Sulfo-Cy5-alkyne; DCM: dichloromethane; TIPS: triisopropylsilyl; tBu: tertiary butyl; DTT: dithiothreitol; DMSO: dimethyl sulfoxide; Trt: trityl; Boc: tertiary butoxycarbonyl; DMF: N,N-dimethylformamide; DIEA or DIPEA: N,N-diisopropylethylamine; DIPCI or DIC: N,N'-diisopropylcarbodiimide; Oxyma pure: ethyl cyanohydroxyiminoacetate; DODT: 3,6-dioxa-1,8-octane-dithiol; Fmoc: 9-fluorenylmethyloxycarbonyl; g: grams (unit); HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HOSu: N-hydroxysuccinimide; HPLC: high performance liquid chromatography; LC-MS or LC / MS: liquid chromatography mass spectrometer; mL: milliliters (unit); M: molar (unit); μL: microliters (unit); mM: millimolar (unit); μM: micromolar (unit); mmol: millimole (unit); mg: milligrams (unit); MeCN or CH ３CN: acetonitrile; min: minute (unit); mm: millimeter (unit); μm: micrometer (unit); nm: nanometer (unit); nM: nanomolar (unit); OSu: succinimide; PEG: polyethylene glycol; rpm: revolutions per minute (unit); tBu: tertiary butyl; TFA: trifluoroacetic acid; TIS: triisopropylsilane; Trt or Tr: trityl group; H-PEG4Me: 2,5,8,11-tetraoxatridecan-13-amine; H-PEG8Me: 2,5,8,11,14,17,20,23-octaoxapentacosan-25-amine; AA: amino acid; PyAOP: 7-((azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate); CSA: 10-camphorsulfonic acid; Fmoc-OSu: N-(9-fluorenylmethoxycarbonyloxy)succinimide; THF: tetrahydrofuran; ClAcOSu: N-(chloroacetoxy)succinimide; Pd ２ (dba) ３ CHCl ３ : tris(dibenzylideneacetone)dipalladium(0)-chloroform complex; SPhos: 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl; EDCI.HCl: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; Pd(PPh ３ ) ４ : tetrakis(triphenylphosphine)palladium(0); ClAcOH: chloroacetic acid; conc: concentration; HFIP: 1,1,1,3,3,3-hexafluoro-2-propanol; Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; Alloc: allyloxycarbonyl.

[0019] Abbreviations (Unnatural Amino Acids) The following abbreviations for unnatural amino acids include those in which the amino group in the main chain is protected with a common protecting group such as a Boc group or an Fmoc group. MeA: (2S)-2-(methylamino)propanoic acid (CAS 3913-67-5) Dap: (2S)-2,3-diaminopropanoic acid (CAS 4033-39-0) ApG: 2-[(3-aminopropyl)amino]acetic acid (CAS 2875-41-4) CrpG: 4-((carboxymethyl)amino)butanoic acid (CAS 4386-04-3) CmG: 2-[(2-carbamoylethyl)amino]acetic acid (CAS 34299-32-6) MeD: (2S)-2-(methylamino)butanedioic acid (CAS 4226-18-0) CrmG: 2,2'-iminodiacetic acid (CAS 142-73-4) MeopG: 2-[(3-methoxypropyl)amino]acetic acid (CAS 807261-81-0) MeeG: 2-[(2-methoxyethyl)amino]acetic acid (CAS 205124-55-6) AcapG: 2-[(3-acetamidopropyl)amino]acetic acid (CAS 1862908-81-3) A4paa: (S)-2-amino-3-(1-(carboxymethyl)piperidin-4-yl)propanoic acid

[0020] KCOpipzaa: (2S)-2-amino-6-{[4-(carboxymethyl)piperazine-1-carbonyl]amino}hexanoic acid

[0021] CeG: 3-((carboxymethyl)amino)propanoic acid (CAS 505-72-6) CrbG: 5-((carboxymethyl)amino)pentanoic acid (CAS 72253-27-1) KMe: (2S)-2-amino-6-(methylamino)hexanoic acid (CAS 1188-07-4) Hly: (2S)-2,7-diaminoheptanoic acid (CAS 37689-89-7) Hgl: (S)-2-aminohexanedioic acid (CAS 1118-90-7) Orn: (2S)-2,5-diaminopentanoic acid (CAS 70-26-8) Dab: (2S)-2,4-diaminobutanoic acid (CAS 1758-80-1) MeE: (2S)-2-(methylamino)pentanedioic acid (CAS 6753-62-4) da: D-alanine (CAS 338-69-2) Aib: 2-amino-2-methylpropanoic acid (CAS 62-57-7) de: D-glutamic acid (CAS 6893-26-1) dkCOpipzaa: (2R)-2-amino-6-{[4-(carboxymethyl)piperazine-1-carbonyl]amino}hexanoic acid

[0022] ddab: (2R)-2,4-diaminobutanoic acid (CAS 26908-94-1) done: (2R)-2,5-diaminopentanoic acid (CAS 348-66-3) MeW: (S)-3-(1H-indol-3-yl)-2-(methylamino)propanoic acid (CAS No. 526-31-8) W7N: (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (CAS 49758-35-2) W4C: (S)-2-amino-3-(4-chloro-1H-indol-3-yl)propanoic acid (CAS 52448-14-3) Bph: (S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid (CAS 155760-02-4) 3Py6Ph: (S)-2-amino-3-(6-phenylpyridin-3-yl)propanoic acid

[0023] W6N: (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propanoic acid (CAS 149704-63-2) F41dMeCmm4Pyz: (S)-2-amino-3-(4-(1-(2-(dimethylamino)-2-oxoethyl)-1H-pyrazol-4-yl)phenyl)propanoic acid

[0024] W1Bn: (2S)-2-amino-3-(1-benzyl-1H-indol-3-yl)propanoic acid (CAS 740-11-4) W6H: (S)-2-amino-3-(6-hydroxy-1H-indol-3-yl)propanoic acid (CAS 13567-14-1) Bph4ms: (S)-2-amino-3-(4'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl)propanoic acid

[0025] W1Ph: (2S)-2-amino-3-(1-phenyl-1H-indol-3-yl)propanoic acid (CAS 1224881-37-1) W1aa: (2S)-2-amino-3-[1-(carboxymethyl)-1H-indol-3-yl]propanoic acid (CAS 773823-50-0) W7Ph3C: (S)-2-amino-3-(7-(3-chlorophenyl)-1H-indol-3-yl)propanoic acid

[0026] W1Ph4OMe: (2S)-2-amino-3-(1-(4-methoxyphenyl)-1H-indol-3-yl)propanoic acid

[0027] W1Ph4COO: 4-{3-[(2S)-2-amino-2-carboxyethyl]-1H-indol-1-yl}benzoic acid

[0028] W6H1Ph4COO: (S)-4-(3-(2-amino-2-carboxyethyl)-6-hydroxy-1H-indol-1-yl)benzoic acid

[0029] 3Py6NH2: (S)-2-amino-3-(6-aminopyridin-3-yl)propanoic acid (CAS 1269968-61-7) Yph: (S)-2-amino-3-(4-phenoxyphenyl)propanoic acid (CAS 150351-64-7) F4CON: (S)-2-amino-3-(4-carbamoylphenyl)propanoic acid (CAS 223593-04-2) YBn: (S)-2-amino-3-(4-(benzyloxy)phenyl)propanoic acid (CAS 16652-64-5) Yae: (S)-2-amino-3-(4-(2-aminoethoxy)phenyl)propanoic acid (CAS 1909283-20-0) Nal1: (S)-2-amino-3-(naphthalen-1-yl)propanoic acid (CAS 55516-54-6) mBph: (S)-3-([1,1'-biphenyl]-3-yl)-2-aminopropanoic acid (CAS 164172-96-7) MeF: (2S)-2-(methylamino)-3-phenylpropanoic acid (CAS No. 2566-30-5) MeY: (2S)-3-(4-hydroxyphenyl)-2-(methylamino)propanoic acid (CAS No. 537-49-5) MeF3Me: (S)-2-(methylamino)-3-(meta-tolyl)propanoic acid

[0030] MeF4CON: (S)-3-(4-carbamoylphenyl)-2-(methylamino)propanoic acid

[0031] MeF3CON: (S)-3-(3-carbamoylphenyl)-2-(methylamino)propanoic acid

[0032] F3CON: (S)-2-amino-3-(3-carbamoylphenyl)propanoic acid (CAS 1217651-22-3) MeCha: (S)-3-cyclohexyl-2-(methylamino)propanoic acid (CAS 2165389-05-7) MeYap: (S)-3-(4-(3-aminopropoxy)phenyl)-2-(methylamino)propanoic acid

[0033] MeA1Ac4pip: (S)-3-(1-acetylpiperidin-4-yl)-2-(methylamino)propanoic acid

[0034] MemBph: (S)-3-([1,1'-biphenyl]-3-yl)-2-(methylamino)propanoic acid

[0035] MeNal1: (S)-2-(methylamino)-3-(naphthalen-1-yl)propanoic acid

[0036] MeNal2: (S)-2-(methylamino)-3-(naphthalen-2-yl)propanoic acid

[0037] MeF4OMe: (S)-3-(4-methoxyphenyl)-2-(methylamino)propanoic acid (CAS 52939-33-0) MeF4C: (S)-3-(4-chlorophenyl)-2-(methylamino)propanoic acid (CAS 347851-70-1) Eva: (S)-2-amino-3-ethylpentanoic acid (CAS 14328-49-5) Cbg: (S)-2-amino-2-cyclobutylacetic acid (CAS 49607-08-1) Gcpe: (S)-2-amino-2-cyclopentylacetic acid (CAS 2521-84-8) TMe: (2S,3R)-2-amino-3-methoxybutanoic acid (CAS 4144-02-9) MeG: 2-(methylamino)acetic acid (CAS 107-97-1) Meda: (2R)-2-(methylamino)propanoic acid (CAS 29475-64-7) dp: D-proline (CAS 344-25-2) EtG: 2-(ethylamino)acetic acid (CAS 627-01-0) PrG: 2-(propylamino)acetic acid (CAS 25303-14-4) F3aa: (S)-2-amino-3-(3-(carboxymethyl)phenyl)propanoic acid (CAS 1270107-31-7) F3aao: (S)-2-amino-3-(3-(carboxymethoxy)phenyl)propanoic acid

[0038] F4aa: (S)-2-amino-3-(4-(carboxymethyl)phenyl)propanoic acid (CAS No. 142348-77-4) F4aao: (S)-2-amino-3-(4-(carboxymethoxy)phenyl)propanoic acid (CAS No. 24558-63-2) F4C: (S)-2-amino-3-(4-chlorophenyl)propanoic acid (CAS No. 14173-39-8) F3OMe: (S)-2-amino-3-(3-methoxyphenyl)propanoic acid (CAS No. 33879-32-2) F3COO: (S)-3-(2-amino-2-carboxyethyl)benzoic acid (CAS No. 13861-02-4) KAc: (2S)-2-amino-6-acetamidohexanoic acid (CAS No. 692-04-6) Bph2C: (S)-2-amino-3-(2'-chloro-[1,1'-biphenyl]-4-yl)propanoic acid

[0039] Bph2OMe: (S)-2-amino-3-(2'-methoxy-[1,1'-biphenyl]-4-yl)propanoic acid

[0040] pBph2aao: (S)-2-amino-3-(2'-(carboxymethoxy)-[1,1'-biphenyl]-4-yl)propanoic acid

[0041] F4COO: (S)-4-(2-amino-2-carboxyethyl)benzoic acid (CAS 126109-42-0) 3Py: (S)-2-amino-3-(pyridin-3-yl)propanoic acid (CAS 64090-98-8) 4Py: (S)-2-amino-3-(pyridin-4-yl)propanoic acid (CAS 37535-49-2) F4F: (S)-2-amino-3-(4-fluorophenyl)propanoic acid (CAS 1132-68-9) F4OMe: (S)-2-amino-3-(4-methoxyphenyl)propanoic acid (CAS 6230-11-1) Cha: (S)-2-amino-3-cyclohexylpropanoic acid (CAS 27527-05-5) MeKMe: (2S)-2,6-bis(methylamino)hexanoic acid (CAS 51876-34-7) MeF4am: (S)-3-(4-(aminomethyl)phenyl)-2-(methylamino)propanoic acid

[0042] MeDap: (S)-3-amino-2-(methylamino)propanoic acid (CAS 904832-42-4) MeF4COO: (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid

[0043] F4am: (S)-2-amino-3-(4-(aminomethyl)phenyl)propanoic acid (CAS 150338-20-8) Ahp: (S)-2-aminoheptanoic acid (CAS number: 1115-90-8) Ano: (S)-2-aminononanoic acid (CAS 133444-84-5) Ado: (S)-2-aminododecanoic acid (CAS 169106-34-7) Ade: (S)-2-aminodecanoic acid (CAS 84277-81-6) Aoc: (S)-2-aminooctanoic acid (CAS 116783-26-7) SPent: (2S)-2-amino-3-(pentyloxy)propanoic acid (CAS 1502644-74-7) HseBu: (2S)-2-amino-4-butoxybutanoic acid (CAS 17673-71-1) AhpOMe: (S)-2-amino-7-methoxyheptanoic acid

[0044] Aoc7r8dH: (2S)-2-amino-7,8-dihydroxyoctanoic acid

[0045] HsePr: (2S)-2-amino-4-propoxybutanoic acid (CAS 18312-28-2) AhpOH: (S)-2-amino-7-hydroxyheptanoic acid (CAS 2136908-21-7) Aun: (S)-2-aminoundecanoic acid (CAS 169106-34-7) MeK: (2S)-6-amino-2-(methylamino)hexanoic acid (CAS 7431-89-2) MeKCOpipzaa: (2S)-6-{[4-(carboxymethyl)piperazine-1-carbonyl]amino}-2-(methylamino)hexanoic acid

[0046] MeDab: (S)-4-amino-2-(methylamino)butanoic acid

[0047] MeF3Et: (S)-3-(3-ethylphenyl)-2-(methylamino)propanoic acid

[0048] Me4Py: (S)-2-(methylamino)-3-(pyridin-4-yl)propanoic acid

[0049] Me3Py: (S)-2-(methylamino)-3-(pyridin-3-yl)propanoic acid (CAS 2651172-69-7) MeC: (2R)-2-(methylamino)-3-sulfanylpropanoic acid (CAS 4026-48-6) dc: D-cysteine ​​(CAS 921-01-7) Medc: (2S)-2-(methylamino)-3-sulfanylpropanoic acid (CAS 95244-61-4) dhcy: (2R)-2-amino-4-sulfanylbutanoic acid (CAS 6027-14-1) Hcy: (2S)-2-amino-4-sulfanylbutanoic acid (CAS 6027-13-0) Aeoac: 2-(2-aminoethoxy)acetic acid (CAS 10366-71-9) Ape: 5-aminopentanoic acid (CAS 660-88-8) dhgl: (R)-2-aminohexanedioic acid (CAS 7620-28-2) Hgl: (S)-2-aminohexanedioic acid (CAS 1118-90-7) MeHgl: (S)-2-(methylamino)hexanedioic acid (CAS 261943-13-9) Medhgl: (R)-2-(methylamino)hexanedioic acid

[0050] MeAeoac: 2-(2-(methylamino)ethoxy)acetic acid (CAS 98137-58-7) MeApe: 5-(methylamino)pentanoic acid (CAS 21382-30-9) pipzAc: 2-(piperazin-1-yl)acetic acid (CAS 37478-58-3) pipAc: 2-(piperidin-4-yl)acetic acid (CAS 51052-78-9) Pip4mAc: 3-(piperidin-4-yl)propanoic acid (CAS 1822-32-8) Pic4: piperidine-4-carboxylic acid (CAS 498-94-2) F42Py: (2S)-2-amino-3-[4-(pyridin-2-yl)phenyl]propanoic acid (CAS 1336207-47-6) F44Py: (2S)-2-amino-3-[4-(pyridin-4-yl)phenyl]propanoic acid

[0051] cC14COO: hexadecanedioic acid (CAS 505-54-4) cC16COO: octadecanedioic acid (CAS 871-70-5)

[0052] 2. Peptides The present invention relates to peptides or pharmaceutically acceptable salts thereof. In this specification, when a "peptide" is mentioned, unless otherwise specified, a reference to a pharmaceutically acceptable salt thereof or a solvate thereof is also included.

[0053] As used herein, the term "amino acid" includes not only natural amino acids but also unnatural amino acids. Unnatural amino acids include, for example, N-alkylamino acids in which the above-described natural amino acids are N-alkylated, and amino acids in which the nitrogen atom forming the peptide bond is modified with a branched or unbranched lower (e.g., C1-C5, preferably C1-C3, more preferably C1) alkyl group. N-Alkylamino acids are preferably N-ethylamino acids, N-butylamino acids, or N-methylamino acids, and more preferably N-methylamino acids. Unnatural amino acids also include chemically modified amino acids such as D-amino acids (also referred to as D-amino acids), β-amino acids, γ-amino acids, amino acid mutants, and amino acid derivatives, as well as amino acids that do not serve as building blocks of proteins in vivo, such as norleucine and ornithine. Furthermore, the amino acids include naturally occurring amino acids having a functional group added to the side chain or substituted with another functional group (for example, amino acids having a substitution or addition in an arylene group, alkylene group, or other portion of the side chain, amino acids having an increased C number in the arylene group, alkylene group, or alkyl group of the side chain, amino acids having a substitution in an aromatic ring of the side chain, and heterocyclized or condensed cyclized amino acids).

[0054] Note that the addition or substitution of a structure such as a functional group to the side chain of a natural amino acid can confer properties different from those of natural amino acids. For example, Dap is an amino acid having an amino group in the side chain of alanine. The addition of this amino group gives Dap the properties of a polar amino acid, which is basic, unlike alanine, which belongs to the nonpolar amino acid group. In other words, unnatural amino acids with similar side chain properties can be included in the aforementioned groups, which are obtained by dividing natural amino acids based on their common side chain properties. For example, N-methylarginine (MeR), an amino acid in which the main chain nitrogen atom of arginine, which belongs to the basic amino acids, is methylated, is an unnatural amino acid, but it exhibits basic properties and can therefore be classified as a basic amino acid. In this way, unnatural amino acids that exhibit side chain properties similar to those of a certain amino acid can also be included as targets for conservative amino acid substitution. Note that D-amino acids such as de (D-glutamic acid) can be classified as D-amino acids, but can also be classified according to the properties of their side chains. N-methylamino acids can also be classified as N-alkylamino acids, or according to the properties of the side chain of the original amino acid that is not N-methylated.

[0055] Non-limiting examples of non-natural amino acids include N-methyl amino acids, MeA, Dap, ApG, CrpG, CmG, MeD, Cr mG, MeopG, MeeG, AcapG, A4paa, KCOpipzaa, CeG, CrbG, KMe, Hly, Hg, Orn, Dab, MeE, da, Aib, de, dkCOpipzaa, ddab, dor, MeW, W7N, W4C, Bph, 3Py6Ph, W6N, F41dMeCmm4Pyz, W1Bn, W6H, Bph4ms, W1Ph, W1aa, W7Ph3C, W1Ph4OMe, W1Ph4COO, W6H1Ph4COO, 3Py6NH2, Yph, F4CON, YBn, Yae, Nal1, F42Py, mBph, MeF, MeY, MeF3Me, MeF4CON, MeF3CON, F3CON, MeCha, MeYap, MeA1Ac4pip, MemBph, MeNal1, MeNal2, MeF4OMe, MeF4C, Eva, Cbg, Gcpe, TMe, MeG, Meda, dp, EtG, PrG, F3aa, F3aa o, F4a, F4aa o, F4C, F3OMe, F3COO, KAc, Bph2C, Bph2OMe, pBph2aa o, F4COO, 3Py, 4Py, F4F, F4OMe, Cha, MeKMe, MeF4am, MeDap, MeF4COO, Ahp, Ano, Ado, Ade, Aoc, Spent, HseBu, AhpOMe, Aoc7r8dH, HsePr, AhpOH, Aun, MeK, MeKCOpipzaa, MeDab, MeF3Et, Me4Py, Me3Py, MeC, dc, Medc, dhcy, Hcy, Aeoa c, Ape, dhgl, Hgl, MeHgl, Medhgl, MeAeoa c, MeApe, pipzAc, pipAc, Pip4mAc, Pic4, F42Py and F44Py, and the like.

[0056] "A pharmaceutically acceptable salt thereof" refers to a salt of any peptide. Examples of pharmaceutically acceptable salts include salts with mineral acids such as sulfuric acid, hydrochloric acid, and phosphoric acid; salts with organic acids such as acetic acid, oxalic acid, lactic acid, tartaric acid, fumaric acid, maleic acid, methanesulfonic acid, and benzenesulfonic acid; salts with amines such as trimethylamine and methylamine; and salts with metal ions such as sodium ions, potassium ions, and calcium ions. For compounds that have come to contain moisture over time, such moisture is also included in the pharmaceutically acceptable salts.

[0057] The peptide may contain additional amino acid residues in addition to the amino acid sequence of SEQ ID NO: 1. The "additional amino acid residues" will be described later.

[0058] In one embodiment, the peptide may include a linker, preferably at the C-terminus. The term "linker" will be described later.

[0059] In one embodiment, the peptide is a cyclic peptide. "Cyclic peptides" will be described in detail below.

[0060] The peptide preferably has myostatin inhibitory activity. The term "having myostatin inhibitory activity" will be explained in more detail below.

[0061] Matters described in other sections also apply to this section unless otherwise stated.

[0062] In one embodiment, the peptide is a cyclic peptide. A "cyclic peptide" refers to a peptide in which two amino acids are bonded together, and the whole or part of the peptide is cyclic. The peptide also includes peptides in which amino acids in the peptide form a cross-linked structure, peptides in which a cyclic structure is formed by lactam ring formation or macrocyclization reaction, and peptides having a lasso peptide-like structure. In other words, the cyclic peptide may be any peptide in which a part thereof forms a cyclic structure, and may also have a linear portion.

[0063] Peptides generally have poor metabolic stability in vivo and, due to their large size, have difficulty penetrating cell membranes. To address these issues, peptide cyclization has been used. Cyclization of peptides improves protease resistance and metabolic stability, and also restricts conformational changes, increasing their rigidity and suggesting improved membrane permeability and affinity for target proteins.

[0064] In one embodiment, the peptide has a cyclic structure in which a chloroacetylated amino acid is bonded to a cysteine ​​residue contained in the peptide. In one embodiment, the peptide has a cyclic structure in which an N-terminal amino acid (the first amino acid residue) is bonded to a cysteine ​​residue contained in the peptide. In one embodiment, the peptide has a cyclic structure in which an N-terminal amino acid (the first amino acid residue) is bonded to a cysteine ​​residue contained in the peptide. In one embodiment, the peptide has a cyclic structure in which a chloroacetylated N-terminal amino acid (the first amino acid residue) is bonded to a cysteine ​​residue contained in the peptide. "Chloroacetylation" may be "halogen acetylation" using another halogen. "Acetylation" may also be "acylation" using an acyl group other than an acetyl group.

[0065] As used herein, some amino acids may be modified to cyclize the peptide. Amino acids with such modifications are also encompassed. For example, as described above, a chloroacetyl group may be added to the N-terminal amino acid, which may then be bonded to a cysteine ​​residue in the peptide to cyclize it. Various (natural / unnatural) amino acids with such a chloroacetyl group added are also encompassed in the amino acids of the present application.

[0066] In one embodiment, the peptide has a cyclic structure in which the N-terminal amino acid (the first amino acid residue; X1) and the C-terminal amino acid are bonded together. In another embodiment, the peptide has a cyclic structure in which the amino group of the N-terminal amino acid (the first amino acid residue) is bonded together with the carboxyl group of the 15th amino acid contained in the peptide. In another embodiment, an additional amino acid may be added to the 15th amino acid residue, thereby forming a cyclic structure in which the N-terminal amino acid (the first amino acid residue) and an additional amino acid residue are added to the 15th amino acid, for example, the 16th or 17th amino acid residues are bonded together. In one embodiment, the peptide has a cyclic structure in which the amino group of the first amino acid residue contained in the peptide is bonded together with the carboxyl group of the 16th amino acid residue.

[0067] In one embodiment, the peptide comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 3, 110 to 140. In one embodiment, the peptide is a cyclic peptide comprising or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 3, 110 to 140. The peptide is a peptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3, 110 to 140.

[0068] The peptide may further comprise additional amino acid residues. Without limitation, the peptide may comprise additional amino acid residues in addition to the amino acid sequences set forth in SEQ ID NOs: 3, 110-140.

[0069] The "additional amino acid residues" may be contained in a peptide forming a cyclic structure, or an amino acid residue may be added to the cyclic peptide in the form of a linker. The number of amide bonds (number and length of amino acids) in the peptide or peptide portion is not particularly limited.

[0070] Furthermore, a linker may be further added to the cyclic peptide. Examples of linkers include the aforementioned amino acid linkers (peptide linkers), chemical linkers, fatty acid linkers, nucleic acid linkers, and sugar chain linkers. Alternatively, a complex of a chemical linker and a peptide linker may be used. A chemical linker may be, for example, a PEG linker consisting of 1 to 24 ethylene glycol units. The linker may also be a fatty acid linker containing a divalent chemical moiety derived from a fatty acid. The amino acid (peptide) linker is a linker containing at least one amino acid, and examples thereof include glycine-rich peptides such as peptides having the sequence [Gly-Gly-Gly-Gly-Ser]n (wherein n is 1, 2, 3, 4, 5, or 6) as described in U.S. Pat. No. 7,271,149, and serine-rich peptide linkers described in U.S. Pat. No. 5,525,491. Furthermore, peptide linkers containing the amino acid sequences set forth in SEQ ID NOs: 315-325 may be used, but are not limited to these. In addition, in peptide linkers, the bond between amino acids or between an amino acid and a chemical linker may be formed via the side chain of the amino acid. Addition of a linker may change the physical properties (e.g., solubility) of the peptide, without limitation.

[0071] The linker may be added at any position. For example, it may be attached to an amino acid located at the C-terminus, or to an amino acid contained in the cyclic peptide. In one embodiment, the peptide contains a linker at the C-terminus. Preferably, the linker is attached to Cys located at the C-terminus, or to the side chain of an amino acid contained in the cyclic peptide.

[0072] Furthermore, the peptides may form a multimer via a linker or the like. The number of peptides contained in the multimer is not limited. In one embodiment, the multimer is a dimer, trimer, tetramer, pentamer, hexamer, octamer, or greater. The multimer may contain multiple identical peptides, or multiple different peptides.

[0073] In one embodiment, preferably, the peptide has myostatin inhibitory activity.

[0074] In one embodiment, preferably, the peptide has GDF-8 inhibitory activity.

[0075] Myostatin is a protein that belongs to the TGF-β (Transforming Growth Factor-β) superfamily, also known as GDF-8 (Glowth Differentiation Factor-8).

[0076] As used herein, the term "myostatin," unless otherwise specified, refers to native myostatin found in mammals, preferably rodents such as mice and primates such as humans. Note that "human myostatin" herein refers to native myostatin found in humans (e.g., Gene ID: 2660). It is known that the amino acid sequences of myostatin in humans, mice, and rats are nearly identical. Unless otherwise specified, "myostatin" and "human myostatin" herein include unprocessed myostatin, processed myostatin, and mutants. Myostatin is known to undergo glycosylation and dimerization, and active myostatin (also known as mature myostatin) is a homodimer in which two C-terminal growth factor domains are disulfide-bonded.

[0077] "Myostatin inhibition" refers to the inhibition, blocking, antagonization, or competitive inhibition of myostatin signaling. Myostatin signaling refers to the binding of myostatin to the type II activin receptor (ActRII: Gene ID: 92), forming a complex with the type I activin receptor (ActRI: Gene ID: 90), and transmitting a signal intracellularly. That is, "inhibition of myostatin signaling" includes, for example, the inhibition of myostatin binding to ActRII due to (a peptide) binding to myostatin or ActRII, the inhibition of ActRI-ActRII complex formation by binding to ActRI, and the inhibition of signaling pathways from the ActRI-ActRII complex, such as the inhibition of signaling pathways mediated by Smad, P13K, Ras, or TAK1. Inhibition of a signaling pathway refers to the reduction, regulation, or inhibition of any of the signaling pathways.

[0078] "Myostatin inhibitory activity" can be evaluated using known methods. For example, as shown in the Examples of this specification, it can be evaluated using an in vitro myostatin inhibitory activity system using a reporter gene, or by evaluating body weight and muscle strength in mice. Alternatively, myostatin inhibitory activity may be evaluated using the methods disclosed in, for example, JP 2020-011965 A and JP 2022-101593 A.

[0079] Without limitation, myostatin inhibitory activity can be expressed, for example, as IC50 in an in vitro myostatin inhibitory activity evaluation system using a reporter gene.

[0080] In one embodiment, the peptide is myostatin-selective (specific), and more preferably, has low inhibitory activity against GDF-11 activity.

[0081] Many molecules with myostatin inhibitory activity are known to inhibit not only myostatin (i.e., GDF-8) but also the activity of GDF-11, which also belongs to the TGF-8 superfamily. Because the amino acid sequence of the aforementioned active myostatin GDF-11 is highly identical, and myostatin, GDF-11, and activin are all signaling transmitters via ActR, molecules such as antibodies with myostatin inhibitory activity often inhibit the activity of all three growth factors: myostatin, GDF-11, and activin. However, because GDF-11 has the effect of improving muscle function as a rejuvenation factor, it is preferable that it is not inhibited, particularly in children and young adults. In one aspect, a myostatin inhibitor that specifically inhibits only myostatin and does not inhibit myostatin signaling substances other than myostatin, such as GDF-11, is preferred. GDF-11 also has high identity among mice, humans, and rats. When "GDF-11" is referred to herein, it is preferably human GDF-11 (Gene ID: 10220).

[0082] The phrase "does not inhibit GDF-11" means that the inhibitory activity against GDF-11 signal transduction is lower than that against myostatin signal transduction. For example, this means that in an in vitro myostatin inhibitory activity evaluation system using a reporter gene, the IC50 of GDF-11 is 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 200-fold, or more times higher than the IC50 of myostatin.

[0083] In one embodiment, the peptide of the present invention has inhibitory activity against myostatin derived from multiple species, preferably against myostatin derived from human.

[0084] The present invention provides novel peptides having myostatin inhibitory activity. The peptides having myostatin inhibitory activity are preferably peptides that do not inhibit GDF-11 as much as they inhibit myostatin. This is suggested by the fact that, as shown in Examples 3 and 4 of the present specification, although they have inhibitory activity against myostatin, their inhibitory activity against GDF-11 is significantly lower than that of myostatin.

[0085] Peptides having myostatin inhibitory activity are effective in treating and preventing various diseases in which BMP is involved, and are useful in various experiments on various diseases in which myostatin is involved.

[0086] Matters described in other sections also apply to this section unless otherwise stated.

[0087] 3. Production of Peptides The peptides of the present invention can be produced by any known method for producing peptides, for example, as follows.

[0088] Matters described in other sections also apply to this section unless otherwise stated.

[0089] Chemical synthesis methods such as liquid phase, solid phase, and hybrid methods that combine liquid and solid phase methods; genetic recombination methods, etc.

[0090] In the solid-phase method, for example, the hydroxyl group of a hydroxyl-containing resin is esterified with the carboxyl group of a first amino acid (usually the C-terminal amino acid of the target peptide) whose α-amino group is protected with a protecting group. Known dehydration condensation agents such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIC) can be used as the esterification catalyst.

[0091] Next, the protecting group of the α-amino group of the first amino acid is removed, and a second amino acid in which all functional groups except the carboxy group of the main chain are protected is added, and the carboxy group is activated to bond the first and second amino acids. Furthermore, the α-amino group of the second amino acid is deprotected, and a third amino acid in which all functional groups except the carboxy group of the main chain are protected is added, and the carboxy group is activated to bond the second and third amino acids. This process is repeated until a peptide of the desired length is synthesized, and then all functional groups are deprotected.

[0092] Examples of resins for solid-phase synthesis include Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGA resin (Merck), HMPA-PEGA resin (Merck), etc. These resins can be used after washing with a solvent (dimethylformamide (DMF), 2-propanol, methylene chloride, etc.).

[0093] Examples of protecting groups for α-amino groups include benzyloxycarbonyl (Cbz or Z) group, tert-butoxycarbonyl (Boc) group, 9-fluorenylmethyloxycarbonyl (Fmoc) group, benzyl group, allyl group, allyloxycarbonyl (Alloc) group, etc. The Cbz group can be deprotected by hydrofluoric acid, hydrogenation, etc., the Boc group can be deprotected by trifluoroacetic acid (TFA), and the Fmoc group can be deprotected by treatment with piperidine or pyrrolidine.

[0094] For protection of the α-carboxy group, for example, methyl ester, ethyl ester, allyl ester, benzyl ester, tert-butyl ester, cyclohexyl ester, etc. can be used.

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

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

[0097] The production of peptides by recombinant DNA techniques (translation synthesis systems) can be carried out using nucleic acids that encode the peptides. The nucleic acids that encode the peptides may be DNA or RNA.

[0098] The nucleic acid encoding the peptide can be prepared by a known method or a method similar thereto. For example, it can be synthesized using an automated synthesizer. A restriction enzyme recognition site may be added to insert the obtained DNA into a vector. Alternatively, a base sequence encoding an amino acid sequence for excising the resulting peptide chain using an enzyme or the like may be incorporated.

[0099] To suppress degradation by proteases derived from the host, a chimeric protein expression method can be used in which the peptide of interest is expressed as a chimeric peptide with another peptide. In this case, the nucleic acid used is a nucleic acid encoding the peptide of interest and a peptide that binds to it.

[0100] Subsequently, an expression vector is prepared using a nucleic acid encoding the peptide. The nucleic acid can be inserted downstream of a promoter in the expression vector either directly or after digestion with a restriction enzyme or after addition of a linker. Examples of vectors include Escherichia coli-derived plasmids (pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pUC118, pBluescript II, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, pC1912, pTP4, pE194, pC194, etc.), yeast-derived plasmids (pSH19, pSH15, YEp, YRp, YIp, YAC, etc.), bacteriophages (e phage, M13 phage, etc.), viruses (retrovirus, vaccinia virus, adenovirus, adeno-associated virus (AAV), cauliflower mosaic virus, tobacco mosaic virus, baculovirus, etc.), and cosmids.

[0101] The promoter can be appropriately selected depending on the type of host. When the host is an animal cell, for example, a promoter derived from SV40 (simian virus 40) or a promoter derived from CMV (cytomegalovirus) can be used. When the host is Escherichia coli, a trp promoter, a T7 promoter, a lac promoter, etc. can be used.

[0102] The expression vector may also incorporate, for example, a DNA replication origin (ori), a selection marker (antibiotic resistance, auxotrophy, etc.), an enhancer, a splicing signal, a polyA addition signal, a nucleic acid encoding a tag (FLAG, HA, GST, GFP, etc.), etc.

[0103] Next, a suitable host cell is transformed with the expression vector. The host can be selected appropriately in relation to the vector. Examples of hosts that can be used include Escherichia coli, Bacillus subtilis, Bacillus sp., yeast, insects or insect cells, and animal cells. Examples of animal cells that can be used include HEK293T cells, CHO cells, COS cells, myeloma cells, HeLa cells, and Vero cells. Transformation can be performed according to known methods, such as lipofection, calcium phosphate, electroporation, microinjection, and particle gun, depending on the type of host. The transformant is cultured according to standard methods to express the desired peptide.

[0104] To purify the peptide from the culture of the transformant, the cultured cells are harvested, suspended in an appropriate buffer, disrupted by sonication, freeze-thawing, or other methods, and then centrifuged or filtered to obtain a crude extract. If the peptide is secreted into the culture medium, the supernatant is collected.

[0105] Purification from the crude extract or culture supernatant can also be carried out by known methods or methods equivalent thereto (e.g., salting out, dialysis, ultrafiltration, gel filtration, SDS-PAGE, ion exchange chromatography, affinity chromatography, reversed-phase high performance liquid chromatography, etc.).

[0106] The obtained peptide may be converted from a free form to a salt, or from a salt to a free form, by a known method or a method similar thereto.

[0107] In one embodiment, the translation synthesis system may be a cell-free translation system. Cell-free translation systems generally allow expression products to be obtained in a highly pure form without purification. Cell-free translation systems contain, for example, ribosomal proteins, aminoacyl-tRNA synthetases (ARSs), ribosomal RNAs, amino acids, rRNAs, GTP, ATP, translation initiation factors (IFs), elongation factors (EFs), release factors (RFs), and ribosome recycling factors (RRFs), as well as other factors necessary for translation. Escherichia coli extracts or wheat germ extracts may be added to improve expression efficiency. Additionally, rabbit erythrocyte extracts or insect cell extracts may be added.

[0108] By continuously supplying energy to a system containing these using dialysis, it is possible to produce protein in amounts ranging from several hundred μg to several mg / mL, but this is not limited to this. A system containing RNA polymerase may also be used to perform transcription from gene DNA. Commercially available cell-free translation systems that can be used include systems derived from Escherichia coli such as Roche Diagnostics' RTS-100 (registered trademark), GeneFrontier's PURESYSTEM, and New England Biolabs' PUREExpress In Vitro Protein Synthesis Kit, as well as systems using wheat germ extract from Zoigene and CellFree Sciences.

[0109] In a cellular translation system, instead of the aminoacyl-tRNA synthesized by a natural aminoacyl-tRNA synthetase, an artificial aminoacyl-tRNA in which a desired amino acid or hydroxy acid is linked (acylated) to the tRNA may be used. Such an aminoacyl-tRNA can be synthesized using an artificial ribozyme.

[0110] Such ribozymes include flexizyme (H. Murakami, H. Saito, and H. Suga, (2003), Chemistry & Biology, Vol. 10, 655-662; and WO2007 / 066627, etc.). Flexizyme is also known by the name of the original flexizyme (Fx), and modified versions thereof, such as dinitrobenzyl flexizyme (dFx), enhanced flexizyme (eFx), and aminoflexizyme (aFx).

[0111] By using a tRNA produced by Flexizyme to which a desired amino acid or hydroxy acid is linked, a desired codon can be translated in association with the desired amino acid or hydroxy acid. A non-standard amino acid may also be used as the desired amino acid. For example, the unnatural amino acid required for the above-mentioned cyclization can also be introduced into the linked peptide by this method.

[0112] The peptides can be chemically synthesized using various methods commonly used in the art, including, for example, stepwise solid-phase synthesis, semi-synthesis of peptide fragments via conformationally assisted religation, and chemical ligation. The peptides are synthesized using various solid-phase techniques, such as those described in K. J. Jensen, P. T. Shelton, and S. L. Pedersen, Peptide Synthesis and Applications, 2nd Edition, Springer, 2013. A preferred strategy is based on the combination of an Fmoc group, which temporarily protects the α-amino group and allows selective base removal, and a protecting group, which temporarily protects side chain functional groups and is stable under Fmoc removal conditions. Selection of such general peptide side chains is described in the aforementioned Peptide Synthesis and Applications, 2nd Edition, and G. B. Fields and R. L. Noble, "Solid Phase Peptide Synthesis Utilizing 9-Fluorenylmethoxycarbonyl Amino Acids," Int. J. Peptide Protein Res. 35, 1990, 161-214, etc., and preferred peptide side chain protecting groups include, for example, a benzyl group, a tert-butyl group, and a trityl (Trt) group for the hydroxy group of serine or threonine, a 2-bromobenzyloxycarbonyl group and a tert-butyl group for the hydroxy group of tyrosine, a Boc group, a methyltetrazolethiol (Mtt) group, an Alloc group, and an ivDde group for the amino group of the lysine side chain, and a methyltetrazolethiol (Mtt) group for the imidazole group of histidine. a 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) group for the guanidyl group of arginine; a tert-butyl, allyl, and 3-methylpentane (Mpe) group for the carboxyl group of glutamic acid, aspartic acid, and the like; a Trt group for the carboxamide group of glutamine or asparagine; and a Trt group and monomethoxytrityl (Mmt) group for the thiol group of cysteine.

[0113] The peptide can be synthesized in a stepwise manner on the solid-phase resin described above. The α-amino protecting group of the C-terminal amino acid used, as well as all amino acids and peptides used in the synthesis, must be selectively removed during the synthesis process. Preferably, the solid-phase resin described above is used, and the C-terminal carboxyl group of a peptide whose N-terminus is appropriately protected with an Fmoc group or the like, or the C-terminal carboxyl group of an amino acid protected with an Fmoc group, is converted into an activated ester with an appropriate reagent, followed by addition to the amino group on the solid-phase resin. Subsequent peptide chain elongation can be achieved by sequentially repeating the removal of the N-terminal protecting group (Fmoc group) and the condensation of a protected amino acid derivative according to the amino acid sequence of the target peptide. Note that this procedure can liberate the target peptide at the final stage. For example, the conditions for liberation can be determined as described in Teixeira, W. E. Benckhuijsen, P. E. de Koning, A. R. P. M. Valentijn, J. W. The cleavage can be achieved by using a TFA solution containing water, silyl hydride, and thiol as scavengers in TFA, as described in Drijfhout, Protein Pept. Lett., 2002, 9, 379-385, etc. A typical example is TFA / Water / TIS / DODT (volume ratio 92.5:2.5:2.5:2.5).

[0114] Synthesis of the peptides described herein can be carried out using a single or multi-channel peptide synthesizer, such as a CEM Liberty Blue synthesizer or a Biotage Syro I synthesizer or their successors.

[0115] The activation of the carboxy group can be carried out using a condensing agent, such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), or 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU).

[0116] Peptide cyclization can be carried out according to known methods. Non-limiting examples include designing a peptide to contain two or more cysteine ​​residues, which allows for the formation of a cyclic structure via disulfide bonds after translation. Cyclization can also be achieved by synthesizing a peptide with a chloroacetyl group at the N-terminus and placing a cysteine ​​residue containing a sulfur molecule in the peptide using genetic code reprogramming technology, according to the method of Goto et al. (Y. Goto, et al. ACS Chem. Biol. 3 120-129 (2008)). This allows spontaneous nucleophilic attack of the mercapto group on the chloroacetyl group after translation, resulting in cyclization of the peptide via a thioether bond. Cyclization can also be achieved by placing other amino acid combinations within the peptide that bond to form a ring using genetic code reprogramming technology. Alternatively, cyclization can be achieved by placing an L-2-aminoadipic acid residue in the peptide and bonding it to the N-terminal main chain amino group. As such, any known cyclization method can be used without particular limitation.

[0117] The peptide may further have a bicyclic structure in which the ring is crosslinked. The formation of the bicyclic structure can be carried out by a known method. For example, but not limited to, a peptide having a bicyclic structure can be obtained by bonding the side chains of two amino acids contained in the cyclic structure with or without an additional amino acid.

[0118] 4. Compositions, etc. The present invention also relates to compositions comprising the peptides of the present invention or pharmaceutically acceptable salts thereof. The compositions include, but are not limited to, pharmaceutical compositions (medical compositions), diagnostic compositions, and therapeutic compositions.

[0119] Matters described in other sections also apply to this section unless otherwise stated.

[0120] In one aspect, the present invention relates to a pharmaceutical composition comprising the peptide or a pharmaceutically acceptable salt thereof. The disease for which the pharmaceutical composition is used is not particularly limited, as long as the peptide itself is effective. In one aspect, the pharmaceutical composition has myostatin inhibitory activity.

[0121] In one aspect, the pharmaceutical composition is a pharmaceutical composition for preventing or treating a myostatin-related disease or a disease or condition associated with decreased muscle function.

[0122] "Myostatin-related diseases or diseases or symptoms associated with decreased muscle function" include known myostatin-related diseases or diseases or symptoms associated with decreased muscle function, including, for example, diseases caused by muscle tissue such as myopathy and myogenic muscular atrophy, and diseases caused by nerves such as neurogenic muscular atrophy.

[0123] In one aspect, "myostatin-related diseases" include muscular dystrophies (including DMD; Duchenne muscular dystrophy, FSHD; facioscapulohumeral muscular dystrophy, LGMD; limb-girdle muscular dystrophy, BMD; Becker muscular dystrophy, etc.), myopathies (including congenital myopathies, inflammatory myopathies, metabolic myopathies, mitochondrial myopathies, endocrine myopathies, myofibrillar myopathies, and distal myopathies), neurogenic muscular atrophy, amyotrophic lateral sclerosis (ALS), muscular atrophy, spinal muscular atrophy (SMA), and the like.

[0124] In one embodiment, the medical composition of the present invention is for preventing a decrease in muscle fiber mass. In one embodiment, it is for increasing muscle fiber mass. In one embodiment, it is for increasing muscle fiber strength. In one embodiment, it is for inhibiting muscle fiber necrosis. In one embodiment, it is for inhibiting fibrosis and fatty acid formation that occur secondary to muscle fiber necrosis. Without being limited thereto, the "disease or symptom associated with muscle function decline" includes a decrease in muscle fiber mass, muscle fiber necrosis, and fibrosis and fatty acid formation that occur secondary to muscle fiber necrosis.

[0125] In one aspect, the pharmaceutical composition is administered to an individual with a myostatin-related disease or a disease or condition involving muscle loss. The "individual" is preferably a human.

[0126] The pharmaceutical composition may contain the peptide itself, or may contain a pharmaceutically acceptable salt of the peptide or a solvate thereof. As used herein, "peptide" may include a pharmaceutically acceptable salt or a solvate thereof, unless otherwise specified. The pharmaceutical composition preferably contains an effective amount of the peptide as an active ingredient.

[0127] In the present specification, the administration form of the pharmaceutical composition is not particularly limited, and may be oral or parenteral. Examples of parenteral administration include injection such as intramuscular injection, intravenous injection, and subcutaneous injection, transdermal administration, and transmucosal administration (nasal, oral, ocular, pulmonary, vaginal, and rectal) administration.

[0128] The pharmaceutical composition can be modified in various ways, taking into account the tendency of polypeptides to be easily metabolized and excreted. For example, polyethylene glycol (PEG) or sugar chains can be added to the polypeptide to increase its blood residence time and reduce its antigenicity. Alternatively, the polypeptide can be encapsulated in a sustained-release base such as a biodegradable polymer compound such as polylactic acid glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, or nanospheres. For transdermal administration, a weak electric current can be applied to the skin surface to penetrate the stratum corneum (iontophoresis).

[0129] The pharmaceutical composition may contain the active ingredient as is, or may be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc. Dosage forms include, for example, liquids (e.g., injections), dispersions, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, lozenges, inhalants, ointments, eye drops, nasal drops, ear drops, and poultices. Formulation can be carried out by conventional methods using, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavorings, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, etc. as appropriate.

[0130] Examples of ingredients used in formulations include, but are not limited to, purified water, saline, phosphate buffer, dextrose, glycerol, ethanol and other pharmaceutically acceptable organic solvents, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, silicic anhydride, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, tragacanth, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, human serum albumin, and the like.

[0131] In view of the fact that peptides are generally poorly absorbed transmucosally, the pharmaceutical composition may contain an absorption enhancer that improves the absorption of poorly absorbed drugs. Examples of such absorption enhancers include surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponin; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylic acids; fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, and mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, and nitric oxide donors.

[0132] When the pharmaceutical composition is in the form of a pill or tablet, it may be coated with a sugar coating, gastric or enteric coating material.

[0133] When the pharmaceutical composition is an injection, it may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. Furthermore, it may also contain a wetting agent, an emulsifier, a dispersant, a stabilizer, a solubilizer, a solubilizing agent, a preservative, etc.

[0134] The pharmaceutical composition may be intended for not only humans but also non-human mammals or birds, including non-human primates (monkeys, chimpanzees, gorillas, etc.), livestock animals (pigs, cows, horses, sheep, etc.), dogs, cats, rats, mice, guinea pigs, rabbits, etc.

[0135] The dosage, particularly when administered to humans, varies depending on the symptoms, the patient's age, sex, weight, sensitivity, administration method, administration interval, type of active ingredient, and type of formulation, and may be, but is not limited to, 30 μg to 100 g, 100 μg to 500 mg, or 100 μg to 100 mg, administered once or in divided doses. In the case of injection, 1 μg / kg to 3000 μg / kg, or 3 μg / kg to 1000 μg / kg may be administered once or in divided doses, depending on the patient's weight.

[0136] In one aspect, the present invention relates to a method for preventing or treating a myostatin-related disease or a disease or condition involving muscle loss by administering a peptide of the present invention.

[0137] In one aspect, the present invention relates to the use of the peptide of the present invention for the prevention or treatment of a myostatin-related disease or a disease or condition associated with decreased muscle function.

[0138] In one aspect, the present invention relates to the use of a peptide of the present invention for the manufacture of a pharmaceutical composition for the prevention or treatment of a myostatin-related disease or a disease or condition associated with decreased muscle function.

[0139] In one aspect, the present invention relates to the use of a peptide of the present invention as a pharmaceutical composition for the prevention or treatment of a myostatin-related disease or a disease or condition associated with decreased muscle function.

[0140] In one aspect, the present invention relates to a peptide of the present invention having myostatin inhibitory activity.

[0141] In one aspect, the present invention relates to a peptide of the present invention for use in a method for preventing or treating a myostatin-related disease or a disease or condition involving muscle loss.

[0142] In one aspect, the present invention relates to a peptide of the present invention for use as a pharmaceutical composition for the prevention or treatment of a myostatin-related disease or a disease or condition associated with muscle dysfunction.

[0143] In one aspect, the present invention relates to methods of inhibiting myostatin signaling using the peptides of the present invention, including in vivo, in vitro, and ex vivo methods.

[0144] Diagnostic Composition The present invention also relates to a diagnostic composition for diagnosing a myostatin-related disease or a disease or condition accompanied by decreased muscle function, comprising the peptide of the present invention or a pharmaceutically acceptable salt thereof.

[0145] The peptides of the present invention can also be used as diagnostic compositions for myostatin-related diseases or diseases or conditions accompanied by muscle dysfunction. The diagnostic agent may be a detection agent for diagnosing whether or not a patient has a myostatin-related disease or a disease or condition accompanied by muscle dysfunction. When used as a detection agent, the peptides may be detectably labeled.

[0146] The peptide may be detectably labeled. Examples of peptide labels include antibodies labeled with enzymes such as peroxidase and alkaline phosphatase, radioactive substances such as 125I, 131I, 35S, and 3H, fluorescent substances such as fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine isothiocyanate, and near-infrared fluorescent materials, and luminescent substances such as luciferase, luciferin, and aequorin. Antibodies labeled with nanoparticles such as gold colloids and quantum dots can also be detected. For example, the severity of myostatin-related diseases or diseases or symptoms associated with muscle dysfunction can be detected by preparing a complex between the peptide and an antibody that binds to a protein related to myostatin-related diseases or diseases or symptoms associated with muscle dysfunction, preparing a complex labeled with the antibody or the peptide, and administering and detecting the complex. Furthermore, in immunoassays, the peptide can be labeled with biotin and then detected by binding to avidin or streptavidin labeled with an enzyme or the like.

[0147] Among immunoassays, ELISA using enzyme labeling is preferred because it allows for simple and rapid antigen measurement. For example, an antibody is immobilized on a solid support, a sample is added and reacted, and then the labeled peptide is added and reacted. After washing, the antibody reacts with an enzyme substrate to develop color, and the absorbance is measured, thereby enabling the severity of myostatin-related diseases or diseases or symptoms associated with muscle dysfunction to be detected. After reacting the antibody immobilized on the solid support with the sample, the unlabeled peptide may be added, and an antibody against the peptide may be enzyme-labeled and then added. The antibody may be immobilized on the surface of the solid support or inside the solid support.

[0148] When the enzyme is peroxidase, 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), etc. can be used as the enzyme substrate, and when the enzyme is alkaline phosphatase, p-nitrophenyl phosphate (pNPP), etc. can be used.

[0149] As used herein, the term "solid phase carrier" is not particularly limited as long as it is a carrier capable of immobilizing an antibody, and examples thereof include microtiter plates made of glass, metal, resin, etc., substrates, beads, nitrocellulose membranes, nylon membranes, and PVDF membranes, and the target substance can be immobilized on these solid phase carriers according to known methods.

[0150] The present invention relates to a diagnostic kit comprising the peptide of the present invention, which comprises reagents and instruments necessary for the above-mentioned detection (including, but not limited to, any or all of the peptide of the present invention, antibody, solid phase support, buffer, enzyme reaction stop solution, microplate reader, etc.).

[0151] The present invention relates to a method for diagnosing a myostatin-related disease or a disease or symptom accompanied by muscle dysfunction using the peptide of the present invention. The "method for diagnosis" includes in vivo or in vitro diagnostic methods. Preferably, it is an in vitro diagnostic method. The present invention also relates to a method for detecting a disease using the peptide of the present invention. Disease detection can be performed by laboratory technicians, researchers, etc. other than physicians, for example, at research institutions (including educational institutions such as universities), companies, etc. In one aspect, the method for detecting a disease does not include medical procedures.

[0152] The present invention relates to the use of the peptides of the present invention for diagnosing myostatin-related diseases or diseases or conditions associated with muscle dysfunction.

[0153] The present invention relates to the use of the peptide of the present invention for the production of a diagnostic composition for diagnosing a myostatin-related disease or a disease or condition associated with muscle dysfunction.

[0154] The present invention relates to the use of the peptide of the present invention as a diagnostic composition for diagnosing a myostatin-related disease or a disease or condition associated with muscle dysfunction.

[0155] The present invention relates to a peptide of the present invention for use in a method for diagnosing a myostatin-related disease or a disease or condition involving muscle loss.

[0156] The present invention relates to a peptide of the present invention for use as a diagnostic composition for diagnosing a myostatin-related disease or a disease or condition associated with muscle dysfunction.

[0157] The present invention relates to a tester comprising the peptide of the present invention.The present invention relates to a diagnostic or detection tester comprising the peptide of the present invention.

[0158] Research Composition The present invention also relates to a research composition comprising the peptide of the present invention. "Research composition" includes those used by researchers, engineers, students, doctors, etc. in research institutions (including educational institutions such as universities), companies, hospitals, etc.

[0159] The research compositions can be used, for example, to detect myostatin, detect myostatin-related diseases, or diseases or conditions involving muscle loss.

[0160] In the present specification, the carrier for immobilizing peptides is not particularly limited, and examples thereof include microtiter plates made of glass, metal, or resin, substrates, beads, nitrocellulose membranes, nylon membranes, and PVDF membranes.

[0161] The present invention includes methods for detecting myostatin using the peptides of the present invention.

[0162] The present invention also includes the use of the peptides of the present invention for detecting myostatin.

[0163] The present invention relates to a diagnostic or detection kit comprising the peptide of the present invention, which comprises reagents and instruments necessary for the detection (including, but not limited to, any or all of the peptide of the present invention, antibody, solid phase support, buffer, enzyme reaction stop solution, microplate reader, etc.).

[0164] The present invention also relates to a method for testing a peptide or a pharmaceutically acceptable salt thereof, which tests at least one of the following for a peptide or a pharmaceutically acceptable salt thereof: a) solubility in a solvent; b) myostatin inhibitory activity; c) toxicity to cells and / or tissues; or d) toxicity to experimental animals, wherein the peptide or a pharmaceutically acceptable salt thereof is the peptide of the present invention or a pharmaceutically acceptable salt thereof.

[0165] "Solubility in a solvent" can be measured using a known method. When measuring solubility, the solvent is not limited and may be freely selected depending on the purpose. Furthermore, as for the method for measuring solubility, a known method can be appropriately selected depending on the type of solvent. The solubility may be measured when the peptide is dissolved in a known solvent such as water, glycerol, PBS, or DMSO, without limitation.

[0166] "Myostatin inhibitory activity" can be measured, for example, as described in "2. Peptides" and the like.

[0167] "Cell and / or tissue toxicity" can be measured using known methods. For example, a test for cell and / or tissue toxicity may be a known toxicity evaluation test using cells and / or tissues, or may be an in vitro method. The cells and / or tissues may be cells and / or tissues typically used in toxicity evaluation tests for pharmaceuticals, and are not limited thereto.

[0168] "Toxicity to laboratory animals" can be measured using known methods. For example, laboratory animals are not particularly limited as long as they are commonly used, and examples include mice, rats, guinea pigs, gerbils, hamsters, ferrets, rabbits, dogs, cats, pigs, goats, horses, cows, birds (e.g., chickens, quails, etc.), monkeys, and primates other than humans (e.g., cynomolgus monkeys, marmosets, rhesus monkeys, etc.).

[0169] Furthermore, the evaluation test for the toxicity described above is not limited to, and may be a safety test that is normally conducted in non-clinical trials of pharmaceuticals, and examples thereof include general toxicity tests (single-dose toxicity tests / repeated-dose toxicity tests), genotoxicity tests (Ames tests / chromosomal aberration tests / in vitro micronucleus tests), carcinogenicity tests, reproductive and developmental toxicity tests (ICH-I, II, III), local irritation tests (eye irritation tests, skin irritation tests, etc.), other toxicity tests (skin sensitization tests, phototoxicity tests, antigenicity tests), chemical analysis / biological analysis (TK / PK), etc.

[0170] 5. Combinations The peptides of the present invention may be used in combination with other drugs for the prevention or treatment of myostatin-related diseases or diseases or conditions accompanied by decreased muscle function. In one aspect, the present invention relates to a combination of the peptides with other drugs for the prevention or treatment of myostatin-related diseases or diseases or conditions accompanied by decreased muscle function.

[0171] The peptide and a drug for preventing or treating other myostatin-related diseases or diseases or symptoms accompanied by muscle decline may be administered simultaneously or sequentially. Preferably, the peptide and a drug for preventing or treating other myostatin-related diseases or diseases or symptoms accompanied by muscle decline are administered so as to achieve an additive effect, preferably a synergistic effect, of both. When administered sequentially, the order of administration is not important. When administered sequentially, it is preferred that both drugs be taken within 2 hours, 1 hour, 30 minutes, 20 minutes, 15 minutes, 10 minutes, or 5 minutes, but this is not a limitation.

[0172] Matters described in other sections also apply to this section unless otherwise stated.

[0173] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited thereto. Those skilled in the art can easily modify and alter the present invention based on the description in this specification, and such modifications and alterations are included in the technical scope of the present invention. Note that the names of compounds shown in the following reference examples and examples do not necessarily conform to the IUPAC nomenclature. Note that abbreviations may be used to simplify the description, but these abbreviations are as described above.

[0174] The raw materials, building blocks, reagents, acids, bases, solid-phase resins, and solvents used in the chemical synthesis of compounds were either commercially available or, if otherwise specified, synthesized using organic chemistry techniques. Amino acids containing protecting groups were commercially available and used as is.

[0175] The peptide residues are numbered such that the amino acid residue to be ClAc-modified is counted as residue 1, followed by residues 2, 3, etc. toward the resin. The common amino acids used are listed below, with side chain protecting groups indicated in parentheses.

[0176] Fmoc-Phe-OH; Fmoc-Val-OH; Fmoc-Trp(Boc)-OH; Fmoc-Arg(Pbf)-OH; Fmoc-Ala-OH H ２ O; Fmoc-Gly-OH; Fmoc-N-Me-Tyr(tBu)-OH; Fmoc-Ile-OH; Fmoc-Leu-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OMpe)-OH; Fmoc-(Dmb)Gly-OH (CAS number 166881-42-1); Fmoc-D-Pro-OH; Fmoc-Cys(Trt)-OH; Fmoc-Lys(Boc)-OH.

[0177] The unnatural amino acids used were as follows:

[0178] Fmoc-dp-OH; Fmoc-de(tBu)-OH; Fmoc-KCOpipzaa(tBu)-OH; Fmoc-W4N-OH; Fmoc-W7N-OH; Fmoc-Me4Py-OH; Fmoc-MeE(tBu)-OH; Fmoc-MeF-OH; Fmoc-MeY(tBu)-OH; Fmoc-MeA-OH; Fmoc-MeG-OH; Fmoc-W4F-OH; Fmoc-W7F-OH; Fmoc-MeF2F-OH; Fmoc-3Py6NH2(Boc)-OH; Fmoc-Kac-OH; Fmoc-W73Pyz1Me(Boc)-OH; Fmoc-W7Ph3C-OH; Fmoc-MeF4C-OH; Fmoc-PEG8c-OH; Fmoc-F4COO(tBu)-OH(CAS:183070-44-2); Fmoc-CrpG(tBu)-OH(CAS:174799-90-7); Fmoc-de(allyl)-OH(CAS:204251-33-2); Fmoc-da-OH(CAS:79990-15-1); Fmoc-CrpG(allyl)-OH(CAS:1403683-46-4); Fmoc-4Py-OH(CAS:169555-95-7); Fmoc-ApG(Boc)-OH(CAS:143192-31-8); Fmoc-Dap(Boc)-OH(CAS:162558-25-0); Fmoc-MeC(Trt)-OH(CAS:944797-51-7)。

[0179] The structure of the chemically synthesized peptide was determined by ESI-MS(+) mass spectrometry, where the molecular weight was calculated taking into account the amino acids used in the target sequence and the building blocks used as needed. "ESI-MS(+)" refers to electrospray ionization mass spectrometry performed in positive ion mode. Detected masses were reported in "m / z" units. Compounds with molecular weights greater than approximately 1000 were frequently detected as doubly or triply charged ions. Analysis Conditions A: Column: Kinetex EVO C18 2.6 μm, 2.1 ID x 150 mm, 100 Å (Phenomenex) Column temperature: 60°C Mobile phase A: 0.025% TFA in water Mobile phase B: 0.025% TFA (CH ３ CN) Gradient: 20-60% over 20 minutes Flow rate: 0.25 mL / min Detection: PDA (225 nm) Analysis condition B Column: Kinetex EVO C18 2.6 μm, 2.1 ID x 150 mm, 100 Å (Phenomenex) Column temperature: 60°C Mobile phase A: 0.025% TFA (in water) Mobile phase B: 0.025% TFA (in CH ３ CN) Gradient: 20-60% over 7.2 minutes Flow rate: 0.5 mL / min Detection: PDA (225 nm) Analysis condition C Column: Kinetex EVO C18 2.6 μm, 2.1 ID x 150 mm, 100 Å (Phenomenex) Column temperature: 60°C Mobile phase A: 0.025% TFA (in water) Mobile phase B: 0.025% TFA (in CH ３ CN) Gradient: 40-80% over 7.2 minutes Flow rate: 0.5 mL / min Detection: PDA (225 nm) Analysis condition D Column: Kinetex EVO C18 2.6 μm, 2.1 ID x 150 mm, 100 Å (Phenomenex) Column temperature: 60°C Mobile phase A: 0.025% TFA (in water) Mobile phase B: 0.025% TFA (in CH ３ CN) Gradient: 5-45% over 7.2 min Flow rate: 0.5 mL / min Detection: PDA (225 nm)

[0180] Example 1 In this example, the following peptides or linker-added peptides were synthesized.

[0181] Example 1-1 Synthesis of Myostatin_99_Variant_07 (SEQ ID NO: 3)

[0182]

[0183] The target peptide was synthesized using H-dc(Trt)-Trt(2-Cl) resin (Watanabe Chemical Co., Ltd., 0.83 mmol / g, 1.20 g) on ​​a CEM Liberty Blue solid-phase synthesizer according to the manufacturer's instructions.

[0184] To introduce each residue, 0.21 M Fmoc-AA in DMF / 0.5 M HATU (in DMF) / 1 M DIEA (in DMF) (4.2 equivalents / 4 equivalents / 8 equivalents) was used per equivalent of resin, and the reaction was carried out once for 30 minutes at 25°C. However, for the 4th, 6th, 10th, 12th, and 13th residues, the reaction was carried out twice for 60 minutes at 25°C. In addition, Fmoc removal was carried out twice by reaction with a 20% piperidine solution in DMF at 25°C for 5 minutes. The introduction of a chloroacetyl group was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin retaining the Fmoc-protected peptide obtained in the previous step by the method described above, followed by shaking the resin with 0.1 M ClAcOH (in DMF) / 0.1 M HATU (in DMF) / 0.2 M DIEA (in DMF) (5 equivalents / 5 equivalents / 10 equivalents) at 25°C for 30 minutes.

[0185] To deprotect the side chains and cleave them from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, then washed with diethyl ether, and dried under reduced pressure. Then, reagent cocktail A (20 mL, TFA / H ２A mixture of 92.5 / 2.5 / 2.5 / 2.5 / volume ratios of 0.01% to 0.2% (DIS / TIS / DODT) was added and the mixture was shaken at room temperature for 40 minutes. The reaction solution was filtered and recovered through a frit. The solid phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate described above. When this filtrate was added to an excess of a diethyl ether / hexane (1 / 1) mixed solvent cooled to 0°C, a cloudy white precipitate formed.

[0186] This mixture was centrifuged (9000 rpm, 0°C, 2 min), and the solution was decanted. The resulting solid was washed again with a small amount of diethyl ether cooled to 0°C and then dried under reduced pressure. The resulting solid (peptide) was used in the subsequent cyclization reaction. The peptide was dissolved in water / acetonitrile (1 / 1) to a final concentration of 5 mM based on the molar number of the solid-phase resin, and then 10 equivalents of triethylamine was added. The mixture was stirred at room temperature for 120 minutes, and then acetic acid was added to quench the reaction, thereby carrying out the cyclization reaction of the peptide. The reaction solution was concentrated under reduced pressure using a Genevac EZ-II Elite.

[0187] The resulting crude product was purified using the following conditions: (Column: Waters Xbridge® C18 5 μm 50×150 mm; Mobile phase: A=0.1% TFA in water, B=0.1% TFA in MeCN; Temperature: 40° C.; Gradient (% B): 33-33% over 3 min, 33-38% over 8 min, 38-60% over 1 min; Flow rate: 20-20 mL / min over 1 min, 20-120 mL / min over 2 min, then 120 mL / min).

[0188] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analytical conditions. The purity of the target product was 97.8%.

[0189] Analytical conditions: Retention time = 5.04 min; Column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% B conc): 20-60% over 7.15 min, then 60-95% over 0.3 min, then 95-95% over 1.55 min; Flow rate: 0.5 mL / min ESI-MS(+) observed m / z = 1051.58 (M+2H). ２＋

[0190] Example 1-2 Synthesis of Myostatin_000099_15meC (SEQ ID NO: 112)

[0191]

[0192] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical Co., Ltd., 0.52 mmol / g, 0.19 g) on ​​a CEM Liberty Blue solid-phase synthesizer in accordance with the manufacturer's instructions.

[0193] To introduce each residue, 0.21 M Fmoc-AA in DMF / 0.5 M HATU (in DMF) / 1 M DIEA (in DMF) (5.3 equivalents / 5 equivalents / 10 equivalents) was used per equivalent of resin, and the reaction was carried out twice for 10 minutes at 75°C. However, for the first, third, eighth, and ninth residues, the reaction was carried out once for 10 minutes at 75°C. For the second and tenth residues, the reaction was carried out twice for 30 minutes at 25°C. For the fifteenth residue, the reaction was carried out once for 30 minutes at 25°C. Furthermore, Fmoc removal was carried out by reacting with a 20% piperidine solution in DMF at 25°C for 5 minutes, followed by a 10-minute reaction. However, for the first, second, third, fifth, seventh, eighth, ninth, and eleventh residues, the reaction was carried out for 3 minutes at 75°C. Introduction of a chloroacetyl group was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin retaining the Fmoc-protected peptide obtained in the previous step by the method described above, followed by shaking ClAcOH / DIPCI / HOSu (10 equivalents / 10 equivalents / 10 equivalents) in DCM (2.5 mL) for 60 minutes, followed by adding a ClAcOSu solution prepared by adding the same amount of NMP as the DCM, and shaking at 25°C for 60 minutes.

[0194] To deprotect the side chains and cleave them from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and three times with methylene chloride, then washed with diethyl ether, and dried under reduced pressure. Then, reagent cocktail A (5 mL, TFA / H ２ A mixture of 2.5% PEG / TIS / DODT (volume ratio 92.5 / 2.5 / 2.5 / 2.5) was added and the mixture was shaken at room temperature for 30 minutes. The reaction solution was filtered and recovered through a frit. The solid phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate described above. When this filtrate was added to an excess of a diethyl ether / hexane (1 / 1) mixed solvent cooled to 0°C, a cloudy white precipitate formed.

[0195] This mixture was centrifuged (9000 rpm, 0°C, 2 min), and the solution was decanted. The resulting solid was washed again with a small amount of diethyl ether cooled to 0°C and then dried under reduced pressure. The resulting solid (peptide) was used in the subsequent cyclization reaction. The peptide was dissolved in DMSO to a final concentration of 2.5 mM based on the molar number of the solid-phase resin, and then 10 equivalents of triethylamine was added. After stirring at room temperature for 7 hours, acetic acid was added to quench the reaction, thereby carrying out the cyclization reaction of the peptide. The reaction solution was concentrated under reduced pressure using a Genevac EZ-II Elite.

[0196] The resulting crude product was purified using the following conditions: Column: Waters Xbridge® C18 5 μm 30×150 mm; Mobile phase: A=0.1% TFA in water, B=0.1% TFA in MeCN; Temperature: 40° C.; Gradient (% B): 8-33% over 3 min, 33-38% over 8 min, 38-60% over 1 min; Flow rate: 45 mL / min.

[0197] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the analytical conditions. The purity of the target product was 97.4%.

[0198] Analytical conditions: Retention time = 12.11 min; Column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60 °C; Gradient (% B conc): 20-60% over 20 min, then 60-95% over 1 min, then 95-95% over 5 min; Flow rate: 0.25 mL / min ESI-MS(+) observed m / z = 1050.59 (M + 2H). ２＋

[0199] Example 2 Analysis of Peptides The peptides of interest were synthesized according to the general method described above and the synthesis methods of Examples 1-1 and 1-2.

[0200]

[0201] Table 1 shows the target peptide, analytical conditions, retention time, and observed values ​​in ESI-MS(+). terminus indicates the C-terminal functional group (in the table, "-OH" indicates COOH, and "-NH" indicates CO(NH ２ ) indicates a cyclic amino acid sequence, and the absence of any description indicates that no functional group is present at the C-terminus. Furthermore, for "Cyclization" in the table, the description of ClAc indicates that the first amino acid to which a chloroacetyl group has been introduced is cyclized by binding with Cys present at the C-terminus. Furthermore, "Additional / Linker" in the table indicates a linker or additional amino acid not included in the cyclic structure. For example, in Myostatin_99_variant1_10 (SEQ ID NO: 126), the amino acid sequence of X1-X15 forms a cyclic structure, and an amino acid sequence called Dap-Dap is further added as a linker from the dc of the C-terminal X15.

[0202] Example 3: Evaluation of in vitro activity using a reporter gene assay for myostatin signaling in HepG2 cell line In this example, the in vitro myostatin inhibitory effects of the peptides synthesized in Examples 1 and 2 were evaluated using a reporter gene assay for myostatin signaling in HepG2 cell line.

[0203] Evaluation method (1) HepG2 cell line (HB-8065, ATCC) cultured in EMEM (055-08975, Wako Industries) containing 10% FBS (10270106, Gibco) and gentamicin 50 μg / mL (11980-14, Nacalai Tesque) in a 96-well plate (167008, Thermo) was cultured at 2.0 × 10 ４ Seed cells / well and CO ２ Incubator (37°C, 5% CO ２ The cells were cultured for 24 hours in a pGL3 (CAGA) vector (E1751, Promega) containing a luciferase gene downstream of a promoter sequence with 12 CAGA repeats. １２The α-luc plasmid was added at a concentration of 0.04 μg / well together with a transfection reagent (X-tremGENE9 DNA Transfection Reagent, 6365809001, Roche). ２ Incubator (37°C, 5% CO ２ The medium was discarded and replaced with 50 μL / well of EMEM containing 0.1% bovine serum albumin (A7030, Sigma) and 50 μg / mL gentamicin. ２ Incubator (37°C, 5% CO ２ ) for 2 hours.

[0204] Myostatin (788-G8 / C, R&D systems) or GDF-11 (1958-GD-G8 / CF, R&D systems) was added to the final concentrations shown in Table 2.

[0205]

[0206] Myostatin_99_Variant_07 (SEQ ID NO: 3) synthesized in Example 1-1 was added in a 4-fold serial dilution series to give concentrations of 0.006-100 nmol / L (myostatin inhibition) and 0.12-2000 nmol / L (GDF-11 inhibition). Blank wells to which neither myostatin, GDF-11, nor peptide was added were also prepared. The wells were incubated for 20-24 hours in CO ２ Incubator (37°C, 5% CO ２ ) and cultured.

[0207] The cell culture medium was removed, and a 1:1 mixture of PBS (14190144, Thermo) and the chromogenic substrate One-Glo Luciferase Assay System (E6120, Promega) was added to each well at 100 μL / well. The mixture was stirred for 5 minutes on a plate shaker and then transferred to a 384-well plate (3570, Corning) at 20 μL / well.

[0208] After centrifugation at 1,000 x g for 1 minute, the luminescence signal (700 nm luminescence) was measured using a plate reader (Envision 2104, PerkinElmer). Based on the measured values, the IC50 value of the peptide was calculated using software (Prism 9, GraphPad). The results are shown in Table 3.

[0209]

[0210] Evaluation method (2) Evaluation by two-concentration assay pGL3 (CAGA) is a pGL3 vector (E1751, Promega) in which a luciferase gene was inserted downstream of a promoter sequence with 12 CAGA repeats linked together. １２ The -luc plasmid was transfected into a HepG2 cell line (HB-8065, ATCC). The HepG2 cell line (HB-8065, ATCC) was cultured in the same manner as in evaluation method (1).

[0211] Myostatin (788-G8 / CF, R&D systems) or GDF-11 (1958-GD-G8 / CF, R&D systems) was added to a final concentration of 10 ng / mL, and the various peptides synthesized in Examples 1 and 2 were added to 30 nmol / L or 100 nmol / L () for myostatin inhibition measurement and GDF-11 inhibition measurement. Blank wells to which neither myostatin, GDF-11 nor peptide was added were also prepared, and the wells were incubated for 20-24 hours in a CO atmosphere. ２ Incubator (37°C, 5% CO ２ ) and cultured.

[0212] The cell culture medium was removed, and a 1:1 mixture of PBS (14190144, Thermo) and the chromogenic substrate One-Glo Luciferase Assay System (E6120, Promega) was added to each well at 100 μL / well. The mixture was then agitated for 5 minutes on a plate shaker and transferred to a 384-well plate (3570, Corning) at 20 μL / well.

[0213] After centrifugation at 1,000 x g for 1 minute, the luminescence signal (700 nm luminescence) was measured using a plate reader (Envision 2104, PerkinElmer). Based on the measurement results, the inhibition rate at each peptide concentration was calculated. The results are shown in Table 4.

[0214]

[0215] As shown in Tables 3 and 4, all of the peptides of the present invention exhibited inhibitory activity against myostatin. Furthermore, it was shown that the GDF-11 inhibitory activity of the peptides of the present invention was lower than the myostatin inhibitory activity.

[0216] Example 4 Distribution of Peptide in Skeletal Muscle In this example, the distribution of Myostatin — 99_Variant — 07 (SEQ ID NO: 3) peptide in skeletal muscle was investigated.

[0217] The Myostatin_99_Variant_07 (SEQ ID NO: 3) peptide synthesized in accordance with Example 1-1 was subcutaneously administered at 10 mg / kg to 6-8 week-old male CD1 mice. Plasma and quadriceps muscle concentrations were measured 24, 48, and 72 hours after administration. The results are shown in Table 5.

[0218]

[0219] The peptide was found to be distributed in muscle tissue at higher concentrations than in plasma at all time points. Plasma was separated from blood by centrifugation, and tissue was homogenized to 25%. Protein was removed using acetonitrile containing 5% formic acid, and the drug concentration was measured using an LC / MS / MS system (LCMS-8060, Shimadzu Corporation).

[0220] Example 5 In Vivo Myostatin Inhibitory Effect in Wild-Type Mice In this example, the in vivo myostatin signal inhibitory effect of the Myostatin — 99_Variant — 07 (SEQ ID NO: 3) peptide synthesized according to Example 1-1 was evaluated.

[0221] Four-week-old DBA / 2NCrl male mice (Jackson Laboratory Japan, Inc.) were used. The mice were housed in groups of five per cage and were given food (CRF-1, Oriental Yeast Co., Ltd.) and water (tap water) ad libitum. Peptides were administered subcutaneously every day for 14 days as shown in Table 6, and the weight of the mice in each group was measured. Weight measurements were performed every day using an electronic balance, starting from the day before administration.

[0222]

[0223] The changes in body weight over time are shown in Figure 1. The mean value ± standard error is shown. *: P<0.05, **: P<0.01 vs. PBS, Student t-test.

[0224] In Figure 1, v07 shows the evaluation results of Myostatin_99_Variant_07 (SEQ ID NO: 3).

[0225] As shown in Figure 1, a dose-dependent increase in body weight was observed, confirming the in vivo myostatin inhibitory effect of the peptide of the present invention.

[0226] Example 6 Muscle Strength-Improving and Muscle Necrosis-Inhibiting Effects of Peptides in B10 mdx Mice In this example, the muscle strength-improving effects of the Myostatin_99_Variant_07 (SEQ ID NO: 3) peptide synthesized according to Example 1-1 in DMD model mice were evaluated using B10 mdx model mice (Non-Patent Document 17).

[0227] As shown in Table 7, peptides or PBS as a solvent control (vehicle) were subcutaneously administered to 5-week-old male C57BL / 10-mdx / Jcl (Nippon Clea) mice three times a week or every day for 5 weeks.

[0228]

[0229] Using a smart grip strength measuring device for rats and mice (MK-380V: Muromachi Kikai), limb grip strength and body weight were measured on the final day of preliminary breeding (day of grouping), Days 7, 14, 21, 27, and 33, with the individual numbers of the mice being blinded so that the examiner would not know the test conditions. Grip strength measurements were performed seven times consecutively, and the average of the five values ​​excluding the highest and lowest values ​​was used as individual data. Body weight was measured using an electronic balance.

[0230] Figure 2 shows the time course of changes in grip strength. In the figure, v07 indicates the evaluation results for Myostatin_99_Variant_07 (SEQ ID NO: 3), and indicates the mean ± standard error. *: P<0.05, **: P<0.01 vs. PBS, Student t-test. A continuous increase in grip strength was observed in the group administered with Myostatin_99_Variant_07 peptide compared to the Vehicle group administered with PBS.

[0231] The time course of changes in body weight is shown in Figure 3. Note that mean ± standard error *: P<0.05, vs. PBS, Student t-test. Administration of the Myostatin_99_Variant_07 peptide resulted in a continuous increase in body weight compared to the vehicle group.

[0232] Furthermore, serum creatine kinase (CK) concentrations (mean ± SEM) on Day 35, when the animals were euthanized, were measured, and the concentrations were 3,836 ± 754 IU / L in the control Vehicle group and 3,247 ± 558 IU / L in the v07 group. Thus, a tendency for a decrease in CK concentrations was observed with administration of Myostatin_99_Variant_07 of the present invention, suggesting an inhibitory effect on skeletal muscle necrosis.

[0233] Only the amino acid sequences contained in the linker or additional amino acids used in the examples are shown in Table 8.

[0234]

[0235] Reference Examples Synthesis of Novel Unnatural Amino Acids This Reference Example provides examples of the synthesis of various unnatural amino acids.

[0236] Reference Example 1 Synthesis of Fmoc-W1Ph4COO(tBu)-OH

[0237]

[0238] Tertiary-butyl 4-iodobenzoate (25.0 g, 82.2 mmol, CAS: 120363-13-5) was dissolved in DMF (82 mL), and 1H-indole (10.6 g, 90.4 mmol, CAS: 120-72-9), copper(I) iodide (3.1 g, 16.4 mmol, CAS: 7681-65-4), and cesium carbonate (40.3 g, 123 mmol, CAS: 534-17-8) were added at room temperature, followed by stirring at 120°C for 1 hour. The mixture was diluted with 0.1 M aqueous hydrochloric acid and extracted twice with ethyl acetate. The combined organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate=100 / 50 to 0 / 100) to give tert-butyl 4-(1H-indol-1-yl)-benzoate.

[0239] At room temperature, tert-butyl 4-(1H-indol-1-yl)benzoate (20.5 g, 69.9 mmol) was dissolved in DMF (233 mL), and N-iodosuccinimide (18.9 g, 83.9 mmol, CAS: 516-12-1) was added, followed by stirring for 10 minutes. The reaction mixture was ice-cooled, and aqueous sodium thiosulfate solution was added, followed by extraction twice with ethyl acetate. The combined organic layer was washed with saturated aqueous sodium bicarbonate and saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 90 / 10-80 / 20) to give tert-butyl 4-(3-iodo-1H-indol-1-yl)benzoate.

[0240] Zinc (1.30 g, 19.9 mmol, CAS: 7440-66-6) was suspended in DMF (13 mL) at room temperature, and 1,2-dibromoethane (0.057 mL, 0.665 mmol, CAS: 106-93-4) and trimethylsilyl chloride (0.088 mL, 0.665 mmol, CAS: 75-77-4) were added, followed by stirring at 50°C for 30 minutes. After cooling the reaction mixture to room temperature, methyl (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-iodopropanoate (3.00 g, 6.65 mmol, CAS: 156017-42-4) was added, followed by stirring at room temperature for 1 hour to prepare an organozinc reagent.

[0241] To a solution of tert-butyl 4-(3-iodo-1H-indol-1-yl)-benzoate (2.79 g, 6.65 mmol) in DMF (13 mL) was added Pd ２ (dba) ３ CHCl ３ (0.344 g, 0.332 mmol, CAS: 52522-40-4) and SPhos (0.546 g, 1.33 mmol, CAS: 657408-07-6) were added at room temperature, followed by the prepared organozinc reagent. After stirring at 60°C for 2 hours, the reaction mixture was diluted with ethyl acetate, and saturated aqueous sodium bicarbonate was added at 0°C. After filtering the solid, the filtrate was extracted twice with ethyl acetate. The combined organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 97 / 3-20 / 80).

[0242] A portion of the obtained product (2.01 g, 3.26 mmol) was dissolved in isopropanol (48.9 mL), and calcium chloride (5.79 g, 52.1 mmol, CAS: 10043-52-4) was added under ice-cooling. Subsequently, an aqueous solution (16.3 mL) of lithium hydroxide (0.312 g, 13.0 mmol, CAS: 1310-65-2) was added dropwise. After the dropwise addition was completed, the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was cooled to 0°C, and the pH was adjusted to 4 with the addition of 1 M aqueous hydrochloric acid, followed by extraction with ethyl acetate. The combined organic layer was washed with saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane / methanol = 95 / 5-80 / 20). W1Ph4COO(tBu) was obtained. ESI-MS(+) Observed value m / z = 603.3 (M + H) ＋

[0243] Reference Example 2 Synthesis of Fmoc-W1Ph4OMe-OH

[0244]

[0245] W1Ph4OMe was obtained by synthesizing in the same manner as W1Ph4COO(tBu) in Reference Example 1. However, 1-iodo-4-methoxybenzene was used as the starting material instead of tert-butyl 4-iodobenzoate. ESI-MS (+) observed value m / z = 533.2 (M+H) ＋

[0246] Reference Example 3 Synthesis of Fmoc-Hgl-O(tBu)

[0247]

[0248] (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-6-(allyloxy)-6-oxohexanoic acid (2.00 g, 4.72 mmol, CAS: 133464-45-6) was dissolved in dichloromethane (47.2 mL) and THF (4.72 mL), and tert-butyl 2,2,2-trichloroacetimidate (2.06 g, 9.45 mmol, CAS: 98946-18-0) was added at room temperature. The reaction mixture was stirred at room temperature for 12 hours and then at 45°C for 20 hours. The reaction mixture was concentrated under reduced pressure, and the solid was filtered and washed with dichloromethane (50 mL). The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 80 / 20).

[0249] The obtained 6-allyl 1-(tert-butyl)(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)hexanedioate (2.26 g, 4.72 mmol) was dissolved in dichloromethane (23.6 mL) and THF (23.6 mL). Then, phenylsilane (1.16 mL, 9.44 mmol, CAS: 694-53-1) and tetrakis(triphenylphosphine)palladium (0.273 g, 0.236 mmol, CAS: 14221-01-3) were added at 0°C.

[0250] The reaction mixture was stirred at 0°C for 1 hour and then at room temperature for 24 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 100 / 0 to 80 / 20-30 / 70). Hgl-O(tBu) was obtained. ESI-MS(+) observed value m / z = 384.3 (M-C4H8). ＋

[0251] Reference Example 4 Synthesis of Fmoc-dhgl-O(tBu)

[0252]

[0253] Using D-α-aminoadipic acid (CAS: 7620-28-2) as a starting material, (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(allyloxy)-6-oxohexanoic acid was obtained by the method described in Angew. Chem. Int. Ed., 2016, 55, 1192-1195.

[0254] dhgl-O(tBu) was obtained by synthesis in the same manner as for Hgl-O(tBu). However, instead of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(allyloxy)-6-oxohexanoic acid, (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(allyloxy)-6-oxohexanoic acid obtained above was used. ESI-MS(+) observed value m / z = 384.1 (M-C4H8) ＋

[0255] Reference Example 5 Synthesis of Fmoc-MeHgl-OtBu

[0256]

[0257] MeHgl-O(tBu) was obtained by synthesizing it in the same manner as for Hgl-O(tBu). However, (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-6-(allyloxy)-6-oxohexanoic acid was used as the starting material instead of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(allyloxy)-6-oxohexanoic acid. ESI-MS(+) observed value m / z = 398.3 (M-C4H8) ＋

[0258] Reference Example 6 Synthesis of Fmoc-Medhgl-OtBu

[0259]

[0260] (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-6-(allyloxy)-6-oxohexanoic acid was obtained by the method described in US 18 / 109702, except that (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(allyloxy)-6-oxohexanoic acid was used as the starting material in place of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(allyloxy)-6-oxohexanoic acid.

[0261] Medhgl-O(tBu) was obtained by synthesis in the same manner as for Hgl-O(tBu). However, instead of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(allyloxy)-6-oxohexanoic acid, (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)(methyl)amino)-6-(allyloxy)-6-oxohexanoic acid obtained above was used. ESI-MS(+) observed value m / z = 398.1 (M-C4H8)

[0262] Reference Example 7 Synthesis of Fmoc-W6H(PMB)1Ph4COO(tBu)-OH

[0263]

[0264] Cesium carbonate (73.4 g, 225 mmol, CAS: 534-17-8) and 4-methoxybenzyl chloride (16.9 mL, 124 mmol, CAS: 824-94-2) were added to a DMF solution (225 mL) of 1H-indol-6-ol (15.0 g, 113 mmol, CAS: 2380-86-1), and the mixture was stirred at room temperature for 24 hours. The reaction mixture was diluted with ethyl acetate and saturated brine, and the solid was filtered. The filtrate was extracted twice with ethyl acetate. The combined organic layer was washed three times with saturated brine and then dried over sodium sulfate. After filtration and concentration under reduced pressure, the resulting solid was suspended in dichloromethane and collected by filtration. 6-((4-methoxybenzyl)oxy)-1H-indole was obtained.

[0265] To a solution of a portion of the obtained product (3.7 g, 14.5 mmol) in DMF (13 mL), 4-iodobenzoic acid tert-butyl ester (4.0 g, 13.2 mmol, CAS: 120363-13-5), cesium carbonate (6.5 g, 19.8 mmol), and copper iodide (0.50 g, 2.6 mmol, CAS: 7681-65-4) were added, and the mixture was stirred at 120°C for 7 hours. The reaction solution was diluted with ethyl acetate and saturated brine, and filtered through Celite. The filtrate was extracted with ethyl acetate, washed twice with saturated brine, and then dried over sodium sulfate. After filtration and concentration under reduced pressure, the obtained residue was purified by silica gel column chromatography (heptane / ethyl acetate = 97 / 3-80 / 20) to give 4-(6-((4-methoxybenzyl)oxy)-1H-indol-1-yl)benzoic acid tert-butyl ester.

[0266] A portion of the obtained product (5.6 g, 12.9 mmol) was dissolved in DMF (431 mL), N-iodosuccinimide (2.9 g, 12.8 mmol, CAS: 516-12-1) was added, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was ice-cooled, quenched with aqueous sodium thiosulfate, and extracted with ethyl acetate. The obtained organic layer was washed successively with saturated aqueous sodium bicarbonate and saturated brine, and dried over sodium sulfate. The mixture was filtered and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (heptane / ethyl acetate = 90 / 10-50 / 50) to give 4-(3-iodo-6-((4-methoxybenzyl)oxy)-1H-indol-1-yl)benzoic acid tert-butyl ester.

[0267] Zinc (2.0 g, 31 mmol, CAS: 7440-66-6) was suspended in DMF (26 mL) at room temperature, and iodine (2.6 g, 10.4 mmol, CAS: 7790-99-0) was added, followed by stirring at room temperature for 20 minutes. Methyl (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-iodopropanoate (5.6 g, 12.5 mmol, CAS: 156017-42-4) was added to the reaction solution, and the mixture was stirred at room temperature for 3 hours to prepare an organozinc reagent.

[0268] A solution of 4-(3-iodo-6-((4-methoxybenzyl)oxy)-1H-indol-1-yl)benzoic acid tert-butyl ester (5.7 g, 10.4 mmol) in DMF (26 mL) was added to Pd ２ (dba) ３ CHCl ３ (0.54 g, 0.52 mmol, CAS: 52522-40-4) and SPhos (0.85 g, 2.1 mmol, CAS: 657408-07-6) were added at room temperature, followed by the prepared organozinc reagent. The reaction solution was stirred at 70°C for 3 hours. The reaction solution was diluted with ethyl acetate and saturated brine and filtered through Celite. The filtrate was extracted with ethyl acetate, and the resulting organic layer was washed successively with saturated aqueous sodium bicarbonate and saturated brine, and then dried over sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 85 / 15-45 / 55) to give (S)-4-(3-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methoxy-3-oxopropyl)-6-((4-methoxybenzyl)oxy-1H-indol-1-yl)benzoic acid tert-butyl ester.

[0269] A portion of the obtained product (5.0 g, 6.7 mmol) was dissolved in tetrahydrofuran (34 mL) and isopropanol (16.7 mL). Under ice cooling, an aqueous solution (11.7 mL) of calcium chloride (11.9 g, 107 mmol, CAS: 10043-52-4) was added, followed by the dropwise addition of an aqueous solution (5.0 mL) of lithium hydroxide (0.64 g, 26.8 mmol, CAS: 1310-65-2). After the dropwise addition was completed, the reaction mixture was stirred at room temperature for 2 days. 1 M hydrochloric acid was added to adjust the pH to 3. The reaction mixture was extracted with ethyl acetate, and the resulting organic layer was washed with saturated brine and dried over sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by silica gel column chromatography (heptane / ethyl acetate = 65 / 35-0 / 100, followed by dichloromethane / methanol = 100 / 0-90 / 10). The fractions containing the target compound were concentrated and then purified by silica gel column chromatography (ethyl acetate / methanol = 100 / 0 - 90 / 10). W6H1Ph4COO was obtained. ESI-MS (+) observed value m / z = 739.3 (M+H). ＋

[0270] Reference Example 8 Synthesis of Fmoc-W7Ph3C-OH

[0271]

[0272] 7-Bromo-1H-indole (19.6 g, 100 mmol, CAS: 51417-51-7), (3-chlorophenyl)boronic acid (18.8 g, 120 mmol, CAS: 63503-60-6), Pd(PPh3)4 (5.8 g, 5.0 mmol, CAS: 14221-01-3), and tripotassium phosphate (63.7 g, 300 mmol, CAS: 7778-53-2) were dissolved in 1,4-dioxane (450 mL) and water (50 mL) and stirred at 100°C for 75 minutes. The reaction solution was diluted with water and extracted twice with ethyl acetate. The combined organic layer was washed with water, washed with saturated brine, and then dried over sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate=90 / 10-70 / 30) to give 7-(3-chlorophenyl)-1H-indole.

[0273] To a solution of a portion of the obtained product (6.8 g, 30 mmol) in DMF (80 mL) was added potassium hydroxide (4.2 g, 75 mmol), and then a DMF solution (20 mL) of iodine (7.6 g, 30 mmol, CAS: 7553-56-2) was added under ice-cooling, followed by stirring at room temperature for 30 minutes. The reaction solution was diluted with ethyl acetate, washed with saturated brine, and then dried over sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by silica gel column chromatography (heptane / ethyl acetate = 90 / 10-80 / 20) to give 7-(3-chlorophenyl)-3-iodo-1H-indole.

[0274] Zinc (3.9 g, 60 mmol) was suspended in DMF (50 mL) at room temperature, and iodine (1.5 g, 6.0 mmol) was added, followed by stirring for 30 minutes at 60° C. Methyl (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-iodopropanoate (10.8 g, 24.0 mmol, CAS: 156017-42-4) was added to the reaction solution, followed by stirring for 90 minutes at room temperature to prepare an organozinc reagent.

[0275] 7-(3-chlorophenyl)-3-iodo-1H-indole (7.1 g, 20 mmol), Pd ２ (dba) ３ CHCl3 (1.0 g, 1.0 mmol, CAS: 52522-40-4) and SPhos (1.6 g, 4.0 mmol, CAS: 657408-07-6) were added at room temperature and stirred at 60°C for 30 minutes, after which the prepared organozinc reagent was added and stirred at 60°C for 1 hour. The reaction solution was diluted with ethyl acetate and quenched by adding 0.5 M hydrochloric acid under ice-cooling. After stirring for 5 minutes under ice-cooling, the solution was filtered through Celite and washed with ethyl acetate. The filtrate was extracted with ethyl acetate, and the resulting organic layer was washed with saturated brine and dried over sodium sulfate. After filtration and concentration under reduced pressure, the solution was purified by silica gel column chromatography (hexane / ethyl acetate = 90 / 10-40 / 60) to obtain (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(7-(3-chlorophenyl)-1H-3-yl)propanoic acid ester.

[0276] A portion of the obtained product (4.4 g, 8.0 mmol) was dissolved in isopropanol (120 mL) and calcium chloride (14.2 g, 128 mmol, CAS: 10043-52-4) was added at 0°C, followed by the dropwise addition of an aqueous solution (40 mL) of lithium hydroxide (0.77 g, 31.9 mmol, CAS: 1310-65-2). After the dropwise addition was completed, the reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was cooled to 0°C, and 1 M hydrochloric acid (30 mL) was added to adjust the pH to 3-4. The reaction solution was extracted three times with ethyl acetate. The combined organic layer was washed with water, then with saturated brine, and dried over sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by silica gel column chromatography (heptane / ethyl acetate = 30 / 70-80 / 20). W7Ph3C was obtained. ESI-MS(+) Observed value m / z = 537.2 (M + H) ＋

[0277] Reference Example 9 Synthesis of Fmoc-pBph2aao(tBu)-OH

[0278]

[0279] 4-Bromo-[1,1'-biphenyl]-2-ol (4.5 g, 18.0 mmol, CAS: 1679324-76-5) was dissolved in acetone (90 mL), and cesium carbonate (8.8 g, 27.0 mmol, CAS: 534-17-8) and tert-butyl 2-bromoacetate (4.0 mL, 27.0 mmol, CAS: 5292-43-3) were added, followed by stirring at room temperature for 19 hours. The mixture was concentrated under reduced pressure and quenched with saturated aqueous sodium bicarbonate. The mixture was extracted with ethyl acetate and washed with saturated brine. The mixture was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (heptane / ethyl acetate = 97 / 3-90 / 10) to give tert-butyl 2-((4-bromo-[1,1'-biphenyl]-2-yl)oxy)acetate.

[0280] Zinc (3.6 g, 55 mmol) was suspended in DMF (45 mL) at room temperature, and iodine (4.6 g, 18.2 mmol) was added, followed by stirring at room temperature for 30 minutes. Methyl (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-iodopropanoate (5.6 g, 12.5 mmol, CAS: 156017-42-4) was added to the reaction solution, followed by stirring at room temperature for 3.5 hours to prepare an organozinc reagent. Pd ２ (dba) ３ CHCl ３ (0.94 g, 0.91 mmol, CAS: 52522-40-4) and SPhos (1.5 g, 3.6 mmol, CAS: 657408-07-6) were added at room temperature, followed by the prepared organozinc reagent. The reaction solution was stirred at 70°C for 3 hours. The reaction solution was diluted with ethyl acetate and saturated brine and filtered through Celite. The filtrate was extracted with ethyl acetate, and the resulting organic layer was washed successively with saturated aqueous sodium bicarbonate and saturated brine, and then dried over sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 90 / 10-40 / 60) to give methyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-(2-tert-butoxy)-2-oxoethoxy)-[1,1'-biphenyl]-4-yl)propanoic acid.

[0281] A portion of the obtained product (2.9 g, 4.7 mmol) was dissolved in tetrahydrofuran (24 mL) and isopropanol (11.9 mL). Under ice-cooling, an aqueous solution (8.3 mL) of calcium chloride (6.3 g, 56.9 mmol, CAS: 10043-52-4) was added, followed by the dropwise addition of an aqueous solution (3.6 mL) of lithium hydroxide (0.34 g, 14.2 mmol, CAS: 1310-65-2). After the dropwise addition was completed, the reaction mixture was stirred at room temperature for 16 hours. 1 M hydrochloric acid was added to adjust the pH to 3. The reaction mixture was extracted with ethyl acetate, and the resulting organic layer was washed with saturated brine and dried over sodium sulfate. After filtration and concentration under reduced pressure, the resulting residue was purified by silica gel column chromatography (heptane / ethyl acetate = 80 / 20-60 / 40, followed by dichloromethane / methanol = 95 / 5-80 / 20). pBph2aao was obtained. ESI-MS(+) Observed value m / z = 538.3 (M + H) ＋

[0282] The peptide of the present invention has myostatin inhibitory activity and can be used as a pharmaceutical composition, diagnostic composition, research composition, etc. for myostatin-related diseases or diseases or symptoms accompanied by decreased muscle function.

Claims

1. A peptide comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 3, 110-140, or a pharma- ceutically acceptable salt thereof.

2. The peptide of claim 1, or a pharma- ceutically acceptable salt thereof, wherein the peptide is a cyclic peptide.

3. The peptide according to claim 1, or a pharma- ceutically acceptable salt thereof, which has a cyclic structure in which a chloroacetylated amino acid and a cysteine ​​residue contained in the peptide are bound.

4. The peptide according to claim 1, or a pharma- ceutically acceptable salt thereof, which has a cyclic structure in which the amino group of the first amino acid residue and the carboxy group of the fifteenth amino acid residue contained in the peptide are bonded.

5. The peptide of claim 1, or a pharma- ceutically acceptable salt thereof, further comprising additional amino acid residues.

6. The peptide of claim 1, or a pharma- ceutically acceptable salt thereof, comprising a linker at the C-terminus.

7. A pharmaceutical composition comprising the peptide according to any one of claims 1 to 6, or a pharma- ceutically acceptable salt thereof.

8. The pharmaceutical composition according to claim 7, having myostatin inhibitory activity.

9. The pharmaceutical composition according to claim 7 for preventing or treating a myostatin-related disease or a disease or condition accompanied by decreased muscle function.

10. A method for inhibiting myostatin signaling using a peptide described in any one of claims 1 to 6, or a pharma- ceutically acceptable salt thereof.

11. A diagnostic composition for diagnosing a myostatin-related disease or a disease or condition accompanied by decreased muscle function, comprising the peptide according to any one of claims 1 to 6, or a pharma-ceutically acceptable salt thereof.

12. A method for testing a peptide or a pharma- ceutically acceptable salt thereof, comprising testing at least one of the following: a) solubility in a solvent; b) myostatin inhibitory activity; c) toxicity to cells and / or tissues; or d) toxicity to experimental animals, wherein the peptide or the pharma-ceutically acceptable salt thereof is a peptide or a pharma-ceutically acceptable salt thereof according to any one of claims 1 to 6.