Peptide and peptide-containing agent

Abstract

[Problem] To provide a novel peptide having a BMP4 binding ability. [Solution] The present invention relates to a peptide and an agent comprising the same. A peptide according to the present invention comprises the following amino acid sequence: E-R-V-F4COO-Atp-D-R-Bph-P-Y-Bph-Y-F4COO-F4COO-S-C (SEQ ID NO: 1), wherein the amino acid sequence of the peptide is as shown in the specification, or an amino acid sequence having a substitution, addition, deletion or insertion in 1-15 amino acid residues selected from a group consisting of the first, second, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth and sixteenth amino acid residues of the above-mentioned amino acid sequence.

Description

Technical Field

[0001] The present invention relates to a peptide and an agent containing the peptide. Background Art

[0002] Stem cells with pluripotency, such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), have been reported so far, and attempts are being made to create regenerative medicine or disease models using cells induced by differentiation from these pluripotent stem cells.

[0003] Growth factors are important information transfer molecules related to embryonic development, tissue repair and regulation of intercellular communication. Many growth factors that are responsible for important tasks in the survival, self-replication, differentiation and dedifferentiation of stem cells have been identified. By combining these growth factors, stem cell culture can be controlled in a variety of ways.

[0004] Bone morphogenetic proteins (BMP) are secretory signaling molecules belonging to the TGF-β superfamily. BMP was originally discovered as a control factor for cartilage tissue or bone formation. It is known that there are at least 20 types of BMP, and they act as growth factors. Noggin is a secretory protein with BMP inhibitory activity. Noggin inhibits BMP signaling by binding to BMP itself to prevent BMP from binding to receptors, thereby affecting cell differentiation and induction. For example, if human ES cells are cultured in Noggin-added culture medium, the action of BMP-2 produced endogenously in ES cells is inhibited, thereby inhibiting the differentiation of ES cells into extraembryonic endoderm cells and maintaining the undifferentiated state of ES cells. Afterwards, by continuing to culture under neural differentiation culture conditions, differentiation induction into neural cells is promoted (Patent Document 1). As for secretory proteins that act as such BMP inhibitors, in addition to Noggin, Chordin, follistatin, etc. are known. However, since these factors are proteins, when used in cell culture, in addition to increasing the culture cost, there are also problems such as lack of reproducibility.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: International Patent Publication No. 01 / 98463 Summary of the invention

[0008] Problems to be solved by the invention

[0009] The present inventors have made intensive research with the purpose of creating a peptide having BMP-binding activity, and have completed the present invention. The present invention provides a novel peptide, various agents containing the peptide, and the use of the peptide.

[0010] Technical means to solve the problem

[0011] The above-mentioned problems are solved by novel peptides having a sequence shown in SEQ ID NO: 1 and the like.

[0012] An invention described in this specification relates to the following peptide or a pharmaceutically acceptable salt thereof.

[0013] A peptide or a pharmaceutically acceptable salt thereof, comprising the following amino acid sequence:

[0014] ERV-F4COO-Atp-DR-Bph-PYP-Bph-Y-F4COO-F4COO-SC (SEQ ID NO: 1),

[0015] Or an amino acid sequence having a substitution, addition, deletion or insertion in 1 to 15 amino acid residues selected from the group consisting of the 1st, 2nd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th and 16th amino acid residues of the above amino acid sequence.

[0016] An invention described in this specification relates to the following peptide or a pharmaceutically acceptable salt thereof.

[0017] A peptide or a pharmaceutically acceptable salt thereof, which has the following amino acid sequence:

[0018] A peptide having an amino acid sequence represented by X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17 (SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof, or a peptide having an amino acid sequence represented by substitution, deletion, addition or insertion of 1 to 10 amino acids from the amino acid sequence represented by SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof,

[0019] X1 is E, A, T, Q or L,

[0020] X2 is R, K, Hgl, any polar amino acid or any aliphatic amino acid,

[0021] X3 is V,

[0022] X4 is an amino acid having an aromatic ring which may be a heterocyclic ring in the side chain,

[0023] X5 is any aliphatic amino acid, Y, D, Atp, Cha4tH, Gthp or A1Ac4pip,

[0024] X6 is D, any basic amino acid or any polar amino acid,

[0025] X7 is any basic amino acid or any polar amino acid,

[0026] X8 is biphenylalanine which may have a substituent and the C on the aromatic ring may be substituted with N,

[0027] X9 is any amino acid,

[0028] X10 is Y, Hty or 4Py,

[0029] X11 is any amino acid,

[0030] X12 is biphenylalanine which may have a substituent and the C on the aromatic ring may be substituted with N,

[0031] X13 is unsubstituted or substituted F, 4Py or 3Py6NH2,

[0032] X14 is F4COO, D or G,

[0033] X15 is F4COO, Y, 4Py, 3Py6NH2, W7N, Hph or N,

[0034] X16 is any amino acid,

[0035] X17 is C.

[0036] The peptide having an amino acid sequence in which 1 to 10 amino acid sequences are substituted, deleted, added or inserted from the amino acid sequence shown in SEQ ID NO: 2 is preferably one having BMP4 binding ability as described below. In addition, it may be a peptide having an amino acid sequence in which 1 to 9 (1 to 8, 1 to 5, 1 to 4, 1 to 3, 2 or 1) amino acid sequences are substituted, deleted, added or inserted from the amino acid sequence shown in SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

[0037] Preferred examples of the above-mentioned peptide or its pharmaceutically acceptable salt are:

[0038] X1 is E,

[0039] X2 is R or any polar amino acid,

[0040] X3 is V,

[0041] X4 is unsubstituted or substituted Y or F4COO,

[0042] X5 is V or Atp,

[0043] X6 is D or KCOpipzaa,

[0044] X7 is R,

[0045] X8 is Bph,

[0046] X9 is P or S,

[0047] X10 is Y or Hty,

[0048] X11 is any aliphatic amino acid,

[0049] X12 is unsubstituted or substituted Bph,

[0050] X13 is unsubstituted or substituted F,

[0051] X14 is F4COO,

[0052] X15 is F4COO or 4Py,

[0053] X16 is any amino acid,

[0054] X17 is C.

[0055] Preferred examples of the above-mentioned peptide or its pharmaceutically acceptable salt are:

[0056] X1 is E, A, T, Q or L,

[0057] X2 is R or KCOpipzaa,

[0058] X3 is V,

[0059] X4 is Y, F4F, F4COO, 4Py, H or F4aaO,

[0060] X5 is any aliphatic amino acid, Atp, Cha4tH, Gthp or A1Ac4pip,

[0061] X6 is D, S or KCOpipzaa,

[0062] X7 is R or K,

[0063] X8 is 3Py6Ph, Bph, Bph4C, pBph2F or Bph3C,

[0064] X9 is S, P, KCOpipzaa or H,

[0065] X10 is Y or Hty,

[0066] X11 is any aliphatic amino acid or Hyp,

[0067] X12 is biphenylalanine which may have a substituent,

[0068] X13 is unsubstituted or substituted F or 4Py,

[0069] X14 is F4COO,

[0070] X15 is F4COO or 4Py,

[0071] X16 is S,

[0072] X17 is C.

[0073] Preferred examples of the above-mentioned peptide or its pharmaceutically acceptable salt are:

[0074] A peptide or a pharmaceutically acceptable salt thereof, which has the following amino acid sequence: a peptide having an amino acid sequence as shown in X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-C (SEQ ID NO: 4), or a peptide having an amino acid sequence obtained by substituting, deleting, adding or inserting 1 to 10 amino acids from the amino acid sequence as shown in SEQ ID NO: 4, or a pharmaceutically acceptable salt thereof,

[0075] X1 is A,

[0076] X2 is R, KAc, Q or KCOpipzaa,

[0077] X3 is V,

[0078] X4 is Y, F4F or 4Py,

[0079] X5 is V,

[0080] X6 is D, KCOpipzaa, K, R, G or H,

[0081] X7 is R, K, KAc, 4Py, S or Cit,

[0082] X8 is 3Py6Ph or Bph,

[0083] X9 is P, S, K, H, G, S or KCOpipzaa,

[0084] X10 is Y,

[0085] X11 is any aliphatic amino acid,

[0086] X12 is Bph or 3Py6Ph,

[0087] X13 is Y or F4F,

[0088] X14 is F4COO,

[0089] X15 is F4COO or 4Py,

[0090] X16 is S.

[0091] The peptide having an amino acid sequence in which 1 to 10 amino acid sequences are substituted, deleted, added or inserted from the amino acid sequence shown in SEQ ID NO: 4, or a pharmaceutically acceptable salt thereof, preferably has BMP4 binding ability as described below. In addition, it may be a peptide having an amino acid sequence in which 1 to 9 (1 to 8, 1 to 5, 1 to 4, 1 to 3, 2 or 1) amino acid sequences are substituted, deleted, added or inserted from the amino acid sequence shown in SEQ ID NO: 4, or a pharmaceutically acceptable salt thereof.

[0092] Preferred examples of the above-mentioned peptide or a pharmaceutically acceptable salt thereof are a peptide having the ability to bind to BMP4 or a pharmaceutically acceptable salt thereof.

[0093] Preferred examples of the above-mentioned peptide or a pharmaceutically acceptable salt thereof are a peptide having BMP4 inhibitory activity or a pharmaceutically acceptable salt thereof.

[0094] Preferred examples of the above-mentioned peptide are a peptide consisting of an amino acid sequence described in any one of SEQ ID NOs: 5 to 187 and SEQ ID NOs: 243 to 421, or a pharmaceutically acceptable salt thereof,

[0095] Or a peptide having an amino acid sequence obtained by substitution, deletion, addition or insertion of 1 to 10 amino acids from the amino acid sequence described in any one of SEQ ID NOs: 5 to 187 and SEQ ID NOs: 243 to 421 and having the ability to bind to BMP4 or the activity to inhibit BMP4, or a pharmaceutically acceptable salt thereof.

[0096] An invention described in this specification relates to the following peptide or a pharmaceutically acceptable salt thereof.

[0097] A peptide or a pharmaceutically acceptable salt thereof, comprising the following amino acid sequence:

[0098] FG-MeF-G-Bph-GDD-Cha-A-MeF-LHC (SEQ ID NO: 241), or an amino acid sequence having a substitution, addition, deletion or insertion in 1 to 12 amino acid residues selected from the group consisting of the 1st, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th and 13th amino acid residues of the above amino acid sequence.

[0099] An invention described in this specification relates to the following peptide.

[0100] A peptide or a pharmaceutically acceptable salt thereof, which has the following amino acid sequence:

[0101] A peptide having an amino acid sequence shown as X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 (SEQ ID NO: 242), or a peptide having an amino acid sequence in which 1 to 12 amino acids are substituted, deleted, added or inserted from the amino acid sequence shown as SEQ ID NO: 242, or a pharmaceutically acceptable salt thereof,

[0102] X1 is phenylalanine which may have a substituent and in which the C on the aromatic ring may be substituted with N,

[0103] X2 is G,

[0104] X3 is N-methylphenylalanine which may have a substituent,

[0105] X4 is G, which may be a D-type aliphatic amino acid or a polar amino acid,

[0106] X5 is biphenylalanine in which the C on the aromatic ring may be substituted with N or phenylalanine which may have a substituent,

[0107] X6 is G or A of D type,

[0108] X7 is any polar amino acid,

[0109] X8 is any acidic amino acid,

[0110] X9 is any aliphatic amino acid,

[0111] X10 is any amino acid,

[0112] X11 is N-methylphenylalanine which may have a substituent,

[0113] X12 is any aliphatic amino acid,

[0114] X13 is any amino acid,

[0115] X14 is C.

[0116] Preferred examples of the above-mentioned peptides are peptides having BMP7 inhibitory activity or pharmaceutically acceptable salts thereof.

[0117] A preferred example of the above peptide is a peptide consisting of an amino acid sequence described in any one of SEQ ID NOs: 322 to 421, or a pharmaceutically acceptable salt thereof.

[0118] Or a peptide having an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, added or inserted into the amino acid sequence described in any one of SEQ ID NOs: 322 to 421 and having BMP7 inhibitory activity, or a pharmaceutically acceptable salt thereof.

[0119] Preferred examples of the above-mentioned peptide are cyclic peptides or pharmaceutically acceptable salts thereof.

[0120] A preferred example of the above-mentioned peptide is a peptide having a cyclic structure in which a chloroacetylated amino acid is bonded to a cysteine ​​residue contained in the above-mentioned peptide, or a pharmaceutically acceptable salt thereof.

[0121] A preferred example of the above-mentioned peptide is a peptide further comprising an additive amino acid residue sequence or a pharmaceutically acceptable salt thereof. An example of an additive amino acid residue sequence is a linker.

[0122] A preferred use example of the above-mentioned peptide is a BMP4 and / or BMP7 signal transduction inhibitor comprising any of the above-mentioned peptides or a pharmaceutically acceptable salt thereof.

[0123] A preferred use example of the above-mentioned peptide is a human stem cell differentiation induction regulator comprising any of the above-mentioned peptides or a pharmaceutically acceptable salt thereof.

[0124] A preferred example of the use of the above-mentioned peptide or its pharmaceutically acceptable salt, BMP4 and / or BMP7 signal transduction inhibitor and human stem cell differentiation induction control agent is a culture medium additive containing any one or two or more of them. By adding these to the culture medium, the BMP4 and / or BMP7 signal transduction of the object can be inhibited or the differentiation induction of human stem cells can be controlled.

[0125] A preferred use example of the above-mentioned peptide or its pharmaceutically acceptable salt is a method of inhibiting the information transmission activity of BMP4 and / or BMP7 using any of the above-mentioned peptides or its pharmaceutically acceptable salt. For example, by administering any of the above-mentioned peptides to a subject (e.g., a human or non-human animal), the information transmission activity of BMP4 and / or BMP7 in the subject can be inhibited. In addition, in a test tube or in vivo experiment, by administering any of the above-mentioned peptides to a subject cell, the information transmission activity of BMP4 and / or BMP7 in the subject cell can be inhibited.

[0126] A preferred use example of the above-mentioned peptide or its pharmaceutically acceptable salt is a method for controlling the differentiation induction of human stem cells using any of the above-mentioned peptides or its pharmaceutically acceptable salt. For example, by administering any of the above-mentioned peptides or its pharmaceutically acceptable salt to a subject (e.g., a human), the differentiation induction of human stem cells in the subject can be controlled. Furthermore, in an in vitro or in vivo experiment, by administering any of the above-mentioned peptides to the subject cells, the differentiation induction of human stem cells in the subject cells can be controlled.

[0127] Invention effect

[0128] The peptide of the present invention or its pharmaceutically acceptable salt has BMP4 binding ability, BMP4 inhibitory activity, preferably has BMP4 information transmission inhibitory activity, and more preferably has human stem cell differentiation induction control activity. The peptide of the present invention or its pharmaceutically acceptable salt has BMP7 information transmission inhibitory activity, preferably has human stem cell differentiation induction control activity. The peptide of the present invention or its pharmaceutically acceptable salt can be used in culture medium for human stem cells, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0129] [ Figure 1 ] Figure 1 It is a graph showing the results of the evaluation of the inhibition of BMP information transmission caused by the phosphorylation of SMAD1 using the peptides of the present invention and Noggin. A represents the evaluation results of the inhibition of BMP4 information transmission, and B represents the evaluation results of the inhibition of BMP7 information transmission. In the figure, the black triangle represents the BMP4 information transmission inhibitory peptide No.177, the white triangle represents the BMP4 information transmission inhibitory peptide No.181, the white circle represents the BMP4 information transmission inhibitory peptide No.87, and the black quadrilateral represents Noggin. The horizontal axis represents the concentration of the peptide (nM), and the vertical axis represents the inhibition rate when the phosphorylation value of SMAD1 in the unstimulated state is set to 100% inhibition and the maximum value of the phosphorylation of SMAD1 induced by BMP is set to 0% inhibition.

[0130] [ Figure 2 ] Figure 2 A graph showing the results of the evaluation of the inhibition of BMP family signaling caused by the phosphorylation of SMAD1 using the peptide of the present invention and Noggin. The triangle in the figure represents the BMP4 signaling inhibitory peptide No. 177, and the quadrangle represents Noggin. The horizontal axis represents the concentration of the peptide (nM), and the vertical axis represents the inhibition rate when the phosphorylation value of SMAD1 in the unstimulated state is set to 100% inhibition and the maximum value of the phosphorylation of SMAD1 induced by BMP is set to 0% inhibition. DETAILED DESCRIPTION

[0131] 1. Abbreviation

[0132] In this specification, unless otherwise specified, the following abbreviations are used in the following meanings.

[0133] Abbreviation (general)

[0134] Angstrom (unit)

[0135] Bovine serum albumin is BSA;

[0136] tert-butyloxycarbonyl is Boc;

[0137] Chloroacetyl is ClAc;

[0138] Dichloromethane or methyl chloride is DCM;

[0139] N, N'-diisopropylcarbodiimide is DIPCI;

[0140] N,N-diisopropylethylamine is DIPEA or DIEA;

[0141] Dimethyl sulfoxide is DMSO;

[0142] N,N-dimethylformamide is DMF;

[0143] 3,6-dioxa-1,8-octanedithiol is DODT;

[0144] Dulbecco's modified Eagle's medium is DMEM;

[0145] The 50% effective concentration is EC50;

[0146] Fetal bovine serum is FBS;

[0147] 9-Fluorenylmethyloxycarbonyl is Fmoc;

[0148] Gram (unit) is g;

[0149] O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate is HATU;

[0150] High performance liquid chromatography is HPLC;

[0151] Liquid chromatography mass spectrometer is LC-MS or LC / MS;

[0152] Molar (unit) is M;

[0153] Milligram (unit) is mg;

[0154] Milliliter (unit) is mL;

[0155] Millimole (unit) is mM;

[0156] Acetonitrile is MeCN;

[0157] Minutes (unit) is min;

[0158] Millimeter (unit) is mm;

[0159] O-3-methyl-pent-3-yl is Mpe group;

[0160] N-hydroxysuccinimide is NHS;

[0161] Nanometer (unit) is nm;

[0162] Micrometer (unit) is μL;

[0163] Succinimide is OSu;

[0164] 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl is Pbf;

[0165] Polyethylene glycol is PEG;

[0166] The number of revolutions per minute (unit) is rpm;

[0167] tert-butyl is tBu;

[0168] Trifluoroacetic acid is TFA;

[0169] Triisopropylsilane is TIS;

[0170] Trityl is Trt or Tr.

[0171] Abbreviation (unnatural amino acid)

[0172] The following abbreviations for non-natural amino acids also include those in which the main chain amino group is protected by a common protecting group such as a Boc group or an Fmoc group.

[0173] 3Py6NH2 or 3Py6-NH2: (S)-2-amino-3-(6-aminopyridin-3-yl)propanoic acid (CAS No.: 1269968-61-7);

[0174] Aib: 2-amino-2-methylpropionic acid (CAS No.: 62-57-7);

[0175] aMeP: (S)-2-methylpyrrolidine-2-carboxylic acid (CAS No.: 42856-71-3);

[0176] Aze: (S)-Azamethine-2-carboxylic acid (CAS No.: 2133-34-8);

[0177] Cit: L-citrulline (CAS No.: 372-75-8);

[0178] F3aa: (S)-2-amino-3-(3-(carboxymethyl)phenyl)propanoic acid (CAS No.: 1270107-31-7);

[0179] F44pip: (S)-2-amino-3-(4-(piperidin-4-yl)phenyl)propanoic acid (CAS No.: 1888705-98-3);

[0180] F4G: (S)-2-amino-3-(4-guanidinophenyl)propionic acid (CAS No. 59574-11-7);

[0181] Hcit: N6-carbamoyl-L-lysine (CAS No.: 1190-49-4);

[0182] Hph: (S)-2-amino-4-phenylbutyric acid (CAS No.: 943-73-7);

[0183] Hpr: (S)-piperidine-2-carboxylic acid (CAS No.: 3105-95-1);

[0184] Hty: Homo-L-tyrosine (CAS No.: 221243-01-2);

[0185] 5Ind: (S)-2-amino-3-(1H-indol-5-yl)propionic acid (CAS No.: 460096-38-2);

[0186] KCOpipzaa: N6-(4-(carboxymethyl)piperidin-1-carbonyl)-L-lysine (KISHIDA CHEMICAL Co., Ltd.);

[0187] MeA: N-methyl-L-alanine (CAS No.: 3913-67-5);

[0188] MeKCOpipzaa: N6-(4-(carboxymethyl)piperidin-1-carbonyl)-N2-methyl-L-lysine (KISHIDACHEMICAL Co., Ltd.);

[0189] MeV: N-methyl-L-valine (CAS No.: 2480-23-1);

[0190] P4F2: (S)-4,4-difluoropyrrolidine-2-carboxylic acid (CAS No.: 52683-81-5);

[0191] Pc4F: (2S, 4S)-4-fluoropyrrolidine-2-carboxylic acid (CAS No.: 2438-57-5);

[0192] Pt4F: (2S, 4R)-4-fluoropyrrolidine-2-carboxylic acid (CAS No.: 2507-61-1);

[0193] W7N: (S)-2-amino-3-(1H-indole[2,3-b]pyridin-3-yl)propanoic acid (CAS No. 49758-35-2);

[0194] Y3COO: (S)-5-(2-amino-2-carboxyethyl)-2-hydroxybenzoic acid (CAS No.: 4303-95-1);

[0195] Y3CONmm: (S)-2-amino-3-(4-hydroxy-3-(methylcarbamoyl)phenyl)propanoic acid;

[0196] OCOmPEG10000: methoxy polyethylene glycol carbonate (average molecular weight 10,000);

[0197] PEG12Me: 2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontane-37-amine;

[0198] 3Py: (S)-2-amino-3-(pyridin-3-yl)propanoic acid (CAS No.: 64090-98-8);

[0199] 3Py6Ph: (S)-2-amino-3-(6-phenylpyridin-3-yl)propanoic acid (CAS No.: 1335566-07-8);

[0200] 4Py: 3-(4-pyridyl)-L-alanine (CAS No.: 37535-49-2);

[0201] 4Py2NH2: (S)-2-amino-3-(2-aminopyridin-4-yl)propanoic acid (CAS No.: 1269969-46-1);

[0202] A1Ac4pip: (S)-3-(1-acetylpiperidin-4-yl)-2-aminopropionic acid (CAS No.: 2349666-59-5);

[0203] Abu: (S)-2-aminobutyric acid (CAS No.: 1492-24-6);

[0204] Acpe: (S)-2-amino-3-cyclopentylpropionic acid (CAS No.: 99295-82-6);

[0205] Ahp: (S)-2-aminoheptanoic acid (CAS No.: 44902-02-5);

[0206] Atp: (2S)-2-amino-3-(tetrahydropyran-4-yl)propanoic acid (CAS No.: 1344910-91-3);

[0207] bA: 3-aminopropionic acid (CAS No.: 107-95-9);

[0208] Bph: (S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropionic acid (CAS No.: 155760-02-4);

[0209] Bph3C: (S)-2-amino-3-(3'-chloro-[1,1'-biphenyl]-4-yl)propanoic acid (Fmoc protected form CAS No.: 1380437-47-7);

[0210] Bph3F: (S)-2-amino-3-(3'-fluoro-[1,1'-biphenyl]-4-yl)propanoic acid (CAS No.: 1380437-41-1);

[0211] Bph3Me: (S)-2-amino-3-(3'-methyl-[1,1'-biphenyl]-4-yl)propanoic acid (CAS No.: 1241680-67-0);

[0212] Bph3OMe: (S)-2-amino-3-(3'-methoxy-[1,1'-biphenyl]-4-yl)propanoic acid (CAS No.: 1417316-70-1);

[0213] Bph4C: (S)-2-amino-3-(4'-chloro-[1,1'-biphenyl]-4-yl)propanoic acid (CAS No.: 1270097-85-2);

[0214] Bph4F: (S)-2-amino-3-(4'-fluoro-[1,1'-biphenyl]-4-yl)propanoic acid (CAS No.: 2230870-07-0);

[0215] Cba: (S)-2-amino-3-cyclobutylpropionic acid (CAS No.: 1201593-65-8);

[0216] CF3A: (S)-2-amino-4,4,4-trifluorobutyric acid (CAS No.: 15960-05-1);

[0217] Cha: (S)-2-amino-3-cyclohexylpropionic acid (CAS No.: 27527-05-5);

[0218] Cha4tH: (S)-2-amino-3-((1r,4S)-4-hydroxycyclohexyl)propanoic acid (CAS No.: 221243-22-7);

[0219] Chg: (S)-2-amino-2-cyclohexylacetic acid (CAS No.: 14328-51-9);

[0220] da: D-alanine (CAS No.: 338-69-2);

[0221] de: D-glutamic acid (CAS number: 6893-26-1);

[0222] ds: D-serine (CAS No.: 312-84-5);

[0223] F3C: (S)-2-amino-3-(3-chlorophenyl)propionic acid (CAS No.: 80126-51-8);

[0224] F3Me: (S)-2-amino-3-(m-toluoyl)propanoic acid (114926-37-3);

[0225] F42Py: (S)-2-amino-3-(4-(pyridin-2-yl)phenyl)propanoic acid (CAS No.: 1336207-47-6);

[0226] F43Py: (S)-2-amino-3-(4-(pyridin-3-yl)phenyl)propanoic acid (CAS No.: 916911-74-5);

[0227] F44Py: (S)-2-amino-3-(4-(pyridin-4-yl)phenyl)propanoic acid (Fmoc protected form CAS number: 1282042-56-1);

[0228] F44thp: (S)-2-amino-3-(4-(tetrahydro-2H-pyran-4-yl)phenyl)propanoic acid (methyl ester protected form CAS number: 938197-48-9);

[0229] F4aao: (S)-2-amino-3-(4-(carboxymethoxy)phenyl)propanoic acid (CAS No.: 24558-63-2);

[0230] F4C: (S)-2-amino-3-(4-chlorophenyl)propionic acid (CAS No.: 14173-39-8);

[0231] F4COO(S)-4-(2-amino-2-carboxyethyl)benzoic acid (CAS No.: 126109-42-0);

[0232] F4F: (S)-2-amino-3-(4-fluorophenyl)propanoic acid (CAS No.: 1132-68-9);

[0233] F4Me: (S)-2-amino-3-(p-toluoyl)propanoic acid (CAS No.: 1991-87-3);

[0234] Gcpr: (S)-2-amino-2-cyclopropylacetic acid (CAS No. 49606-99-7);

[0235] Gthp: (S)-2-amino-2-(tetrahydro-2H-pyran-4-yl)acetic acid (CAS No.: 811842-25-8);

[0236] Hgl: L-2-aminoadipic acid (CAS No.: 1118-90-7);

[0237] Hgn: (S)-2,6-diamino-6-oxohexanoic acid (CAS No.: 7433-32-1);

[0238] Hty: Homo-L-tyrosine (CAS No.: 221243-01-2)

[0239] Hyp: (2S, 4R)-4-hydroxypyrrolidine-2-carboxylic acid (CAS No.: 51-35-4);

[0240] KCOpipzaa: N6-(4-(carboxymethyl)piperidin-1-carbonyl)-L-lysine (Fmoc protection / tert-butyl ester protection CAS number: 2680607-34-3);

[0241] MeF: methyl-L-phenylalanine (CAS number: 2566-30-5);

[0242] MeF3C: (S)-3-(3-chlorophenyl)-2-(methylamino)propanoic acid (CAS No.: 2255324-91-3);

[0243] MeF3Me: (S)-2-(methylamino)-3-(m-toluoyl)propanoic acid (Fmoc protected form CAS number: 2642331-20-0);

[0244] MeF4C: (S)-3-(4-chlorophenyl)-2-(methylamino)propanoic acid (CAS No.: 347851-70-1);

[0245] MeF4Me: (S)-2-(methylamino)-3-(p-toluoyl)propanoic acid (CAS No.: 2307782-25-6);

[0246] MeY: methyl-L-tyrosine (CAS No.: 537-49-5);

[0247] P4Sh: (2S, 4S)-4-hydroxypyrrolidine-2-carboxylic acid (CAS No.: 618-27-9);

[0248] pBph2F: (S)-2-amino-3-(2-fluoro-[1,1'-biphenyl]-4-yl)propanoic acid (CAS No.: 1389900-88-2);

[0249] pBph3F: (S)-2-amino-3-(3-fluoro-[1,1'-biphenyl]-4-yl)propanoic acid.

[0250] 2. Peptide (A)

[0251] One embodiment of the present invention relates to a peptide or a pharmaceutically acceptable salt thereof.

[0252] In one embodiment, the peptide of the present invention comprises the following amino acid sequence:

[0253] ERV-F4COO-Atp-DR-Bph-PYP-Bph-Y-F4COO-F4COO-SC (SEQ ID NO: 1),

[0254] Or an amino acid sequence having a substitution, addition, deletion or insertion in 1 to 15 amino acid residues selected from the group consisting of the 1st, 2nd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th and 16th amino acid residues of the above amino acid sequence.

[0255] ERV-F4COO-Atp-DR-Bph-PYP-Bph-Y-F4COO-F4COO-SC (SEQ ID NO: 1) is a peptide confirmed to have BMP binding activity in the Examples of the present specification.

[0256] It may also have substitution, addition, deletion or insertion in 1 to 15 amino acid residues selected from the group consisting of the 1st, 2nd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th and 16th amino acid residues of the amino acid sequence of SEQ ID NO: 1. The number of amino acids substituted, deleted, added and / or inserted can be 1 or more and 10 or less, and the lower limit is 1. The upper limit is 10, 9, 8, 7, 6, 5, 4, 3, 2, and the minimum is 1. Preferably, it is "substitution" of amino acids. The amino acid substitution is preferably a conservative amino acid substitution.

[0257] By "conservative amino acid substitution" is meant a substitution with an amino acid that is functionally equivalent or similar. Conservative amino acid substitutions in a peptide generally do not affect the function and / or activity of the peptide. In general, substitutions within a group are considered to be conservative with respect to structure and function. However, as will be appreciated by one of ordinary skill in the art, the role played by a particular amino acid residue can be determined by its significance in the three-dimensional structure of the molecule containing the amino acid. For example, cysteine ​​residues are less polar than the reduced (thiol) form and can be in the oxidized (disulfide) form. The long aliphatic portion of the arginine side chain can constitute a structurally and functionally important feature. In addition, side chains containing aromatic rings (tryptophan, tyrosine, phenylalanine) can contribute to ion-aromatic interactions or cation-pi interactions. In this case, even if an amino acid having such a side chain is substituted with an amino acid belonging to an acidic or non-polar group, it can still be conservative in terms of structure and functionality. Residues such as proline, glycine, and cysteine ​​(in disulfide form) may have a direct effect on the three-dimensional structure of the main chain, and generally cannot be substituted without causing structural distortion.

[0258] Conservative amino acid substitutions include specific substitutions based on side chain similarity (e.g., substitutions described in Lehninger, Biochemistry, Revised 2nd edition, 1975, pp. 73-75: L. Lehninger, Biochemistry, 2nd edition, pp. 73-75, Worth Publisher, New York (1975)) and typical substitutions as shown below. In addition, conservative amino acid substitutions are preferably substituted with an amino acid belonging to the same group as the group to which an amino acid belongs, in a group formed by distinguishing natural amino acids based on the properties of their common side chains, as shown below.

[0259] Hydrophobic (also known as non-polar) amino acids: Amino acids that exhibit hydrophobicity (non-polarity), including alanine ("Ala" or simply "A"), glycine ("Gly" or simply "G"), valine ("Val" or simply "V"), leucine ("Leu" or simply "L"), isoleucine ("Ile" or simply "I"), proline ("Pro" or simply "P"), phenylalanine ("Phe" or simply "F"), tryptophan ("Trp" or simply "W"), tyrosine ("Tyr" or simply "Y"), and methionine ("Met" or simply "M").

[0260] In addition, hydrophobic amino acids can also be classified into the following groups.

[0261] Aliphatic amino acids: Amino acids that have fatty acids or hydrogen in their side chains, including Ala, Gly, Val, Ile, Leu.

[0262] Aliphatic branched-chain amino acids: Amino acids that have branched fatty acids in their side chains, including Val, Ile, Leu.

[0263] Aromatic amino acids: Amino acids that have aromatic rings in their side chains, including Trp, Tyr, Phe.

[0264] Hydrophilic (also known as polar) amino acids: Amino acids that exhibit hydrophilicity (polarity), including serine ("Ser" or simply "S"), threonine ("Thr" or simply "T"), cysteine ("Cys" or simply "C"), asparagine ("Asn" or simply "N"), glutamine ("Gln" or simply "Q"), aspartic acid ("Asp" or simply "D"), glutamic acid ("Glu" or simply "E"), lysine (also known as Lysine. "Lys" or simply "K"), arginine ("Arg" or simply "R"), and histidine ("His" or simply "H").

[0265] In addition, hydrophilic amino acids can be further classified into the following groups.

[0266] Acidic amino acids: Amino acids whose side chains exhibit acidity, including Asp, Glu.

[0267] Basic amino acids: Amino acids whose side chains exhibit basicity, including Lys, Arg, His.

[0268] Neutral amino acids: Amino acids whose side chains exhibit neutrality, including Ser, Thr, Asn, Gln, Cys.

[0269] Furthermore, Gly and Pro can also be classified as "amino acids that affect the direction of the main chain", and amino acids containing sulfur molecules in the side chains, Cys and Met can also be classified as "sulfur-containing amino acids".

[0270] In the present specification, "amino acid" includes not only natural amino acids but also non-natural amino acids. Non-natural amino acids include, for example, N-alkyl amino acids obtained by N-alkylation of the natural amino acids described above, and those in which the nitrogen forming the peptide bond is modified by a branched or unbranched low-alkyl group (for example, C1 to C5, preferably C1 to C3, and more preferably C1). Among N-alkyl amino acids, N-ethyl amino acids, N-butyl amino acids or N-methyl amino acids are preferred, and N-methyl amino acids are more preferred. Furthermore, non-natural amino acids include chemically modified amino acids such as D-amino acids (also called D-amino acids), β-amino acids, γ-amino acids, amino acid variants, amino acid derivatives, and amino acids such as norleucine and ornithine that cannot serve as constituent materials of proteins in vivo. Furthermore, it includes amino acids in which functional groups are further added to the side chains of natural amino acids or replaced by other functional groups (for example: amino acids with substitution or addition to the arylene group, alkylene group, etc. in the side chain; amino acids in which the number of carbon atoms in the arylene group, alkylene group or alkyl group in the side chain has been increased; amino acids with substitution in the aromatic ring of the side chain; or amino acids formed by heterocyclization or cyclization, etc.).

[0271] In addition, by adding or replacing structures such as functional groups on the side chains of natural amino acids, properties different from those of natural amino acids can be imparted. For example, although A4p is an amino acid having a piperidinyl group on the side chain of alanine, by adding the piperidinyl group, it exhibits the properties of a polar amino acid with alkalinity, unlike alanine belonging to the non-polar amino acid group. In other words, in the aforementioned groups that are divided into natural amino acids based on the properties of their common side chains, non-natural amino acids with the same side chain properties can be included. For example, although the amino acid N-methylarginine (MeR), which is an amino acid in which the main chain nitrogen atom of arginine, which belongs to the basic amino acids, is methylated, it is a non-natural amino acid, but because it exhibits alkalinity, it can be classified as a basic amino acid. In this way, non-natural amino acids that exhibit the same side chain properties as an amino acid can also be included as objects of conservative amino acid substitution. In addition, D-amino acids such as de can also be classified as D-amino acids, but can also be classified according to the properties of their side chains. For N-methyl amino acids, they can be classified as N-alkyl amino acids, or they can be classified according to the properties of the side chains of the original amino acids that are not N-methylated.

[0272] In this specification, "without limitation" and "in one embodiment" can be used synonymously.

[0273] "Pharmaceutically acceptable salts thereof" means salts 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, trifluoromethanesulfonic acid, and benzenesulfonic acid; salts with amines such as trimethylamine and methylamine; or salts with metal ions such as sodium ions, potassium ions, and calcium ions. For compounds that contain water due to changes over time, such water is also included in pharmaceutically acceptable salts.

[0274] The peptides of the present invention include peptides belonging to Group A1, peptides belonging to Group A2, peptides belonging to Group A3, and peptides belonging to Group A4 described below.

[0275] The peptide of the present invention belonging to group A1, in one embodiment, is a peptide comprising the following amino acid sequence:

[0276] ERV-F4COO-Atp-DR-Bph-PYP-Bph-Y-F4COO-F4COO-SC (SEQ ID NO: 1),

[0277] Alternatively, the amino acid sequence may have five or less amino acid substitutions selected from the following (1-i) to (1-xii) in the above amino acid sequence.

[0278] (1-i) The first amino acid sequence of SEQ ID NO: 1 is substituted with A.

[0279] (1-ii) The second amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with any polar amino acid.

[0280] (1-iii) The fourth amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with unsubstituted or substituted Y.

[0281] (1-iv) The fifth amino acid sequence of SEQ ID NO: 1 is substituted with V.

[0282] (1-v) The sixth amino acid sequence of SEQ ID NO: 1 is substituted with KCOpipzaa.

[0283] (1-vi) The 9th amino acid sequence of SEQ ID NO: 1 is substituted with S.

[0284] (1-vii) The tenth amino acid sequence of SEQ ID NO: 1 is substituted with Hty.

[0285] (1-viii) The 11th amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with any aliphatic amino acid.

[0286] (1-ix) The 12th amino acid sequence of SEQ ID NO: 1 is substituted with substituted Bph.

[0287] (1-x) The 13th amino acid sequence of SEQ ID NO: 1 is substituted with F4F.

[0288] (1-xi) The 15th amino acid sequence of SEQ ID NO: 1 is substituted with 4Py.

[0289] (1-xii) The 16th amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with any amino acid excluding S.

[0290] Although not limited, any polar amino acid in (1-ii) above may be Hcit or KCOpipzaa, the unsubstituted or substituted Y in (1-iii) above may be unsubstituted Y, any aliphatic amino acid in (1-viii) above may be V, and any amino acid excluding S in (1-xii) above may be H.

[0291] Furthermore, although not limited, the peptides of the present invention belonging to group A1 may be peptides comprising an amino acid sequence having 4 or less, 3 or less, 2 or less, or 1 amino acid substitution selected from the above (1-i) to (1-xii) in the amino acid sequence described in SEQ ID NO: 1.

[0292] Furthermore, the peptide of the present invention belonging to group A2 is, in one embodiment, a peptide comprising the following amino acid sequence:

[0293] ERVYVDR-3Py6Ph-SYV-Bph-Y-F4COO-F4COO-SC (SEQ ID NO: 2),

[0294] Alternatively, the amino acid sequence may comprise five or less amino acid substitutions selected from the following (2-i) to (2-xi).

[0295] (2-i) The first amino acid sequence of SEQ ID NO: 2 is substituted with A.

[0296] (2-ii) The second amino acid sequence of SEQ ID NO: 2 is substituted with KCOpipzaa.

[0297] (2-iii) The fourth amino acid sequence of SEQ ID NO: 2 is substituted with F4F or F4COO.

[0298] (2-iv) The sixth amino acid sequence of SEQ ID NO: 2 is substituted with KCOpipzaa.

[0299] (2-v) The 7th amino acid sequence of SEQ ID NO: 2 is substituted with K.

[0300] (2-vi) The 8th amino acid sequence of SEQ ID NO: 2 is substituted with Bph.

[0301] (2-vii) The 9th amino acid sequence of SEQ ID NO: 2 is substituted with P or KCOpipzaa.

[0302] (2-viii) The 11th amino acid in the amino acid sequence of SEQ ID NO: 2 is substituted with any aliphatic amino acid.

[0303] (2-ix) The 12th amino acid sequence of SEQ ID NO: 2 is substituted with substituted Bph.

[0304] (2-x) The 13th amino acid sequence of SEQ ID NO: 2 is substituted with substituted F.

[0305] (2-xi) The 15th amino acid sequence of SEQ ID NO: 2 is substituted with 4Py.

[0306] Although not limited, any aliphatic amino acid in (2-viii) above may be P, Hyp, P4Sh, Pc4F, Pt4F, P4F2 or aMePV, and the substituted F in (2-x) above may be F4F.

[0307] Furthermore, although not limited, the peptide of the present invention belonging to group A2 may be a peptide comprising an amino acid sequence having 4 or less, 3 or less, 2 or less, or 1 amino acid substitution selected from the above (2-i) to (2-xi) in the amino acid sequence described in SEQ ID NO: 2.

[0308] Furthermore, the peptide of the present invention belonging to group A3, in one embodiment, is a peptide comprising the following amino acid sequence:

[0309] ARVYVDR-Bph-PYA-Bph-Y-F4COO-F4COO-SC (SEQ ID NO: 3),

[0310] Alternatively, the amino acid sequence may comprise five or less amino acid substitutions selected from the following (3-i) to (3-x) in the above amino acid sequence.

[0311] (3-i) The second amino acid sequence of SEQ ID NO: 3 is substituted with K, KAc, Q or KCOpipzaa.

[0312] (3-ii) The fourth amino acid sequence of SEQ ID NO: 3 is substituted with F4F or 4Py.

[0313] (3-iii) The sixth amino acid sequence of SEQ ID NO: 3 is substituted with KCOpipzaa, K, R, G or H.

[0314] (3-iv) The seventh amino acid sequence of SEQ ID NO: 3 is substituted with K, KAc, 4Py, S or Cit.

[0315] (3-v) The 8th amino acid sequence of SEQ ID NO: 3 is substituted with 3Py6Ph.

[0316] (3-vi) The 9th amino acid sequence of SEQ ID NO: 3 is substituted with S, K, H, G, S or KCOpipzaa.

[0317] (3-vii) The 11th amino acid in the amino acid sequence of SEQ ID NO: 3 is substituted with any aliphatic amino acid.

[0318] (3-viii) The 12th amino acid sequence of SEQ ID NO: 3 is substituted with 3Py6Ph.

[0319] (3-ix) The 13th amino acid sequence of SEQ ID NO: 3 is substituted with F4F.

[0320] (3-x) The 15th amino acid sequence of SEQ ID NO: 3 is substituted with 4Py.

[0321] Although not limited thereto, the arbitrary aliphatic amino acid in (3-vii) above may be V, P or A.

[0322] Furthermore, although not limited, the peptide of the present invention belonging to group A3 may be a peptide comprising an amino acid sequence having 4 or less, 3 or less, 2 or less, or 1 amino acid substitution selected from the above (3-i) to (3-x) in the amino acid sequence described in SEQ ID NO: 3.

[0323] Furthermore, the peptide of the present invention belonging to group A4, in one embodiment, is a peptide comprising the following amino acid sequence:

[0324] FG-MeF-G-Bph-GDD-Cha-A-MeF-LHC (SEQ ID NO: 241),

[0325] Alternatively, the amino acid sequence has four or less amino acid substitutions selected from the following (4-i) to (4-xii) in the above amino acid sequence.

[0326] (4-i) The first amino acid sequence of SEQ ID NO: 241 is substituted with substituted phenylalanine.

[0327] (4-ii) The third amino acid sequence of SEQ ID NO: 241 is substituted with substituted N-methylphenylalanine.

[0328] (4-iii) The fourth amino acid sequence of SEQ ID NO: 241 is substituted with da or ds.

[0329] (4-iv) The fifth amino acid sequence of SEQ ID NO: 241 is substituted with biphenylalanine, wherein C on the aromatic ring is substituted with N.

[0330] (4-v) The sixth amino acid sequence of SEQ ID NO: 241 is substituted with A or da.

[0331] (4-vi) The 7th amino acid sequence of SEQ ID NO: 241 is substituted with N.

[0332] (4-vii) The 8th amino acid sequence of SEQ ID NO: 241 is substituted with E.

[0333] (4-viii) The 9th amino acid sequence of SEQ ID NO: 241 is substituted with Acpe.

[0334] (4-ix) The tenth amino acid in the amino acid sequence of SEQ ID NO: 241 is substituted with any polar amino acid.

[0335] (4-x) The 11th amino acid sequence of SEQ ID NO: 241 is substituted with substituted N-methylphenylalanine.

[0336] (4-xi) The 12th amino acid in the amino acid sequence of SEQ ID NO: 241 is substituted with any aliphatic amino acid.

[0337] (4-xii) The 13th amino acid in the amino acid sequence of SEQ ID NO: 241 is substituted with any polar amino acid.

[0338] Although not limited, any polar amino acid in (4-ix) above may be Hgl or Q, any aliphatic amino acid in (4-xi) above may have a side chain carbon number of 7 or less, and any polar amino acid in (4-xii) above may be P4Sh or F4COO.

[0339] Furthermore, although not limited, the peptide of the present invention belonging to group A4 may be a peptide comprising an amino acid sequence having 3 or less, 2 or less, or 1 amino acid substitution selected from the above (4-i) to (4-xii) in the amino acid sequence described in SEQ ID NO: 241.

[0340] In one embodiment, the peptide is a cyclic peptide. The "cyclic peptide" is described in detail in "3. Peptide (B)".

[0341] The aforementioned peptides may contain additional amino acid residues in addition to the amino acid sequences of SEQ ID NOs: 5 to 187, SEQ ID NOs: 188 to 240, SEQ ID NOs: 243 to 321, and SEQ ID NOs: 322 to 421. The "additive amino acid residues" are described in detail in "3. Peptide (B)".

[0342] The peptides of the present invention belonging to the aforementioned groups A1 to A3 bind to BMP4. The peptides of the present invention belonging to group A1 preferably further bind to BMP7. Moreover, the peptides of the present invention belonging to group A2 preferably do not bind to BMP7.

[0343] The peptides of the present invention belonging to the aforementioned groups A1 to A3 have BMP4 inhibitory activity. The aforementioned peptides more preferably have human stem cell differentiation induction control activity. "Binding to BMP4", "having BMP4 inhibitory activity", "having human stem cell differentiation induction control activity" are described in detail in "3. Peptide (B)".

[0344] The peptides belonging to group A4 have BMP7 inhibitory activity. The peptides more preferably have human stem cell differentiation induction control activity. "Having BMP7 inhibitory activity" and "having human stem cell differentiation induction control activity" are described in detail in "3. Peptide (B)".

[0345] 3. Peptide (B)

[0346] An embodiment of the present invention which is different from the above one relates to the following peptide (B).

[0347] The peptide of the present invention of this embodiment comprises the following amino acid sequence:

[0348] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-C (SEQ ID NO: 4),

[0349] in,

[0350] X1 is E, A, T or Q,

[0351] X2 is R, K, Hgl, any polar amino acid or any aliphatic amino acid,

[0352] X3 is V,

[0353] X4 is an amino acid having an aromatic ring which may be a heterocyclic ring in the side chain,

[0354] X5 is any aliphatic amino acid, Y, D or Atp,

[0355] X6 is D, any basic amino acid or any polar amino acid,

[0356] X7 is any basic amino acid or any polar amino acid,

[0357] X8 is biphenylalanine which may have a substituent and the C on the aromatic ring may be substituted with N,

[0358] X9 is any amino acid,

[0359] X10 is Y, Hty or 4Py,

[0360] X11 is any amino acid,

[0361] X12 is biphenylalanine which may have a substituent and the C on the aromatic ring may be substituted with N,

[0362] X13 is unsubstituted or substituted F, 4Py or 3Py6NH2,

[0363] X14 is F4COO, D or G,

[0364] X15 is F4COO, Y, 4Py, 3Py6NH2, W7N, Hph or N,

[0365] X16 is any amino acid.

[0366] The above options of X1 to X16 can be combined and selected arbitrarily.

[0367] Furthermore, the substituted F includes an amino acid having a substituent on the benzene ring on the phenylalanine side chain, an amino acid derivative of F having a heterocyclic ring, or an amino acid derivative of F having a condensed polycyclic structure. Therefore, the substituted F includes Y.

[0368] In one embodiment, X2 is R, K, Hgl, Hcit, KCOpipzaa or Tic.

[0369] In one embodiment, X5 is V, Y, D or Atp.

[0370] In one embodiment, X6 is D, R, H, K, KCOpipzaa, V, Y, D or Atp.

[0371] In one embodiment, X7 is R, K or any polar amino acid.

[0372] In one embodiment, X8 is unsubstituted Bph or unsubstituted 3Py6Ph.

[0373] In one embodiment, X12 is unsubstituted Bph or unsubstituted 3Py6Ph.

[0374] In one embodiment, X13 is unsubstituted or substituted F.

[0375] In one embodiment, X16 is S or H.

[0376] The peptides of the present invention include peptides belonging to group B1, peptides belonging to group B2, peptides belonging to group B3, and peptides belonging to group B4 described below.

[0377] Although not limited, the peptides of the present invention belonging to group B1 include the following amino acid sequence:

[0378] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-C (SEQ ID NO: 4),

[0379] in,

[0380] X1 is E,

[0381] X2 is R or any polar amino acid,

[0382] X3 is V,

[0383] X4 is unsubstituted or substituted Y or F4COO,

[0384] X5 is V or Atp,

[0385] X6 is D or KCOpipzaa,

[0386] X7 is R,

[0387] X8 is Bph,

[0388] X9 is P or S,

[0389] X10 is Y or Hty,

[0390] X11 is any amino acid,

[0391] X12 is unsubstituted or substituted Bph,

[0392] X13 is unsubstituted or substituted F,

[0393] X14 is F4COO,

[0394] X15 is F4COO or 4Py,

[0395] X16 is any amino acid.

[0396] The above options of X1 to X16 can be combined and selected arbitrarily.

[0397] In one embodiment, X2 is R, Hcit or KCOpipzaa.

[0398] In one embodiment, X4 is Y or F4COO.

[0399] In one embodiment, X11 is any aliphatic amino acid.

[0400] In one embodiment, X12 is Bph.

[0401] In one embodiment, X13 is Y or F4F.

[0402] In one embodiment, X15 is F4COO.

[0403] In one embodiment, X16 is S or H.

[0404] In one embodiment, X2 is KCOpipzaa, and X15 is 4Py.

[0405] Furthermore, although not limited thereto, the peptides of the present invention belonging to group B2 include the following amino acid sequence:

[0406] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-C (SEQ ID NO: 4),

[0407] in,

[0408] X1 is E or A,

[0409] X2 is R or KCOpipzaa,

[0410] X3 is V,

[0411] X4 is Y, F4F or F4COO,

[0412] X5 is V,

[0413] X6 is D or KCOpipzaa,

[0414] X7 is R or K,

[0415] X8 is 3Py6Ph or Bph,

[0416] X9 is S, P or KCOpipzaa,

[0417] X10 is Y,

[0418] X11 is any aliphatic amino acid,

[0419] X12 is Bph,

[0420] X13 is unsubstituted or substituted F,

[0421] X14 is F4COO,

[0422] X15 is F4COO or 4Py,

[0423] X16 is S.

[0424] The above options of X1 to X16 can be combined and selected arbitrarily.

[0425] In one embodiment, X11 is V, P, Hyp, P4Sh, Pc4F, Pt4F, P4F2 or aMeP.

[0426] In one embodiment, X12 is Bph.

[0427] In one embodiment, X13 is Y or F4F.

[0428] In one embodiment, X15 is F4COO.

[0429] Furthermore, although not limited thereto, the peptides of the present invention belonging to group B3 include the following amino acid sequence:

[0430] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-C (SEQ ID NO: 4),

[0431] in,

[0432] X1 is A,

[0433] X2 is R, KAc, Q or KCOpipzaa,

[0434] X3 is V,

[0435] X4 is Y, F4F or 4Py,

[0436] X5 is V,

[0437] X6 is D, KCOpipzaa, K, R, G or H,

[0438] X7 is R, K, KAc, 4Py, S or Cit,

[0439] X8 is 3Py6Ph or Bph,

[0440] X9 is P, S, K, H, G, S or KCOpipzaa,

[0441] X10 is Y,

[0442] X11 is any aliphatic amino acid,

[0443] X12 is Bph or 3Py6Ph,

[0444] X13 is Y or F4F,

[0445] X14 is F4COO,

[0446] X15 is F4COO or 4Py,

[0447] X16 is S.

[0448] The above options of X1 to X16 can be combined and selected arbitrarily.

[0449] In one embodiment, X11 is V, P or A.

[0450] Furthermore, although not limited thereto, the peptides of the present invention belonging to group B4 include the following amino acid sequence:

[0451] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 (SEQ ID NO: 242),

[0452] in,

[0453] X1 is phenylalanine which may have a substituent and in which the C on the aromatic ring may be substituted with N,

[0454] X2 is G,

[0455] X3 is N-methylphenylalanine which may have a substituent,

[0456] X4 is G, which may be a D-type aliphatic amino acid or a polar amino acid,

[0457] X5 is biphenylalanine in which the C on the aromatic ring may be substituted with N or phenylalanine which may have a substituent,

[0458] X6 is G or A of D type,

[0459] X7 is any polar amino acid,

[0460] X8 is any acidic amino acid,

[0461] X9 is any aliphatic amino acid,

[0462] X10 is any amino acid,

[0463] X11 is N-methylphenylalanine which may have a substituent,

[0464] X12 is any aliphatic amino acid,

[0465] X13 is any amino acid,

[0466] X14 is C.

[0467] In one embodiment, the aforementioned peptide is a cyclic peptide. "Cyclic peptide" refers to a peptide in which two amino acids are bound and all or part of the peptide is cyclic. The aforementioned peptide also includes a peptide in which amino acids form a cross-linked structure, a peptide in which a cyclic structure is formed by lactam ring formation or macrocyclization reaction, a peptide having a lasso peptide-like structure, etc. In other words, the aforementioned cyclic peptide only needs to form a cyclic structure for a part of it, and may also have a straight chain part.

[0468] Peptides generally have poor metabolic stability in vivo and have difficulty penetrating cell membranes due to their large size. To address this problem, a method of cyclizing the peptide can be adopted. If the peptide is cyclized, protease resistance and metabolic stability are improved, and because conformational changes are also restricted, it is suggested that rigidity is increased and membrane permeability or affinity with the target protein is improved.

[0469] In one embodiment, the peptide has a ring structure formed by binding a chloroacetylated amino acid to a cysteine ​​residue contained in the peptide. In one embodiment, the peptide has a ring structure formed by binding an N-terminal amino acid (the first amino acid residue) to a cysteine ​​residue contained in the peptide. In one embodiment, the peptide has a ring structure formed by binding an N-terminal amino acid (the first amino acid residue) to the 17th cysteine ​​residue contained in the peptide. In one embodiment, the peptide has a ring structure formed by binding a chloroacetylated N-terminal amino acid (the first amino acid residue) to the 17th cysteine ​​residue contained in the peptide. "Chloroacetylation" may be "halogen acetylation" caused by other halogens. Furthermore, "acetylation" may be "acylation" caused by an acyl group other than an acetyl group.

[0470] In one embodiment, the peptide comprises or consists of an amino acid sequence as described in any one of SEQ ID NOs: 5 to 187, SEQ ID NOs: 188 to 240, SEQ ID NOs: 243 to 321, and SEQ ID NOs: 322 to 421. In one embodiment, the peptide is a cyclic peptide comprising or consisting of an amino acid sequence as described in any one of SEQ ID NOs: 5 to 187, SEQ ID NOs: 188 to 240, SEQ ID NOs: 243 to 321, and SEQ ID NOs: 322 to 421. The peptide is a peptide consisting of an amino acid sequence as described in any one of SEQ ID NOs: 5 to 187, SEQ ID NOs: 188 to 240, SEQ ID NOs: 243 to 321, and SEQ ID NOs: 322 to 421, or a peptide consisting of an amino acid sequence in which 1 to 10 (preferably 1 to 4, 1 to 3, 2 or 1) amino acid residues are substituted, deleted, inserted or added. In addition, as long as the added part is a linker, a sequence of multiple amino acid residues (e.g., 1 to 10 residues) may be further added to the sequence of added amino acid residues. In addition, the peptide composed of the amino acid sequence formed by substitution, deletion, insertion or addition of amino acid residues is preferably one having the ability to bind to BMP4 or having BMP4 and / or BMP7 inhibitory activity.

[0471] The above-mentioned peptide may contain additional amino acid residues in addition to the amino acid sequences described in SEQ ID NO: 4 and SEQ ID NO: 242. The additional amino acid residues may be contained in the peptide forming a cyclic structure, and amino acid residues may be further added in a linker form from the cyclic peptide. The number of amide bonds (the number of amino acids and the length) of the peptide or peptide part is not particularly limited.

[0472] In addition, a linker can be further added from the cyclic peptide. As a linker, for example, the aforementioned amino acid linker (peptide linker), chemical linker, fatty acid linker, nucleic acid linker, sugar chain linker, etc. can be listed, and, for example, a complex of a chemical linker and a peptide linker can be used. As a chemical linker, for example, a PEG linker composed of 1 to 24 ethylene glycol units can be used. In addition, the linker can also be a fatty acid linker containing a divalent chemical part induced by a fatty acid. The amino acid (peptide) linker is a linker containing at least one amino acid, and for example, a peptide having a sequence [Gly-Gly-Gly-Gly-Ser]n (where n is 1, 2, 3, 4, 5 or 6) as described in U.S. Patent No. 7,271,149 can be used, such as a glycine-rich peptide (glycine-rich peptides), and a serine-rich peptide linker described in U.S. Patent No. 5,525,491. In addition, in the peptide linker, the amino acid-amino acid or amino acid-chemical linker bond can also be bonded through the side chain of the amino acid. In a non-limiting manner, the physical properties (such as solubility) of the peptide may be changed by the addition of the linker.

[0473] The addition position of the linker can be any position. For example, it can be combined with Cys located at the C-terminal side, or it can be combined with an amino acid contained in a cyclic peptide. It is preferably combined with Cys located at the C-terminal side or with the side chain of an amino acid contained in a cyclic peptide.

[0474] Furthermore, the aforementioned peptides may also be formed into multimers via a linker or the like.

[0475] The peptides belonging to the aforementioned groups B1 to B3 have the ability to bind to BMP4. The peptides of the present invention belonging to group B1 preferably further have the ability to bind to BMP7. Moreover, the peptides of the present invention belonging to group B2 preferably do not have the ability to bind to BMP7.

[0476] The peptides belonging to group B4 have BMP7 inhibitory activity, but preferably do not have BMP4 inhibitory activity.

[0477] Regarding the combination with BMP

[0478] The binding of the aforementioned peptide to the BMP protein can be measured by any method for measuring known intermolecular binding. In a non-limiting manner, for example, it can be determined by any appropriate format known per se, and the any appropriate format known per se includes: surface plasmon resonance (SPR) analysis, scatchard analysis and / or radioimmunoassay (RIA), enzyme immunoassay (EIA), Sandwich competitive assay and other competitive binding assays and different variants known in the art. Although not limited, for peptides and BMP proteins, the results measured by any known method for measuring intermolecular binding (such as the method shown in Example 15), in the case where the KD value is less than 100nM, the peptide may be a person with binding ability to the BMP protein.

[0479] In addition, this specification provides novel peptides. The peptide having the inhibitory activity of BMP is preferably a peptide having the binding activity of BMP. The following situation also implies this: the peptide having the inhibitory activity of BMP4 and / or BMP7 shown in Examples 11 and 12 has the binding activity for BMP as shown in the results of Example 15 (SPR measurement). Peptides having the inhibitory activity of BMP are effective for the treatment or prevention of various diseases related to BMP. Peptides having the binding activity of BMP are effective for various experiments for various diseases related to BMP. Osteogenic factor 4 (BMP4) mainly acts on the formation and development of bone and cartilage, and is also related to various bone diseases (Japanese Patent No. 6516724). BMP4 is a substance acting on the BMP information transmission pathway (Japanese Patent No. 7120585). Osteogenic factor 7 (BMP-7) is a substance acting on the BMP information transmission pathway (Japanese Patent No. 7120585).

[0480] Regarding the BMP4 inhibitory activity

[0481] BMP4 inhibitory activity can be evaluated by phosphorylation of SMAD1 caused by BMP4 stimulation. A specific method, for example, can be evaluated based on the method described in Example 11. As a BMP4 activity inhibitor, Noggin is known. A peptide having BMP4 inhibitory activity preferably has the same degree of BMP4 inhibitory activity as a known BMP4 activity inhibitor.

[0482] In the present specification, BMP4 inhibitory activity and BMP4 signal transduction inhibitory activity have the same meaning.

[0483] Regarding the BMP7 inhibitory activity

[0484] BMP7 inhibitory activity can be evaluated by phosphorylation of SMAD1 caused by BMP7 stimulation. A specific method, for example, can be evaluated based on the method described in Example 12. As a BMP7 activity inhibitor, Noggin is known. A peptide having BMP7 inhibitory activity preferably has the same degree of BMP7 inhibitory activity as a known BMP7 activity inhibitor.

[0485] In the present specification, BMP7 inhibitory activity and BMP7 signal transduction inhibitory activity have the same meaning.

[0486] Regarding the activity of inducing and controlling human stem cell differentiation

[0487] As described above, by evaluating whether the protein has BMP4 and / or BMP7 inhibitory activity, it is possible to evaluate whether the protein has human stem cell differentiation induction control activity (inhibitory activity).

[0488] In the present specification, "peptide" includes both "2. Peptide (A)" and "3. Peptide (B)" unless otherwise specified. In the present specification, peptide also includes pharmaceutically acceptable salts thereof.

[0489] 4. Peptide Production

[0490] The peptide of the present invention can be produced by any known method for producing a peptide, for example, as follows.

[0491] Chemical synthesis methods such as liquid phase method, solid phase method, hybrid method combining liquid phase method and solid phase method, gene recombination method, etc.

[0492] The solid phase method is, for example, to subject the hydroxyl group of a resin having a hydroxyl group to an esterification reaction 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 by a protecting group. As an esterification catalyst, a known dehydration condensation agent such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), or diisopropylcarbodiimide (DIC) can be used.

[0493] 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 other than the carboxyl group of the main chain are protected is added, and the carboxyl group is activated, so that the first and second amino acids are bonded. Furthermore, the α-amino group of the second amino acid is deprotected, and a third amino acid in which all functional groups other than the carboxyl group of the main chain are protected is added, and the carboxyl group is activated, so that the second and third amino acids are bonded. This is repeated, and when a peptide of the target length is synthesized, all functional groups are deprotected.

[0494] 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). These resins can be used after washing with a solvent (dimethylformamide (DMF), 2-propanol, methyl chloride, etc.).

[0495] Examples of the protecting group of the α-amino group include a benzyloxycarbonyl (Cbz or Z) group, a tert-butyloxycarbonyl (Boc) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a benzyl group, an allyl group, an 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.

[0496] The α-carboxyl group can be protected by using, for example, methyl ester, ethyl ester, allyl ester, benzyl ester, tert-butyl ester, cyclohexyl ester and the like.

[0497] The activation of the carboxyl group can be carried out using a condensing agent. Examples of the condensing agent include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU).

[0498] The peptide chain from the resin can be cleaved by treating with an acid such as TFA or hydrogen fluoride (HF).

[0499] The production of the peptide by genetic recombination (translation synthesis system) can be carried out using a nucleic acid encoding the above-mentioned peptide. The nucleic acid encoding the above-mentioned peptide may be DNA or RNA.

[0500] The nucleic acid encoding the aforementioned peptide can be prepared by known methods or methods following the same. For example, it can be synthesized by an automatic synthesis device. In order to insert the obtained DNA into a vector, a restriction enzyme recognition site can also be added. Alternatively, a base sequence encoding an amino acid sequence can also be incorporated, and the amino acid sequence is used to cut out the prepared peptide chain with an enzyme or the like.

[0501] In order to inhibit degradation by proteases derived from the host, a chimeric protein expression method can also be used, wherein the target peptide is expressed as a chimeric peptide with other peptides. In this case, as the aforementioned nucleic acid, a nucleic acid encoding the target peptide and a peptide bound thereto can be used.

[0502] Next, the expression vector is prepared using a nucleic acid encoding the peptide. The nucleic acid can be directly or by restriction enzyme digestion or addition of a linker and inserted downstream of the promoter of the expression vector. As a vector, for example, plasmids derived from Escherichia coli (pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pUC118, pBluescript II, etc.), plasmids derived from Bacillus subtilis (pUB110, pTP5, pC1912, pTP4, pE194, pC194, etc.), plasmids derived from yeast (pSH19, pSH15, YEp, YRp, YIp, YAC, etc.), bacteriophages (e phage, M13 phage, etc.), viruses (retrovirus, vaccinia virus, adenovirus (Adenoviridae), adeno-associated virus (AAV), cauliflower mosaic virus, tobacco mosaic virus, baculovirus, etc.), cosmids, etc. can be cited.

[0503] The promoter can be appropriately selected according to 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.

[0504] The expression vector may also include nucleic acids encoding, for example, a DNA replication origin (ori), a selection marker (antibiotic resistance, nutrient requirement, etc.), an enhancer, splicing information, a polyadenylic acid addition signal, and a tag (FLAG, HA, GST, GFP, etc.).

[0505] Next, the appropriate host cell is transformed using the aforementioned expression vector. The host can be appropriately selected in relation to the vector. As a host, for example, Escherichia coli, Bacillus subtilis, Bacillus, yeast, insects or insect cells, animal cells, etc. can be used. As animal cells, for example, HEK293T cells, CHO cells, COS cells, myeloma cells, HeLa cells, Vero cells can be used. Transformation can be carried out according to known methods such as lipid transfection, calcium phosphate method, electroporation, microinjection, particle gun method, etc., depending on the type of host. The target peptide is expressed by culturing the transformant according to the usual method.

[0506] The peptides from the culture of the transformant are purified by collecting the cultured cells, suspending them in an appropriate buffer, disrupting the cells by ultrasonic treatment, freeze-thaw, etc., and obtaining a crude extract by centrifugation and filtration. In the case where the peptide is secreted in the culture medium, the supernatant is collected.

[0507] Purification from the crude extract or culture supernatant can also be carried out using known methods or methods in accordance therewith (e.g., salting out, dialysis, ultrafiltration, gel filtration, SDS-PAGE, ion exchange chromatography, affinity chromatography, reversed-phase high performance liquid chromatography, etc.).

[0508] The obtained peptide can also be converted from a free form to a salt or from a salt to a free form by a known method or a method in accordance therewith.

[0509] In one embodiment, the translation synthesis system can also be a cell-free translation system. If according to the cell-free translation system, generally speaking, the expression product can be obtained in a highly pure form without refining. The cell-free translation system, for example, includes ribosomal proteins, aminoacyl tRNA synthetase (ARS), ribosomal RNA, amino acids, rRNA, GTP, ATP, translation initiation factor (IF), elongation factor (EF), termination factor (RF), ribosome recycling factor (RRF) and other factors required for translation. In order to improve the expression efficiency, Escherichia coli extract or wheat germ extract can also be added. In addition, rabbit red blood cell extract or insect cell extract can also be added.

[0510] In a system including such, energy can be continuously supplied by using dialysis, and several 100 μg to several mg / mL of protein can be produced non-limitingly. In order to perform transcription from gene DNA at the same time, it can also be a system including RNA polymerase. As a commercially available cell-free translation system, as a system derived from Escherichia coli, RTS-100 (registered trademark) of Roche Diagnostics, PURESYSTEM of GeneFrontier Corporation, PURExpress In Vitro Protein Synthesis Kit of NEW ENGLAND Biolabs, etc. can be used, and as a system using wheat germ extract, products of ZOEGENE Corporation and CellFree Sciences Co., Ltd. can be used.

[0511] In a cell translation system, an artificial aminoacyl tRNA in which a desired amino acid or hydroxy acid is linked (acylated) to tRNA can be used instead of the aminoacyl tRNA synthetase synthesized by the natural aminoacyl tRNA synthetase. This aminoacyl tRNA can be synthesized using an artificial ribozyme.

[0512] As such ribozymes, there are exemplified flexizymes (H. Murakami, H. Saito, and H. Suga, (2003), Chemistry & Biology, Vol. 10, 655-662; and WO2007 / 066627, etc.) The flexizymes are known as prototype flexizymes (Fx) and the modified flexizymes thereof, such as dinitrobenzyl flexizymes (dFx), enhanced flexizymes (eFx), and amino flexizymes (aFx).

[0513] By using tRNA generated by elastase and linked to the desired amino acid or hydroxy acid, the desired codon can be linked to the desired amino acid or hydroxy acid for translation. As the desired amino acid, a special amino acid can also be used. For example, the non-natural amino acid required for the above-mentioned cyclization can also be introduced into the binding peptide by this method.

[0514] The chemical synthesis of the aforementioned peptide can be synthesized using various methods commonly used in this technical field, such as staged solid phase synthesis, semi-synthesis of peptide fragments reconnected by conformational support, and chemical connection. The synthesis of the aforementioned peptide is, for example, chemical synthesis using various solid phase techniques such as those described in KJ Jensen, PT Shelton, SL Pedersen, Peptide Synthesis and Applications, 2nd Edition, Springer, 2013. As a preferred strategy, it is based on a combination of the following two: an Fmoc group that temporarily protects the α-amino group and can be selectively removed by a base; and a protective group that temporarily protects the side chain functional group and is stable under Fmoc removal conditions. This general selection of peptide side chains is recorded in the aforementioned Peptide Synthesis and Applications, 2nd Edition, GB Fields, RL Noble, Solid Phase Peptide Synthesis Utilizing 9-FluorenylmethoxycarbonylAmino Acids, Int. J. Peptide Protein Res.35, 1990, 161-214, etc., but as preferred peptide side chain protecting groups, for example, benzyl or tert-butyl and trityl (Trt) groups for the hydroxyl groups of serine or threonine, 2-bromobenzyloxycarbonyl or tert-butyl groups for the hydroxyl groups of tyrosine, Boc groups for the amino groups of the lysine side chain, methyltetrazolylthiol (Mtt) groups, Alloc groups and ivDde groups for the side chain of histidine, The invention also includes a Trt group or a Boc group of an imidazole group, a 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) group for the guanidine group of arginine, a tert-butyl group, an allyl group and a 3-methylpentane (Mpe) group for the carboxyl group headed by glutamic acid or aspartic acid, a Trt group for the acylamino group of glutamic acid or asparagine, and a Trt group and a monomethoxytrityl (Mmt) group for the thiol group of cysteine.

[0515] The aforementioned peptide can be synthesized on the aforementioned solid phase resin in a staged method. The C-terminal amino acid used and all amino acids or peptides used in the synthesis must selectively remove the α-amino protecting group during the synthesis process. Preferably, the aforementioned solid phase resin is used, and the C-terminal carboxyl group of the peptide whose N-terminus is appropriately protected by the Fmoc group or the C-terminal carboxyl group of the amino acid protected by the Fmoc group is made into an activated ester by an appropriate reagent, and then added to the amino group on the solid phase resin, thereby starting. The elongation of the subsequent peptide chain can be achieved by following the amino acid sequence of the target peptide, repeating the removal of the N-terminal protecting group (Fmoc group) and the condensation of the protected amino acid derivative in sequence. In addition, this can free the target peptide in the final stage. For example, as the conditions for releasing it, Teixeira, WE Benckhuijsen, PE de Koning, ARP Valentijn, JW Drijfhout, Protein Pept. Lett., 2002, 9, 379-385, etc. can be cited, and it can be released in a TFA solution containing water / silylhydride / thiol as a scavenger in TFA. As a typical example, TFA / Water / TIS / DODT (volume ratio 92.5:2.5:2.5:2.5) can be cited.

[0516] The synthesis of the peptides described in the present specification can be carried out by using a single-channel or multi-channel peptide synthesizer, such as the Liberty Blue synthesizer of CEM or the Syro I synthesizer of Biotage or their successor models.

[0517] The activation of the carboxyl group can be carried out using a condensing agent. Examples of the condensing agent include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU).

[0518] For the cyclization of peptides, known methods can be followed. In a non-limiting manner, for example, by designing to include more than two cysteine ​​residues in the peptide, after translation, a cyclic structure is formed by a disulfide bond. In addition, by the method of Goto et al. (Y. Goto et al., ACS Chem. Biol. 3 120-129 (2008)), and by the reprogramming technology of the genetic code, a peptide with a chloroacetyl group at the N-terminus can be synthesized, and a cysteine ​​residue containing a sulfur molecule is configured in the peptide, thereby cyclizing. By this, after translation, the sulfhydryl group spontaneously attacks the chloroacetyl group, and the peptide is cyclized by a thioether bond. By the reprogramming technology of the genetic code, a combination of other amino acids that are combined and formed into a ring can be configured in the peptide for cyclization. Alternatively, it can also be cyclized by configuring an L-2-aminoadipic acid residue in the peptide and combining it between the main chain amino group at the N-terminus. As described above, any known cyclization method can be used without particular limitation.

[0519] 5. BMP4 and / or BMP7 signal transduction inhibitors, human stem cell differentiation induction control agents, culture medium additives, and culture medium containing the peptide of the present invention

[0520] The peptide of the present invention has the ability to bind to BMP4. BMP4 is known to strongly bind to its receptor, BMPR1A, and has the function of transmitting information into cells to control cell differentiation and induction. The peptide of the present invention inhibits the binding of BMP4 to the BMP4 receptor by binding to BMP4, thereby having the activity of inhibiting the transmission of BMP4 information in cells.

[0521] The peptide of the present invention has BMP7 signal transduction inhibitory activity.

[0522] Therefore, the present invention provides, in one embodiment, a BMP4 and / or BMP7 signal transduction inhibitor comprising the aforementioned peptide.

[0523] In one embodiment, the present invention provides a composition for inhibiting BMP4 and / or BMP7 signal transduction, comprising the aforementioned peptide.

[0524] In one embodiment, the present invention provides a method for inhibiting the signal transduction activity of BMP4, comprising the step of contacting the peptide with BMP4. Although not limited to the present invention, the step of contacting the peptide with BMP4 may be a step of contacting the peptide with BMP4 in vitro.

[0525] In one embodiment, the present invention provides a method for inhibiting the signal transduction activity of BMP7.

[0526] Furthermore, since BMP4 and / or BMP7 control the differentiation and induction of human stem cells through the aforementioned signal transduction, the aforementioned peptide having the activity of inhibiting the signal transduction of BMP4 and / or BMP7 has the activity of controlling the differentiation induction of human stem cells.

[0527] Therefore, the present invention provides, in one embodiment, an agent for controlling the differentiation induction of human stem cells comprising the aforementioned peptide.

[0528] In one embodiment, the present invention provides a composition comprising the aforementioned peptide and having the ability to control the differentiation induction of human stem cells.

[0529] In one embodiment, the present invention provides a method for controlling differentiation induction of human stem cells using the peptide. Although not limited thereto, the method can be implemented by adding the peptide to a culture medium for culturing human stem cells. The method is preferably a method that can be implemented in vitro.

[0530] Here, the human stem cells include pluripotent stem cells such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and multipotent stem cells such as mesenchymal stem cells.

[0531] Furthermore, the aforementioned differentiation induction control ability may be a differentiation induction promoting ability or a differentiation induction inhibiting ability, which varies according to the type of the aforementioned human stem cells.

[0532] The composition for inhibiting the transmission of BMP4 and / or BMP7 signals and / or the agent for controlling the differentiation induction of human stem cells, or the composition having the ability to control the differentiation induction of human stem cells may be a pharmaceutical composition as one embodiment, or may be an active ingredient for the manufacture of a pharmaceutical composition.

[0533] Furthermore, the present invention provides a culture medium additive and a culture medium for human stem cells containing the aforementioned culture medium additive in one embodiment, wherein the culture medium additive is a culture medium additive for human stem cells and contains the aforementioned peptide, the aforementioned BMP4 and / or BMP7 information transmission inhibitor and / or the aforementioned human stem cell differentiation induction inhibitor. The culture medium additive may be in the form of a solution or in the form of a dry solid (e.g., solid, powdered, etc.). In the case of a solution, it can be used directly as a culture medium, or it can be used as a culture medium that is diluted with a solvent and added with the aforementioned additives as needed. As the solvent used for dilution, for example, water, buffer, physiological saline, various culture media that can be used for cell or tissue culture, etc. can be cited. These can be used alone or in combination of two or more.

[0534] The culture medium of the present invention is not particularly limited as long as it is a medium for culturing cells or tissues, but can be prepared by appropriately using a medium commonly used for culturing target human stem cells as its basic medium.

[0535] As secretory proteins that play the role of BMP inhibitors and have differentiation induction control ability, Noggin, Chordin, follistatin, etc. are known, but since these are proteins, when used in cell culture, in addition to consuming culture costs, there are also problems such as lack of reproducibility. Compared with the above-mentioned proteins, the human stem cell differentiation induction control agent containing the above-mentioned peptide of the present invention has a low manufacturing cost and contains less impurities because it is easy to purify. Therefore, by using the culture medium of the present invention, compared with the previous ones, the culture cost can be suppressed, and the human stem cell differentiation induction experiment with high reproducibility can be performed.

[0536] The composition comprising the peptide of the present invention may contain a known carrier (eg, water, physiological saline or excipient) as appropriate in addition to the peptide of the present invention.

[0537] The agent containing the peptide of the present invention contains the peptide of the present invention as an active ingredient in an effective amount. The agent containing the peptide of the present invention can be prepared, for example, by mixing the peptide of the present invention with a known carrier. Examples of dosage forms of such agents are injections and oral agents. The dosage of the agent of the present invention to mammals (e.g., humans, mice, rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs, sheep, etc.), especially humans, varies according to symptoms, patient age, sex, weight, sensitivity differences, administration methods, administration intervals, types of active ingredients, and types of preparations. It is not particularly limited, but for example, 30 μg to 1000 mg, 100 μg to 500 mg, 100 μg to 100 mg can be administered once or divided into several times. In the case of injection administration, 1 μg / kg to 3000 μg / kg, 3 μg / kg to 1000 μg / kg can be administered once or divided into several times according to the patient's weight.

[0538] [Example]

[0539] The present invention is described in detail below based on the embodiments, but the present invention is not limited to these embodiments. A person skilled in the art can easily add modifications and changes to the present invention based on the description of this specification, which are also included in the technical scope of the present invention.

[0540] Chemical synthesis

[0541] In the following examples, all raw materials, building blocks, reagents, acids, bases, solid phase resins, and solvents used in chemical synthesis are commercial products or can be synthesized by a person skilled in the art using organic chemical techniques. In addition, amino acids containing protective groups are commercial products unless otherwise specified.

[0542] The elongation of the peptide chain on the solid phase resin is carried out using the resin described in each example as a starting material, using generally used peptide coupling reaction conditions and Fmoc removal reaction conditions. The reaction is carried out using a peptide automatic synthesizer, i.e., Biotage's Syro I, Biotage's Syro II, CEM's Liberty Blue, CEM's Liberty Blue HT12, or CEM's Liberty Prime, in accordance with the manufacturer's manual.

[0543] The resin used was Fmoc-D-Glu(tBu)-Wang resin, NovaPEG Rink Amide resin or Seiber Amide resin, and the amount used was in the range of 4 mg to 2 g according to each peptide.

[0544] The amount of the reagent cocktail used for the deprotection of the side chain and the cleavage from the solid phase resin was 0.2 mL to 50 mL depending on the peptide, and a solution having the following composition was used.

[0545] Composition A: TFA / H2O / TIS / DODT (92.5 / 2.5 / 2.5 / 2.5)

[0546] Composition B: TFA / H2O / TIS / DODT (90 / 2.5 / 2.5 / 5)

[0547] The commonly used amino acids are listed below, with the side chain protecting groups shown in brackets.

[0548] Fmoc-Ala-OH;

[0549] Fmoc-Arg(Pbf)-OH;

[0550] Fmoc-Ash(Trt)-OH;

[0551] Fmoc-Asp(OMpe)-OH;

[0552] Fmoc-Asp(OtBu)-OH;

[0553] Fmoc-Cys(Trt)-OH;

[0554] Fmoc-Gln(Trt)-OH;

[0555] Fmoc-Glu(OtBu)-OH;

[0556] Fmoc-Gly-OH;

[0557] Fmoc-Ile-OH;

[0558] Fmoc-His(Boc)-OH;

[0559] Fmoc-His(Trt)-OH;

[0560] Fmoc-Leu-OH;

[0561] Fmoc-Lys(Boc)-OH;

[0562] Fmoc-Phe-OH;

[0563] Fmoc-Pro-OH;

[0564] Fmoc-Ser(tBu)-OH;

[0565] Fmoc-Ser(Trt)-OH;

[0566] Fmoc-Thr(tBu)-OH;

[0567] Fmoc-Thr(Trt)-OH;

[0568] Fmoc-Tyr(tBu)-OH;

[0569] Fmoc-Val-OH.

[0570] The crude peptide was purified by using a Waters AutoPurification System-SQD2 single quadruple mass spectrometer and reversed-phase preparative HPLC to elute the target while monitoring the m / z ions derived from the target. It was confirmed that the mass spectrum obtained by the ESI-positive scanning mode was consistent with the mass spectrum containing multivalent ions calculated from the molecular formula of the target within the error range of the mass spectrometer used. In addition, the purification conditions including the column used are shown in each example.

[0571] The structure of chemically synthesized peptides is determined by the molecular weight calculated by considering the amino acids used in the target sequence and the building blocks used as needed, and is confirmed by ESI-MS(+) in mass spectrometry. In addition, the so-called "ESI-MS(+)" means electrospray ionization mass spectrometry performed in positive ion mode. The detected mass is reported by the "m / z" unit label. In addition, compounds with a molecular weight greater than about 1000 are detected at a high frequency as multivalent ions.

[0572] In the mass spectrometry analysis of the peptides synthesized in the following Examples, the following basic analysis apparatus and basic conditions were used unless otherwise specified: Gradient B (%) was analyzed using any of the X / Y / Z conditions.

[0573] Equipment: Shimadzu LC / MS system (LC-20ADXR, CTO-20AC, SPD-M20A, SIL-20AXR, CBM-20A and LCMS-2020)

[0574] Column temperature: 60℃

[0575] Mobile phase A: 0.025% TFA in water

[0576] Mobile phase B: 0.025% TFA in MeCN

[0577] Flow rate: 0.5mL / min

[0578] Wavelength: 225nm PDA

[0579] Gradient B (%): X: 20-60% / 7.15 min, 60-95% / 0.3 min, 95-95% / 1.55 ​​min;

[0580] Y: 40-80% / 7.15min, 80-95% / 0.3min, 95-95% / 1.55min;

[0581] Z: 5-45% / 7.15min, 45-95% / 0.3min, 95-95% / 1.55min

[0582] Example 1

[0583] Synthesis of BMP4 signal transduction inhibitory peptide No.5 (SEQ ID NO: 5)

[0584] In this example, BMP4 signal transduction inhibitory peptide No. 5 (SEQ ID NO: 5) having the following structure was synthesized.

[0585] [Chemistry 1]

[0586]

[0587] Using Sieber amide resin (Merck, 0.49 mmol / g), the target peptide was synthesized by the general method starting from the removal of the Fmoc group. At this time, the Syro II of Biotage was used as a solid phase synthesizer and the synthesis was carried out according to the manufacturer's manual. In the introduction of each residue, Fmoc-AA / HATU / DIEA (4.2 equivalents / 3.9 equivalents / 8.4 equivalents) was used relative to 1 equivalent of the resin, and the reaction was carried out twice in DMF at 75°C for 10 minutes. However, the second residue, the seventh residue, and the 17th residue were reacted twice at 50°C for 30 minutes.

[0588] Fmoc removal was carried out by reacting with a 20% piperidine solution in DMF at room temperature for 5 minutes, removing the solution, and then reacting again with a 20% piperidine solution in DMF at room temperature for 15 minutes.

[0589] The introduction of the chloroacetyl group was carried out in the following manner: for the solid phase resin holding the Fmoc-protected peptide obtained in the previous step, the Fmoc group of the α-amino group was removed by the above-mentioned method, and then a 0.3 M chloroacetic acid solution in DMF (4.2 equivalents), a 0.28 M HCTU solution in DMF (3.92 equivalents), and a 1.05 M DIPEA solution in DMF (8.4 equivalents) were added to the solid phase resin, and the mixture was shaken at room temperature for 30 minutes and repeated twice.

[0590] The deprotection of the side chain and the excision from the solid phase resin are firstly to wash the resin obtained after the chloroacetyl group introduction step with DMF 5 times and methyl chloride 3 times, and then dry it under reduced pressure. Then, the reactant mixture-A (a mixture of TFA / H2O / TIS / DODT with a volume ratio of 92.5:2.5:2.5:2.5) is added to the reaction container with the solid phase resin, and shaken at room temperature for 60 minutes. The reaction solution is filtered and recovered by glass frit. If the filtrate is added to the cooled excess diisopropyl ether, a precipitate will be generated. The mixture is centrifuged (8500 rpm, 30 seconds) and the solution is decanted. The obtained solid is washed again with a cooled mixed solvent of diethyl ether / hexane (1 / 1), and then dried under reduced pressure. The obtained solid is used for the subsequent cyclization reaction.

[0591] The peptide cyclization reaction was carried out by dissolving the peptide in DMSO at a final concentration of 2.5 mM based on the molar number of the solid phase resin, adding triethylamine (0.01 mL), and shaking at room temperature overnight. The obtained reaction solution was concentrated under reduced pressure, and DMSO (0.1 mL) was added to the obtained residue.

[0592] The obtained solution is subjected to solid phase extraction as follows. (1) A Gilson ASPEC (registered trademark) C18 500 mg 3 mL (Gilson) column is washed with extracting solution A (0.1% TFA dissolved in 95% MeCN / H2O, 3 mL). (2) The column is equilibrated with extracting solution B (0.1% TFA dissolved in 5% MeCN / H2O, 3 mL). (3) 0.1 mL of the reaction solution is added to the column. (4) The column is washed with extracting solution B (4 mL). (5) Extraction is performed with extracting solution A (4 mL). The obtained extract is concentrated under reduced pressure.

[0593] The purity of the main peak of the target substance was calculated from the area ratio of the LC / MS (UV wavelength 220 nm) chromatogram under the following analysis conditions and was 43.4%.

[0594] Analysis conditions: holding time = 1.39 minutes;

[0595] Column: Kinetex (registered trademark) EVO C18 1.7μm 2.1X50mm;

[0596] Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN;

[0597] Temperature: 60℃;

[0598] Gradient (%B conc): 5-95% in 2.10 min, then 95-95% in 0.75 min;

[0599] Flow rate: 0.6mL / min;

[0600] ESI-MS (+) observed value m / z = 1258.0 (M + 2H) 2+ .

[0601] Example 2

[0602] Synthesis of BMP4 signal transduction inhibitory peptide No. 177 (SEQ ID NO: 177)

[0603] In this example, a BMP4 signal transduction inhibitory peptide No. 177 (SEQ ID NO: 177) having the following structure was synthesized.

[0604] [Chemistry 2]

[0605]

[0606] The target peptide was synthesized using Fmoc-D-Glu(tBu)-Wang resin (Watanabe Chemical, 0.62 mmol / g) by a general method starting from the removal of the Fmoc group. Synthesis was performed using Syro I from Biotage as a solid phase synthesizer in accordance with the manufacturer's manual. In the introduction of each residue, Fmoc-AA / HATU / DIPEA (4.2 equivalents / 4 equivalents / 8.4 equivalents) was used relative to 1 equivalent of the resin, and the reaction was performed twice in DMF at 75°C for 20 minutes. However, the second and seventh residues were reacted twice at room temperature for 30 minutes. The 17th residue was reacted once at room temperature for 30 minutes. The 18th residue was reacted once at 75°C for 20 minutes.

[0607] For removal of the Fmoc group, the reaction was performed with a 20% piperidine solution in DMF at room temperature for 5 minutes, the solution was removed, and then the reaction was performed again with a 20% piperidine solution in DMF at room temperature for 15 minutes.

[0608] The introduction of the chloroacetyl group is carried out in the following manner: for the solid phase resin holding the Fmoc-protected peptide obtained in the previous step, the Fmoc group of the α-amino group is removed by the above-mentioned method, and then a DCM / DMF solution of ClAcOSu prepared with chloroacetic acid (10 equivalents), DIPCI (10 equivalents) and HOSu (10 equivalents) is added to the solid phase resin and shaken at room temperature for 60 minutes.

[0609] The deprotection of the side chain and the excision from the solid phase resin are firstly to wash the resin obtained after the chloroacetyl group introduction step with DMF 5 times and with methyl chloride 3 times, and then dry it under reduced pressure. Then, the reactant mixture-A (a mixture of TFA / H2O / TIS / DODT with a volume ratio of 92.5:2.5:2.5:2.5) is added to the reaction container in which the solid phase resin has been placed, and it is shaken at room temperature for 90 minutes. The reaction solution is filtered and recovered by a glass frit. The solid phase resin remaining in the reaction container is shaken again with the excision mixture, and the solution components are recovered by a glass frit and mixed with the above-mentioned filtrate. If this filtrate is added to a mixed solvent of diethyl ether / hexane (1 / 1), a precipitate will be generated. This mixture is centrifuged and the solution is decanted. The obtained solid is washed again with diethyl ether and dried under reduced pressure. The obtained solid is used for the subsequent cyclization reaction.

[0610] The peptide cyclization reaction was carried out by dissolving the peptide in MeCN / H2O (1 / 1) at a final concentration of 5 mM based on the molar number of the solid phase resin, adding triethylamine (10 equivalents), and shaking at room temperature for 15 hours. Acetic acid was added to the resulting reaction solution, and the mixture was concentrated under reduced pressure using Genevac EZ-II elite.

[0611] The obtained crude product was purified under the following conditions.

[0612] Column: Waters XBridge (registered trademark) C18, 30X150mm;

[0613] Mobile phase: A = 0.1% TFA in water, B = 0.1% TFA in MeCN; Temperature: 50°C; Gradient (%Bconc.): 9-34% in 3 minutes, then 34-39% in 8 minutes, then 39-60% in 1 minute; Flow rate: 45 mL / min.

[0614] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analysis conditions and was 93.5%.

[0615] Analysis conditions: holding time = 4.49 minutes;

[0616] Column: Kinetex (registered trademark) EVO C18 2.6μm 2.1X150mm,

[0617] Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN;

[0618] Temperature: 60°C; Gradient (%B conc): 20-60% in 7.15 min, then 60-95% in 0.30 min, then 95-95% in 1.55 min;

[0619] Flow rate: 0.5mL / min

[0620] ESI-MS (+) observed value m / z = 1400.9 (M + 2H) 2+ .

[0621] Example 3

[0622] Synthesis of BMP4 signal transduction inhibitory peptide No. 84 (SEQ ID NO: 84)

[0623] In this example, a BMP4 signal transduction inhibitory peptide No. 84 (SEQ ID NO: 84) having the following structure was synthesized.

[0624] [Chemistry 3]

[0625]

[0626] The target peptide was synthesized using Fmoc-D-Glu(OtBu)-Wang resin (Watanabe Chemical, 0.79 mmol / g) by a general method starting with the removal of the Fmoc group. Synthesis was performed using Syro I (Biotage's solid phase synthesizer) according to the manufacturer's manual. In the introduction of each residue, Fmoc-AA / HATU / DIPEA (4.2 equivalents / 4 equivalents / 8.4 equivalents) was used relative to 1 equivalent of the resin, and the reaction was carried out twice in DMF at 75°C for 20 minutes. However, the second residue was reacted twice at 75°C for 30 minutes. The seventh residue was reacted twice at 50°C for 30 minutes. The 17th residue was reacted twice at room temperature for 30 minutes.

[0627] For removal of the Fmoc group, the reaction was performed with a 20% piperidine solution in DMF at room temperature for 5 minutes, the solution was removed, and then the reaction was performed again with a 20% piperidine solution in DMF at room temperature for 15 minutes.

[0628] The introduction of the chloroacetyl group was carried out as follows: a solution of ClAcOSu in NMP / DCM prepared with ClAcOH / DIPCI / HOSu (5.3 eq. / 5.25 eq. / 5.3 eq.) was added to the solid phase resin relative to 1 eq. of the solid phase resin holding the Fmoc-protected peptide obtained in the previous step, and the mixture was shaken at room temperature for 90 minutes.

[0629] The deprotection of the side chain and the excision from the solid phase resin are firstly to wash the resin obtained after the chloroacetyl group introduction step with DMF 5 times and methyl chloride 3 times, and then dry it under reduced pressure. Then, the reactant mixture-A (a mixture of TFA / H2O / TIS / DODT with a volume ratio of 92.5:2.5:2.5:2.5) is added to the reaction container with the solid phase resin, and it is shaken at room temperature for 90 minutes. The reaction solution is filtered and recovered by the glass frit. The solid phase resin remaining in the reaction container is shaken again with the excision mixture, and the solution components are recovered by the glass frit and mixed with the above-mentioned filtrate. If this filtrate is added to the cooled excess diisopropyl ether, a precipitate will be generated. The mixture is centrifuged and the supernatant is removed. The obtained solid is washed again with cooled diethyl ether and then dried under reduced pressure. The obtained solid is used for the subsequent cyclization reaction.

[0630] The peptide cyclization reaction was carried out by dissolving the peptide in DMSO / H2O (9 / 1) at a final concentration of 2.5 mM based on the molar number of the solid phase resin, adding triethylamine (10 equivalents), and shaking at room temperature for 2 hours. After adding acetic acid to the resulting reaction solution, it was concentrated under reduced pressure using Genevac EZ-II elite.

[0631] The obtained crude product was purified under the following conditions.

[0632] Column: Jeanious One-Column, 20X150mm (ChromaJean Company);

[0633] Mobile phase: A = 0.1% TFA in water, B = 0.1% TFA in MeCN; Temperature: room temperature; Gradient (% Bconc): 25.9-45.9% in 9 minutes, then 45.9-90% in 0.01 minutes;

[0634] Flow rate: 20mL / min.

[0635] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analysis conditions and was 82.8%.

[0636] Analysis conditions: retention time = 4.14 minutes; column: Kinetex (registered trademark) EVO C18 2.6 μm 2.1X150 mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN Temperature: 60°C; Gradient (%B conc): 20-60% in 7.15 min, then 60-95% in 0.30 min, then 95-95% in 1.55 min; Flow rate: 0.5 mL / min

[0637] ESI-MS (+) observed value m / z = 1359.9 (M+2H) 2+.

[0638] Example 4

[0639] Synthesis of BMP4 signal transduction inhibitory peptide No.87 (SEQ ID NO: 87)

[0640] In this example, BMP4 signal transduction inhibitory peptide No. 87 (SEQ ID NO: 87) having the following structure was synthesized.

[0641] [Chemistry 4]

[0642]

[0643] Using Sieber Amide resin (Merck, 0.48 mmol / g), the target peptide was synthesized by the aforementioned general method starting from the removal of the Fmoc group. Using Syro I of Biotage as a solid phase synthesizer, the synthesis was carried out in accordance with the manufacturer's manual. In the introduction of each residue, Fmoc-AA / HATU / DIPEA (4.2 equivalents / 4 equivalents / 8.4 equivalents) was used relative to 1 equivalent of the resin, and the reaction was carried out twice in DMF at 75°C for 20 minutes. However, the second residue was reacted twice at 75°C for 30 minutes. The seventh residue was reacted twice at 50°C for 30 minutes. The 17th residue was reacted twice at room temperature for 30 minutes.

[0644] For removal of the Fmoc group, the reaction was performed with a 20% piperidine solution in DMF at room temperature for 5 minutes, the solution was removed, and then the reaction was performed again with a 20% piperidine solution in DMF at room temperature for 15 minutes.

[0645] The introduction of the chloroacetyl group was carried out as follows: a solution of ClAcOSu in NMP / DCM prepared with ClAcOH- / DIPCI / HOSu (5.3 eq. / 5.25 eq. / 5.3 eq.) was added to the solid phase resin relative to 1 eq. of the solid phase resin holding the Fmoc-protected peptide obtained in the previous step, and the mixture was shaken at room temperature for 90 min.

[0646] The deprotection of the side chain and the excision from the solid phase resin are firstly to wash the resin obtained after the chloroacetyl group introduction step with DMF 5 times and methyl chloride 3 times, and then dry it under reduced pressure. Then, the reactant mixture-A (a mixture of TFA / H2O / TIS / DODT with a volume ratio of 92.5:2.5:2.5:2.5) is added to the reaction container with the solid phase resin, and it is shaken at room temperature for 90 minutes. The reaction solution is filtered and recovered by the glass frit. The solid phase resin remaining in the reaction container is shaken again with the excision mixture, and the solution components are recovered by the glass frit and mixed with the above-mentioned filtrate. If this filtrate is added to the cooled excess diisopropyl ether, a precipitate will be generated. The mixture is centrifuged and the supernatant is removed. The obtained solid is washed again with cooled diethyl ether and then dried under reduced pressure. The obtained solid is used for the subsequent cyclization reaction.

[0647] The peptide cyclization reaction was carried out by dissolving the peptide in DMSO / H2O (9 / 1) at a final concentration of 2.5 mM based on the molar number of the solid phase resin, adding triethylamine (10 equivalents), and shaking at room temperature for 2 hours. Acetic acid was added to the resulting reaction solution, and the mixture was concentrated under reduced pressure using Genevac EZ-II elite.

[0648] The obtained crude product was purified under the following conditions.

[0649] Column: Jeanious One-Column, 20X150mm (ChromaJean Company);

[0650] Mobile phase: A = 0.1% TFA in water, B = 0.1% TFA in MeCN; Temperature: room temperature; Gradient (% Bcorc): 18.7-38.7% in 9 minutes, then 38.7-90% in 0.01 minutes; Flow rate: 20 mL / min.

[0651] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analysis conditions and was 90.3%.

[0652] Analysis conditions: retention time = 3.15 minutes; column: Kinetex (registered trademark) EVO C18 2.6 μm 2.1X150 mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (%B conc): 20-60% in 7.15 min, then 60-95% in 0.30 min, then 95-95% in 1.55 min; Flow rate: 0.5 mL / min

[0653] ESI-MS (+) observed value m / z = 1283.6 (M + 2H) 2+ .

[0654] Example 5

[0655] Synthesis of BMP7 signal transduction inhibitory peptide

[0656] In this example, a BMP7 signal transduction inhibitory peptide having the following structure (ClAc-FG-MeF-G-Bph-GDD-Cha-A-MeF-LHCG-NH2, SEQ ID NO: 371, -NH2 is an addition sequence) was synthesized.

[0657] [Chemistry 5]

[0658]

[0659] Using Sieber Amide resin (Watanabe Chemical, 0.57 mmol / g), the target peptide was synthesized by the general method starting from the removal of the Fmoc group. At this time, the Syro II of Biotage was used as a solid phase synthesizer, and the synthesis was carried out according to the manufacturer's manual. In the introduction of each residue, Fmoc-AA / HATU / DIEA (4.2 equivalents / 3.9 equivalents / 8.4 equivalents) was used relative to 1 equivalent of the resin, and the reaction was carried out twice in DMF at 75°C for 20 minutes. However, the 11th residue and the 13th residue were reacted twice at 50°C for 30 minutes. The 14th residue was reacted twice at 25°C for 20 minutes.

[0660] Fmoc removal was carried out by reacting with a 20% piperidine solution in DMF at room temperature for 5 minutes, removing the solution, and then reacting again with a 20% piperidine solution in DMF at room temperature for 15 minutes.

[0661] The introduction of the chloroacetyl group was carried out in the following manner: for the solid phase resin holding the Fmoc-protected peptide obtained in the previous step, the Fmoc group of the α-amino group was removed by the above-mentioned method, and then a 0.3 M chloroacetic acid solution in DMF (4.2 equivalents), a 0.28 M HCTU solution in DMF (3.9 equivalents), and a 1.05 M DIEA solution in DMF (8.4 equivalents) were added to the solid phase resin, and the mixture was shaken at room temperature for 30 minutes, and repeated twice.

[0662] The side chain is deprotected and cut from the solid phase resin. First, the resin obtained after the chloroacetyl group introduction step is washed with DMF 3 times and methyl chloride 3 times, and then dried under reduced pressure. Then, the reactant mixture-A (a mixture of TFA / H2O / TIS / DODT with a volume ratio of 92.5:2.5:2.5:2.5) is added to the reaction container with the solid phase resin, and shaken at room temperature for 60 minutes. The reaction solution is filtered and recovered by glass frit. If the filtrate is added to the cooled excess diisopropyl ether, a precipitate will be generated. The mixture is centrifuged (8500 rpm, 30 seconds), and the solution is decanted. The obtained solid is washed again with a cooled diethyl ether / hexane (1 / 1) mixed solvent. The solid is centrifuged again (8500 rpm, 30 seconds), and the solution is decanted and dried under reduced pressure. The obtained solid is used for the subsequent cyclization reaction.

[0663] The peptide cyclization reaction was carried out by dissolving the peptide in 30% aqueous DMSO so that the final concentration of the peptide was 2.5 mM based on the molar number of the solid phase resin, adding triethylamine (10 equivalents), and shaking at room temperature overnight. The obtained reaction solution was concentrated under reduced pressure using Genevac EZ-2 Elite, and DMSO (0.1 mL) was added to the obtained residue.

[0664] The obtained solution was subjected to solid phase extraction using a column from GILSO (column: Gilson ASPEC C18 500 mg 3 mL): (1) The column was washed with extract A (0.1% TFA dissolved in 95% MeCN / H2O, 3 mL). (2) The column was balanced with extract B (0.1% TFA dissolved in 5% MeCN / H2O, 3 mL). (3) 0.1 mL of the above reaction solution was added to the column. (4) The column was washed with extract B (4 mL). (5) Extraction was performed with extract A (4 mL). The obtained extract was concentrated under reduced pressure using EZ-2Elite.

[0665] The purity of one of the main peaks of the target product was calculated from the area ratio of the LC / MS (UV wavelength 220 nm) chromatogram under the following analysis conditions and was 60.1%.

[0666] Analysis conditions: hold time = 1.58 minutes; column: KineteXEVO C18 1.7 μm 2.1X50 mm; mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; temperature: 60°C; gradient (% B conc): 5-95% in 2.10 minutes, then 95-95% in 0.75 minutes; flow rate: 0.6 mL / min

[0667] ESI-MS (+) observed value m / z = 894.2 (M + 2H) 2+ .

[0668] Example 6

[0669] Synthesis of BMP7 signal transduction inhibitory peptide

[0670] In this example, a BMP7 signal transduction inhibitory peptide having the following structure was synthesized (ClAc-F4F-G-MeF-G-3Py6Ph-GDE-Cha-Hgl-MeF4Me-L-P4Sh-C-bA-de-OH, SEQ ID NO: 417, bA-de-OH is an addition sequence).

[0671] [Chemistry 6]

[0672]

[0673] The target peptide was synthesized using Fmoc-d-Glu(tBu)-Wang resin (Watanabe Chemical, 0.62 mmol / g) by a general method, starting with the removal of the Fmoc group. The synthesis was performed using CEM's Liberty Prime as a solid phase synthesizer in accordance with the manufacturer's manual. For the introduction of each residue, Fmoc-AA / DIC / Oxyma pure (5.25 equivalents / 10 equivalents / 5 equivalents) was used relative to 1 equivalent of the resin, and one reaction was performed in DMF at 105°C for 2 minutes. However, for the 5th residue, Fmoc-AA dissolved in NMP was used. The 2nd and 10th residues were reacted twice at 90°C for 10 minutes. The 14th residue was reacted once at 50°C for 15 minutes.

[0674] The removal of the Fmoc group was carried out using the following conditions as the basic conditions: reaction with a 4% pyrrolidine DMF solution at 110°C for 1 minute. However, the 1st, 3rd, 4th, 5th, 6th and 7th residues were reacted with a 10% pyrrolidine DMF solution at 50°C for 90 seconds. The 2nd and 10th residues were reacted with a 10% pyrrolidine DMF solution for 1 minute, the solution was removed, and then the residue was reacted again with a 10% pyrrolidine DMF solution at room temperature for 1 minute.

[0675] The introduction of chloroacetyl group was carried out as follows: a DCM / DMF solution of ClAcOSu (10 equivalents) was added to the solid phase resin and shaken at room temperature for 60 minutes.

[0676] The side chain was deprotected and cut from the solid phase resin. First, the resin obtained after the chloroacetyl group introduction step was washed with DMF and methyl chloride and then with diethyl ether, and then dried under reduced pressure. Then, the reactant mixture-A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction container in which the solid phase resin was placed, and the mixture was shaken at room temperature for 75 minutes. The reaction solution was filtered and recovered through a glass frit.

[0677] If the obtained filtrate is added to a cooled excess diethyl ether / hexane (1 / 1) mixed solvent, a precipitate will be generated. The mixture is centrifuged and the solution is decanted. The obtained solid is washed again with diethyl ether and dried under reduced pressure. The obtained solid is used in the subsequent cyclization reaction.

[0678] The peptide cyclization reaction was carried out by dissolving the peptide in DMSO at a final concentration of 5 mM based on the molar number of the solid phase resin, adding triethylamine (10 equivalents), and shaking at room temperature for 20 hours. Acetic acid was added to the resulting reaction solution, and the mixture was concentrated under reduced pressure using Genevac EZ-II elite.

[0679] The obtained crude product was purified using the following conditions. (Column: Waters XBridge (registered trademark) C18, 19X150 mm; Mobile phase: A = 0.1% TFA dissolved in water, B = 0.1% TFA dissolved in MeCN; Temperature: 50°C; Gradient (%Bconc.): 12-37% in 3 minutes, then 37-42% in 8 minutes, then 42-60% in 1 minute; Flow rate: 17 mL / min.

[0680] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analysis conditions and was 97.93%.

[0681] Analysis conditions: retention time = 5.48 minutes; column: KineteXEVO C18 2.6μm 2.1X150mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (%B conc): 20-60% in 7.15 min, then 60-95% in 0.30 min, then 95-95% in 1.55 min; Flow rate: 0.5 mL / min

[0682] ESI-MS (+) observed value m / z = 1013.5 (M + 2H) 2+ .

[0683] Example 7

[0684] Synthesis of BMP7 signal transduction inhibitory peptide

[0685] In this example, a BMP7 signal transduction inhibitory peptide having the following structure was synthesized (ClAc-FG-MeF-G-3Py6Ph-GDE-Cha-Hgl MeF-L-P4Sh-C-bA-de-OH, SEQ ID NO: 415, bA-de-OH is an addition sequence).

[0686] [Chemistry 7]

[0687]

[0688] The target peptide was synthesized using Fmoc-d-Glu(tBu)-Wang resin (Watanabe Chemical, 0.62 mmol / g) by a general method, starting with the removal of the Fmoc group. The synthesis was performed using CEM's Liberty Prime as a solid phase synthesizer in accordance with the manufacturer's manual. For the introduction of each residue, Fmoc-AA / DIC / Oxyma pure (5.25 equivalents / 10 equivalents / 5 equivalents) was used relative to 1 equivalent of the resin, and the reaction was performed once in DMF at 105°C for 2 minutes. However, for the 5th residue, Fmoc-AA dissolved in NMP was used. The 2nd and 10th residues were reacted twice at 90°C for 10 minutes. The 14th residue was reacted once at 50°C for 15 minutes. The 15th residue was reacted once at 105°C for 1 minute.

[0689] The removal of the Fmoc group was performed using the following conditions as basic conditions: reaction with a 4% pyrrolidine solution in DMF at 110°C for 1 minute.

[0690] However, the 1st, 3rd, 4th, 5th, 6th and 7th residues were reacted with a 10% pyrrolidine DMF solution at 50°C for 90 seconds. The 2nd and 10th residues were reacted with a 10% pyrrolidine DMF solution for 1 minute, the solution was removed, and then reacted again with a 10% pyrrolidine DMF solution at room temperature for 1 minute.

[0691] The introduction of chloroacetyl group was carried out as follows: a DCM / DMF solution of ClAcOSu (10 equivalents) was added to the solid phase resin and shaken at room temperature for 60 minutes.

[0692] The side chain was deprotected and cut from the solid phase resin. First, the resin obtained after the chloroacetyl group introduction step was washed with DMF and methyl chloride and then with diethyl ether, and then dried under reduced pressure. Then, the reactant mixture-A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction container in which the solid phase resin was placed, and the mixture was shaken at room temperature for 75 minutes. The reaction solution was filtered and recovered through a glass frit.

[0693] If the obtained filtrate is added to a cooled excess diethyl ether / hexane (1 / 1) mixed solvent, a precipitate will be generated. The mixture is centrifuged and the solution is decanted. The obtained solid is washed again with diethyl ether and dried under reduced pressure. The obtained solid is used in the subsequent cyclization reaction.

[0694] The peptide cyclization reaction was carried out by dissolving the peptide in DMSO at a final concentration of 5 mM based on the molar number of the solid phase resin, adding triethylamine (10 equivalents), and shaking at room temperature for 20 hours. Acetic acid was added to the resulting reaction solution, and the mixture was concentrated under reduced pressure using Genevac EZ-II elite.

[0695] The crude product was purified using the following conditions. (Column: Jeanious One-Column 20 mm I.D. x 150 mm L; Mobile phase: A = 0.1% TFA dissolved in water, B = 0.1% TFA dissolved in MeCN; Temperature: 60°C; Gradient (% B conc.): 27.6-47.6% in 9 minutes; Flow rate: 20 mL / min.

[0696] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analysis conditions and was 94.88%.

[0697] Analysis conditions: holding time = 5.40 minutes; column: KineteXEVO C18 2.6μm 2.1X150mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (%B conc): 20-60% in 7.15 min, then 60-95% in 0.30 min, then 95-95% in 1.55 min; Flow rate: 0.5 mL / min

[0698] ESI-MS (+) observed value m / z = 997.5 (M + 2H) 2+ .

[0699] Example 8

[0700] Synthesis of BMP4 signal transduction inhibitory peptide

[0701] In this example, a BMP4 signal transduction inhibitory peptide having the following structure (ClAc-ERV-F4COO-V-KCOpipzaa-R-3Py6Ph-KCOpipzaa-Y-Hyp-Bph-Y-F4COO-F4COO-SCG-NH2, SEQ ID NO: 277) was synthesized.

[0702] [Chemistry 8]

[0703]

[0704] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.57 mmol / g) by a general method, starting with the removal of the Fmoc group. The synthesis was performed using CEM's Liberty Prime as a solid phase synthesizer in accordance with the manufacturer's manual. In the introduction of each residue, Fmoc-AA / DIC / Oxyma pure (4.2 equivalents / 16 equivalents / 6 equivalents) was used relative to 1 equivalent of the resin, and one reaction was performed in DMF at 105°C for 90 seconds. However, Fmoc-AA dissolved in NMP was used for the 8th residue. The 2nd and 7th residues were reacted twice at 50°C for 15 minutes. The 17th residue was reacted once at 50°C for 15 minutes.

[0705] The Fmoc group was removed by reacting with a 4% pyrrolidine solution in DMF at 110°C for 1 minute.

[0706] The introduction of chloroacetyl group was carried out as follows: a DMF solution of ClAcOSu (5 equivalents) was added to the solid phase resin and shaken at room temperature for 60 minutes.

[0707] The side chain was deprotected and cut from the solid phase resin. First, the resin obtained after the chloroacetyl group introduction step was washed with DMF and methyl chloride and then with diethyl ether, and then dried under reduced pressure. Then, the reactant mixture-A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction container in which the solid phase resin was placed, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was filtered and recovered by glass frit.

[0708] If the obtained filtrate is added to a cooled excess diisopropyl ether / hexane (1 / 1) mixed solvent, a precipitate will be generated. The mixture is centrifuged and the solution is decanted. The obtained solid is washed again with diethyl ether and dried under reduced pressure. The obtained solid is used in the subsequent cyclization reaction.

[0709] The peptide cyclization reaction was carried out by dissolving the peptide in DMSO at a final concentration of 2.5 mM based on the molar number of the solid phase resin, adding triethylamine (10 equivalents), and shaking at room temperature for 8 hours. Acetic acid was added to the resulting reaction solution, and the mixture was concentrated under reduced pressure using Genevac EZ-II elite.

[0710] The obtained crude product was purified using the following conditions. (Column: Waters XBridge (registered trademark) C18, 30X150 mm; Mobile phase: A = 0.1% TFA dissolved in water, B = 0.1% TFA dissolved in MeCN; Temperature: 50°C; Gradient (%Bconc.): 5-22% in 3 minutes, then 22-27% in 8 minutes, then 27-60% in 1 minute; Flow rate: 45 mL / min.

[0711] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analysis conditions and was 93.29%.

[0712] Analysis conditions: retention time = 5.36 minutes; column: KineteXEVO C18 2.6μm 2.1X150mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (%B conc): 5-45% in 7.15 min, then 45-95% in 0.30 min, then 95-95% in 1.55 min; Flow rate: 0.5 mL / min

[0713] ESI-MS (+) observed value m / z = 1001.3 (M + 3H) 3+ .

[0714] Example 9

[0715] Synthesis of BMP4 signal transduction inhibitory peptide

[0716] In this example, a BMP4 signal transduction inhibitory peptide having the following structure (ClAc-ERV-F4COO-V-KCOpipzaa-R-3Py6Ph-KCOpipzaa-Y-Hyp-Bph3F-Y-F4COO-F4COO-SCG-NH2, SEQ ID NO: 311) was synthesized.

[0717] [Chemistry 9]

[0718]

[0719] The target peptide was synthesized using Sieber Amide resin by general methods, starting with the removal of the Fmoc group. The synthesis was performed using CEM's Liberty Prime as a solid phase synthesizer in accordance with the manufacturer's manual. In the introduction of each residue, Fmoc-AA / DIC / Oxyma pure (4.2 equivalents / 16 equivalents / 6 equivalents) was used relative to 1 equivalent of the resin, and one reaction was performed in DMF at 105°C for 90 seconds. However, Fmoc-AA dissolved in NMP was used for the 8th residue. The 2nd and 7th residues were reacted twice at 50°C for 15 minutes.

[0720] The Fmoc group was removed by reacting with a 4% pyrrolidine solution in DMF at 110°C for 1 minute.

[0721] The introduction of chloroacetyl group was performed as follows: a DMF solution of ClAcOH / HATU / DIEA (5 equivalents / 5 equivalents / 10 equivalents) was added to the solid phase resin and shaken at room temperature for 30 minutes.

[0722] The side chain was deprotected and cut from the solid phase resin. First, the resin obtained after the chloroacetyl group introduction step was washed with DMF and methyl chloride and then with diethyl ether, and then dried under reduced pressure. Then, the reactant mixture-A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction container in which the solid phase resin was placed, and the mixture was shaken at room temperature for 60 minutes. The reaction solution was filtered and recovered through a glass frit.

[0723] If the obtained filtrate is added to a cooled excess diethyl ether / hexane (1 / 1) mixed solvent, a precipitate will be generated. The mixture is centrifuged and the solution is decanted. The obtained solid is washed again with diethyl ether and dried under reduced pressure. The obtained solid is used in the subsequent cyclization reaction.

[0724] The peptide cyclization reaction was carried out by dissolving the peptide in MeCN / H2O (1 / 1) at a final concentration of 2 mM based on the molar number of the solid phase resin, adding triethylamine (20 equivalents), and shaking at room temperature overnight. After adding acetic acid (30 equivalents) to the resulting reaction solution, it was concentrated under reduced pressure using Genevac HT-12.

[0725] The obtained crude product was purified using the following conditions. (Column: Waters XBridge (registered trademark) C18, 30X150 mm; Mobile phase: A = 0.1% TFA dissolved in water, B = 0.1% TFA dissolved in MeCN; Temperature: 50°C; Gradient (%Bconc.): 5-23% in 3 minutes, then 23-28% in 8 minutes, then 28-60% in 1 minute; Flow rate: 45 mL / min.

[0726] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analysis conditions and was 95.52%.

[0727] Analysis conditions: retention time = 5.04 minutes; column: KineteXEVO C18 2.6μm 2.1X150mm, Mobile phase: A = 0.025% TFA in water, B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (%B conc): 5-45% in 7.15 min, then 45-95% in 0.30 min, then 95-95% in 1.55 min; Flow rate: 0.5 mL / min

[0728] ESI-MS (+) observed value m / z = 1007.7 (M + 3H) 3+ .

[0729] Example 10

[0730] Various peptide synthesis

[0731] In this example, various cyclic peptides were chemically synthesized in the same manner as in Example 1 or 2. The sequences of the synthesized peptides are shown in Table 1, Table 5, Table 6 and Table 7. In addition, the peptide synthesized and purified in the same manner as in Example 1 is indicated as 1 in the synthesis row of Table 1, Table 6 and Table 7, and the peptide synthesized and purified in the same manner as in Example 2 is indicated as 2 in the synthesis row. In Table 1, the peptides having an amino acid sequence other than SEQ ID NO: 46 have a cyclized structure in which the first amino acid is chloroacetylated and the 17th amino acid Cys is bonded, and furthermore, the Cys end thereof has a structure in which Gly, bA or PEG is bonded. In addition, although SEQ ID NO: 46 has the amino acid sequence described in SEQ ID NO: 47 to 50, it does not have an addition structure such as glycine (G) at the C-terminus. The peptides in Table 6 are those in which the amino acid residue at the 5' end is substituted with a chloroacetyl (ClAc) group. The synthesized peptide was analyzed using the basic analysis equipment and basic conditions and the analysis conditions described in Examples 1 to 9, and the structure was confirmed by ESI-MS(+) in mass spectrometry. The obtained ESI-MS(+) observation value (ESI(m / z)) and the valence ([M+XH]X+) in this case are shown in Table 1.

[0732] Table 1

[0733] [Table 1-1]

[0734]

[0735] [Table 1-2]

[0736]

[0737] [Table 1-3]

[0738]

[0739] [Table 1-4]

[0740]

[0741] [Table 1-5]

[0742]

[0743] [Table 1-6]

[0744]

[0745] [Table 1-7]

[0746]

[0747] [Table 1-8]

[0748]

[0749] [Table 1-9]

[0750]

[0751] [Table 1-10]

[0752]

[0753] [Table 1-11]

[0754]

[0755] [Table 1-12]

[0756]

[0757] [Table 1-13]

[0758]

[0759] [Table 1-14]

[0760]

[0761] [Table 1-15]

[0762]

[0763] Embodiment 11

[0764] BMP4 signal transduction inhibitory activity evaluation test

[0765] In this example, the BMP4 signal transduction inhibitory activity of each peptide composed of the amino acid sequence described in SEQ ID NOs: 5 to 240 and 243 to 421 synthesized in Example 10 was verified by phosphorylation of SMAD1 caused by BMP4 stimulation.

[0766] A549 cells were cultured in MEM (Thermo Fisher Scientific) containing 10% FBS (Thermo Fisher Scientific), 1× non-essential amino acids (Thermo Fisher Scientific), and 50 ug / mL gentamicin. The cells were detached using TrypLE (Gibco) and seeded in a 96-well plate with 20,000 cells per well, and cultured for 48 hours.

[0767] After culturing the cells as described above, the medium was replaced with a nutrient starvation medium containing 0.1% BSA (Sigma) instead of FBS in the culture medium, and the peptide solution and recombinant human Noggin protein (R&D) solution were diluted to various concentrations. After culturing for 30 minutes, 0.2 nM (final concentration) of BMP4 was added and stimulated for 60 minutes. The medium was removed, and the cells were lysed using the lysis buffer attached to the AlphaLISA (registered trademark) SureFire (registered trademark) Ultra SMAD1 (p-ser463 / 465) Assay Kit (PerkinElmer). The operation procedures of the kit were followed, and the SpectraMax (registered trademark) Paradigm (registered trademark) multimode microplate reader (Molecular Devices) was used for the detection of information. The obtained information was analyzed by GraphPad Prism (GraphPad Software), and the inhibition rate was calculated by setting no stimulation as 100% inhibition and BMP stimulation alone as 0% inhibition.

[0768] In addition, the concentrations of the peptides of SEQ ID NO: 5 to 187 were 0.02, 0.2, and 2 nM for the peptides synthesized by the same method as in Example 2 (shown as 2 in the synthesis row), and 0.2, 2, and 20 nM for the peptides synthesized by the same method as in Example 1 (shown as 1 in the synthesis row).

[0769] The evaluation results were evaluated using peptides synthesized by the same method as in Example 2. Those showing an inhibitory activity of 50% or more in the area where the peptide was added in a manner to become 0.2 nM were denoted as A, those showing an inhibitory activity of less than 50% in the 0.2 nM added area and 50% or more in the 2 nM added area were denoted as B or C, and those showing an inhibitory activity of less than 50% in the 2 nM added area were denoted as D.

[0770] In addition, regarding the results of evaluation using the peptides synthesized by the same method as in Example 1, those showing an inhibitory activity of 50% or more in the 0.2 nM addition region are indicated as D, those showing an inhibitory activity of less than 50% in the 0.2 nM addition region and 50% or more in the 2 nM addition region are indicated as E, and those showing an inhibitory activity of less than 50% in the 2 nM addition region and 50% or more in the 20 nM addition region are indicated as F. The evaluation results of each peptide are shown in Table 1.

[0771] The test was performed for SEQ ID NOs: 243 to 321 at the concentrations described in Table 6. As the evaluation results, the peptides were evaluated by indicating an inhibitory activity of 30% or more in the region where the peptide was added at 0.02 nM as A, the inhibitory activity of less than 30% in the region where the peptide was added at 0.02 nM as B, and the inhibitory activity of less than 30% in the region where the peptide was added at 0.2 nM as C.

[0772] Table 6 shows the evaluation results of each peptide.

[0773] For SEQ ID NOs: 322 to 421, the test was performed at the concentrations described in Table 7. First, the evaluation results for BMP4 were expressed as A for peptides showing an inhibitory activity of less than 30% in the 2nM addition region and 30% or more in the 20nM addition region, and B for peptides showing an inhibitory activity of less than 30% in the 20nM addition region and 30% or more in the 200nM addition region. The evaluation results of each peptide are shown in Table 7.

[0774] In addition, the result of ND means that the inhibitory activity is less than 30% at the highest concentration evaluated.

[0775] This result shows that the peptide of the present invention has BMP4 signal transduction inhibitory activity.

[0776] Example 12

[0777] BMP7 signal transduction inhibitory activity evaluation test

[0778] In this example, the BMP7 signal transduction inhibitory activity of each peptide composed of the amino acid sequence described in SEQ ID NOs: 5 to 240 and 243 to 421 synthesized in Example 10 was verified by phosphorylation of SMAD1 caused by BMP7 stimulation.

[0779] The evaluation was performed using 10 nM BMP7 instead of BMP4.

[0780] In addition, for the concentration of the peptides of SEQ ID NO: 5 to 240, as in Example 11, the peptides synthesized by the same method as in Example 2 (denoted as 2 in the synthesis row) were implemented at 1, 10, and 100 nM, and the peptides synthesized by the same method as in Example 1 (denoted as 1 in the synthesis row) were implemented at 10, 100, and 1000 nM.

[0781] For the peptides synthesized in the same manner as in Example 2, the verification using A549 cells was performed in the same manner as in Example 11, except that the peptides were added at molar concentrations of 1 nM, 10 nM, and 100 nM, respectively. As the evaluation results, the peptides showing an inhibitory activity of 50% or more in the region where the peptide was added at 1 nM were indicated as A, the peptides showing an inhibitory activity of less than 50% in the 1 nM addition region and 50% or more in the 10 nM addition region were indicated as B, the peptides showing an inhibitory activity of less than 50% in the 10 nM addition region and 50% or more in the 100 nM addition region were indicated as C, and the peptides showing an inhibitory activity of less than 50% in the 100 nM addition region were indicated as D.

[0782] In addition, the results of the evaluation using the peptides synthesized by the same method as in Example 1 were similarly performed except that the peptides were added at molar concentrations of 10 nM, 100 nM, and 1000 nM, respectively. The evaluation results were expressed as E for those showing 50% or more inhibitory activity in the region added in such a manner as to become 10 nM, F for those showing less than 50% in the 10 nM added region and 50% or more in the 100 nM added region, G for those showing less than 50% in the 100 nM added region and 50% or more in the 1000 nM added region, and H for those showing 30% or more and less than 50% inhibitory activity in the 1000 nM added region. The evaluation results of each peptide are shown in Table 1.

[0783] In addition, the result of ND means that the inhibitory activity is less than 30% at the highest concentration evaluated.

[0784] The test was performed for SEQ ID NOs: 243 to 273 and SEQ ID NOs: 299 to 321 at the concentrations (10, 100, 1000 nM) listed in Table 6. The evaluation results were expressed as D for those showing an inhibitory activity of less than 30% in the region where the peptide was added at 10 nM and 30% or more in the region where the peptide was added at 100 nM, and E for those showing an inhibitory activity of less than 30% in the region where the peptide was added at 100 nM and 30% or more in the region where the peptide was added at 1000 nM.

[0785] In addition, the test was carried out with the concentrations described in Table 6 (100, 300, 1000 nM) for SEQ ID NOs: 274 to 298.

[0786] Those showing 30% or more inhibitory activity in the 100 nM addition region were indicated as F, those showing less than 30% in the 100 nM addition region and 30% or more inhibitory activity in the 300 nM addition region were indicated as G, and those showing less than 30% in the 300 nM addition region and 30% or more inhibitory activity in the 1000 nM addition region were indicated as H. The evaluation results of each peptide are shown in Table 6.

[0787] In addition, the result of ND means that the inhibitory activity is less than 30% at the highest concentration evaluated.

[0788] The test was conducted at concentrations (1, 10, 100 nm) for SEQ ID NOs: 322 to 370 and SEQ ID NOs: 393 to 421. The evaluation results were expressed as C for those showing an inhibitory activity of less than 30% in the region where the peptide was added at 1 nM and 30% or more in the region where the peptide was added at 10 nM, and D for those showing an inhibitory activity of less than 30% in the region where the peptide was added at 10 nM and 30% or more in the region where the peptide was added at 100 nM.

[0789] In addition, the test was conducted at concentrations (10, 30, and 90 nM) for SEQ ID NOs: 371 to 391. As a result of the evaluation, those showing an inhibitory activity of 30% or more in the 10 nM addition region were indicated as E, those showing an inhibitory activity of less than 30% in the 10 nM addition region and 30% or more in the 30 nM addition region were indicated as F, and those showing an inhibitory activity of less than 30% in the 30 nM addition region and 30% or more in the 90 nM addition region were indicated as G.

[0790] Table 7 shows the evaluation results of each peptide.

[0791] This result shows that the peptides of the present invention include those having both BMP4 and BMP7 signal transduction inhibitory activity or those having BMP7 signal transduction inhibitory activity.

[0792] Embodiment 13

[0793] IC50 values ​​for BMP4 and BMP7

[0794] In this example, the BMP4 and BMP7 signal transduction inhibitory peptides synthesized in Examples 1 to 10 were used to calculate the BMP4 and BMP7 signal transduction inhibitory activity (IC50).

[0795] The evaluation test of the signal transduction inhibitory activity of SEQ ID NO: 87, 177 and 181 was carried out in the same manner as in Example 11 using A549 cells, except that the peptide concentration was changed from 2 nM to 8 points at a 1 / 3 ratio, and the evaluation of the inhibitory activity against BMP7 was carried out in the same manner as in Example 12, except that the peptide concentration was changed from 100 nM to 8 points at a 1 / 3 ratio. In addition, as a positive control group, recombinant human Noggin protein (R&D Co.) was used at the same concentration as the peptide.

[0796] In addition, for SEQ ID NO: 277 (Example 8), the evaluation of BMP4 inhibitory activity was carried out in the same manner as in Example 11 at 8 points with the peptide concentration increasing from 20 nM in a 1 / 3 ratio, and the evaluation of BMP7 inhibitory activity was carried out in the same manner as in Example 12 at 8 points with the peptide concentration increasing from 1000 nM in a 1 / 3 ratio.

[0797] In addition, for SEQ ID NO: 417 (Example 6), the evaluation of BMP4 inhibitory activity was carried out in the same manner as in Example 11 at 8 points with the peptide concentration increased from 300 nM to 1 / 4, and the evaluation of BMP7 inhibitory activity was carried out in the same manner as in Example 12 at 8 points with the peptide concentration increased from 300 nM to 1 / 4.

[0798] The results will be published in Figure 1 , Table 2-1 and Table 2-2.

[0799] In addition, the result "ND" means that IC50 was not calculated in this evaluation system.

[0800] [Table 2-1]

[0801]

[0802] [Table 2-2]

[0803]

[0804] The results in Table 2-1 show that BMP4 signaling inhibitory peptides No. 87, No. 177, and No. 181 have the same degree of BMP4 inhibitory activity as Noggin. In addition, it is shown that BMP4 signaling inhibitory peptides No. 177 and No. 181 have the same degree of BMP7 inhibitory activity as Noggin. In addition, it is shown that No. 87 does not inhibit the signaling of BMP7.

[0805] The results in Table 2-2 show that the BMP4 signaling inhibitory peptide SEQ ID NO: 277 (Example 8) has the same degree of BMP4 inhibitory activity as Noggin. In addition, the BMP7 signaling inhibitory peptide SEQ ID NO: 417 (Example 6) has the same degree of BMP7 inhibitory activity as Noggin, but does not inhibit BMP4 signaling.

[0806] Embodiment 14

[0807] Other BMP family signal transduction inhibitory activity evaluation assay

[0808] In this example, the BMP4 information transmission inhibitory peptide No. 177, peptide No. 29986 (SEQ ID NO: 277 (Example 8)), peptide SEQ ID NO: 417 (Example 6) synthesized by Examples 2, 8, and 6, and the presence or absence of information transmission inhibitory activity caused by BMP2, 5, 6, 9, 12, and 14 belonging to each BMP family were verified in the same manner as in Example 13.

[0809] In addition, regarding the concentration of each BMP under evaluation, BMP2 was added and stimulated at 0.3 nM, BMP5 was added and stimulated at 2 nM, BMP6 was added and stimulated at 3 nM, BMP9 and BMP12 were added and stimulated at 10 nM, and BMP14 was added and stimulated at 0.6 nM.

[0810] In addition, for No. 177, the concentration of the peptide was 10 nM for BMP2, 200 nM for BMP5, 100 nM for BMP6, 1000 nM for BMP9 and BMP12, and 60 nM for BMP14, and the concentrations were respectively 1 / 3 times the common ratio at 8 points. Except for the above points, the evaluation was carried out in the same manner as in Example 11. In addition, as a positive control group, recombinant human Noggin protein (R&D Co.) was used at the same concentration as the peptide.

[0811] Furthermore, for peptide SEQ ID NO: 277 (Example 8), the concentration of the peptide was 30 nM for BMP2, 200 nM for BMP5, 300 nM for BMP6, 1000 nM for BMP12, and 60 nM for BMP14, and the concentrations were respectively increased at 8 points with a 1 / 3 ratio.

[0812] In addition, for peptide SEQ ID NO: 417 (Example 6), the concentration of the peptide was 300 nM for BMP2, 200 nM for BMP5, 300 nM for BMP6, 1000 nM for BMP12, and 60 nM for BMP14, and the concentrations were respectively increased at 8 points with a 1 / 3 ratio.

[0813] The IC50 of each BMP family was calculated from the obtained measured values, and the results are shown in Table 3.

[0814] In addition, in this evaluation system, the result of ND means that IC50 was not calculated, and the result of NT means that the test was not carried out.

[0815] In addition, the inhibition curve of BMP4 signal transduction inhibitory peptide SEQ ID NO: No.177 is recorded in Figure 2 The triangle in the figure represents BMP4 signal transduction inhibitory peptide No. 177, and the quadrangle represents Noggin. The results show that BMP4 signal transduction inhibitory peptide No. 177 has signal transduction inhibitory activity not only against BMP4 and 7 but also against BMP2, 5, and 6.

[0816] [Table 3]

[0817]

[0818] Embodiment 15

[0819] Evaluation of the molecular interaction between BMP4 / BMP7 and peptides by surface plasmon resonance (SPR)

[0820] In this example, the intermolecular interaction of the peptides synthesized in Examples 2 and 3 with respect to BMP4 / BMP7 was tested by the method described below using surface plasmon resonance (SPR). The specific test method is described below.

[0821] [SPR measurement]

[0822] Insert a CM5 sensor chip (Cytiva) into BiacoreT200 (Cytiva), perform three prime operations with electrophoresis buffer: HBS-EP+ (Cytiva), and perform equilibrium at a flow rate of 30μL / min. Mix 50μL of 60mM EDC solution (Cytiva) and 650mM NHS solution (Cytiva), respectively, and react at a flow rate of 10μL / min for 420 seconds. Dilute with 10mM acetic acid solution (pH5.0) to prepare 150μL of 0.2μM BMP4 / BMP7 solution, react at a flow rate of 10μL / min for 420 seconds, and immobilize BMP4 / BMP7 on the CM5 sensor chip. After immobilization, react with 1.0M ethanolamine aqueous solution (Cytiva) at a flow rate of 10μL / min for 420 seconds for capping. The peptide solution prepared at 10 mM in DMSO was diluted with electrophoresis buffer to prepare peptide solutions at a final concentration of 10 μM. The peptide solutions were 100 nM, 50 nM, 25 nM, 10 nM, and 5 nM. The peptide kinetics for BMP4 / BMP7 were measured by SPR using the above samples.

[0823] The kinetic evaluation model was set to Single Cycle Kinetics and curve fitting was performed using Biacore T200 Evaluation Software Version 3.0 (Cytiva). For the obtained sensorgram, curve fitting was performed using the least square method to obtain the KD value, thereby evaluating the binding of the peptide to BMP4 / BMP7. The obtained results are shown in Table 4.

[0824] [Table 4]

[0825]

[0826] Example 16

[0827] Synthesis of various peptides

[0828] The peptides prepared in Example 10 are shown in Tables 5, 6 and 7.

[0829] Table 5

[0830] [Table 5-1]

[0831]

[0832] [Table 5-2]

[0833]

[0834] [Table 5-3]

[0835]

[0836] [Table 5-4]

[0837]

[0838] Table 6

[0839] [Table 6-1-1]

[0840]

[0841] [Table 6-1-2]

[0842]

[0843] [Table 6-1-3]

[0844]

[0845] [Table 6-2-1]

[0846]

[0847] [Table 6-2-2]

[0848]

[0849] [Table 6-2-3]

[0850]

[0851] [Table 6-3-1]

[0852]

[0853] [Table 6-3-2]

[0854]

[0855] [Table 6-3-3]

[0856]

[0857] [Table 6-4-1]

[0858]

[0859] [Table 6-4-2]

[0860]

[0861] [Table 6-4-3]

[0862]

[0863] Table 7

[0864] [Table 7-1-1]

[0865]

[0866] [Table 7-1-2]

[0867]

[0868] [Table 7-1-3]

[0869]

[0870] [Table 7-2-1]

[0871]

[0872] [Table 7-2-2]

[0873]

[0874] [Table 7-2-3]

[0875]

[0876] [Table 7-3-1]

[0877]

[0878] [Table 7-3-2]

[0879]

[0880] [Table 7-3-3]

[0881]

[0882] [Table 7-4-1]

[0883]

[0884] [Table 7-4-2]

[0885]

[0886] [Table 7-4-3]

[0887]

[0888] Embodiment 17

[0889] Synthesis of (2S)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-{3-fluoro-[1,1'-biphenyl]-4-yl}propanoic acid (pBph3F)

[0890] In this example, amino acids having the following structures used in the present invention were synthesized.

[0891] [Chemistry 10]

[0892]

[0893] Under nitrogen atmosphere, sodium carbonate (10.6 g, 54.9 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane adduct (2.04 g, 5.50 mmol) were added to a mixed solution of 1-bromo-2-fluoro-4-iodobenzene (15.0 g, 49.9 mmol) and phenylboronic acid in toluene (200 mL), water (200 mL), and ethanol (50 mL). The mixture was stirred at 110°C for 16 hours and then extracted three times with ethyl acetate (100 mL). The combined organic extracts were washed three times with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The obtained residue was purified by silica gel chromatography (ethyl acetate: petroleum ether = 1:25).

[0894] Under nitrogen atmosphere, methyl (2R)-2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)-3-iodopropionate (17.3 g, 38.2 mmol) and iodine (2.44 g, 9.61 mmol) were added to a suspension of zinc (6.25 g, 95.6 mmol) in DMF (200 mL), and the mixture was stirred at room temperature for 30 minutes. To this reaction solution were added a portion of the product obtained above (8.00 g, 31.9 mmol), tris((1E,4E)-1,5-diphenylpenta-1,4-dien-3-one)dipalladium (0.730 g, 0.797 mmol), and dicyclohexyl({2',6'-dimethoxy-[1,1'-biphenyl]-2-yl})phosphane (0.640 g, 1.56 mmol), and the mixture was stirred at 50°C for about 16 hours. The solid was filtered off, 600 mL of water was added to the filtrate to stop the reaction, and then extracted three times with ethyl acetate (200 mL). The combined organic extracts were washed three times with saturated brine (200 mL), dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The obtained residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:3).

[0895] To a mixed solution of the obtained product (12 g, 24.4 mmol) in 2-propanol (120 mL), water (40 mL), and THF (40 mL), calcium chloride (43.0 g, 387 mmol) was added, and the mixture was stirred at 0°C for 10 minutes. Lithium hydroxide monohydrate (4.06 g, 96.8 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for about 16 hours. After adjusting the reaction solution to pH 7 using an aqueous citric acid solution, the solid was filtered out and washed with methanol: dichloromethane = 1 / 10 (200 mL). The filtrate was combined with the washing solution, the organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The obtained residue was washed with diethyl ether and dried under reduced pressure to obtain the title compound. 1 H NMR (500MHz, DMSO-d6) 87.88 (d, J=7.5Hz, 2H), 7.69-7.53 (m, 4H), 7.48-7.21 (m, 11H), 6.76- 6.14 (m, 1H), 4.25-3.85 (m, 4H), 3.31-3.11 (m, 1H), 2.88-2.66 (m, 1H). LC-MS (ESI, m / z): [MH] - 480.

[0896] In the event of any discrepancy between the Table in this specification and the sequence listing, the description in the Table shall prevail.

[0897] Industrial Availability

[0898] This invention can be used in the pharmaceutical industry and the bioengineering industry.

Claims

1. A peptide or a pharmaceutically acceptable salt thereof, comprising the following amino acid sequence: ERV-F4COO-Atp-DR-Bph-PYP-Bph-Y-F4COO-F4COO-SC (SEQ ID NO: 1), Or an amino acid sequence having a substitution, addition, deletion or insertion in 1 to 15 amino acid residues selected from the group consisting of the 1st, 2nd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th and 16th amino acid residues of the above amino acid sequence.

2. A peptide or a pharmaceutically acceptable salt thereof having the following amino acid sequence: A peptide having an amino acid sequence represented by X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17 (SEQ ID NO: 2) or a pharmaceutically acceptable salt thereof, or a peptide having an amino acid sequence represented by substitution, deletion, addition or insertion of 1 to 10 amino acids from the amino acid sequence represented by SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof, X1 is E, A, T, Q or L, X2 is R, K, Hgl, any polar amino acid or any aliphatic amino acid, X3 is V, X4 is an amino acid having an aromatic ring which may be a heterocyclic ring in the side chain, X5 is any aliphatic amino acid, Y, D, Atp, Cha4tH, Gthp or A1Ac4pip, X6 is D, any basic amino acid or any polar amino acid, X7 is any basic amino acid or any polar amino acid, X8 is biphenylalanine which may have a substituent and the C on the aromatic ring may be substituted with N, X9 is any amino acid, X10 is Y, Hty or 4Py, X11 is any amino acid, X12 is biphenylalanine which may have a substituent and the C on the aromatic ring may be substituted with N, X13 is unsubstituted or substituted F, 4Py or 3Py6NH2, X14 is F4COO, D or G, X15 is F4COO, Y, 4Py, 3Py6NH2, W7N, Hph or N, X16 is any amino acid, X17 is C.

3. The peptide or pharmaceutically acceptable salt thereof according to claim 3, wherein: X1 is E, X2 is R or any polar amino acid, X3 is V, X4 is unsubstituted or substituted Y or F4COO, X5 is V or Atp, X6 is D or KCOpipzaa, X7 is R, X8 is Bph, X9 is P or S, X10 is Y or Hty, X11 is any aliphatic amino acid, X12 is unsubstituted or substituted Bph, X13 is unsubstituted or substituted F, X14 is F4COO, X15 is F4COO or 4Py, X16 is any amino acid, X17 is C.

4. The peptide or a pharmaceutically acceptable salt thereof according to claim 2, wherein: X1 is E, A, T, Q or L, X2 is R or KCOpipzaa, X3 is V, X4 is Y, F4F, F4COO, 4Py, H or F4aaO, X5 is any aliphatic amino acid, Atp, Cha4tH, Gthp or A1Ac4pip, X6 is D, S or KCOpipzaa, X7 is R or K, X8 is 3Py6Ph, Bph, Bph4C, pBph2F or Bph3C, X9 is S, P, KCOpipzaa or H, X10 is Y or Hty, X11 is any aliphatic amino acid or Hyp, X12 is biphenylalanine which may have a substituent, X13 is unsubstituted or substituted F or 4Py, X14 is F4COO, X15 is F4COO or 4Py, X16 is S, X17 is C.

5. The peptide or pharmaceutically acceptable salt thereof according to claim 2, which has the following amino acid sequence: a peptide having an amino acid sequence as shown in X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-C (SEQ ID NO: 4), or a peptide having an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, added or inserted from the amino acid sequence as shown in SEQ ID NO: 4, X1 is A, X2 is R, KAc, Q or KCOpipzaa, X3 is V, X4 is Y, F4F or 4Py, X5 is V, X6 is D, KCOpipzaa, K, R, G or H, X7 is R, K, KAc, 4Py, S or Cit, X8 is 3Py6Ph or Bph, X9 is P, S, K, H, G, S or KCOpipzaa, X10 is Y, X11 is any aliphatic amino acid, X12 is Bph or 3Py6Ph, X13 is Y or F4F, X14 is F4COO, X15 is F4COO or 4Py, X16 is S. 6 . The peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 , which has the ability to bind to BMP4. 7 . The peptide or a pharmaceutically acceptable salt thereof according to claim 1 , which has an inhibitory activity against BMP4.

8. The peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, which is a peptide consisting of an amino acid sequence described in any one of SEQ ID NOs: 5 to 187 and SEQ ID NOs: 243 to 421, or a pharmaceutically acceptable salt thereof, Or a peptide or a pharmaceutically acceptable salt thereof having an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, added or inserted into the amino acid sequence described in any one of SEQ ID NOs: 5 to 187 and SEQ ID NOs: 243 to 421 and having the ability to bind to BMP4 or the activity to inhibit BMP4.

9. A peptide or a pharmaceutically acceptable salt thereof, comprising the following amino acid sequence: FG-MeF-G-Bph-GDD-Cha-A-MeF-LHC (SEQ ID NO: 241), Or an amino acid sequence having a substitution, addition, deletion or insertion in 1 to 12 amino acid residues selected from the group consisting of the 1st, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th and 13th amino acid residues of the above amino acid sequence.

10. A peptide or a pharmaceutically acceptable salt thereof having the following amino acid sequence: a peptide having the amino acid sequence shown in X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 (SEQ ID NO: 242), or a peptide having an amino acid sequence in which 1 to 12 amino acids are substituted, deleted, added or inserted from the amino acid sequence shown in SEQ ID NO: 245, or a pharmaceutically acceptable salt thereof, X1 is phenylalanine which may have a substituent and the C on the aromatic ring may be substituted with N, X2 is G, X3 is N-methylphenylalanine which may have a substituent, X4 is G, which may be a D-type aliphatic amino acid or a polar amino acid, X5 is biphenylalanine in which the C on the aromatic ring may be substituted with N or phenylalanine which may have a substituent, X6 is G or A of D type, X7 is any polar amino acid, X8 is any acidic amino acid, X9 is any aliphatic amino acid, X10 is any amino acid, X11 is N-methylphenylalanine which may have a substituent, X12 is any aliphatic amino acid, X13 is any amino acid, X14 is C. The peptide or a pharmaceutically acceptable salt thereof according to claim 9 or 10, which has an inhibitory activity against BMP7.

12. The peptide or pharmaceutically acceptable salt thereof according to claim 9 or 10, which is a peptide consisting of the amino acid sequence described in any one of SEQ ID NOs: 243 to 421. Or a peptide having an amino acid sequence in which 1 to 10 amino acids are substituted, deleted, added or inserted into the amino acid sequence described in any one of SEQ ID NOs: 243 to 421 and having BMP7 inhibitory activity, or a pharmaceutically acceptable salt thereof. 13 . The peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12 , which is a cyclic peptide.

14. The peptide or a pharmaceutically acceptable salt thereof according to claim 13, wherein It has a ring structure formed by combining a chloroacetylated amino acid with a cysteine ​​residue contained in the above-mentioned peptide.

15. The peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 14, further comprising an additive amino acid residue sequence. 16 . A BMP4 and / or BMP7 signal transduction inhibitor comprising the peptide according to claim 1 or a pharmaceutically acceptable salt thereof. 17 . A human stem cell differentiation induction controlling agent comprising the peptide according to claim 1 or a pharmaceutically acceptable salt thereof.

18. A culture medium additive for culturing human stem cells, comprising one or more of the peptide according to any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof, the BMP4 and / or BMP7 signal transduction inhibitor according to claim 16, and the human stem cell differentiation induction controller according to claim 17.

19. A method for inhibiting the signal transduction activity of BMP4 and / or BMP7 using the peptide according to any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof.

20. A method for controlling differentiation induction of human stem cells using the peptide according to any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof.

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