Vipr2 antagonistic peptides

Abstract

The present application provides a novel VIPR2 antagonistic peptide for use as a pharmaceutical, a diagnostic and / or a research reagent. A cyclic peptide consisting of an amino acid sequence represented by formula (1) or formula (2) or a pharmacologically acceptable salt thereof: c[X N -X 4 -X 5 -X 6 -c(X 7 -X 8 -X 9 -X 10 ] -X 11 ) -X 12 -X 13 -X 14 -X 15 -X 16 (1); c[X N -X 4 -X 5 -X 6 -c(X 7 -X 8 -X 9 -X 10 ] -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 (2).

Description

[0001] Cross-references

[0002] This application claims priority based on Japanese Patent Application No. 2020-059721, filed on March 30, 2020, the entire contents of which are incorporated herein by reference. Furthermore, all patents, patent applications, and documents cited in this application are incorporated herein by reference in their entirety. Technical Field

[0003] This invention relates to peptides that inhibit the activity of VIPR2, and more specifically, to cyclic peptides having specific amino acid sequences and cyclic structures. Background Technology

[0004] Vasoactive intestinal peptide receptor 2 (VIPR2), also known as VPAC2, is a class B G protein conjugate receptor (GPCR). It is expressed throughout the body, including the central nervous system, peripheral nerves, sensory organs, digestive organs, and reproductive organs. It exhibits various physiological functions through interactions with its ligands, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). For example, signaling via VIPR2 or VIP or PACAP, which are its receptor subtypes, is known to have neuroprotective effects (Non-Patent Literature 1 and Non-Patent Literature 2). On the other hand, interestingly, recent reports suggest that high expression and overactivation of VIPR2 may be associated with the pathogenesis of schizophrenia and autism spectrum disorders (Non-Patent Literature 3, Non-Patent Literature 4, and Non-Patent Literature 5). Furthermore, reports indicate that blocking VIP signaling can inhibit cancer proliferation and activate immunity (Non-Patent Literature 6 and Non-Patent Literature 7).

[0005] In 2011, Vacic et al. reported that some patients with integration disorder and some with autism spectrum disorder had duplications in the gene region encoding VIPR2, and that the mRNA expression level of VIPR2 in their lymphocytes was increased, leading to hypersensitivity to VIP (Non-Patent Literature 3). Furthermore, in 2015, Ago et al. reported that administering the VIPR2-selective agonist Ro25-1553 to newborn mice artificially induced a state of VIPR2 hyperactivation, resulting in mature individuals exhibiting reduced cognitive function (Non-Patent Literature 4). Subsequently, in 2019, Tian et al. reported the creation of transgenic mice with duplications in the gene region encoding VIPR2, which exhibited symptoms such as dopamine dysfunction, cognitive impairment, and social behavioral disorders (Non-Patent Literature 5). These insights strongly suggest that excessive VIPR2 signal transduction is associated with the pathogenesis of mental illnesses such as schizophrenia and autism spectrum disorder.

[0006] It is estimated that 24 million people worldwide suffer from schizophrenia. Schizophrenia is a mental illness that causes positive symptoms (hallucinations, delusions, etc.), negative symptoms (decreased libido, etc.), and cognitive impairment, leading not only to reduced social functioning but also a high suicide rate. Its heritability is as high as 80%, but the specific pathogenic gene shared by all patients has not yet been identified, and there is hope for the development of new drugs and treatments. It is estimated that approximately 1-2 out of every 1,000 people worldwide suffer from autism spectrum disorder, which causes social impairment and difficulty in communication. It is primarily caused by congenital factors, and no clear prevention or treatment methods have yet been established.

[0007] As mentioned above, some patients with schizophrenia and some patients with autism spectrum disorder have been reported to overexpress VIPR2, suggesting that overactivation of VIPR2 may be associated with the pathogenesis of these conditions. That is, given the confirmed duplication of the gene region encoding VIPR2 through genomic analysis, the administration of VIPR2 inhibitors is expected to be effective as a means of preventing and / or treating the onset of schizophrenia and autism spectrum disorder.

[0008] In 2018, the inventors reported the discovery of a cyclic antagonistic peptide VIPep-3 that binds to the extracellular region of VIPR2 and inhibits VIP and PACAP signaling via the VIPR2 signaling pathway in cell assays: Ac-c(Cys-Pro-Pro-Tyr-Leu-Pro-Arg-Arg-Leu-Cys)-Thr-Leu-Leu-Arg-Ser-OH (Serial No. 11) (Non-Patent Literature 8).

[0009] Existing technical documents

[0010] Non-patent literature

[0011] Non-patent literature 1: Vaudry D, Falluel-Morel A, Bourgault S, Basille M, Burel D, Wurtz O, Fournier A, Chow BK, Hashimoto H, Galas L, and Vaudry H. PharmacolRev.2009, 61(3):283-357.

[0012] Non-patent literature 2: Passemard S, Sokolowska P, Schwendimann L, and Gressens P. Curr Pharm Des. 2011, 17(10): 1036-1039.

[0013] Non-patent literature 3: Vacic V, McCarthy S, Malhotra D, Murray F, Chou HH, Peoples A, Makarov V, Yoon S, Bhandari A, Corominas R, Iakocheva LM, Krastoshevsky O, KrauseV, Larach-Walters V, Welsh DK, Craig D, Kelsoe JR, Gershon ES, Leal SM, Dell AquilaM, Morris DW, Gill M, Corvin A, Insel PA, McClellan J, King MC, Karayiorgou M, LevyDL, DeLisi LE, and Sebat J. Nature. 2011, 471(7339):499-503.

[0014] Non-patent literature 4: Ago Y, Condro MC, Tan YV, Ghiani CA, Colwell CS, Cushman JD, Fanselow MS, Hashimoto H, and Waschek JA. Psychopharmacology (Berl). 2015, 232(12):2181-2189.

[0015] Non-patent literature 5: Tian X, Richard A, EI-Saadi MW, Bhandari A, Latimer B, VanSavage I, Holmes K, Klein RL, Dwyer D, Goeders NE, Yang XW, and Lu

[0016] Non-patent literature 6: Moody TW, Chan D, Fahrenkrug J, and Jensen RT. Curr PharmDes. 2003, 9(6):495-509.

[0017] Non-patent literature 7: Li JM, Petersen CT, Li JX, Panjwani R, Chandra DJ, Giver CR, Blazar BR, and Waller EK. Cancer Res. 2016, 76(23):6802-6815.

[0018] Non-patent literature 8: Sakamoto K, Koyama R, Kamada Y, Miwa M, and Tani A. Biochem Biophys Res Commun. 2018, 503: 1973-1979.

[0019] Non-patent literature 9: Fairlie DP and Dantas de Araujo. Biopolymers 2016, 106(6):843-852.

[0020] Non-patent literature 10: Lau YH, de Andrade P, Wu Y, and Spring DR. Chem Soc Rev. 2015, 44(1)91-102.

[0021] Non-patent literature 11: Chen S, Gopalakrishnan R, Schaer T, Marger F, Hovius R, Bertrand D, Pojer F, and Heinis C. Nat Chem 2014, 6(11)1009-1016.

[0022] Non-patent literature 12: Hara Y, Ago Y, Taruta A, Katashiba K, Hasebe S, Takano E, Onaka Y, Hashimoto H, Matsuda T, and Takuma K. Autism Res. 2016, 9(9)926-939. Summary of the Invention

[0023] The technical problem that the invention aims to solve

[0024] The inventors discovered that the above-mentioned VIPEP-3 is a cyclic antagonistic peptide that binds firmly to the extracellular domain of human VIPR2 and blocks the VIP-VIPR2 signaling pathway. However, since it is composed of only natural amino acids, there are concerns about its low resistance to protease hydrolysis.

[0025] The present invention was made in view of the above-mentioned problems, and its object is to provide a novel VIPR2 antagonistic peptide suitable for use as a pharmaceutical, diagnostic and / or research reagent.

[0026] Means for solving technical problems

[0027] During the optimization study of VIPEP-3, the inventors discovered a novel VIPR2 antagonistic peptide sequence. To address the aforementioned issues, they investigated (1) amino acid residue substitution, (2) reducing the molecular weight by deleting N-terminal and / or C-terminal amino acid residues, and (3) introducing a bicyclic structure. As a result, they discovered a novel VIPR2 antagonistic peptide sequence with protease resistance and VIPR2 antagonistic activity, thus completing this invention.

[0028] That is, the present invention includes the following embodiments.

[0029] [1] A cyclic peptide consisting of an amino acid sequence of formula (1) or formula (2) or a pharmacologically acceptable salt thereof.

[0030] c[X N -X 4 -X 5 -X 6 -c(X 7 -X 8 -X 9 -X 10 ]-X 11 )-X 12 -X 13 -X 14 -X 15 -X 16 (1)

[0031] c[X N -X4 -X 5 -X 6 -c(X 7 -X 8 -X 9 -X 10 )]-X 11 -X 12 -X 13 -X 14 -X 15 -X 16 (2)

[0032] Here, X in equation (1) N With X 10 Between, X 7 With X 11 Between, and X in equation (2) N With X 7 With X 10 Each chain independently forms a covalent bond, resulting in two ring structures within the molecule. These covalent bonds can be direct covalent bonds between the main chain and side chains or between side chains, or indirect covalent bonds formed through a linker.

[0033] In equations (1) and (2) above, X N For X 1 ~X 2 ~X 3 X 1 X 2 X 3 X 7 X 10 and X 11 When involved in intramolecular cyclization of the peptide molecule, each group independently represents an amino, carboxyl, thiol, allyl, alkynyl, azide, amino acid residue with a halogen atom, or their derivatives; when not involved in intramolecular cyclization of the peptide molecule, X 1 and X 2 Each can independently represent any amino acid residue or deletion, X 3 and X 11 Each can be any amino acid residue independently. Additionally, X 5 X 9 X 12 and X 13 Each independently represents an amino acid residue containing a hydrocarbon group that may have substituents, or a derivative thereof. 4 X represents an amino acid residue containing an aromatic carbocyclic group that may have substituents, or a derivative thereof. 6 and X 8 X represents any amino acid residue. 14 X15 and X 16 Each can independently represent any amino acid residue or deletion; the N-terminal amino group and the C-terminal carboxyl group can be modified or deleted.

[0034] The following describes preferred or other embodiments of the present invention, and X in the above-described cyclic peptide. 1 To X 16 The preferred methods for each amino acid residue are described in detail, but each preferred method is independent of the others, and each embodiment can be combined arbitrarily.

[0035] Invention Effects

[0036] According to the present invention, novel cyclic peptides for using VIPR2 antagonists as pharmaceuticals, diagnostic agents and / or research reagents can be provided. Attached Figure Description

[0037] Figure 1 The amino acid sequence of VIPEP-3 associated with its VIPR2 binding activity (Farmacora) and cyclic restriction (SS bond) is shown. Additionally, a structural outline of the peptide of the present invention, derived from VIPEP-3, is shown.

[0038] Figure 2 Examples of amino acids for amino acid residue substitution studies and positions of amino acid residues for bicyclization studies are shown at the positions of each amino acid in VIPEP-3.

[0039] Figure 3 This diagram illustrates a competitive binding assay based on cell ELISA used to confirm the binding activity of peptides derived from substituted amino acid residues to VIPR2. In the figure, SA represents streptavidin, B represents biotin, and HRP represents horseradish peroxidase.

[0040] Figure 4 The results show the comparison of the VIPR2 binding activity of peptide sequences provided for amino acid residue substitution studies and representative examples of peptides of the present invention with the VIPR2 binding activity of the parent peptide VIPep-3.

[0041] Figure 5 The results show the addition of representative examples of the peptides of the present invention and VIP to VIPR2-expressing cells, and the evaluation of changes in intracellular calcium concentration, which is one of the downstream signals of VIPR2.

[0042] Figure 6 The results of mixing representative examples of Vipep-3 and the peptides of the present invention with rat plasma and comparing resistance to protease degradation are shown.

[0043] Figure 7The results show the addition of representative examples of the peptides of the present invention and VIP to VIPR1-expressing cells and the evaluation of changes in intracellular calcium concentration as one of the downstream signals of VIPR1; the addition of representative examples of the peptides of the present invention and VIP to VIPR2-expressing cells and the evaluation of changes in intracellular calcium concentration as one of the downstream signals of VIPR2; and the addition of representative examples of the peptides of the present invention and PACAP to PAC1-expressing cells and the evaluation of changes in intracellular calcium concentration as one of the downstream signals of PAC1.

[0044] Figure 8 The results show the phosphorylation of intracellular circulating AMP response element binding protein (CREB) (one of the downstream signals of VIPR2) in the prefrontal cortex of ICR mice (12 days old) after subcutaneous administration of Ro25-1553 (represented as “Vehicle” in the figure), a selective VIPR2 agonist as a VIP analog, or a representative example of the peptide of the present invention, to Ro25-15532 (represented as peptide concentrations of 1 nmol / g or 10 nmol / g in the figure).

[0045] Figure 9 (A) shows the evaluation results of the cognitive function of mice given Ro25-1553, a selective agonist of VIPR2, which is an analog of VIP, in terms of novel objects. Figure 9 (B) shows the evaluation results of the cognitive function of mice given Ro25-1553 alone or given a mixture of a representative example of the peptide of the present invention and Ro25-15532.

[0046] Figure 10 The structural formulas of representative examples of the peptides of the present invention used in the embodiments are shown. Detailed Implementation

[0047] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings. Furthermore, the embodiments described below do not limit the invention as defined in the claims, and not all elements and combinations thereof described in the embodiments are essential to the solutions of the present invention. All patent and non-patent disclosures referenced herein are incorporated herein by reference in their entirety.

[0048] (definition)

[0049] In this specification, a peptide refers to a substance composed of two or more amino acids bonded together by an amide bond (peptide bond), for example, a substance composed of 2 to 20 amino acids bonded together by an amide bond. Furthermore, according to the conventions for describing peptides, the left end is the N-terminus (amino terminus), and the right end is the C-terminus (carboxyl terminus). The first carbon atom adjacent to the carbonyl group forming the peptide bond is called the Cα carbon.

[0050] In this specification, "any amino acid or derivative thereof" is used in its broadest sense, including not only natural amino acids, but also artificial amino acids having non-natural structures, chemically synthesized compounds having the characteristic properties of amino acids known in the art, and carboxylic acids having functional groups. Examples of non-natural amino acids include D-type amino acids, α / α-disubstituted amino acids with a main chain structure different from the natural type (such as α-methylated amino acids like 2-aminoisobutyric acid), N-alkyl-amino acids (such as N-methylated amino acids), N-substituted glycine (peptide-like amino acids), amino acids with extended main chains (β-high amino acids, γ-high amino acids), amino acids with side chain structures different from the natural type (such as cyclohexylalanine, allylglycine, 2-(2-pyridyl)-glycine, 3-(1H-benzimidazol-2-yl)-alanine), amino acids with a partially substituted side chain (such as ortholeucine, diaminopropionic acid, 3-(2-pyridyl)-alanine), amino acids with redundant functional groups in the side chain; amino acids with redundant C, alkyl, or methyl groups in the side chain (such as high-ortholeucine, γ-methylleucine), and amino acids with halogen atoms (F, Cl, Br, I) in the side chain. Amino acids with halogen atoms (F, Cl, Br, I) in their side chains (such as 3-chloro-alanine), carboxylic acids with functional groups in their side chains (such as 3-butenoic acid), amino acids with extra N or amino groups in their side chains (such as β-azidoalanine and ornithine), amino acids with extra O or methoxy groups in their side chains (such as O-methyl-serine and O-methyl-threonine), amino acids with extra hydroxyl groups in their side chains (such as 3-hydroxy-phenylalanine), amino acids with extra carboxyl groups (-COOH) in their side chains (such as 3-carboxy-phenylalanine), amino acids with extra S groups in their side chains (such as ethionine), amino acids whose carboxylic acid functional groups in their side chains are protected by esters (such as aspartic acid-4-methyl ester), and amino acids whose sulfhydryl groups (-S-) in their side chains are oxidized to sulfinyl groups (-S(=O)-) or sulfonyl groups (-S(=O)2-) (such as methionine sulfoxide), etc., but not limited to these.

[0051] In this specification, examples of hydrocarbon groups that may have substituents include: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 2-ethylbutyl, etc. 1-10Alkyl, vinyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 4-methyl-3-pentenyl, 1-hexenyl, 3-hexenyl, 5-hexenyl, etc. 2-10 Alkenyl, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 4-methyl-2-pentynyl, etc. (C) 2-10 Alkyne, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclic [2.2.1]octyl, bicyclic [2.2.2]octyl, bicyclic [3.2.1]octyl, adamantyl, etc. (C) 3-10 Cycloalkyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, etc. C 3-10 Cycloalkenyl groups, which may have substituents, are aromatic carbocyclic groups such as phenyl and naphthyl. Aromatic heterocyclic groups that may have substituents include pyridinyl, thiopheneyl, furanyl, pyrroloyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, triazolyl, tetrazolyl, triazinyl, etc., which are 5- to 6-membered monocyclic aromatic heterocyclic groups. Further, they include benzofuranyl, benzothiopheneyl, benzimidazolyl, benzoxazolyl, benzoisoxazolyl, benzothiazolyl, benzoisothiazolyl, benzotriazolyl, imidazopyridyl, thiophene-pyridyl, etc. The aromatic heterocyclic groups of 8 to 14 members, including pyridyl, furanopyridyl, pyrrolopyridyl, pyrazolopyridyl, oxazolopyridyl, thiazopyridyl, imidazopyrazinyl, imidazopyrimidinyl, thiophenepyrimidinyl, furanopyrimidine, pyrrolopyrimidine, pyrazolopyrimidinyl, oxazolopyrimidinyl, thiazopyrimidinyl, pyrazolotriazinyl, naphtho[2,3-b]thiophene, phenoxathioyl, indolyl, isoindolyl, 1H-inzolyl, purine, isoquinolinyl, quinolinyl, phthalazinyl, naphridyl, quinoxolinyl, quinazolinyl, cenolinyl, carbazoyl, β-carolinyl, phenanthrene, acridineyl, phenazinyl, phenthiazinyl, phenoxazinyl, etc., are not limited to these.

[0052] In this specification, "VIPR2" refers to proteins from mammals such as mice, rats, dogs, monkeys, and humans.

[0053] In this specification, the phrase "having VIPR2 antagonistic activity" refers to the following: in in vitro experiments, the peptide of the present invention (1) inhibits the binding of the reported VIPR2 antagonistic peptide Vipep-3 to biotin-labeled recombinant VIPR2 protein; (2) inhibits the binding of the reported VIPR2 antagonistic peptide Vipep-3 to biotin-labeled VIPR2-expressing cells; (3) binds to the VIPR2 recombinant protein in a concentration-dependent manner; (4) binds to the VIPR2-expressing cells in a concentration-dependent manner; (5) when added before or together with VIP, it inhibits the increase in calcium concentration in VIPR2-expressing cells; (6) when added before or together with VIP, it inhibits the increase in cAMP concentration in VIPR2-expressing cells; (7) when added before or together with VIP, it inhibits the recruitment of β-retention protein in VIPR2-expressing cells, etc., and any of these effects are described as "having VIPR2 antagonistic activity". Regarding the presence or absence of VIPR2 antagonistic activity, reference can be made to non-patent literature 7, etc., which can be confirmed by those skilled in the art using known methods, but are not limited to these.

[0054] (Cyclic peptide)

[0055] based on Figure 1 The structure of a cyclic peptide according to one embodiment of the present invention will be described. Figure 1The diagram schematically illustrates the features (farmacopha) related to VIPR2 binding activity and the functionally related to cyclic structure restriction (SS binding) of VIPep-3, which forms the basis for the design of the cyclic peptide, and their relationship to the cyclic peptide of this embodiment, which is derived from VIPep-3. The first to 13th amino acid residues of VIPep-3 correspond to the first to 13th amino acid residues of the cyclic peptide of this embodiment, but in the cyclic peptide of this embodiment, the first amino acid, or the first and second amino acid residues, may be omitted. In Vipep-3, the cysteine ​​residues at positions 1 and 10 are cyclized, forming the cyclic peptide of this embodiment: (1) two cyclic structures based on bond A between amino acid residues at positions 1 and 10 and bond B between amino acid residues at positions 7 and 11; (2) two cyclic structures based on bond C between amino acid residues at positions 2 and 10 and bond D between amino acid residues at positions 7 and 11; or two cyclic structures based on bond G between amino acid residues at positions 2 and 10 and bond H between amino acid residues at positions 7 and 11; (3) two cyclic structures based on amino acid residues at positions 3 and 10. Two ring structures based on the bond E between amino acid residues and the bond F between amino acid residues at positions 7 and 11, or based on the bond I between amino acid residues at positions 3 and 10 and the bond J between amino acid residues at positions 7 and 11, (4) based on the bond K between amino acid residues at positions 1 and 7 and 10, (5) based on the bond L or bond N between amino acid residues at positions 2 and 7 and 10, (6) based on the bond M or bond O between amino acid residues at positions 3 and 7 and 10, are further stabilized. Furthermore, examples of amino acids for amino acid residue substitution at each amino acid position of VIPEP-3 and the positions of amino acid residues for which bicyclization studies were conducted are shown. Figure 2 .

[0056] [1] More specifically, Figure 1 The cyclic peptide shown is represented by either formula (1) or formula (2).

[0057] c[X N -X 4 -X 5 -X 6 -c(X 7 -X 8 -X 9 -X 10 ]-X 11 )-X 12 -X 13 -X 14 -X 15 -X 16 (1)

[0058] c[XN -X 4 -X 5 -X 6 -c(X 7 -X 8 -X 9 -X 10 )]-X 11 -X 12 -X 13 -X 14 -X 15 -X 16 (2)

[0059] Here, X in equation (1) N With X 10 Between, X 7 With X 11 Between, and X in equation (2) N With X 7 With X 10 Each chain independently forms a covalent bond, resulting in two ring structures within the molecule. These covalent bonds can be direct covalent bonds between the main chain and the side chain, or between side chains, or indirect bonds via linkers.

[0060] In equations (1) and (2) above, X N For X 1 -X 2 -X 3 X 1 X 2 X 3 X 7 X 10 and X 11 When involved in intramolecular cyclization of the peptide molecule, each group independently represents an amino, carboxyl, thiol, allyl, alkynyl, azide, amino acid residue with a halogen atom, or their derivatives; when not involved in intramolecular cyclization of the peptide molecule, X 1 and X 2 Each can independently represent any amino acid residue or deletion, X 3 and X 11 Each can be any amino acid residue independently. Additionally, X 5 X 9 X 12 and X 13 Each independently represents an amino acid residue containing a hydrocarbon group that may have substituents, or a derivative thereof. 4 X represents an amino acid residue containing an aromatic carbocyclic group that may have substituents, or a derivative thereof. 6 and X 8 X represents any amino acid residue. 14 X15 and X 16 Each can independently represent any amino acid residue or deletion; the N-terminal amino group and the C-terminal carboxyl group can be modified or deleted.

[0061] [2] The preferred embodiment included in Formula (1) is a cyclic peptide consisting of the amino acid sequence shown in Formula (3) below or a pharmacologically acceptable salt thereof.

[0062] c[X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -c(X 7 -X 8 -X 9 -X 10 ]-X 11 )-X 12 -X 13 (3)

[0063] In equation (3), X 1 With X 10 Between, X 7 With X 11 Each element independently forms a covalent bond, resulting in two intramolecular ring structures. This covalent bond is located at X. 1 Main chain or side chain with X 10 Between the side chains and X 7 The side chain and X 11 X is formed between the side chains. 1 ~X 13 As recorded in [1].

[0064] In one implementation, X 1 Indicates cysteine, D-cysteine, 3-mercaptopropionic acid, 2,3-diaminopropionic acid, D-2,3-diaminopropionic acid, β-alanine, aspartic acid, or glutamic acid, X 10 Indicates cysteine, D-cysteine, 2,3-diaminopropionic acid, D-2,3-diaminopropionic acid, aspartic acid, or glutamic acid, X 2 and X 3 Each can independently represent any amino acid residue, preferably proline, X 4 and X 8 Each of these terms independently represents an amino acid residue having an aromatic carbocyclic group, preferably tyrosine, phenylalanine, or a derivative thereof. 5 X 9 X 12 and X 13 Each can independently represent leucine, isoleucine, or their derivatives, X7 and X 11 Each of these can be independently represented as ornithine, ornithine, arginine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, aspartic acid, glutamic acid, cysteine, homocysteine, or their derivatives.

[0065] [3] Where, X 1 With X 10 The bonds between Cα carbon atoms preferably have the structure shown in the following formula (4).

[0066] Cα 1 -(CH2) A -L 1 -(CH2) B -Cα 10 (4)

[0067] In the formula, A and B each independently represent any integer from 0 to 2. The sum of A and B is any integer from 1 to 4. Connector L 1 It represents S, SS, CH2-CH2, S-CH2, CH2-S, O-CH2, CH2-O, CH=CH, NH-C(=O), N(CH3)-C(=O), NH-C(=S), N(CH3)-C(=S), OC(=O), C(=O)-NH, C(=O)-N(CH3), C(=S)-NH, C(=S)-N(CH3), C(=O)-O, CH2-CH2-CH2, S-CH2-CH2, CH2-CH2-S, CH2-S-CH2, O-CH2-CH2, CH2-CH2-O, CH2-O-CH2, S-CH2-S, SC(=CH2)-S, S-(CH2)2-S, or triazole.

[0068] Among them, L 1 Preferred orientations are SS, NH-C(=O), CH2-S-CH2, CH=CH, and CH2-CH2, with L being even more preferred. 1 This indicates the case of SS or NH-C (=O). L 1 The case of S~S refers to, for example, X 1 and X 10 All of them are amino acid residues or their derivatives with thiol groups, forming disulfide bonds under oxidative conditions. 1 The case representing NH-C (=O) refers to, for example, X 1 Amino acid residues with an amino group on their side chain, X 10This refers to a cyclization structure formed by the dehydration condensation of amino and carboxyl groups on an amino group of an amino acid residue with a carboxyl group on its side chain. Examples of amino acid residues with a thiol group on their side chain include cysteine, D-cysteine, 3-mercaptopropionic acid, homocysteine, and D-homocysteine. Examples of amino acid residues with an amino group on their side chain include 2,3-diaminopropionic acid, D-2,3-diaminopropionic acid, or β-alanine. Examples of amino acid residues with a carboxyl group on their side chain include aspartic acid and glutamic acid.

[0069] [4] Other preferred embodiments included in the above formula (1) are: cyclic peptides consisting of the amino acid sequence shown in formula (5) or pharmacologically acceptable salts thereof.

[0070] c[X 2 -X 3 -X 4 -X 5 -X 6 -c(X 7 -X 8 -X 9 -X 10 ]-X 11 )-X 12 -X 13 (5)

[0071] In equation (5), X 2 With X 10 Between, X 7 With X 11 Each element independently forms a covalent bond, thereby creating two intramolecular ring structures. This covalent bond is formed at X. 2 Main chain or side chain with X 10 The side chains and X 7 The side chain and X 11 Between the side chains, X 2 ~X 13 As described in [1].

[0072] [5] Where, X 2 With X 10 The bonds between Cα carbon atoms preferably have the structure shown in the following formula (6).

[0073] Cα 2 -(CH2) A -L 2 -(CH2) B -Cα 10 (6)

[0074] In the formula, A and B each independently represent any integer from 0 to 6, and the sum of A and B is any integer from 4 to 7. 2Indicates S, SS, CH2-CH2, S-CH2, CH2-S, O-CH2, CH2-O, CH=CH, NH-C(=O), N(CH3)-C(=O), NH-C(=S), N(CH3)-C(=S), OC(=O), C(=O)-NH, C(=O)-N(CH3), C(=S)-NH, C(=S)-N(CH3), C(=O)-O, CH2-CH2-CH2, S-CH2-CH2, CH2-CH2-S, CH2-S-CH2, O-C H2-CH2, CH2-CH2-O, CH2-O-CH2, S-CH2-S, SC(=CH2)-S, S-(CH2)2-S, S-(CH2)3-S, S-(CH2)4-S, S-(CH2)5-S, S-CH2-CH=CH-CH2-S, S-CH2-C(=O)-NH, NH-C(=O)-CH2-S, S-CH2-C6H4-CH2-S (the methylene group can be bonded to the phenylene ring at any of the ortho, meta, or para positions), S-CH2-C 10 H6-CH2-S, S-CH2-C(=O)-CH2-S, or S-CH2-C(=CH2)-CH2-S, triazole or dithiotetrafluorobenzene.

[0075] Among them, L 2 Preferred representations are S-(CH2)2-S, S-(CH2)3-S, S-(CH2)4-S, and S-(CH2)5-S. For example, X 2 and X 10 All of them are amino acid residues or their derivatives with thiol groups, which can form thioether bonds between the thiol groups of these residues and halogenated alkyl linkers such as 1,2-diiodoethane, 1,3-diiodopropane, and 1,4-diiodobutane.

[0076] [6] Another preferred embodiment of the above formula (1) is a cyclic peptide composed of the amino acid sequence shown in formula (7) or a pharmacologically acceptable salt thereof.

[0077] c[X 3 -X 4 -X 5 -X 6 -c(X 7 -X 8 -X 9 -X 10 ]-X 11 )-X 12 -X 13 (7)

[0078] In equation (7), X 3 With X 10Between, X 7 With X 11 Each element independently forms a covalent bond, thereby creating two intramolecular ring structures. This covalent bond is formed at X. 3 Main chain or side chain with X 10 The side chains and X 7 The side chain and X 11 Between the side chains, X 3 ~X 13 As described in [1].

[0079] In one implementation, X 3 X represents cysteine, D-cysteine, homocysteine, D-homocysteine, 3-mercaptopropionic acid, 6-mercaptohexanoic acid, or 8-mercaptohexanoic acid. 10 Indicates cysteine, D-cysteine, homocysteine, or D-homocysteine, X 4 and X 8 Each of the following independently represents an amino acid residue having an aromatic carbocyclic group, preferably tyrosine, phenylalanine, or their derivatives, X 5 X 9 X 12 and X 13 Each can independently represent leucine, isoleucine, or their derivatives, X 7 and X 11 Each of these can be used independently to represent lysine, ornithine, arginine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, aspartic acid, glutamic acid, or their derivatives.

[0080] [7] Where, X 3 With X 10 The bonds between Cα carbon atoms preferably have the structure shown in the following formula (8).

[0081] Cα 3 -(CH2) A -L 3 -(CH2) B -Cα 10 (8)

[0082] In the formula, A and B each independently represent any integer from 0 to 10, and the sum of A and B is any integer from 7 to 10. 3Represents S, SS, CH2-CH2, S-CH2, CH2-S, O-CH2, CH2-O, CH=CH, NH-C(=O), N(CH3)-C(=O), NH-C(=S), N(CH3)-C(=S), OC(=O), C(=O)-NH, C(=O)-N(CH3 ), C(=S)-NH, C(=S)-N(CH3), C(=O)-O, CH2-CH2-CH2, S-CH2-CH2, CH2-CH2-S, CH2-S-CH2, O-CH2-CH2, CH2-CH2-O, CH2-O-CH2, S-CH2-S, SC(= CH2)-S, S-(CH2)2-S, S-(CH2)3-S, S-(CH2)4-S, S-(CH2)5-S, S-(CH2)6-S, S-(CH2)7-S, S-(CH2)8-S, S-CH2-CH=CH-CH2-S, S-CH2-C(=O)-NH, NH-C(=O)-CH2-S, S-CH2-C6H4-CH2-S (the methylene group can be ortho, meta, or para on the phenylene ring), S-CH2-C6H4-C6H4-CH2-S, S-CH2-C5NH3-C5NH3-CH2-S, S-CH2-C 10 H6-CH2-S, S-CH2-C(=O)-CH2-S, or S-CH2-C(=CH2)-CH2-S, triazole or dithiotetrafluorobenzene).

[0083] Among them, L 3 Preferably, it represents S-(CH2)3-S, S-(CH2)4-S, S-(CH2)5-S, S-(CH2)6-S, or S-CH2-C6H4-CH2-S (the bonding site of the methylene group on the phenylene ring can be any of ortho, meta, or para). For example, X 3 and X 10 All of them are amino acid residues or their derivatives with thiol groups, which can form thioether bonds between the thiol groups of these residues and halogenated alkyl linkers such as 1,3-diiodopropane, 1,4-diiodobutane, 1,5-diiodopentane, 1,6-diiodohexane, 1,2-dibromomethylbenzene, 1,3-dibromomethylbenzene or 1,4-dibromomethylbenzene.

[0084] [8] As another preferred embodiment contained in the above formulas (1), (3), (5), (7), X is preferred. 7 With X 11 The bonds between Cα carbon atoms have the structure shown in the following formula (9).

[0085] Cα 7 -(CH2)A -L 7 -(CH2) B -Cα 11 (9)

[0086] In the formula, A and B each independently represent any integer from 0 to 7, and the sum of A and B is any integer from 2 to 7. 7 Represents S, SS, CH2-CH2, S-CH2, CH2-S, O-CH2, CH2-O, CH=CH, NH-C(=O), N(CH3)-C(=O), NH-C(=S), N(CH3)-C(=S), OC(=O), C(=O)-NH , C(=O)-N(CH3), C(=S)-NH, C(=S)-N(CH3), C(=O)-O, CH2-CH2-CH2, S-CH2-CH2, CH2-CH2-S, CH2-S-CH2, O-CH2-CH2, CH2-CH2- O, CH2-O-CH2, S-CH2-S, SC(=CH2)-S, S-(CH2)2-S, S-(CH2)3-S, S-(CH2)4-S, S-CH2-CH=CH-CH2-S, S-CH2-C(=O)-NH, NH-C(=O)-CH2-S, S-CH2-C6H4-CH2-S (the methylene group can be bonded to the ortho, meta, or para position on the phenylene ring), S-CH2-C(=O)-CH2-S, or S-CH2-C(=CH2)-CH2-S, triazole or dithiotetrafluorobenzene.

[0087] Among them, L 7 Preferably, it represents NH-C(=O), SS, or S-(CH2)3-S. 7 The case representing NH-C (=O) refers to, for example, X 7 X is an amino acid residue with an amino group on its side chain. 11 This refers to a structure formed by the cyclization of amino acid residues with carboxyl groups on their side chains through the dehydration condensation of these amino and carboxyl groups. 7 The case of S~S refers to, for example, X 7 and X 11 All of them are amino acid residues or their derivatives with thiol groups, forming disulfide bonds under oxidative conditions. 7 The case representing S-(CH2)3-S refers to, for example, X 7 and X 11These are all amino acid residues or their derivatives containing a thiol group, which can form thioether bonds between the thiol group and a haloalkyl linker such as 1,3-diiodopropane. Examples of amino acid residues with an amino group in their side chain include lysine and ornithine. Examples of amino acid residues with a carboxyl group in their side chain include aspartic acid and glutamic acid. Examples of amino acid residues with a thiol group in their side chain include cysteine, D-cysteine, homocysteine, and D-homocysteine.

[0088] [9] The preferred embodiment included in the above formula (2) is a cyclic peptide composed of the amino acid sequence shown in formula (10) or a pharmacologically acceptable salt thereof.

[0089] c[X 3 -X 4 -X 5 -X 6 -c(X 7 -X 8 -X 9 -X 10 )]-X 11 -X 12 -X 13 (10)

[0090] In equation (10), X 3 With X 7 and X 10 Two ring structures are formed within the molecule through covalent bonds, X 3 ~X 13 As described in [1].

[0091] In one implementation, X 3 X represents cysteine, D-cysteine, homocysteine, D-homocysteine, or 3-mercaptopropionic acid. 7 and X 10 Each can be independently represented as cysteine, D-cysteine, homocysteine, or D-homocysteine, X 4 and X 8 Each of the following independently represents an amino acid residue having an aromatic carbocyclic group, preferably tyrosine, phenylalanine, or their derivatives, X 5 X 9 X 12 and X 13 Each can independently represent leucine, isoleucine, or their derivatives, X 11 It refers to serine, threonine, diaminopropionic acid, diaminobutyric acid, or their derivatives.

[0092]

[10] Wherein, in the cyclic peptide shown in formula (2) or formula (10) above, X 1 With X7 With X 10 X 2 With X 7 With X 10 、 or X 3 With X 7 With X 10 The bonds between Cα carbon atoms preferably have the structure shown in the following formula (11).

[0093]

[0094] In the formula, N represents any one of 1, 2 or 3, A, B and D each independently represent any integer of 1 or 2, and Y represents [(S-CH3)3-C6H3], [(S-CH2-CH2-C(=O))3-C3N3H6], [(S-CH2-C(=O)-NH)3-C6H3], [(NH-C(=O))3-C6H3], [(S-CH3)3-C3N3H6] or [(S-CH2-C(=O))3-C3N3H6].

[0095] Wherein, Y preferably represents [(S-CH3)3-C6H3]. For example, in X 3 3-Mercaptopropionic acid (MPa), X 7 and X 10 When all residues are amino acid residues with thiol groups, such as cysteine ​​(Cys), peptide cyclization can be achieved through thioether bonds between the thiol groups of these residues and halogenated alkyl linkers such as 1,3,5-tris(bromomethyl)benzene.

[0096]

[11] In addition, in the cyclic peptides shown in formula (2) or formula (10) above, X 1 With X 7 With X 10 X 2 With X 7 With X 10 、 or X 3 With X 7 With X 10 The bonds between Cα carbon atoms preferably have the structure shown in the following formula (12).

[0097]

[0098] In the formula, N represents any one of 1, 2, or 3; A, B, D, E, and F each independently represent any integer in 1 or 2; Z and Z' each independently represent S, SS, S-CH2-S, SC(=CH2)-S, S-(CH2)2-S, S-(CH2)3-S, S-(CH2)4-S, S-CH2-CH=CH-CH2-S, S-CH2-C(=O)-NH, NH-C(=O)-CH2-S, S-CH2-C6H4-CH2-S (the bonding site of the methylene group on the phenylene ring can be any one of ortho, meta, or para), S-CH2-C6H4-C6H4-CH2-S, S-CH2-C 10 H6-CH2-S, S-CH2-C(=O)-CH2-S or S-CH2-C(=CH2)-CH2-S, or dithiotetrafluorobenzene.

[0099]

[12] In the cyclic peptides shown in formulas (1) to (3), (5), (7) and (10) above, X 5 X 9 X 12 and X 13 Each is preferably an amino acid residue represented by the following formula (13).

[0100]

[0101] In the formula, the wavy line represents the connection point with the carbonyl or nitrogen atom that forms the amide bond in the main chain, R 1 R 2 R 6 and R 7 Each can independently represent a hydrogen atom or a methyl group, R 3 R 4 R 5 Each element independently represents a hydrogen atom, a methyl group, or a halogen atom (F, Cl, Br, I), and n represents any integer from 0 to 10. Among these, leucine, N-methylleucine, α-methylleucine, isoleucine, β-homoleucine, valine, leucine, 2-aminoheptanoic acid, 2-aminooctanoic acid, and 2-aminononanoic acid are particularly preferred.

[0102] Or, X 5 X 9 X 12 AndびX 13 It can be an amino acid residue as shown in the following formula (14).

[0103]

[0104] In the formula, the wavy line represents the connection point with the carbonyl or nitrogen atom that forms the amide bond in the main chain, Z represents C or N, and R represents the carbonyl or nitrogen atom.8 and R 9 This represents a hydrogen atom or a methyl group, and n represents any integer from 1 to 6. Among them, cyclopropylalanine and cyclobutylalanine are particularly preferred.

[0105]

[13] In the cyclic peptides shown in formulas (1) to (3), (5), (7) and (10) above, X 4 Preferably, the amino acid residues are those shown in the following formula (15).

[0106]

[0107] In the formula, the wavy line represents the connection point with the carbonyl or nitrogen atom that forms the amide bond in the main chain, R 10 R 11 R 12 R 13 and R 14 Each of these groups independently represents a hydrogen atom, hydroxyl group, hydroxymethyl group, methoxy group, methoxymethyl group, amino group, aminomethyl group, monomethylated amino group, dimethylated amino group, trimethylated amino group, monomethylated aminomethyl group, dimethylated aminomethyl group, trimethylated aminomethyl group, acetyl group, amide group, methyl group, tert-butyl group, halomethyl group, or halogen atom (F, Cl, Br, I), R 15 and R 16 It represents a hydrogen atom or a methyl group.

[0108] Among them, X 4 Preferably, it represents tyrosine, 3-hydroxyphenylalanine, 4-fluorophenylalanine, 4-aminophenylalanine, 4-aminomethylphenylalanine, 4-amidephenylalanine, N-methyltyrosine, α-methyltyrosine, O-methyltyrosine, 3-methoxyphenylalanine, and 4-acetylphenylalanine.

[0109]

[14] Furthermore, in the cyclic peptides shown in formulas (1) to (3), (5), (7) and (10) above, X 8 Preferably, it comprises amino acid residues or derivatives thereof that contain aromatic carbocyclic groups that may have substituents.

[0110]

[15] X as an aromatic carbocyclic group 8 The preferred embodiment is the amino acid residue shown in the following formula (16).

[0111]

[0112] In the formula, the wavy line represents the connection point with the carbonyl or nitrogen atom that forms the amide bond in the main chain, R 17 R 18 R 19 R 20 and R 21Each of these groups independently represents a hydrogen atom, hydroxyl group, hydroxymethyl group, methoxy group, methoxymethyl group, amino group, aminomethyl group, monomethylated amino group, dimethylated amino group, trimethylated amino group, monomethylated aminomethyl group, dimethylated aminomethyl group, trimethylated aminomethyl group, acetyl group, amide group, methyl group, tert-butyl group, halomethyl group, or halogen atom (F, Cl, Br, I), R 22 and R 23 It represents a hydrogen atom or a methyl group.

[0113] Among them, X is further optimized. 8 The terms "tyrosine", "3-hydroxyphenylalanine", "4-fluorophenylalanine", "4-aminophenylalanine", "4-aminomethylphenylalanine", "4-amidephenylalanine", "N-methylated tyrosine", "α-methylated tyrosine", "O-methyl-tyrosine", "3-methoxy-phenylalanine", and "4-acetyl-phenylalanine" are used to represent tyrosine, 3-hydroxyphenylalanine, 4-aminophenylalanine, 4-aminomethyl ...

[0114]

[16] Furthermore, in the cyclic peptides shown in formulas (1) to (3), (5), (7) and (10) above, X 6 Preferred ingredients include glycine, N-methylglycine, alanine, N-methylalanine, D-alanine, N-methyld-D-alanine, 2-aminoisobutyric acid, N-methyld-2-aminoisobutyric acid, 2-azacyclobutane-2-carboxylic acid, D-azacyclobutane-2-carboxylic acid, proline, D-proline, thioproline, D-thioproline, 3,4-dehydroproline, D-3,4-dehydroproline, 2-piperidine acid, D-2-piperidine acid, or derivatives thereof.

[0115] Among them, X is further optimized. 6 This indicates proline, N-methylglycine, N-methylalanine, 2-azacyclobutane-2-carboxylic acid, and 2-piperidinic acid.

[0116]

[17] In one embodiment of the present invention, the cyclic peptide is composed of the amino acid sequence shown in the following formula (17).

[0117] c[X 1 -Pro 2 -X 3 -Tyr 4 -Leu 5 -Pro 6 -c(X 7 -X 8 -Leu 9 -Cys 10 )-X 11 )-X 12 -X 13 (17)

[0118] In equation (17), X 1Indicates cysteine, MPa (3-mercaptopropionic acid), or D-cysteine, X 3 X represents N-methylated glycine, N-methylated alanine, 2-azacyclobutane-2-carboxylic acid, proline, hydroxyproline, 3,4-dehydroproline, 2-piperidinic acid, serine, or lysine. 8 X represents tyrosine, proline, or arginine. 7 and X 11 X represents any combination selected from lysine and aspartic acid, ornithine and glutamic acid, aspartic acid and lysine, glutamic acid and ornithine, lysine and glutamic acid, or glutamic acid and lysine. 12 and X 13 Each can be independently represented as leucine, isoleucine, or ortholeucine, X 1 With Cys 10 Disulfide bonds are formed between their respective side chains, X 7 With X 11 Amide bonds are formed between their respective side chains, so the peptide of formula (17) has two cyclic structures in the molecule. The N-terminal amino group and the C-terminal carboxyl group can be modified or omitted.

[0119]

[18] In a further preferred embodiment of the cyclic peptide, in formula (17) above, X 1 X represents cysteine. 3 X represents proline or serine. 7 Indicates lysine, X 8 X represents tyrosine. 11 It represents aspartic acid, and X 12 and X 13 Each can be used independently to represent leucine, isoleucine, or ortholeucine. The N-terminal amino group is acetylated, and the C-terminal carboxyl group is amidated.

[0120] Examples of cyclic peptides included in formula (17) or formula (18) above include the following.

[0121] Ac-c[Cys 1 -Pro 2 -Pro 3 -Tyr 4 -Leu 5 -Pro 6 -c(Lys 7 -Tyr 8 -Leu 9 -Cys 10 -Asp 11 )-Leu 12 -Ile 13 -NH2 (Serial Number 10)

[0122] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Tower 8 -Leu 9 -Cys 10 ]-Asp 11 )-Leu 12 -Where 13 -NH2

[0123] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Orn 7 - Tower 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Where 13 -NH2

[0124] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Orn 7 - Tower 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Where 13 -NH2

[0125] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Asp 7 - Tower 8 -Leu 9 -Cys 10 ]-Court 11 )-Leu 12 -Where13 -NH2

[0126] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Asp 7 - Tower 8 -Leu 9 -Cys 10 ]-Court 11 )-Leu 12 -Where 13 -NH2

[0127] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Tower 8 -Leu 9 -Cys 10 ]-Orn 11 )-Leu 12 -Where 13 -NH2

[0128] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Tower 8 -Leu 9 -Cys 10 ]-Orn 11 )-Leu 12 -Where 13 -NH2

[0129] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Tower 8 -Leu 9 -Cys 10 ]-Asp11 )-Leu 12 -Leu 13 -NH2

[0130] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Tower 8 -Leu 9 -Cys 10 ]-Asp 11 )-Leu 12 -Leu 13 -NH2

[0131] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Orn 7 - Tower 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Leu 13 -NH2

[0132] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Orn 7 - Tower 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Leu 13 -NH2

[0133] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Asp 7 - Tower 8 -Leu9 -Cys 10 ]-Court 11 )-Leu 12 -Leu 13 -NH2

[0134] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Asp 7 - Tower 8 -Leu 9 -Cys 10 ]-Court 11 )-Leu 12 -Leu 13 -NH2

[0135] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Tower 8 -Leu 9 -Cys 10 ]-Orn 11 )-Leu 12 -Leu 13 -NH2

[0136] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Tower 8 -Leu 9 -Cys 10 ]-Orn 11 )-Leu 12 -Leu 13 -NH2

[0137] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Court7 -Tyr 8 -Leo 9 -Cys 10 ]-Asp 11 )-No 12 -Ile 13 -NH2

[0138] Ac-c[Cys 1 -Pro 2 -Ser 3 -Tyr 4 -Leo 5 -Pro 6 -c(Lys 7 -Tyr 8 -Leo 9 -Cys 10 ]-Asp 11 )-No 12 -Ile 13 -NH2

[0139] Ac-c[Cys 1 -Pro 2 -Pro 3 -Tyr 4 -Leo 5 -Pro 6 -vase 7 -Tyr 8 -Leo 9 -Cys 10 ]-Glu 11 )-No 12 -Ile 13 -NH2

[0140] Ac-c[Cys 1 -Pro 2 -Ser 3 -Tyr 4 -Leo 5 -Pro 6 -vase 7 -Tyr 8 -Leo 9 -Cys 10 ]-Glu 11 )-No 12 -Ile 13 -NH2

[0141] Ac-c[Cys 1 -Pro 2 -Pro 3 -Tyr 4 -Leo5 -Pro 6 -c(Asp 7 -Tyr 8 -Leo 9 -Cys 10 ]-Lys 11 )-No 12 -Ile 13 -NH2

[0142] Ac-c[Cys 1 -Pro 2 -Ser 3 -Tyr 4 -Leo 5 -Pro 6 -c(Asp 7 -Tyr 8 -Leo 9 -Cys 10 ]-Lys 11 )-No 12 -Ile 13 -NH2

[0143] Ac-c[Cys 1 -Pro 2 -Pro 3 -Tyr 4 -Leo 5 -Pro 6 -c(Glu 7 -Tyr 8 -Leo 9 -Cys 10 ]-Orn 11 )-No 12 -Ile 13 -NH2

[0144] Ac-c[Cys 1 -Pro 2 -Ser 3 -Tyr 4 -Leo 5 -Pro 6 -c(Glu 7 -Tyr 8 -Leo 9 -Cys 10 ]-Orn 11 )-No 12 -Ile 13 -NH2

[0145] Ac-c[Cys 1 -Pro 2 -Pro3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Tower 8 -Leu 9 -Cys 10 ]-Asp 11 ) - Place 12 -Leu 13 -NH2

[0146] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Tower 8 -Leu 9 -Cys 10 ]-Asp 11 ) - Place 12 -Leu 13 -NH2

[0147] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Orn 7 - Tower 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Leu 13 -NH2

[0148] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Orn 7 - Tower 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Leu 13 -NH2

[0149] Ac-c[Cons1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Asp 7 - Tower 8 -Leu 9 -Cys 10 ]-Court 11 ) - Place 12 -Leu 13 -NH2

[0150] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Asp 7 - Tower 8 -Leu 9 -Cys 10 ]-Court 11 ) - Place 12 -Leu 13 -NH2

[0151] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Tower 8 -Leu 9 -Cys 10 ]-Orn 11 ) - Place 12 -Leu 13 -NH2

[0152] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Tower 8 -Leu 9 -Cys 10 ]-Orn 11 ) - Place 12 -Leu 13 -NH2

[0153] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Court 7 - Rec 8 -Leu 9 -Cys 10 ]-Asp 11 )-Leu 12 -Where 13 -NH2

[0154] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Court 7 - Rec 8 -Leu 9 -Cys 10 ]-Asp 11 )-Leu 12 -Where 13 -NH2

[0155] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Orn 7 - Rec 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Where 13 -NH2

[0156] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Orn 7 - Rec 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu12 -Where 13 -NH2

[0157] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Asp 7 - Rec 8 -Leu 9 -Cys 10 ]-Court 11 )-Leu 12 -Where 13 -NH2

[0158] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Asp 7 - Rec 8 -Leu 9 -Cys 10 ]-Court 11 )-Leu 12 -Where 13 -NH2

[0159] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Glu 7 - Rec 8 -Leu 9 -Cys 10 ]-Orn 11 )-Leu 12 -Where 13 -NH2

[0160] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Glu 7 - Rec 8 -Leu 9 -Cys10 ]-Orn 11 )-Leu 12 -Where 13 -NH2

[0161] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Court 7 - Rec 8 -Leu 9 -Cys 10 ]-Asp 11 )-Leu 12 -Leu 13 -NH2

[0162] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Court 7 - Rec 8 -Leu 9 -Cys 10 ]-Asp 11 )-Leu 12 -Leu 13 -NH2

[0163] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Orn 7 - Rec 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Leu 13 -NH2

[0164] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Orn 7 - Rec8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Leu 13 -NH2

[0165] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Asp 7 - Rec 8 -Leu 9 -Cys 10 ]-Court 11 )-Leu 12 -Leu 13 -NH2

[0166] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Asp 7 - Rec 8 -Leu 9 -Cys 10 ]-Court 11 )-Leu 12 -Leu 13 -NH2

[0167] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Glu 7 - Rec 8 -Leu 9 -Cys 10 ]-Orn 11 )-Leu 12 -Leu 13 -NH2

[0168] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro6 -c (Glu 7 - Rec 8 -Leu 9 -Cys 10 ]-Orn 11 )-Leu 12 -Leu 13 -NH2

[0169] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Court 7 - Rec 8 -Leu 9 -Cys 10 ]-Asp 11 ) - Place 12 -Where 13 -NH2

[0170] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Court 7 - Rec 8 -Leu 9 -Cys 10 ]-Asp 11 ) - Place 12 -Where 13 -NH2

[0171] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Orn 7 - Rec 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Where 13 -NH2

[0172] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec4 -Leu 5 - Pro 6 -c (Orn 7 - Rec 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Where 13 -NH2

[0173] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Asp 7 - Rec 8 -Leu 9 -Cys 10 ]-Court 11 ) - Place 12 -Where 13 -NH2

[0174] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Asp 7 - Rec 8 -Leu 9 -Cys 10 ]-Court 11 ) - Place 12 -Where 13 -NH2

[0175] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Glu 7 - Rec 8 -Leu 9 -Cys 10 ]-Orn 11 ) - Place 12 -Where 13 -NH2

[0176] Ac-c[Cons 1 - Pro2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Glu 7 - Rec 8 -Leu 9 -Cys 10 ]-Orn 11 ) - Place 12 -Where 13 -NH2

[0177] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Court 7 - Rec 8 -Leu 9 -Cys 10 ]-Asp 11 ) - Place 12 -Leu 13 -NH2

[0178] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Court 7 - Rec 8 -Leu 9 -Cys 10 ]-Asp 11 ) - Place 12 -Leu 13 -NH2

[0179] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Orn 7 - Rec 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Leu 13 -NH2

[0180] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Orn 7 - Rec 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Leu 13 -NH2

[0181] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Asp 7 - Rec 8 -Leu 9 -Cys 10 ]-Court 11 ) - Place 12 -Leu 13 -NH2

[0182] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Asp 7 - Rec 8 -Leu 9 -Cys 10 ]-Court 11 ) - Place 12 -Leu 13 -NH2

[0183] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Glu 7 - Rec 8 -Leu 9 -Cys 10 ]-Orn 11 ) - Place 12-Leu 13 -NH2

[0184] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Glu 7 - Rec 8 -Leu 9 -Cys 10 ]-Orn 11 ) - Place 12 -Leu 13 -NH2

[0185] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Court 7 - Pro 8 -Leu 9 -Cys 10 ]-Asp 11 )-Leu 12 -Where 13 -NH2

[0186] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Court 7 - Pro 8 -Leu 9 -Cys 10 ]-Asp 11 )-Leu 12 -Where 13 -NH2

[0187] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Orn 7 - Pro 8 -Leu 9 -Cys 10]-Glu 11 )-Lion 12 -Ile 13 NH2

[0188] Ac-c[Cys 1 -Pro 2 -Serum 3 -Arg 4 -Lion 5 -Pro 6 -horn 7 -Pro 8 -Lion 9 -Cys 10 ]-Glu 11 )-Lion 12 -Ile 13 NH2

[0189] Ac-c[Cys 1 -Pro 2 -Pro 3 -Arg 4 -Lion 5 -Pro 6 -c(Asp 7 -Pro 8 -Lion 9 -Cys 10 ]-Lys 11 )-Lion 12 -Ile 13 NH2

[0190] Ac-c[Cys 1 -Pro 2 -Serum 3 -Arg 4 -Lion 5 -Pro 6 -c(Asp 7 -Pro 8 -Lion 9 -Cys 10 ]-Lys 11 )-Lion 12 -Ile 13 NH2

[0191] Ac-c[Cys 1 -Pro 2 -Pro 3 -Arg 4 -Lion 5 -Pro 6 -c(Glu 7 -Pro 8-Leu 9 -Cys 10 ]-Orn 11 )-Leu 12 -Where 13 -NH2

[0192] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Glu 7 - Pro 8 -Leu 9 -Cys 10 ]-Orn 11 )-Leu 12 -Where 13 -NH2

[0193] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Court 7 - Pro 8 -Leu 9 -Cys 10 ]-Asp 11 )-Leu 12 -Leu 13 -NH2

[0194] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Court 7 - Pro 8 -Leu 9 -Cys 10 ]-Asp 11 )-Leu 12 -Leu 13 -NH2

[0195] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6-c (Orn 7 - Pro 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Leu 13 -NH2

[0196] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Orn 7 - Pro 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Leu 13 -NH2

[0197] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Asp 7 - Pro 8 -Leu 9 -Cys 10 ]-Court 11 )-Leu 12 -Leu 13 -NH2

[0198] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Asp 7 - Pro 8 -Leu 9 -Cys 10 ]-Court 11 )-Leu 12 -Leu 13 -NH2

[0199] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4-Leu 5 - Pro 6 -c (Glu 7 - Pro 8 -Leu 9 -Cys 10 ]-Orn 11 )-Leu 12 -Leu 13 -NH2

[0200] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Glu 7 - Pro 8 -Leu 9 -Cys 10 ]-Orn 11 )-Leu 12 -Leu 13 -NH2

[0201] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Court 7 - Pro 8 -Leu 9 -Cys 10 ]-Asp 11 ) - Place 12 -Where 13 -NH2

[0202] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Court 7 - Pro 8 -Leu 9 -Cys 10 ]-Asp 11 ) - Place 12 -Where 13 -NH2

[0203] Ac-c[Cons 1 - Pro 2-Pro 3 -Arg 4 -Lion 5 -Pro 6 -horn 7 -Pro 8 -Lion 9 -Cys 10 ]-Glu 11 )-No 12 -Ile 13 NH2

[0204] Ac-c[Cys 1 -Pro 2 -Serum 3 -Arg 4 -Lion 5 -Pro 6 -horn 7 -Pro 8 -Lion 9 -Cys 10 ]-Glu 11 )-No 12 -Ile 13 NH2

[0205] Ac-c[Cys 1 -Pro 2 -Pro 3 -Arg 4 -Lion 5 -Pro 6 -c(Asp 7 -Pro 8 -Lion 9 -Cys 10 ]-Lys 11 )-No 12 -Ile 13 NH2

[0206] Ac-c[Cys 1 -Pro 2 -Serum 3 -Arg 4 -Lion 5 -Pro 6 -c(Asp 7 -Pro 8 -Lion 9 -Cys 10 ]-Lys 11 )-No 12 -Ile 13 NH2

[0207] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Glu 7 - Pro 8 -Leu 9 -Cys 10 ]-Orn 11 ) - Place 12 -Where 13 -NH2

[0208] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Glu 7 - Pro 8 -Leu 9 -Cys 10 ]-Orn 11 ) - Place 12 -Where 13 -NH2

[0209] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Court 7 - Pro 8 -Leu 9 -Cys 10 ]-Asp 11 ) - Place 12 -Leu 13 -NH2

[0210] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Court 7 - Pro 8 -Leu 9 -Cys 10 ]-Asp 11 ) - Place 12 -Leu13 -NH2

[0211] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Orn 7 - Pro 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Leu 13 -NH2

[0212] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Orn 7 - Pro 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Leu 13 -NH2

[0213] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Asp 7 - Pro 8 -Leu 9 -Cys 10 ]-Court 11 ) - Place 12 -Leu 13 -NH2

[0214] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Asp 7 - Pro 8 -Leu 9 -Cys 10 ]-Court11 ) - Place 12 -Leu 13 -NH2

[0215] Ac-c[Cons 1 - Pro 2 - Pro 3 - Rec 4 -Leu 5 - Pro 6 -c (Glu 7 - Pro 8 -Leu 9 -Cys 10 ]-Orn 11 ) - Place 12 -Leu 13 -NH2

[0216] Ac-c[Cons 1 - Pro 2 - Star 3 - Rec 4 -Leu 5 - Pro 6 -c (Glu 7 - Pro 8 -Leu 9 -Cys 10 ]-Orn 11 ) - Place 12 -Leu 13 -NH2

[0217] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Tower 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Where 13 -NH2

[0218] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Tower 8 -Leu9 -Cys 10 ]-Glu 11 )-Leu 12 -Leu 13 -NH2

[0219] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Tower 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Where 13 -NH2

[0220] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Tower 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Leu 13 -NH2

[0221] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Tower 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Where 13 -NH2

[0222] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Court7 - Tower 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Leu 13 -NH2

[0223] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Tower 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Where 13 -NH2

[0224] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Tower 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Leu 13 -NH2

[0225] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Rec 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Where 13 -NH2

[0226] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu5 - Pro 6 -c (Court 7 - Rec 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Leu 13 -NH2

[0227] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Rec 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Where 13 -NH2

[0228] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Rec 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Leu 13 -NH2

[0229] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Rec 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Where 13 -NH2

[0230] Ac-c[Cons 1 - Pro 2 - Star3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Rec 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Leu 13 -NH2

[0231] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Rec 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Where 13 -NH2

[0232] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Rec 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Leu 13 -NH2

[0233] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Pro 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Where 13 -NH2

[0234] Ac-c[Cons1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Pro 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Leu 13 -NH2

[0235] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Pro 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Where 13 -NH2

[0236] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Pro 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Leu 13 -NH2

[0237] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Pro 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Where 13 -NH2

[0238] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Pro 8 -Leu 9 -Cys 10 ]-Glu 11 )-Leu 12 -Leu 13 -NH2

[0239] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Pro 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Where 13 -NH2

[0240] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Court 7 - Pro 8 -Leu 9 -Cys 10 ]-Glu 11 ) - Place 12 -Leu 13 -NH2

[0241] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Tower 8 -Leu 9 -Cys 10 ]-Court 11 )-Leu12 -Where 13 -NH2

[0242] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Tower 8 -Leu 9 -Cys 10 ]-Court 11 )-Leu 12 -Leu 13 -NH2

[0243] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Tower 8 -Leu 9 -Cys 10 ]-Court 11 ) - Place 12 -Where 13 -NH2

[0244] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Tower 8 -Leu 9 -Cys 10 ]-Court 11 ) - Place 12 -Leu 13 -NH2

[0245] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Tower 8 -Leu 9 -Cys10 ]-Court 11 )-Leu 12 -Where 13 -NH2

[0246] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Tower 8 -Leu 9 -Cys 10 ]-Court 11 )-Leu 12 -Leu 13 -NH2

[0247] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Tower 8 -Leu 9 -Cys 10 ]-Court 11 ) - Place 12 -Where 13 -NH2

[0248] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Tower 8 -Leu 9 -Cys 10 ]-Court 11 ) - Place 12 -Leu 13 -NH2

[0249] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Rec8 -Leu 9 -Cys 10 ]-Court 11 )-Leu 12 -Where 13 -NH2

[0250] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Rec 8 -Leu 9 -Cys 10 ]-Court 11 )-Leu 12 -Leu 13 -NH2

[0251] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Rec 8 -Leu 9 -Cys 10 ]-Court 11 ) - Place 12 -Where 13 -NH2

[0252] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Rec 8 -Leu 9 -Cys 10 ]-Court 11 ) - Place 12 -Leu 13 -NH2

[0253] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro6 -c (Glu 7 - Rec 8 -Leu 9 -Cys 10 ]-Court 11 )-Leu 12 -Where 13 -NH2

[0254] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Rec 8 -Leu 9 -Cys 10 ]-Court 11 )-Leu 12 -Leu 13 -NH2

[0255] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Rec 8 -Leu 9 -Cys 10 ]-Court 11 ) - Place 12 -Where 13 -NH2

[0256] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Rec 8 -Leu 9 -Cys 10 ]-Court 11 ) - Place 12 -Leu 13 -NH2

[0257] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower4 -Leu 5 - Pro 6 -c (Glu 7 - Pro 8 -Leu 9 -Cys 10 ]-Court 11 )-Leu 12 -Where 13 -NH2

[0258] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Pro 8 -Leu 9 -Cys 10 ]-Court 11 )-Leu 12 -Leu 13 -NH2

[0259] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Pro 8 -Leu 9 -Cys 10 ]-Court 11 ) - Place 12 -Where 13 -NH2

[0260] Ac-c[Cons 1 - Pro 2 - Pro 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Pro 8 -Leu 9 -Cys 10 ]-Court 11 ) - Place 12 -Leu 13 -NH2

[0261] Ac-c[Cons 1 - Pro2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Pro 8 -Leu 9 -Cys 10 ]-Court 11 )-Leu 12 -Where 13 -NH2

[0262] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Pro 8 -Leu 9 -Cys 10 ]-Court 11 )-Leu 12 -Leu 13 -NH2

[0263] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Pro 8 -Leu 9 -Cys 10 ]-Court 11 ) - Place 12 -Where 13 -NH2

[0264] Ac-c[Cons 1 - Pro 2 - Star 3 - Tower 4 -Leu 5 - Pro 6 -c (Glu 7 - Pro 8 -Leu 9 -Cys 10 ]-Court 11 ) - Place 12 -Leu 13 -NH2

[0265] The peptides of the present invention include: peptides that are homologous and have VIPR2 binding activity, having one or more amino acids deleted, added, and / or substituted in the amino acid sequences shown in [1] to

[17] above. In this specification, when referring to "peptides with one or more amino acids deleted, added, and / or substituted," the number of these amino acids is not particularly limited as long as the peptide has VIPR2 binding activity; preferably, it is 1 to 5, more preferably 1 or 2. The deletion, addition, and / or substitution can be at the end or in the middle of the peptide, and can be at one or more locations.

[0266] Examples of amino acid sequences that have one or more amino acids missing, added, and / or substituted in the above-mentioned amino acid sequence include amino acid sequences that, when calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or similar methods (for example, using the default or initially set parameters), have at least 50%, preferably 70%, more preferably 80%, and particularly preferably 90% or more homology with the above-mentioned amino acid sequence.

[0267] Furthermore, peptides and protein domains with low primary structural homology but highly similar conformations are also known. Therefore, peptides with at least 50%, preferably 70%, more preferably 80%, and particularly preferably 90% conformational homology to the above-mentioned amino acid sequences and possessing VIPR2 binding activity are also included in this embodiment. Regarding the conformational homology of such peptides, their conformations can be predicted from the amino acid sequences of peptides with unknown conformations using homology modeling methods, etc. For example, when an arbitrary amino acid sequence (target sequence) with a similar sequence to the cyclic peptide (reference peptide) of this embodiment is given, an alignment between the target sequence and the reference sequence is given (the sequences are set side by side). If an alignment calculated by FASTA, PSI-BLAST, LIBRA, etc. is used, the correspondence between each amino acid between the target sequence and the reference sequence is determined, and therefore, based on this relationship, the 3D coordinates of each amino acid on the target sequence are generated from the 3D coordinates of the reference peptide. In the construction of 3D coordinates, structurally inappropriate gaps, conflicts, and deformations sometimes occur between amino acid residues. Therefore, energy minimization calculations are used to eliminate these structural deformations. According to modeling software, to successfully eliminate these structural deformations, the process is sometimes performed in stages, rather than simultaneously on all atoms of the peptide. That is, the α-carbon atoms forming the peptide backbone are processed first, followed by the main chain atoms containing the α-carbon atoms, and finally the entire peptide containing the side chain atoms. If an alignment of the target array is obtained in this way, its conformation can be predicted. Indicators of conformational homology, such as the difference between the XYZ coordinates at optimal superposition (RMSD), can be used to compare conformational homology.

[0268] As long as the problem of this invention can be solved, the peptide of this invention also includes various derivatives and / or modifications thereof. Examples of such derivatives include substances in which the saturated fatty chain of the peptide is replaced by an unsaturated fatty chain; substances in which a portion of the peptide's atoms is replaced by other atoms, including radioactive or non-radioactive isotopic atoms; substances in which the amide bond of the peptide is replaced by a thioamide bond (-NH-C(=S)-); substances in which the amide bond of the peptide is replaced by an alkene (-C=C-); substances in which the amide bond of the peptide is replaced by an alkyl group (-CC-); substances in which the amide bond of the peptide is replaced by a hydroxyethylidene group (-C(-OH)-C-); and substances in which the amide bond of the peptide is replaced by an ester (-OC(=S)-). Examples of such modifications include substances with α-carbon substitution (-C=C-), substances with amide bonds of peptides substituted with olefins (-C=C-), substances with amide bonds of peptides substituted with (-C-NH-), or substances with amide bonds of peptides substituted with (-C(=O)-C-). Other examples of such modifications include substances with α-carbon substitution (-C=C-), substances with amide bonds of peptides N-alkylated, and substances where a portion of the functional group of a peptide accepts halogenation, cyanation, nitration, oxidation, hydroxylation, amination, deamination, dehydrogenation, amidation, acetylation, methoxylation, isopreneation, and alkylation. Examples of substances that have undergone modifications such as alkylation (e.g., substances in which a portion of the amino group of a peptide is acetylated, formylated, myristylated, palmitoylated, pyroglutamicized, alkylated, or deaminated; substances in which a portion of the carboxyl group of a peptide is N-pyrrolealkylated or N-piperidinized, or substances that become amides (amides, methylamides, ethylamides, p-nitroaniline, β-naphthylamides, etc.) or esters (methyl esters, ethyl esters, thioesters, etc.)); substances in which the S group of a peptide becomes sulfoxide S (=O) or sulfone S (=O)2; polymerized substances in which peptides are polymerized via chemical linkers; substances in which peptides are biotin-labeled; substances in which peptides are fluorescently labeled; substances in which peptides are luminescently labeled; and substances formed by fusing peptides with alkyl chains, polyethylene glycol, antibodies, lectins, glycans, enzymes, membrane-permeable peptides, low-molecular-weight compounds, or molecules that induce protein ubiquitination, etc., but are not limited to these.

[0269] The peptides of this invention also include peptide salts. Salts of peptides can be physiologically acceptable bases or acids, such as addition salts of inorganic acids (hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc.), addition salts of organic acids (p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carboxylic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc.), addition salts of inorganic bases (ammonium hydroxide, or bases, or alkaline earth metal hydroxides, carbonates, bicarbonates, etc.), and addition salts of amino acids.

[0270] The peptide of this invention can be a prodrug. A prodrug refers to a compound that is converted into the peptide of this invention under physiological conditions in an organism through reactions with enzymes, gastric acid, etc., that is, a compound that is converted into the peptide of this invention through enzymatic oxidation, reduction, hydrolysis, etc., or a compound that is converted into the peptide of this invention through hydrolysis by gastric acid, etc.

[0271] Examples of prodrugs for the peptides of the present invention include compounds in which the amino group of the peptides of the present invention is acylated, alkylated, or phosphorylated (e.g., compounds in which the amino group of the peptides of the present invention is icosanolylated, alanylated, pentylaminocarbonylated, (5-methyl-2-oxo-1,3-dioxolane-4-yl)methoxycarbonylated, tetrahydrofuranylated, pyrrolidinylmethylated, neopentyloxymethylated, or tert-butylated); compounds in which the hydroxyl group of the peptides of the present invention is acylated, alkylated, phosphorylated, or borated (e.g., compounds in which the hydroxyl group of the peptides of the present invention is acetylated, palmitoylated, propionylated, neopentylacylated, succinylated, fumarylated, propionylated, or dimethylaminomethylcarbonylated); and compounds in which the hydroxyl and carboxyl groups of the peptides of the present invention are esterified or amidated (e.g., compounds in which the hydroxyl and carboxyl groups of the peptides of the present invention are C-terminated). 1-6 Compounds may be alkylated, phenylated, carboxymethylated, dimethylaminomethylated, neopentanoyloxymethylated, ethoxycarbonyloxyethylated, phthaloyl esterified, (5-methyl-2-oxo-1,3-dioxolane-4-yl)methylated, cyclohexyloxycarbonylethylated, or methylamidated, etc. They are not limited to these. These compounds can be prepared from the peptides of this invention by methods known per se.

[0272] The prodrug of the peptide of the present invention can be converted into the peptide of the present invention under the physiological conditions described in the molecular design section of Volume 7 of "Pharmaceutical Development" published by Hirokawa Shoten in 1990, on pages 163-198.

[0273] In this specification, the prodrug may form a salt, and examples of such salts as salts of the peptides of the present invention can be cited.

[0274] The peptides of this invention can be crystals, whether single crystals or mixtures of crystals. Crystals can be manufactured by crystallization using methods known per se.

[0275] The peptides of this invention can be pharmaceutically acceptable eutectic or eutectic salts. Here, a eutectic or eutectic salt refers to a crystalline substance composed of two or more distinct solids having different physical properties (e.g., structure, melting point, heat of fusion, hygroscopicity, solubility, and stability) at room temperature. Eutectic or eutectic salts can be manufactured according to eutectic methods known per se.

[0276] (Effects of cyclic peptides)

[0277] As described in the examples below, Seq-1 to 10, which are representative examples of the amino acid sequence sets shown in this embodiment, exhibit binding activity against VIPR2-expressing cells. Seq-1, 6, 9, and 10 possess antagonistic activity against VIPR2-expressing cells and resistance to protease hydrolysis. The amino acid sequence sets shown in this embodiment have similar amino acid sequences and conformational features to Seq-1 to 10, therefore it is highly likely that they also possess resistance to protease hydrolysis, VIPR2 binding activity, and VIPR2 antagonistic activity.

[0278] The VIPR2 binding activity of the peptides shown in the examples described below is as follows: (1) The three C-terminal residues of VIPEP-3 (amino acid residues at positions 14-16: Leu-Arg-Ser) do not lose their VIPR2 binding activity even if they are missing. (2) If the 7th and 11th positions are crosslinked, the VIPR2 binding activity is increased. (3) VIPEP-3 is cyclized by an SS bond formed between the cysteine ​​residues at positions 1 and 10. However, the cyclization for VIPR2 binding activity is not limited to the 1st and 10th positions. As long as amino acid residues with long side chains are introduced at positions 2 and 3 to directly crosslink with position 10, or linkers of different lengths and structures are used to indirectly crosslink, so that the interatomic distance between positions 2 and 10 and the interatomic distance between positions 3 and 10 are close to the interatomic distance between positions 1 and 10, the crosslinks between positions 2 and 10 and between positions 3 and 10 can be used instead. (4) Peptides bicyclic between three amino acid residues also exhibit VIPR2 binding activity, and it is believed that crosslinking modes between positions 1-7-10, 2-7-10, and 3-7-10 are all permissible. More specific explanations of (3) and (4) above will be provided. As shown in the examples described later, the cyclic structure formed between the 1st to 3rd amino acid residues and the 10th amino acid residue of VIPEP-3: Cα 3 -NH-C(=O)-Cα 2 -NH-C(=O)-Cα 1 -CH2-SS-CH2-Cα 10 For example, a ring structure can be formed between the 3rd and 10th amino acid residues of a side chain with a non-natural structure: Cα 3 -CH2-CH2-CH2-CH2-CH2-CH2-CH2-SS-CH2-CH2-Cα 10Instead, the amino acid residues at positions 1-3 of VIPEP-3 are not significantly related to VIPEP-2 binding activity, suggesting that the ring size restriction of the peptide is important for VIPEP-2 binding activity. The peptides of this invention can cyclize not only between positions 1-10, but also between positions 2-10 and 3-10. It is believed that as long as the ring size of the peptide converges within a certain range, it can accept a wide range of chemical structures, including amide bonds, disulfide bonds, thioether bonds, C=C bonds, C / C bonds, binding via triazoles, and binding via dithiotetrafluorobenzene. It is believed that this characteristic is also maintained when the cyclization occurs at position 7 (between positions 1-7-10, 2-7-10, and 3-7-10).

[0279] The amino acid sequence groups shown above [1] to

[16] have similar amino acid sequences and conformational features to Vipep-3, which has antagonistic activity against VIPR2 in humans, mice, and rats. Therefore, it is believed that the possibility of binding to VIPR2 in mammals other than humans, such as mice and rats, and blocking its function is extremely high.

[0280] (Preparation method of cyclic peptides)

[0281] The peptides of this embodiment can be manufactured using known peptide manufacturing methods such as liquid-phase methods, solid-phase methods, or chemical synthesis methods that combine liquid-phase and solid-phase methods.

[0282] Solid-phase synthesis can be performed using commercially available automated synthesis equipment. For example, the hydroxyl group of a resin containing hydroxyl groups is esterified with the carboxyl group of the first amino acid (usually the C-terminal amino acid of the target peptide) protected by a protecting group such as an Fmoc group at the α-position. Known dehydrating condensing agents such as 1-mesinesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIPCDI) can be used as esterification catalysts. Next, the protecting group at the α-position of the first amino acid is deprotected, and a second amino acid with all functional groups protected except for the carboxyl group of the main chain is added, activating the carboxyl group and bonding the first and second amino acids. Then, the α-position of the second amino acid is deprotected, and a third amino acid with all functional groups protected except for the carboxyl group of the main chain is added, activating the carboxyl group and bonding the second and third amino acids. This process is repeated to synthesize a peptide of the target length. The linear peptide is cleaved from the resin, purified, and then the functional groups used for peptide cyclization are deprotected. The peptide is then cyclized using conventional methods.

[0283] Examples of resins synthesized in the solid phase include Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGA resin (Merck), and HMPA-PEGA resin (Merck). These resins can be used after washing with solvents such as dimethylformamide (DMF), 2-propanol, and dichloromethane. Examples of protecting groups for the α-amino group include benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), fluorenylmethoxycarbonyl (Fmoc), benzyl, allyl, and allyloxycarbonyl (Alloc). The Cbz group can be deprotected by hydrofluoric acid or hydrogenation, the Boc group by trifluoroacetic acid (TFA), and the Fmoc group by treatment with piperidine. The α-carboxyl group can be protected using methyl ester, ethyl ester, benzyl ester, tert-butyl ester, and cyclohexyl ester. Other functional groups of amino acids include the hydroxyl groups of serine and threonine, which can be protected with benzyl or tert-butyl groups; and the hydroxyl groups of tyrosine, which can be protected with 2-bromobenzyloxycarbonyl or tert-butyl groups. The amino groups of the side chains of lysine, and the carboxyl groups of glutamic acid and aspartic acid, can be protected in the same way as the α-amino and α-carboxyl groups.

[0284] Carboxyl activation can be achieved using condensing agents. Examples of condensing agents 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. Peptide chain cleavage from the resin can be achieved by treatment with acids such as TFA or hydrogen fluoride (HF).

[0285] As one method, the peptide of this embodiment is cyclized. In this specification, cyclization means that within a peptide, two or more amino acids separated by one or more amino acids form a covalent bond directly or indirectly via a linker, thereby forming one or more cyclic structures within the molecule. Cyclization can be carried out according to the methods described in Non-Patent Documents 9, 10, and 11. For example, amide bonds between amino and carboxyl groups, disulfide bonds between thiol groups, thioether bonds between thiol groups and halogen groups, thioether bonds based on the thiol-alkene reaction between thiol and allyl groups, C=C bonds based on the alkene metathesis reaction between allyl and allyl groups (the C=C bond can be converted to a C-C bond through a reduction reaction), triazole bonds based on the click reaction between alkynyl and azide groups, thioether bonds between a linker having a halogen group and two thiol groups, etc., but are not limited to these. The direct or indirect covalent bonds used for cyclization can be any of the following: main chain-to-main chain, main chain-to-side chain, side chain-to-main chain, or side chain-to-side chain.

[0286] In the cyclization of the peptide of the present invention, for example, (1) cysteine, D-cysteine, homocysteine, and D-homocysteine ​​with thiol groups can each be independently designated as amino acid 1 and amino acid 2, using disulfide bonds formed between these thiol groups; (2) amino acids having a halogen atom (chlorine, bromine, or iodine) as a nucleophilic group (e.g., 3-chloroalanine) or carboxylic acids having a halogen atom (chlorine, bromine, or iodine) (e.g., 3-chloropropionic acid) can be designated as amino acid 1, using thioether bonds formed between them and amino acid 2 with thiol groups; (3) [The text abruptly ends here, so the translation stops as well.] (3) A thioether bond formed between amino acid 1 and amino acid 2, and a linker having a halogen atom (chlorine, bromine, or iodine) as a nucleophile (e.g., 1,3-diiodopropane, 1,4-diiodobutane, 1,5-diiodopentane, 1,6-diiodohexane, 1,2-bis(bromomethyl)benzene, 1,3-bis(bromomethyl)benzene, 1,4-bis(bromomethyl)benzene, etc.); (4) β-azidoalanine having an azide group can be designated as amino acid 1, and 2-amino-5-hexynic acid having an alkyne group can be designated as amino acid 2, using a triazole covalent bond formed via a click reaction between the two. (5) An amino acid with an allyl group (e.g., allyl glycine, D-allyl glycine, homoallyl glycine, D-homonyl glycine, etc.) or a carboxylic acid with an allyl group (e.g., 3-butenoic acid) can be used as amino acid 1, and a thioether bond formed by a thiol-alkene reaction between amino acid 2 with a thiol group can be used; (6) An amino acid with an allyl group or a carboxylic acid with an allyl group can be used independently as amino acid 1 and amino acid 2, and a C=C bond formed by an alkene metathesis reaction between these allyl groups can be used; (7) A CC bond formed by reducing the C=C bond formed by an alkene metathesis reaction can be used; (8) An amide bond can be used between an amino acid 1 with an amino group (e.g., 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine, lysine, their D-amino acids, etc.) and an amino acid 2 with a carboxyl group (aspartic acid, glutamic acid, their D-amino acids, etc.); (9) An amide bond can be used between an N-terminal amino group (β-alanine, γ-aminobutyric acid, etc.) and an amino acid with a carboxyl group. Either of these amino acids 1 and 2 can be located on the N-terminal side. Furthermore, a thioether bond (10) can be used between amino acids 1, 2, and 3 having a thiol group and a linker (e.g., 1,3,5-tris(bromomethyl)benzene) having a halogen atom (chlorine, bromine, or iodine) as a nucleophile. Amino acids 1, 2, and 3 can all be located on the N-terminal side.

[0287] (Drugs, diagnostic drugs, and research reagents containing cyclic peptides)

[0288] The pharmaceutical composition of the present invention contains the above-mentioned amino acid sequence as an active ingredient. By binding the peptide to VIPR2, it can block the binding of the natural ligand to VIPR2 and inhibit signal transduction via VIPR2. The administration method of the above pharmaceutical composition is not particularly limited; it can be administered orally or non-orally. Examples of non-oral administration include mucosal administration (nasal, oral, ocular, pulmonary, vaginal, or rectal administration), injection (intravenous, subcutaneous, intramuscular, etc.), and transdermal administration. Due to its readily metabolizable and excreted nature, the peptide in the pharmaceutical composition can be modified in various ways. For example, by adding alkyl chains, polyethylene glycol, or sugar chains to the peptide, its retention time in the blood can be prolonged, reducing its antigenicity. Furthermore, biodegradable polymers such as polylactic acid glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared from unsaturated fatty acids, nanoparticles, and nanospheres can be used as sustained-release agents to encapsulate the peptide. In the case of transdermal drug delivery, a weaker current can be flowed across the skin surface to allow it to penetrate the stratum corneum (iontophoresis).

[0289] The above-mentioned pharmaceutical compositions can use the active ingredient directly, or they can be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc. Examples of dosage forms include: liquids (injections, etc.), dispersants, suspensions, tablets, pills, powders, suppositories, powders, fine granules, capsules, syrups, tablet lozenges, inhalers, ointments, eye drops, nasal drops, ear drops, poultices, etc. These formulations can be immediate-release or sustained-release formulations, such as controlled-release formulations (sustained-release microcapsules, etc.). Formulation can be carried out using appropriate excipients, binders, disintegrants, lubricants, solubilizers, co-solvents, colorants, taste and odor modifiers, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, etc., through conventional methods. Examples of ingredients used in formulations include, but are not limited to, pharmaceutically acceptable organic solvents such as purified water, saline, phosphate buffer, glucose, glycerol, and ethanol; animal and vegetable oils; lactose; mannitol; glucose; sorbitol; crystalline cellulose; hydroxypropyl cellulose; starch; corn starch; anhydrous silicate; magnesium aluminum silicate; collagen; polyvinyl alcohol; polyvinylpyrrolidone; carboxyvinyl polymers; sodium carboxymethyl cellulose; sodium polyacrylate; sodium alginate; water-soluble dextran; sodium carboxymethyl starch; lectins; methylcellulose; ethylcellulose; xanthan gum; gum arabic; tragacanth; casein; agar; polyethylene glycol; diglycerides; glycerol; propylene glycol; petrolatum; paraffin; octyl dodecyl myristate; isopropyl myristate; higher alcohols; stearyl alcohol; stearic acid; and human serum albumin. When peptides are difficult to absorb through mucous membranes, absorption enhancers that improve the absorption of poorly absorbed drugs can be used as surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponins; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylic acids; fatty acids such as hexanoic acid, decanoic acid, lauric acid, oleic acid, linoleic acid, and mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, and nitric oxide donors.

[0290] Pills or tablets can also be coated with sugar, gastric-soluble, or enteric-soluble substances. Injectable preparations may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. Furthermore, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, solvent aids, preservatives, etc., can be added. Depending on the needs, appropriate amounts of common preservatives, antioxidants, colorants, sweeteners, adsorbents, wetting agents, etc., can also be used.

[0291] The pharmaceutical compositions of the present invention are used to prevent or treat the following diseases associated with the activation of VIR2: central nervous system diseases, such as mental disorders (schizophrenia, schizoaffective disorder, schizophrenia-like disorders, delusional disorder, etc.), childhood mental disorders (attention deficit disorder, attention deficit / hyperactivity disorder, behavioral disorders, autism, etc.), neurodegenerative disorders, neural stem cell disorders, neural progenitor disorders, ischemic disorders, neurotraumatic disorders, mood disorders, psychomotor disorders, sleep disorders (hypersomnia, circadian rhythm sleep disorder, insomnia, abnormal sleep behavior, sleep blocking, etc.), mental disorders such as anxiety (acute stress disorder, generalized anxiety disorder, social anxiety disorder, panic disorder, post-traumatic stress disorder, agoraphobia, obsessive-compulsive disorder, etc.), pseudopsychiatric disorders (acute hallucinogenic mania, etc.), impulse control disorders (compulsive gambling, intermittent burst disorder, etc.), mood disorders (bipolar I disorder, bipolar II disorder, mania, mixed mood state, etc.), major depressive disorder, chronic depression, seasonal affective disorder, psychotic depression, seasonal affective disorder, dementia (amnesia, Alzheimer's disease). HIV-related dementia, Alzheimer's disease, Huntington's disease, Lewy body dementia, vascular dementia, drug-related dementia, tardive dyskinesia, clonic muscle spasms, dystonia, delirium, Pick's disease, Creutzfeldt-Jakob disease, AIDS, Gilles de La dementia, Tourette syndrome, epilepsy, muscle spasms, mild cognitive impairment, etc.; intellectual disability (spasticity, Down syndrome, Fragile X syndrome, etc.); premenstrual syndrome (PMS), premenstrual mood disorder (PDD), postpartum depression, neuronal damage disorders (eye damage, retinal diseases). This includes, but is not limited to, conditions such as macular degeneration, tinnitus, hearing impairment, cerebral edema, Parkinson's disease, Parkinson's-like disorders, migraines, epilepsy, Alzheimer's disease, brain injury, stroke, cerebrovascular diseases (cerebral arteriosclerosis, cerebral amyloid angiopathy, hereditary cerebral hemorrhage, hypoxic-ischemic cerebral disease, etc.), drug dependence (narcotic dependence, alcoholism, amphetamine dependence, cocaine addiction, nicotine dependence, drug withdrawal syndrome, etc.), and eating disorders (anorexia, bulimia, recreational eating disorder, binge eating, obesity, compulsive eating, ice addiction, etc.). Additional diseases are not specifically limited to these; for example, inhibition of cancer cell proliferation expressing VIPR2 and activation of the immune system by blocking VIPR2 function can be cited.

[0292] The pharmaceutical composition of the present invention can be used in combination with other drugs or treatments such as various chemotherapy, surgical treatment, and radiotherapy that are useful for the above-mentioned diseases.

[0293] The dosage of the pharmaceutical composition of the present invention administered to mammals (e.g., humans, mice, rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs, etc.), particularly humans, varies depending on the symptoms, the patient's age, sex, weight, sensitivity differences, method of administration, dosing interval, type of active ingredient, and type of formulation, and is not particularly limited. For example, 30 μg to 1000 mg, 100 μg to 500 mg, or 100 μg to 100 mg may be administered once or in several doses.

[0294] The embodiments shown below are merely illustrative and intended only to illustrate the invention in detail with the above-described embodiments, and are not intended to limit the invention. Those skilled in the art can modify the invention in various ways without departing from its meaning, and such modifications are also included within the scope of the invention.

[0295] Example

[0296] The abbreviations used in this specification have the following meanings.

[0297] VIPR2: Vasoactive intestinal peptide receptor 2

[0298] VIP: Vasoactive Intestinal Peptides

[0299] BSA: Bovine serum albumin

[0300] RP-HPLC: Reversed-phase high-performance liquid chromatography

[0301] HRP: Horseradish peroxidase

[0302] SA: Streptavidin

[0303] HBSS: Hank's equilibrium salt solution

[0304] ELISA: Enzyme-linked immunosorbent assay

[0305] Ac: Acetyl group

[0306] Cys: L-cysteine

[0307] hmC: L-homocysteine

[0308] MPa: 3-Mercaptopropionic acid

[0309] Pro: L-proline

[0310] Try: L-tyrosine

[0311] Leu: L-Leucine

[0312] Ile: L-Isoleucine

[0313] Arg: L-arginine

[0314] Asp: L-Aspartic acid

[0315] Glu: L-glutamic acid

[0316] Dap: (S)-2,3-diaminopropionic acid

[0317] Dab: (S)-2,4-Diaminobutyric acid

[0318] Tyr(Ome): O-methyl-L-tyrosine

[0319] 4aF: 4-Amino-L-phenylalanine

[0320] 4AcF: 4-Acetyl-L-phenylalanine

[0321] 4amdF: 4-Amino-L-phenylalanine

[0322] 3hF: 3-Hydroxy-L-phenylalanine

[0323] 3(Ome)F: 3-Methoxy-L-phenylalanine

[0324] 4amF: 4-Aminomethyl-L-phenylalanine

[0325] 4fF: 4-Fluoro-L-phenylalanine

[0326] Glycine

[0327] Ala: L-alanine

[0328] Aze: L-azacyclobutanecarboxylic acid

[0329] thioP: L-thioproline

[0330] Dhp: 3,4-Dehydro-L-proline

[0331] Pip: L-piperidinic acid

[0332] Val: L-valine

[0333] Nle: L-Leucine

[0334] Ahep: (S)-2-aminoheptanoic acid

[0335] Aoc: (S)-2-aminooctanoic acid

[0336] Anon: (S)-2-aminononanoic acid

[0337] CprA: L-Cyclopropylalanine

[0338] CbuA: L-Cyclobutylalanine

[0339] βhmLeu: β-Hylo-L-Leucine

[0340] M6a: 6-Mercaptohexanoic acid

[0341] M8a: 8-Mercaptooctanoic acid

[0342] DIBut: 1,4-Diiodobutane

[0343] DIPen: 1,5-Diiodopentane

[0344] DIHex: 1,6-Diiodohexane

[0345] DBoXy: 1,2-bis(bromomethyl)benzene

[0346] DBmXy: 1,3-bis(bromomethyl)benzene

[0347] DBpXy: 1,4-bis(bromomethyl)benzene

[0348] TBMB: 1,3,5-Tris(bromomethyl)benzene

[0349] Nme-X: N-methylated amino acid X

[0350] aMe-X: α-methylated amino acid X

[0351] D X: D-amino acid X

[0352] c[XY], c(XY): Cyclization between amino acid X and amino acid Y

[0353] (Peptide synthesis)

[0354] In this embodiment, the chemical synthesis of all peptides was outsourced to SCRUM Co., Ltd. (Tokyo, Japan), and a standard solid-phase synthesis method using a SyroII automated synthesizer (manufactured by Biotage) was performed, employing a 9-fluorenylmethoxycarbonyl (Fmoc) group as the protecting group for the α-amino group. The C-terminal protected amino acid-resin was placed into a synthesis column, and the apparatus was set up. Next, as the next amino acid protected with the Fmoc group, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) / diisopropylethylamine (DIEA) was added for activation, and the mixture was placed into the column to react. After the reaction, the mixture was washed, and the Fmoc group was deprotected using 20% ​​piperidine. By repeating this process, the peptide chain was extended, and after deprotecting the Fmoc group of the final amino acid, the peptide resin was removed from the apparatus.

[0355] The cyclization of peptides was performed according to the methods described in Non-Patent Literature 8, Non-Patent Literature 9, Non-Patent Literature 10, and Non-Patent Literature 11. As examples, the cyclization of Seq-1, 6, and 9 is shown below. For Seq-1 and Seq-6, the resin containing the linear side-chain protected peptides was swollen with dimethylformamide (DMF) and reacted in a 2% hydrazine solution for 5–10 minutes to deprotect the protecting groups of the lysine side chain (Dde) and the aspartic acid side chain (ODmab). Then, Oxima Pure and diisopropylcarbodiimide (DIC) were added as coupling agents, and the reaction was carried out at 50°C for 3 hours. After washing the resin, TFA was added to deprotect the thiol protecting groups, simultaneously cleaving the monocyclic peptides from the resin. The monocyclic peptides were purified by RT-HPLC using a SunFire C18 column (10 × 150 mm) (Waters Corporation) and then lyophilized.

[0356] For Seq-1, the monocyclic peptide was dissolved in a mixture of Tris-HCl buffer (pH 8.5) and acetonitrile, followed by the addition of DMSO and stirring at room temperature for 36 hours, thereby cyclizing the peptide via disulfide bonds. For Seq-6, the monocyclic peptide was dissolved in dimethyl sulfoxide (DMSO), and 3 equivalents of 1,3-dibromomethylbenzene dissolved in DMF were added, along with 0.1 M NaHCO3 containing 10 mM tris(2-carboxyethyl)phosphine (TCEP) and acetonitrile. The reaction was carried out overnight at room temperature, thereby cyclizing the peptide via thioether bonds between the thiol group and the haloalkyl linker. Seq-1 and 6, as bicyclic peptides, were purified by RT-HPLC using a SunFire C18 column (10 × 150 mm) (Waters Corporation) and then lyophilized. The molecular weights of the final peptides were determined using a microflex (Bruker Corporation) to identify the target compounds.

[0357] For Seq-9, after washing the resin, TFA was added to deprotect the thiol protecting group and simultaneously cleave the linear peptide from the resin. After purification using RT-HPLC on a SunFire C18 column (10×150 mm) (Waters Corporation), the peptide was lyophilized. The linear peptide was dissolved in dimethyl sulfoxide (DMSO), and 3 equivalents of 1,3,5-tris(bromomethyl)benzene (TBMB) dissolved in DMF, 0.1 M NaHCO3 containing 10 mM tris(2-carboxyethyl)phosphine (TCEP), and acetonitrile were added. The reaction was carried out at 80 °C for 3 hours, thereby cyclizing the peptide through a thioether bond between the thiol group and the haloalkyl linker. The bicyclic peptide was purified by RT-HPLC using a SunFire C18 column (10×150 mm) (Waters Corporation) and then lyophilized. The molecular weight of the final peptide was determined using a microflex (Bruker Corporation) to identify the target analyte. The theoretical molecular weight, measured molecular weight, purity, cyclization type, and sequence of the peptides synthesized in this embodiment are shown in Table 1. Additionally, the amino acid sequences of Seq-1 to Seq-9 are shown below, and their structural formulas are shown below. Figure 7 It should be noted that in the amino acid sequences in Table 1 and below, amino acids without the D-type symbol represent L-type amino acids.

[0358] [Table 1-1]

[0359]

[0360] [Table 1-2]

[0361]

[0362] Seq-1: c(Mpa-Pro-Pro-Tyr-Leu-Pro-c[Lys-Tyr-Leu-Cys)-Asp]-Leu-Ile-NH2 (Serial Number 1)

[0363] Seq-2: c(Mpa-NmeTyr-Leu-Pro-c[Lys-Tyr-Leu-Cys)-Asp]-Leu-Ile-NH2 (Serial Number 2)

[0364] Seq-3: c(Mpa-NmeTyr-Leu-Pro-c[Lys-Tyr-Leu-Cys)-Asp]-Leu-Ile-NH2 (Serial Number 3)

[0365] Seq-4: c(Mpa-NmeTyr-Leu-Pro-c[Lys-Tyr-Leu-Cys)-Asp]-Leu-Ile-NH2 (Serial Number 4)

[0366] Seq-5: c(Mpa-NmeTyr-Leu-Pro-c[Lys-Tyr-Leu-Cys)-Asp]-Leu-Ile-NH2 (Serial Number 5)

[0367] Seq-6: c(Mpa-NmeTyr-Leu-Pro-c[Lys-Tyr-Leu-Cys)-Asp]-Leu-Ile-NH2 (Serial Number 6)

[0368] Seq-7: c(Mpa-NmeTyr-Leu-Pro-c[Lys-Tyr-Leu-Cys)-Asp]-Leu-Ile-NH2 (Serial Number 7)

[0369] Seq-8: c(Mpa-NmeTyr-Leu-NmeGly-c(Cys)-Tyr-Leu-Cys)-Ser-Leu-Ile-NH2 (Serial Number 8)

[0370] Seq-9: c(Mpa-NmeTyr-Leu-NmeGly-c(Cys)-Tyr-Leu-Cys)-Dap-Leu-Ile-NH2 (Serial Number 9)

[0371] Seq-10: Ac-c[Cys] 1 -Pro 2 -Pro 3 -Tyr 4 -Leu 5 -Pro 6 -c(Lys 7 -Tyr 8 -Leu 9 -Cys 10 -Asp 11 )-Leu 12 -Ile 13 -NH2 (Serial Number 10)

[0372] Seq-1 and Seq-10 are peptides that have undergone bicyclization by removing the C-terminal 3 residues (Leu-Arg-Ser) of Vipep-3, via an SS bond between positions 1 and 10 and an amide bond between positions 7 and 11. (Seq-2 to 7) are peptides that have undergone bicyclization by removing the N-terminal 2 residues (Cys-Pro) and the C-terminal 3 residues (Leu-Arg-Ser) of Vipep-3, via a thioether bond via a chemical linker between positions 3 and 10 and an amide bond between positions 7 and 11. (Seq-8 to 9) are peptides that have undergone bicyclization by removing the N-terminal 2 residues (Cys-Pro) and the C-terminal 3 residues (Leu-Arg-Ser) of Vipep-3, via a thioether bond via a TBMB linker between positions 3, 7, and 10.

[0373] (Constructing a competitive binding assay using cell ELISA)

[0374] To confirm the binding activity of the amino acid-substituted peptide to VIPR2, a [structure / method / etc.] was constructed. Figure 3The competitive binding assay based on cell ELISA is shown below. The cell ELISA method is briefly described below. Human VIPR2 expression cell line (catalog No. HTS079RTA, manufactured by Eurofins) was inoculated into 96-well plates using the manufacturer's provided medium and cultured overnight at 37°C in a CO2 incubator. After washing the plate with HBSS containing 0.1% BSA, a mixture of Biotin-VIpep-3 (1000 nM) diluted with HBSS containing 0.1% BSA and any concentration of amino acid substitution peptide or Vipep-3 was added at 50 μL / well. After reacting on ice for 60 minutes, the plate was washed with HBSS containing 0.1% BSA. The binding of Biotin-VIpep-3 to VIPR2 expression cells adhered to the plate was detected using SA-HRP (catalog No. ab7403, manufactured by Avecam). In HRP quantification, chemiluminescence values ​​were measured using the SuperSignal ELISA Pico Chemiluminescent Substrate (catalog No. 37070, ThermoFischer). The binding of Biotin-VIpep-3 to VIPR2 on the cell surface competes with the binding of coexisting amino acid substitution peptides and Vipep-3 to VIPR2 in solution; therefore, the binding of amino acid substitution peptides and Vipep-3 is blocked in a concentration-dependent manner. Specifically, the binding activity of amino acid substitution peptides or Vipep-3 to VIPR2 was detected as a competitive inhibitory activity against Biotin-VIpep-3. The luminescence value of wells without Biotin-VIpep-3 was set as 100% inhibition activity, and the luminescence value of wells without amino acid substitution peptides or Vipep-3 was set as 0% inhibition activity, calculating the 50% inhibitory activity values ​​for both amino acid substitution peptides and Vipep-3. Then, the 50% inhibitory activity value of Vipep-3 was set to 1, calculating the relative value of the inhibitory activity of the amino acid substitution peptides.

[0375] (Evaluation of the binding activity of amino acid substitution peptides to VIPR2)

[0376] The results of the competitive binding assay of VIPR2-expressing cells at N=4 are shown in Figure 4 Representative examples of the peptides of the present invention (Seq-1 to 9) all compete with Biotin-VIpep-3. These results show that the peptides of the present invention, which incorporate amino acid substitutions and bicyclization, possess VIPR2 binding activity. In particular, the binding activities of Seq-1 to 4 and Seq-6 are equal to or greater than those of VIPep-3.

[0377] (Evaluation of the antagonistic activity of the peptides of this invention against VIPR2)

[0378] Changes in intracellular calcium concentration are known to be one of the downstream signals of VIPR2. For the human VIPR2-expressing cell line (catalog No. HTS079RTA, manufactured by Eurofins), Scree Quest was used... TM The Fluo-8 No Wash Calucium Assay Kit (Catalogue No. 36315, manufactured by AAT Bioquest) was used to introduce Fluo-8, an indicator reagent for measuring changes in intracellular calcium concentration using fluorescence. VIP (final concentration 150 nM) and Vipep-3 (final concentration 750 nM) as natural ligands, or representative examples of the peptide of this invention (Seq-1, 5-9) (final concentration 750 nM), were added together, and changes in fluorescence intensity were observed over time using a fluorescence microscope. The difference between the fluorescence intensity before and after the addition of VIP was set to 100%, and compared with the difference in fluorescence intensity when Vipep-3 or the peptide of this invention was added together.

[0379] like Figure 5 As shown, VIPEP-3 and representative examples of the peptides of the present invention (Seq-1, 5-9) blocked the receptor agonistic activity of VIP on VIPR2. In particular, the inhibitory activity of Seq-1 and Seq-6 was equal to or greater than that of VIPEP-3.

[0380] (Evaluation of the protease degradation resistance of the peptides of this invention)

[0381] To confirm that the peptides of this invention exhibit protease degradation resistance compared to Vipep-3, Vipep-3 or representative peptides of this invention (Seq-1, 6, 9, 10) were mixed in rat plasma. After incubation for a certain period, the presence or absence of peptide peaks was evaluated by RP-HPLC. 20 μL of rat plasma was added to each 10 mM peptide, and the mixture was incubated at 37°C for any time (0 or 24 hours). Then, 80% acetonitrile was added to each of the peptides (Vipep-3 and all others), and the mixture was thoroughly mixed. The mixture was then incubated on ice for 10 minutes and centrifuged at 15,000 rpm for 10 minutes at 4°C to remove plasma proteins. The supernatant was recovered, and the residual peptide was detected using RP-HPLC (solution A: 0.1% TFA / water, solution B: 0.1% TFA / acetonitrile, gradient of 80% solution A → 10% solution A at 1 mL / min for 20 min) on a SunFire C18 column (5 μm 4.6 × 150 mm). Figure 6As shown, the peak derived from VIPEP-3 decreased significantly after 24 hours of culture, suggesting that any part of the sequence was subjected to protease hydrolysis. On the other hand, the peaks from the representative peptides of this invention (Seq-1, 6, 9, 10) remained approximately the same even after 24 hours compared to the culture time of 0 hours. These results demonstrate that the peptides of this invention, which incorporate amino acid substitutions and bicyclization, possess resistance to protease hydrolysis.

[0382] (Evaluation of the selective antagonistic activity of the peptides of this invention against VIPR2)

[0383] Representative examples (Seq-10) of the peptides of the present invention at arbitrary concentrations were added to VIPR1 expression cells, VIPR2 expression cells, or PAC1 expression cells that had been introduced with an intracellular calcium concentration indicator, and cultured for 30 minutes. Then, changes in intracellular calcium concentration were measured 2 minutes after the addition of VIP (25 nM and 150 nM) to VIPR1 and VIPR2 cells, and the addition of PACAP (35 nM) to PAC1 cells, using FLIPRTetra. The change in intracellular calcium concentration when only the ligand of VIP or PACAP was added was set as an inhibition rate of 0%, and the change in intracellular calcium concentration without simultaneous addition of the antagonistic peptide and ligand was set as an inhibition rate of 100%. The inhibition rate of the antagonistic peptide was calculated.

[0384] like Figure 7 As shown, in the antagonistic activity of the representative example (Seq-10) of the peptide of the present invention, the antagonistic activity against VIP-VIPR2 interaction is significantly stronger than the antagonistic activity against VIP-VIPR1 interaction and PACAP-PAC1 interaction.

[0385] (Evaluation of the VIPR2 antagonistic activity of the peptides of this invention in vivo)

[0386] Ro25-1553 (Peptide Research Institute Co., Ltd.) (0.4 nmol / g body weight), a selective agonist of VIPR2 and an analog of VIP, was dissolved in PBS and then mixed with a representative example of the peptide of the present invention (Seq-10) (1 nmol / g body weight or 10 nmol / g body weight) or DMSO (vehicle group) dissolved in DMSO at a ratio of 9:1 (final concentration of DMSO 10%). These solutions were administered subcutaneously to ICR mice (12 days old, male). One hour later, the brain was removed, and the frontal cortex was harvested. Western blotting was used to evaluate the phosphorylation of circulating AMP response sequence binding protein (CREB) (one of the downstream signals of VIPR2) in the cells. It should be noted that the antibodies used in the Western blotting were anti-phosphorylated CREB antibody (#9198) and anti-CREB antibody (#9197) purchased from Cell Signaling Technology Co., Ltd.

[0387] like Figure 8 As shown, in the presence of Seq-10, phosphorylation of CREB was clearly inhibited, thus demonstrating that representative examples of the peptides of the present invention have antagonistic activity against VIPR2 in vivo.

[0388] (An evaluation of the improvement of mice's cognitive function of novel objects by administering the peptides of the present invention)

[0389] As reported in Non-Patent Literature 12, mice that were given Ro25-1553 subcutaneously once daily for 14 days after birth showed a significant decrease in their ability to recognize novel objects as they continued to grow (8 weeks old). Figure 9 (A)). The efficacy of the peptides of the present invention was evaluated to improve the recognition of novel objects, which is reduced due to VIPR2 activation. Ro25-1553 (PEPTIDE INSTITUTE, INC.) (0.07 nmol / g body weight), a selective VIPR2 agonist and VIP analog, was dissolved in PBS and then mixed with a representative example of the peptides of the present invention (Seq-10) (1 nmol / g body weight) or DMSO (vehicle group) dissolved in DMSO at a ratio of 99:1 (final DMSO concentration 1%). These solutions were administered subcutaneously to male ICR mice on day 1 for 14 days, followed by continued feeding, and were used for novel object recognition tests at 8 weeks of age. The novel object recognition test was conducted according to Non-Patent Literature 12 during the light period (8:00-20:00). First, in a soundproof laboratory with an illuminance of 30 lux, mice were trained for 10 minutes for one day and for three days in test cages (30cm × 30cm × 35cm) made of acrylic-modified polyvinyl chloride with only sterilized wooden sheets (SANKYO LABO SERVICE Co., Ltd.) as the flooring. On the fourth day, two different objects (objects a and b, randomly selected from golf balls, building blocks, plastic cylinders, and sockets) were placed 8cm from the wall, and the mice were allowed to explore freely for 10 minutes (training experiment). After 24 hours, the mice were allowed to explore freely for 5 minutes in a test cage where object b was replaced with object c as a novelty object (training experiment). The animals' actions during the training and experiment were video-recorded, and the exploration time for each of the two objects was measured. The percentage (%) of the difference in exploration time between object c and object a during the training experiment was used as the recognition index.

[0390] like Figure 9As shown in (B), mice given Ro25-1553 exhibited a significant decrease in their ability to recognize novel objects. On the other hand, mice treated with a combination of the present invention's peptide (Seq-10) showed a significant improvement in this decreased ability to recognize novel objects (p < 0.001 obtained by Student's t-test). Furthermore, this cognitive function was substantially the same as that of normal mice. These results demonstrate that administration of the present invention's peptide can significantly prevent and improve the decrease in novel object recognition in mice caused by VIPR2 activation.

[0391] Industrial availability

[0392] The peptides of this invention possess protease resistance and, by binding to VIPR2, block VIPR2 signal transduction via its natural ligand. Therefore, the peptides of this invention are considered useful for the prevention and treatment of diseases associated with VIPR2 activation, such as schizophrenia and autism spectrum disorder.

[0393] The peptides of the present invention can be used not only as pharmaceutical agents, but also as delivery molecules to tissues expressing VIPR2, or as molecules for detecting VIPR2 expression.

[0394] Furthermore, by combining the peptides of the present invention with drugs or therapies having other mechanisms of action, it is expected to exert further preventive or therapeutic effects on schizophrenia or autism spectrum disorders. sequence list <110> Ichimaru Pharchs Co., Ltd. <120> VIPR2 antagonistic peptide <130> FP222612JP <150> JP 2020-059721 <151> 2020-03-30 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 13 <212> PRT <213> Artificial sequence <220> <223> VIPR2 antagonist dicyclic peptide 1 <220> <221> MOD_RES <222> (1)...(1) <223> 3-Mercaptopropionic acid (MPa) <220> <221> DISULFID <222> (1)...(10) <223> S-S(1-10) <220> <221> MISC_FEATURE <222> (7)...(11) <223> Cycloning via amide bond (7-11) <220> <221> MOD_RES <222> (13)...(13) <223> amidation <400> 1 Xaa Pro Pro Tyr Leu Pro Lys Tyr Leu Cys Asp Leu Ile 1 5 10 <210> 2 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VIPR2 antagonist dicyclic peptide 2 <220> <221> MOD_RES <222> (1)...(1) <223> 3-Mercaptopropionic acid (MPa) <220> <221> THIOETH <222> (1)...(8) <223> Cycloning (1-8) via reaction with 1,4-diiodobutane (DIBut) <220> <221> MOD_RES <222> (2)...(2) <223> N-methyltyrosine (NmeTyr) <220> <221> MISC_FEATURE <222> (5)...(9) <223> Cycloning via amide bonds (5-9) <220> <221> MOD_RES <222> (11)...(11) <223> amidation <400> 2 Xaa Tyr Leu Pro Lys Tyr Leu Cys Asp Leu Ile 1 5 10 <210> 3 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VIPR2 antagonist dicyclic peptide 3 <220> <221> MOD_RES <222> (1)...(1) <223> 3-Mercaptopropionic acid (MPa) <220> <221> THIOETH <222> (1)...(8) <223> Cycloning (1-8) via reaction with 1,5-diiodopentane (DIPen) <220> <221> MOD_RES <222> (2)...(2) <223> N-methyltyrosine (NmeTyr) <220> <221> MISC_FEATURE <222> (5)...(9) <223> Cycloning via amide bonds (5-9) <220> <221> MOD_RES <222> (11)...(11) <223> amidation <400> 3 Xaa Tyr Leu Pro Lys Tyr Leu Cys Asp Leu Ile 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VIPR2 antagonist dicyclic peptide 4 <220> <221> MOD_RES <222> (1)...(1) <223> 3-Mercaptopropionic acid (MPa) <220> <221> THIOETH <222> (1)...(8) <223> Cycloning (1-8) via reaction with 1,6-diiodohexane (DIHex) <220> <221> MOD_RES <222> (2)...(2) <223> N-methyltyrosine (NmeTyr) <220> <221> MISC_FEATURE <222> (5)...(9) <223> Cycloning via amide bonds (5-9) <220> <221> MOD_RES <222> (11)...(11) <223> amidation <400> 4 Xaa Tyr Leu Pro Lys Tyr Leu Cys Asp Leu Ile 1 5 10 <210> 5 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VIPR2 antagonist dicyclic peptide 5 <220> <221> MOD_RES <222> (1)...(1) <223> 3-Mercaptopropionic acid (MPa) <220> <221> THIOETH <222> (1)...(8) <223> Cycloning (1-8) via reaction with 1,2-bis(bromomethyl)benzene (DBoXy) <220> <221> MOD_RES <222> (2)...(2) <223> N-methyltyrosine (NmeTyr) <220> <221> MISC_FEATURE <222> (5)...(9) <223> Cycloning via amide bonds (5-9) <220> <221> MOD_RES <222> (11)...(11) <223> amidation <400> 5 Xaa Tyr Leu Pro Lys Tyr Leu Cys Asp Leu Ile 1 5 10 <210> 6 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VIPR2 antagonist dicyclic peptide 6 <220> <221> MOD_RES <222> (1)...(1) <223> 3-Mercaptopropionic acid (MPa) <220> <221> THIOETH <222> (1)...(8) <223> Cycloning (1-8) via reaction with 1,3-bis(bromomethyl)benzene (DBmXy) <220> <221> MOD_RES <222> (2)...(2) <223> N-methyltyrosine (NmeTyr) <220> <221> MISC_FEATURE <222> (5)...(9) <223> Cycloning via amide bonds (5-9) <220> <221> MOD_RES <222> (11)...(11) <223> amidation <400> 6 Xaa Tyr Leu Pro Lys Tyr Leu Cys Asp Leu Ile 1 5 10 <210> 7 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VIPR2 antagonist dicyclic peptide 7 <220> <221> MOD_RES <222> (1)...(1) <223> 3-Mercaptopropionic acid (MPa) <220> <221> THIOETH <222> (1)...(8) <223> Cycloning (1-8) via reaction with 1,4-bis(bromomethyl)benzene (DBpXy) <220> <221> MOD_RES <222> (2)...(2) <223> N-methyltyrosine (NmeTyr) <220> <221> MISC_FEATURE <222> (5)...(9) <223> Cycloning via amide bonds (5-9) <220> <221> MOD_RES <222> (11)...(11) <223> amidation <400> 7 Xaa Tyr Leu Pro Lys Tyr Leu Cys Asp Leu Ile 1 5 10 <210> 8 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VIPR2 antagonist dicyclic peptide 8 <220> <221> MOD_RES <222> (1)...(1) <223> 3-Mercaptopropionic acid (MPa) <220> <221> MISC_FEATURE <222> (1)...(5) <223> Trifurcate linkage via TBMB (1-5-8) <220> <221> MOD_RES <222> (2)...(2) <223> N-methyltyrosine (NmeTyr) <220> <221> MOD_RES <222> (4)...(4) <223> Methylation, (MeGly) <220> <221> MISC_FEATURE <222> (5)...(8) <223> Three-way connection via TBMB (1-5-8) <220> <221> MOD_RES <222> (11)...(11) <223> amidation <400> 8 Xaa Tyr Leu Gly Cys Tyr Leu Cys Ser Leu Ile 1 5 10 <210> 9 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VIPR2 antagonist dicyclic peptide 9 <220> <221> MOD_RES <222> (1)...(1) <223> 3-Mercaptopropionic acid (MPa) <220> <221> MISC_FEATURE <222> (1)...(5) <223> Three-way connection via TBMB (1-5-8) <220> <221> MOD_RES <222> (2)...(2) <223> N-methyltyrosine (NmeTyr) <220> <221> MOD_RES <222> (4)...(4) <223> Methylation, (MeGly) <220> <221> MISC_FEATURE <222> (5)...(8) <223> Three-way connection via TBMB (1-5-8) <220> <221> MOD_RES <222> (9)...(9) <223> (S)-2,3-Diaminopropionic acid (Dap) <220> <221> MOD_RES <222> (11)...(11) <223> amidation <400> 9 Xaa Tyr Leu Gly Cys Tyr Leu Cys Xaa Leu Ile 1 5 10 <210> 10 <211> 13 <212> PRT <213> Artificial sequence <220> <223> VIPR2 antagonist dicyclic peptide 10 <220> <221> MOD_RES <222> (1)...(1) <223> acetylation <220> <221> DISULFID <222> (1)...(10) <223> S-S(1-10) <220> <221> MISC_FEATURE <222> (7)...(11) <223> Cycloning via amide bonds (7-11) <220> <221> MOD_RES <222> (13)...(13) <223> amidation <400> 10 Cys Pro Pro Tyr Leu Pro Lys Tyr Leu Cys Asp Leu Ile 1 5 10 <210> 11 <211> 16 <212> PRT <213> Artificial sequence <220> <223> cyclic peptide VIPEP-3 <220> <221> MOD_RES <222> (1)...(1) <223> acetylation <220> <221> DISULFID <222> (1)...(10) <400> 11 Cys Pro Pro Tyr Leu Pro Arg Arg Leu Cys Thr Leu Leu Leu Arg Ser 1 5 10 15

Claims

1. A cyclic peptide or a pharmacologically acceptable salt thereof, characterized in that, The cyclic peptide is a cyclic peptide having the amino acid sequence described in sequence number 1 and any one of sequence numbers 5 to 10.

2. A drug, diagnostic agent, and / or research reagent comprising the cyclic peptide of claim 1 or a pharmacologically acceptable salt thereof.

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