Hemagglutinin-binding peptide

Abstract

An object of the present invention is to provide a compound having significantly higher antiviral activity than iHA100, an intermediate for producing the compound, a drug containing the highly active compound, etc. A solution of the present invention is a hemagglutinin-binding peptide, a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein the hemagglutinin-binding peptide relates to: (1) a polypeptide composed of the amino acid sequence shown in SEQ ID NO: 1 or 2: Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala-Cys (SEQ ID NO: 1), Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala-Cys-Lys (SEQ ID NO: 2), etc.

Description

Technical Field

[0001] The present invention relates to a hemagglutinin-binding peptide, and more specifically, to a hemagglutinin-binding peptide having extremely high anti-influenza virus activity, a drug for preventing or treating influenza, a drug for detecting influenza, and the like. Background Art

[0002] Influenza viruses are highly infectious and pathogenic viruses, and their spread as an epidemic shows a wide range of pathogenicity to the human body.

[0003] As drugs against influenza viruses, zanamivir (trade name: Relenza (trademark)), oseltamivir (trade name: Tamiflu (trademark)), peramivir (trade name: Rapiacta (trademark)), Laninamivir (trade name: INAVIR (trademark)), which inhibit neuraminidase required for influenza virus release, are widely used. Also, Symmetrel (trade name: amantadine) or Flumadine (trade name: rimantadine), which inhibit the virus enucleation process, and baloxavir marboxil (trade name: Xofluza (trademark)), which inhibits cap-dependent endonuclease, are known drugs. The above drugs, especially anti-influenza drugs against neuraminidase, are widely used as drugs, but drug resistance caused by virus mutations has become a problem.

[0004] On the other hand, hemagglutinin is known as a protein required for influenza viruses to invade host cells, and an antiviral molecule targeting this target has been reported. As an existing antiviral drug, an antiviral molecule targeting hemagglutinin is effective against drug-resistant viruses. Therefore, antiviral molecules targeting target molecules, different from existing drugs, are also highly useful from the viewpoint of drug resistance.

[0005] As an antiviral molecule targeting hemagglutinin, antibody molecules that bind to hemagglutinin have been reported (for example, Patent Documents 1 to 3). On the other hand, antibody drugs have high activity, but neutralizing antibodies generated due to the recognition of antibody drugs as foreign substances in the body sometimes significantly reduce the potency of the drug.

[0006] In recent years, as new molecular groups, peptide molecules having special skeletons such as N-methyl amino acids or D-amino acids have been reported, which not only show high binding ability and in vivo stability, but also have an extremely small molecular weight compared to antibodies. Therefore, they have attracted attention as molecular groups that can solve the problems of conventional antibody drugs (for example, Non-Patent Documents 1 to 4).

[0007] I focused on peptide molecules with this special skeleton and searched for peptide molecules corresponding to hemagglutinin. iHA100 is a peptide molecule corresponding to hemagglutinin, which has a molecular weight and a drug administration route quite different from those of general antibodies, and is an anti-influenza virus molecule that also shows antiviral activity in nasal administration (Patent Document 4). On the other hand, there have been no reports on cases where the anti-influenza virus activity of iHA100 is significantly improved and its structure. The improvement of its anti-influenza virus activity is very helpful for enhancing the efficacy of the drug.

[0008] Prior Art Documents

[0009] Patent Documents

[0010] Patent Document 1: Pamphlet of International Publication No. 2018 / 108086

[0011] Patent Document 2: Pamphlet of International Publication No. 2018 / 015012

[0012] Patent Document 3: Pamphlet of International Publication No. 2017 / 122087

[0013] Patent Document 4: Pamphlet of International Publication No. 2013 / 071904

[0014] Non-Patent Documents

[0015] Non-Patent Document 1: Nature Reviews Drug Discovery 17, 531 - 533 (2018)

[0016] Non-Patent Document 2: Current opinion in chemical biology, 34, 44 - 52 (2016)

[0017] Non-Patent Document 3: Annual Review of Biochemistry, 83, 727 - 752 (2014)

[0018] Non-Patent Document 4: Chemistry, 19, 6530 - 6536 (2013) Summary of the Invention

[0019] Problems to be Solved by the Invention

[0020] An object of the present invention is to provide a compound having significantly higher antiviral activity than iHA100, an intermediate for producing the compound, and a drug containing the above high-activity compound, etc.

[0021] Technical Solutions for Solving the Problems

[0022] The present invention is based on the following insight: The newly synthesized hemagglutinin-binding peptide has significant activity compared to known hemagglutinin-binding peptides with anti-influenza virus activity.

[0023] One of the embodiments disclosed in this specification relates to a hemagglutinin-binding peptide, a pharmaceutically acceptable salt thereof, or a solvate thereof (these will also be referred to as the peptides of the present invention).

[0024] The hemagglutinin-binding peptide is any one of the following peptides (1) to (7).

[0025] (1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2:

[0026] Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala-Cys (SEQ ID NO: 1),

[0027] Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala-Cys-Lys (SEQ ID NO: 2).

[0028] (2) A polypeptide consisting of an amino acid sequence in which Trp at the N-terminus in SEQ ID NO: 1 or 2 is chloroacetyl-Trp.

[0029] (3) A polypeptide consisting of an amino acid sequence in which Trp at the N-terminus in SEQ ID NO: 1 is chloroacetyl-Trp and the C-terminal Cys is modified with the formula (I) by an amide bond.

[0030] (4) A polypeptide consisting of an amino acid sequence in which Trp at the N-terminus in SEQ ID NO: 2 is chloroacetyl-Trp and the C-terminal Lys is replaced with a lysine derivative modified with the formula (II).

[0031] (5) A polypeptide consisting of an amino acid sequence in which the C-terminal Cys in SEQ ID NO: 1 is modified with the formula (I) by an amide bond.

[0032] (6) A polypeptide consisting of a sequence in which the side chain of Lys in SEQ ID NO: 2 contains a lysine derivative of the formula (II) modified with an acyl group.

[0033] (7) A peptide having any one of the amino acid sequences (1) to (6) above and having an amino acid sequence in which 1 or 2 amino acids are deleted, added, substituted, or inserted (wherein, deletion of the C-terminal Cys in SEQ ID NO: 1 and deletion of the C-terminal Lys in SEQ ID NO: 2 are excluded).

[0034] Formula (I)

[0035] [Chemical Formula 9]

[0036]

[0037] (In formula (I), * represents the linking portion to the carbonyl group of the C-terminal Cys, and A 1 represents C8-C 12 alkyl).

[0038] Formula (II)

[0039] [Chemical Formula 10]

[0040]

[0041] (In formula (II), * represents the linking portion to the carbonyl group of the C-terminal Cys, and A 1 represents C8-C 12 alkyl).

[0042] Preferred examples of the hemagglutinin-binding peptide are as follows.

[0043] (1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2.

[0044] (5) A polypeptide consisting of the amino acid sequence in SEQ ID NO: 1 in which the C-terminal Cys is modified as shown in formula (I) by an amide bond.

[0045] (6) A polypeptide consisting of the sequence in SEQ ID NO: 2 in which the C-terminal Lys is replaced with a lysine derivative modified as shown in formula (II); or

[0046] (7) A peptide having any one of the amino acid sequences in (1), (5), and (6) above, and having an amino acid sequence in which 1 or 2 amino acids are deleted, added, substituted, or inserted (wherein, deletion of the C-terminal Cys in SEQ ID NO: 1 and deletion of the C-terminal Lys in SEQ ID NO: 2 are excluded).

[0047] A preferred example of the hemagglutinin-binding peptide is that the hemagglutinin-binding peptide is cyclic.

[0048] A preferred example of the hemagglutinin-binding peptide is represented by the following formula (III) or (IV).

[0049] [Chemical Formula 11]

[0050]

[0051] (In formula (III), formula A 2 represents the group represented by -NH2 or the group represented by formula (I)).

[0052] [Chemical Formula 12]

[0053]

[0054] (In formula (IV), formula A 3 represents a group represented by -NH2 or a group represented by formula (II)).

[0055] Another preferred example of a hemagglutinin-binding peptide different from the above is represented by the following formula (V) or (VI).

[0056] [Chemical Formula 13]

[0057]

[0058] (In formula (V), formula A 4 represents a group represented by -NH2 or a group represented by formula (IIa)).

[0059] [Chemical Formula 14]

[0060]

[0061] (IIa)

[0062] (In formula (IIa), * represents the linking moiety, and A 1 represents C8 - C 12 alkyl).

[0063] [Chemical Formula 15]

[0064]

[0065] In formula (VI), formula A 5 represents a group represented by -NH2 or a group represented by formula (I). Another preferred example of a hemagglutinin-binding peptide different from the above is represented by the following formula (VII).

[0066] [Chemical Formula 16]

[0067]

[0068] Another aspect different from the above in this specification is a drug for preventing or treating viral infectious diseases, which comprises any one of the above hemagglutinin-binding peptides, a pharmaceutically acceptable salt thereof, or a solvate thereof. Another aspect is a drug for preventing or treating influenza (a prophylactic or therapeutic agent for influenza, a medicine), which comprises any one of the above hemagglutinin-binding peptides, a pharmaceutically acceptable salt thereof, or a solvate thereof.

[0069] Another embodiment disclosed in this specification is directed to a virus detection drug. As another aspect, it is an influenza virus detection drug. This virus detection drug contains the above-mentioned hemagglutinin-binding peptide.

[0070] Another embodiment disclosed in this specification is directed to a virus detection kit, which contains the above-mentioned virus detection drug.

[0071] Advantages of the Invention

[0072] As shown in the examples, this application can provide a peptide with significantly higher activity than iHA100, a drug for preventing or treating influenza using this peptide, a detection drug for influenza, and an intermediate for synthesizing the above peptide. Brief Description of the Drawings

[0073] Figure 1-1 A graph showing the results of the activity evaluation of iHA100 and HA152 using influenza virus A / Nagasaki / HA-58 / 2009 (H1N1) in in vitro experiments.

[0074] Figure 1-2 A graph showing the results of the activity evaluation of iHA100 and HA152 using influenza virus A / Puerto Rico / 8 / 34 (H1N1) in in vitro experiments.

[0075] Figure 1-3 A graph showing the results of the activity evaluation of iHA100 and HA152 using influenza virus A / Duck / Pennsylvania / 84 (H5N2) in in vitro experiments.

[0076] Figure 2 For iHA100 and HA119, HA145, HA146, HA151, HA152 using influenza virus A / Duck / Pennsylvania / 84 (H5N2), the results of their antiviral activities in in vitro experiments are shown by EC50 values.

[0077] Figure 3 A graph showing the results of analyzing the antiviral activity of HA152 in in vivo experiments using an influenza virus A / Puerto Rico / 8 / 34 (H1N1) infection model. Detailed Description of the Invention

[0078] Hereinafter, the modes for carrying out the present invention will be described. The present invention is not limited to the modes described below, and also includes those appropriately modified by those skilled in the art within the known scope from the following embodiments.

[0079] One of the embodiments disclosed in this specification is directed to a hemagglutinin-binding peptide, its pharmaceutically acceptable salt, or its solvate.

[0080] In this specification, hemagglutinin refers to an antigenic glycoprotein present on the surface of most bacteria or viruses represented by influenza virus, and is denoted by "HA". Hemagglutinin is involved in the adhesion process of the virus to host cells. Specifically, if the hemagglutinin on the virus surface binds to sialic acid present on the surface of the target host cell, the virus is enveloped by the cell membrane and enters the cell in the form of an endosome containing the virus. Subsequently, the endosomal membrane fuses with the viral membrane, and the viral genome enters the cell and begins to replicate.

[0081] Influenza viruses are classified into three types: type A, type B, and type C. Among the hemagglutinins of type A influenza viruses, which are particularly prone to causing epidemics, there are at least 16 subtypes, designated H1 to H16. In addition, H1, 2, 5, 6, 8, 9, 11, 12, 13, 16, 17, 18 are referred to as group 1, and the other hemagglutinins (H3, 4, 7, 10, 14, 15) are referred to as group II. The H in the subtype name of influenza represents hemagglutinin.

[0082] A hemagglutinin-binding peptide means a peptide that can bind to hemagglutinin. Whether it can bind to hemagglutinin can be confirmed by those skilled in the art according to known methods.

[0083] Its pharmaceutically acceptable salt means a pharmaceutically acceptable salt of the hemagglutinin-binding peptide. Examples of salts include: 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-bromobenzenesulfonic acid, carboxylic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc.), inorganic bases (ammonium hydroxide or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, etc.), addition salts of amino acids, etc.

[0084] Its pharmaceutically acceptable solvate means a pharmaceutically acceptable solvate of the hemagglutinin-binding peptide or a pharmaceutically acceptable solvate of a salt of the hemagglutinin-binding peptide. The solvent molecule can be one that has coordinated with the compound or its salt, and examples of solvates are hydrates and alcoholates.

[0085] The hemagglutinin-binding peptide is any one of the following peptides (1) to (7).

[0086] (1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2:

[0087] Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala-Cys (SEQ ID NO: 1),

[0088] Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala-Cys-Lys (SEQ ID NO: 2).

[0089] (2) A polypeptide consisting of an amino acid sequence in which Trp at the N-terminus in SEQ ID NO: 1 or 2 is chloroacetyl-Trp.

[0090] (3) A polypeptide consisting of an amino acid sequence in which Trp at the N-terminus in SEQ ID NO: 1 is chloroacetyl-Trp and the C-terminal Cys is modified by an amide bond with the modification shown in Formula (I).

[0091] (4) A polypeptide consisting of an amino acid sequence in which Trp at the N-terminus in SEQ ID NO: 2 is chloroacetyl-Trp and the C-terminal Lys is replaced with a lysine derivative modified by the modification shown in Formula (II).

[0092] (5) A polypeptide consisting of an amino acid sequence in which the C-terminal Cys in SEQ ID NO: 1 is modified by an amide bond with the modification shown in Formula (I).

[0093] (6) A polypeptide consisting of an amino acid sequence in which the side chain of Lys in SEQ ID NO: 2 contains a lysine derivative shown in Formula (II) modified with an acyl group.

[0094] (7) A peptide having any one of the amino acid sequences in (1) to (6) above and being an amino acid sequence in which 1 or 2 amino acids are deleted, added, substituted or inserted (wherein, deletion of C-terminal Cys in SEQ ID NO: 1 and deletion of C-terminal Lys in SEQ ID NO: 2 are excluded).

[0095] Formula (I)

[0096] [Chemical Formula 17]

[0097]

[0098] (In Formula (I), * represents the linking portion to the carbonyl group of the C-terminal Cys, and A 1 represents C8-C 12 alkyl). C8-C 12 alkyl is an alkyl having 8 to 12 carbon atoms. C8-C 12 alkyl can be a straight-chain alkyl or a branched alkyl. C8-C 12 alkyl can be any one of C8 alkyl, C9 alkyl, C 10 alkyl, C 11 alkyl and C 12 alkyl. A 1 in each of the following groups is also the same.

[0099] Formula (II)

[0100] [Chemical Formula 18]

[0101]

[0102] (In formula (II), * represents the connecting part to the carbonyl group of the C-terminal Cys, and A 1 represents C8-C 12 alkyl).

[0103] Preferred examples of the hemagglutinin-binding peptide are as follows.

[0104] (1) A polypeptide composed of the amino acid sequence shown in SEQ ID NO: 1 or 2.

[0105] (5) A polypeptide composed of the amino acid sequence in which the C-terminal Cys in SEQ ID NO: 1 is modified with the formula (I) by an amide bond.

[0106] (6) A polypeptide composed of the sequence in which the C-terminal Lys in SEQ ID NO: 2 is replaced with a lysine derivative modified with the formula (II); or

[0107] (7) A peptide having any one of the amino acid sequences in (1), (5), and (6) above, and having an amino acid sequence in which 1 or 2 amino acids are deleted, added, substituted, or inserted (wherein, those in which the C-terminal Cys in SEQ ID NO: 1 is deleted and those in which the C-terminal Lys in SEQ ID NO: 2 is deleted are excluded).

[0108] Examples of the specific amino acid sequences of this peptide are as follows.

[0109] Chloroacetyl-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala-Cys-NH2 (SEQ ID NO: 3)

[0110] Chloroacetyl-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala-Cys-Lys[gamma-C(=O)n-C 11 H 23 -NH2 (SEQ ID NO: 4)

[0111] Chloroacetyl-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala-Cys-Lys[gamma-C(=O)n-C9H19 -NH2 (SEQ ID NO: 5)

[0112] Chloroacetyl-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala-Cys-[NHCH2CH2NHC(=O)n-C 11 H 23 (SEQ ID NO: 6)

[0113] Chloroacetyl-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala-Cys-[NHCH2CH2NHC(=O)n-C9H 19 (SEQ ID NO: 7)

[0114] In this specification, the amino acid (protein amino acid) residues of the protein that constitutes the peptide and polypeptide of the present invention are represented by the three-letter symbols or single-letter symbols accepted in the art.

[0115] As another amino acid disclosed in this specification, examples include: amino acids that do not constitute proteins (also known as non-protein amino acids, simply referred to as non-natural amino acids); or chemically synthesized compounds having the characteristics of amino acids known in the art. Examples of non-natural amino acids include: α,α-disubstituted amino acids (such as α-methylalanine) with a backbone structure different from the natural type, N-alkyl-α-amino acids, N-alkyl-α-D-amino acids, β-amino acids; and amino acids with a side-chain structure different from the natural type (such as norleucine, homohistidine, hydroxyproline, etc.), but are not limited thereto.

[0116] As an example of the non-natural amino acids disclosed in this specification, N-methylglycine, an N-alkyl-α-amino acid, is represented as MeGly, N-methylalanine is represented as MeAla, and N-methylphenylalanine is represented as MePhe. Also, as an example of an amino acid with a side-chain structure different from the natural type, 4R-hydroxyproline is represented as hydPro.

[0117] Chloroacetyl- means chloroacetylated, and Chloroacetyl-Trp represents chloroacetyl-Trp.

[0118] In this specification, a polypeptide refers to two or more amino acids bonded by peptide bonds. For example, it can be formed by peptide bonding of 8-30 amino acids, and can be linear or cyclic. Preferably, it is a cyclic amino acid of 15 or 16 amino acids.

[0119] Furthermore, the hemagglutinin-binding peptide of the present invention can also be cyclized (macrocylized). In the present specification, cyclization means that in one peptide, two amino acids separated by more than one amino acid are directly or indirectly bound through a linker or the like to form a cyclic structure within the molecule.

[0120] Cyclization can be carried out by a known method. For example, it can be carried out according to the method described in the pamphlet of International Publication WO2016 / 063969.

[0121] A preferred example of the hemagglutinin-binding peptide is represented by the following formula (III) or (IV).

[0122] [Chemical formula 19]

[0123]

[0124] (In formula (III), formula A 2 represents a group represented by -NH2 or a group represented by formula (I).)

[0125] [Chemical formula 20]

[0126]

[0127] (In formula (IV), formula A 3 represents a group represented by -NH2 or a group represented by formula (II).)

[0128] Another preferred example of the hemagglutinin-binding peptide different from the above is represented by the following formula (V) or (VI).

[0129] [Chemical formula 21]

[0130]

[0131] (In formula (V), formula A 4 represents a group represented by -NH2 or a group represented by formula (IIa).)

[0132] [Chemical formula 22]

[0133]

[0134] (In formula (IIa), * represents a linking part, and A 1 represents C8-C 12 alkyl.)

[0135] [Chemical formula 23]

[0136]

[0137] In formula (VI), formula A5 represents a group represented by -NH2 or a group represented by formula (I). Another preferred example of a hemagglutinin-binding peptide different from the above is represented by the following formula (VII).

[0138] [Chemical formula 24]

[0139]

[0140] The specific structure of the cyclic peptide is as follows.

[0141] [Chemical formula 25]

[0142]

[0143] [Chemical formula 26]

[0144]

[0145] [Chemical formula 27]

[0146]

[0147] [Chemical formula 28]

[0148]

[0149] [Chemical formula 29]

[0150]

[0151] The peptide of the present invention can be produced by known peptide production methods such as chemical synthesis methods such as liquid phase method, solid phase method, and a mixed method combining liquid phase method and solid phase method; genetic recombination method, etc.

[0152] In the solid phase method, for example, an esterification reaction is carried out between the hydroxyl group of a resin having a hydroxyl group and the carboxyl group of a first amino acid (usually the C-terminal amino acid of the target peptide) whose α-amino group is protected by a protecting group. As the esterification catalyst, known dehydrating condensing agents such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIPCDI) can be used.

[0153] Next, the protecting group of the α-amino group of the first amino acid is removed, and a second amino acid in which all functional groups other than the carboxyl group of the main chain are protected is added, and the carboxyl group is activated so that the first and second amino acids are combined. Then, the α-amino group of the second amino acid is deprotected, and a third amino acid in which all functional groups other than the carboxyl group of the main chain are protected is added, and the carboxyl group is activated so that the second and third amino acids are combined. This operation is repeated, and after synthesizing a peptide of the target length, all functional groups are deprotected.

[0154] Examples of the resin for solid-phase methods include Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGA resin (Merck KGaA), HMPA-PEGA resin (Merck KGaA), etc. These resins can be used after being washed with solvents (dimethylformamide (DMF), 2-propanol, dichloromethane, etc.).

[0155] The protecting group for the α-amino group is not particularly limited as long as it is a known protecting group. Examples include benzyloxycarbonyl (Cbz or Z), tert-butoxycarbonyl (Boc), fluorenylmethoxycarbonyl (Fmoc), benzyl, allyl, allyloxycarbonyl (Alloc), etc. In addition, the Cbz group can be deprotected by hydrofluoric acid, hydrogenation, etc., the Boc group can be deprotected by trifluoroacetic acid (TFA), and the Fmoc group can be deprotected by treatment with piperidine.

[0156] The protecting group for the α-carboxyl group is not particularly limited as long as it is a known protecting group. Examples include methyl ester, ethyl ester, benzyl ester, tert-butyl ester, cyclohexyl ester, etc.

[0157] The other functional groups of the amino acid are not particularly limited. For example, the hydroxyl group of serine or threonine can be protected with benzyl or tert-butyl, and the hydroxyl group of tyrosine is protected with 2-bromobenzyloxycarbonyl or tert-butyl. The amino group in the side chain of lysine and the carboxyl group of glutamic acid or aspartic acid can be protected in the same manner as the α-amino group and α-carboxyl group.

[0158] A condensing agent can be used to activate the carboxyl group. Examples of the condensing agent include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU), etc.

[0159] The peptide chain can be cleaved from the resin by treatment with an acid such as TFA, hydrogen fluoride (HF), etc.

[0160] To produce a peptide using genetic recombination methods (translation synthesis systems), a nucleic acid encoding the peptide of the present invention can be used. The nucleic acid encoding the peptide of the present invention can be DNA or RNA.

[0161] Nucleic acids encoding the peptides of the present invention can be prepared by known methods or methods based thereon. For example, they can be synthesized by an automatic synthesizer. Restriction enzyme recognition sites can also be added to insert the resulting DNA into a vector; or nucleic acid sequences can be incorporated to encode amino acid sequences for excising the formed peptide chain with an enzyme or the like.

[0162] As described above, when the peptide of the present invention is fused with a cell-penetrating peptide or the like, the above nucleic acid also contains a nucleic acid encoding the cell-penetrating peptide.

[0163] In order to inhibit degradation caused by proteases from the host, a chimeric protein expression method in which the target peptide is expressed as a chimeric peptide with another peptide can also be used. In this case, as the above nucleic acid, a nucleic acid encoding the target peptide and a peptide bound thereto can be used.

[0164] Next, an expression vector is prepared using the nucleic acid encoding the peptide of the present invention. The nucleic acid can be inserted directly, digested with a restriction enzyme, or ligated with a linker, etc., downstream of the promoter of the expression vector. Examples of vectors include: plasmids from Escherichia coli (pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pUC118, pBluescript II, etc.), plasmids from Bacillus subtilis (pUB110, pTP5, pC1912, pTP4, pE194, pC194, etc.), plasmids from yeast (pSH19, pSH15, YEp, YRp, YIp, YAC, etc.), phages (e phage, M13 phage, etc.), viruses (retrovirus, poxvirus, adenovirus, adeno-associated virus (AAV), cauliflower mosaic virus, tobacco mosaic virus, rod-shaped virus, etc.), cosmids, etc.

[0165] The promoter can be appropriately selected according to the type of host. When the host is an animal cell, for example, a promoter from SV40 (simian virus 40), a promoter from CMV (cytomegalovirus) can be used. When the host is Escherichia coli, the trp promoter, T7 promoter, lac promoter, etc. can be used.

[0166] The expression vector can also incorporate nucleic acids encoding a DNA replication origin (ori), a selection marker (antibiotic resistance, auxotrophy, etc.), an enhancer, a splicing signal, a poly(A) tailing signal, a tag (FLAG, HA, GST, GFP, etc.), etc.

[0167] Next, transform an appropriate host cell with the above expression vector. The host can be appropriately selected according to the relationship with the vector. For example, Escherichia coli, Bacillus subtilis, Bacillus spp., yeast, insects or insect cells, animal cells, etc. can be used. As animal cells, for example, HEK293T cells, CHO cells, COS cells, myeloma cells, HeLa cells, Vero cells can be used. Transformation can be carried out according to known methods such as transfection, calcium phosphate method, electroporation method, microinjection method, particle gun method, etc. according to the type of host. By culturing the transformant according to the conventional method, the target peptide can appear.

[0168] To purify the peptide from the culture of the transformant, the cultured cells are recovered, suspended in an appropriate buffer, and then the cells are disrupted by methods such as ultrasonic treatment, freeze-thaw, etc., and a crude extract is obtained by centrifugation or filtration. When the peptide is secreted into the culture medium, the supernatant is recovered.

[0169] Purification from the crude extract or culture supernatant can also be carried out by known methods or methods based on them (for example, salting out, dialysis, ultrafiltration, gel filtration, polyacrylamide gel electrophoresis, ion exchange chromatography, affinity chromatography, reverse phase high performance liquid chromatography, etc.).

[0170] The obtained peptide can be converted from the free state to a salt or from a salt to the free state by known methods or methods based on them.

[0171] The translation synthesis system can also be a cell-free translation system. The cell-free translation system includes, for example, ribosomal proteins, aminoacyl-tRNA synthetase (ARS), ribosomal RNA, amino acids, rRNA, GTP, ATP, translation initiation factor (IF), elongation factor (EF), termination factor (RF), and ribosome recycling factor (RRF), as well as other factors required for translation. To improve the expression efficiency, an Escherichia coli extract or wheat germ extract can also be added. In addition, a rabbit red blood cell extract or an insect cell extract can also be added.

[0172] By continuously supplying energy to the system containing these components by using dialysis, peptides can be produced at a concentration of several hundred micrograms to several milligrams per milliliter. To perform transcription from gene DNA at the same time, it can also be a system containing RNA polymerase. As commercially available cell-free translation systems, the RTS-100 (registered trademark) from Roche Diagnostics, the PURESYSTEM from GeneFrontier, or the PURExpress in vitro protein synthesis kit from NEW ENGLAND Biolabs, which are systems from Escherichia coli, can be used, as well as products from ZOEGENE or Cell-Free Sciences Co., Ltd., which are systems using wheat germ extract.

[0173] According to the cell-free translation system, a highly pure form can be obtained without refining the expressed product.

[0174] In the cell-free translation system, an artificial aminoacyl-tRNA in which the required amino acid or hydroxy acid is linked (acylated) to tRNA can also be used instead of the aminoacyl-tRNA synthesized by a natural aminoacyl-tRNA synthetase. This aminoacyl-tRNA can be synthesized using an artificial ribozyme.

[0175] Examples of the ribozyme include: flexizyme (H. Murakami, H. Saito, and H. Suga, (2003), Chemistry & Biology, Vol. 10, 655-662; H. Murakami, D. Kourouklis, and H. Suga, (2003), Chemistry & Biology, Vol. 10, 1077-1084; H. Murakami, A. Ohta, H. Ashigai, H. Suga (2006) Nature Methods 3, 357-359 “The flexizyme system: a highly flexible tRNA aminoacylation tool for the synthesis of nonnatural peptides”; N. Niwa, Y. Yamagishi, H. Murakami, H. Suga (2009) Bioorganic & Medicinal Chemistry Letters 19, 3892-3894 “A flexizyme that selectively charges amino acids activated by a water-friendly leaving group”; and WO2007 / 066627, etc.). Flexizyme is also known by names such as prototype flexizyme (Fx), dinitrobenzyl flexizyme (dFx) which is a variant thereof, enhanced flexizyme (eFx), amino flexizyme (aFx), etc.

[0176] By using tRNA prepared with flexizyme and linked to the required amino acid or hydroxy acid, the required codon can be associated with the required amino acid or hydroxy acid and translation can be carried out. As the required amino acid, special amino acids can also be used. For example, the non-natural amino acid required for the above cyclization can also be introduced into the hemagglutinin-binding peptide by this method.

[0177] The chemical synthesis of the macrocyclic peptides and their analogs of the present invention can be carried out using various methods commonly used in this technical field, including stepwise solid-phase synthesis, semi-synthesis of peptide segments via conformationally supported ligation, and chemical ligation. The synthesis of the peptides and their analogs described in this specification is, for example, chemical synthesis using various solid-phase techniques described in K.J. Jensen, P.T. Shelton, S.L. Pedersen, Peptide Synthesis and Applications, 2nd Edition, Springer, 2013, etc. As a preferred strategy, it is based on the Fmoc group that can temporarily protect the α-amino group and be selectively removed by a base, and a protecting group that can temporarily protect the side-chain functional groups and is stable under Fmoc-removing conditions. The selection of such general peptide side chains is known in the above-mentioned Peptide Synthesis and Applications, 2nd edition or G.B. Fields, R.L. Noble, Solid Phase Peptide Synthesis Utilizing 9-Fluorenylmethoxycarbonyl Amino Acids, Int. J. Peptide Protein Res. 35, 1990, 161-214, etc. However, preferred peptide side-chain protecting groups include: the Boc group or the Mtt group for the amino group represented by lysine, the tert-butyl group for the carboxyl group of glutamic acid or aspartic acid, and the Trt and Mmt groups for the thiol group of cysteine.

[0178] The resin (or simply referred to as resin) that serves as a precursor for peptide synthesis in the present invention can be prepared by using, for example, commercially available PAL-PEG-resin or PAL-PEG-PS, referring to the reported examples in Biopolymers 2011; 96(6): 715-22, etc. For example, as outlined in Scheme 1, n represents the length of the carbon number; R1 represents hydrogen or an alkyl group with 1-4 carbon atoms; R2 represents various alkyl chains or alkyl chains with substituents. In the presence of an appropriate base, the amino group of resin 1 is sulfonylated with o-Ns-Cl to prepare 2. An amino alcohol 3 with the N-terminal amino group protected by Fmoc, such as Fmoc-glycol, is combined with the reagents used in the Mitsunobu reaction, for example, made to react in the presence of triphenylphosphine and diisopropyl azodicarboxylate, etc., whereby the solid-phase resin 4 can be obtained. The deprotection of Fmoc can be carried out in an appropriate combination of secondary amine and solvent, for example, in a piperidine DMF solution to form 5, and various acyl groups are introduced into the regenerated amine using a known peptide coupling method to form 6. Regarding R2, it can be introduced by making, for example, decanoic acid with a long-chain alkyl group (formula CH3(CH2)8COOH) react in the presence of a condensing agent HATU and DIPEA. Finally, the o-Ns group is removed by treating with an appropriate base and thiol group, such as a combination of DBU and DODT, whereby the resin 7 serving as a precursor for peptide synthesis can be prepared.

[0179] [Chemical formula 30]

[0180]

[0181] For peptide synthesis in the present invention, commercially available resins can also be used as other methods. For example, as outlined in scheme-2, as another preparation method for a preferred precursor resin, commercially available RinkAmide MBHA resin (Sigma-Aldrich), Fmoc-Rink Amide NovaPEGresin (Merck Millipore), or Fmoc-NH-SAL-PEG resin (Watanabe Chemical) can be used as the solid-phase resin (8). After removing Fmoc, it is condensed with Fmoc amino acid 9, and the Fmoc amino acid 9 has a primary or secondary amino group in the side chain, and the primary or secondary amino group has a protecting group that can be removed under mild acidic conditions, such as Fmoc-Lys(Mtt)-OH (m = 3, R 3 = H, S-configuration). The obtained solid-phase resin is treated with a solution of TFA / TIS / CH2Cl2 in a volume ratio of 1:4:95, whereby the Mmt group can be selectively removed to form 11. Then, the above R2COOH can be introduced to form 12, and the Fmoc group is removed to form 13, which can be used for the target peptide synthesis.

[0182] [Chemical Formula 31]

[0183]

[0184] The peptides and their analogs described in the present invention can be synthesized on the above solid-phase resin in a stepwise method. The C-terminal amino acid used and all the amino acids or peptides used for synthesis must selectively remove the α-amino protecting group during the synthesis process. Preferably, the above solid-phase resin is used, and after the C-terminal carboxyl group of the peptide with the N-terminal appropriately protected with a protecting group such as Fmoc or the C-terminal carboxyl group of the amino acid appropriately protected with a protecting group such as Fmoc is formed into an activated ester by an appropriate reagent, it is started by adding to the amino group on the solid-phase resin. Then, the extension of the peptide chain can be achieved by sequentially repeating the removal of the N-terminal protecting group (for example, the Fmoc group), followed by the condensation of the protected amino acid derivative, according to the amino acid sequence of the target peptide. In addition, these operations can free the target peptide in the final stage. For example, as the conditions for freeing it, as listed in Teixeira, W.E., Benckhuijsen, P.E., de Koning, A.R.P.M., Valentijn, J.W., Drijfhout, Protein Pept. Lett., 2002, 9, 379 - 385, etc., it can be freed in TFA under a TFA solution containing water / silyl hydride / thiol group as a scavenger. As a typical example, TFA / Water / TIS / DODT (volume ratio 92.5:2.5:2.5:2.5) can be cited.

[0185] The synthesis of the peptide analogs described in this specification can be carried out by using a single or multi-channel peptide synthesizer, such as the Liberty Blue synthesizer of CEM Corporation or the Syro I synthesizer of Biotage Company.

[0186] A condensing agent can be used for the activation of the carboxyl group. As the condensing agent, for example, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU), etc. can be cited.

[0187] Another embodiment disclosed in this specification is about a drug. The drug contains the above hemagglutinin-binding peptide, its pharmaceutically acceptable salt or solvate (for convenience, these will also be abbreviated as hemagglutinin-binding peptides hereinafter). The drug preferably uses the above hemagglutinin-binding peptide as the active ingredient and contains an effective amount.

[0188] When using a hemagglutinin-binding peptide for pharmaceutical use, in order to impart functions or various changes to one or more amino acids of the peptide, chemical modifications can be applied, for example, using hydrocarbons, fatty acids, polyethylene glycol (PEG), etc. Also, these chemical modifications can also be carried out using a linker. Through these modifications, for example, a peptide that is more chemically and metabolically stable can be obtained. The linker is a cyclic peptide that can exhibit antiviral activity (such as the basic structure shown in (II)) and a substructure that can undergo chemical modifications for function imparting. Specific examples of the basic structure in the present invention are the 1,2-ethylenediamine structure shown in (I) or the lysine amide structure with an NH2 at the C-terminus shown in (II).

[0189] As shown in the following examples, the peptide of the present invention exhibits antiviral activity by binding to hemagglutinin on the virus surface. In addition, as long as it exhibits antiviral activity against viruses that infect humans, etc. through hemagglutinin or a similar protein, the type of virus is not particularly limited. Examples of the type of virus are, for example, enveloped viruses, and more preferably viruses having class I fusion proteins. Specific examples of the virus are seasonal influenza (including human, avian, swine influenza viruses or novel influenza viruses, also simply referred to as influenza virus), highly pathogenic avian influenza, swine epidemic diarrhea influenza, HIV-1, Ebola virus, yellow fever virus, and novel coronavirus. The virus to be targeted is preferably the influenza virus. Also, although the influenza virus to be targeted can be any of types A, B, and C, type A is preferred, and more preferably an influenza virus having hemagglutinin belonging to group I. In addition, in this specification, anti-influenza virus activity is also referred to as antiviral activity.

[0190] The composition containing the peptide of the present invention is very useful as a prophylactic agent for virus infection or a therapeutic agent for virus infectious diseases, and preferably as a prophylactic or therapeutic agent for influenza. Similarly, the peptide of the present invention is very useful in the method for preventing virus infection or treating virus infectious diseases, and preferably in the method for preventing or treating influenza.

[0191] In this specification, influenza refers to an acute infectious disease caused by the influenza virus. When infected with the influenza virus, the human body will show cold symptoms accompanied by fever, muscle soreness, etc. Sometimes it will be accompanied by gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea, and the complications are pneumonia and influenza encephalitis.

[0192] In this specification, infection is used as a term that means either the process by which a virus invades a living body through the skin or mucous membrane, or the process by which a virus invades a cell through membrane fusion. Also, in this specification, virus infection refers to the state in which a virus invades the body regardless of whether there are symptoms or not. Also, in this specification, infection symptoms refer to various symptoms caused by virus infection.

[0193] In this specification, the treatment or prevention of influenza is used in its broadest sense, for example, it means producing alleviation of one or more symptoms associated with infection by influenza virus or preventing its deterioration, suppressing the onset of symptoms after infection, preventing (delaying or stopping) the infection of cells by the virus in the body, preventing (delaying or stopping) the reproduction of the virus in the body, reducing the number of viruses in the body, etc. When at least one of these effects is shown, it is judged to be useful for the treatment or prevention of influenza.

[0194] Furthermore, as shown in the following examples, the present invention is such that the peptide has neutralizing activity against hemagglutinin, and thus it is understood that the same effects as those of an influenza vaccine can be obtained.

[0195] In this specification, the dosage form of the pharmaceutical composition is not particularly limited and may be oral administration or parenteral administration. Examples of parenteral administration include injection administration such as intramuscular injection, intravenous injection, subcutaneous injection, transdermal administration, transmucosal administration (administration via nose, mouth, eye, lung, vagina, rectum), etc.

[0196] In view of the property that polypeptides are easily metabolized and excreted, the above-mentioned pharmaceutical composition can be subjected to various modifications. For example, polyethylene glycol (PEG) or sugar chains can be added to the polypeptide to prolong the retention time in the blood and reduce antigenicity. Furthermore, biodegradable high molecular compounds such as polylactic acid - glycolic acid (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, nanospheres, etc. can be used as sustained-release bases and the polypeptide can be incorporated therein. When performing transdermal administration, a weak electric current can also be applied to the skin surface to penetrate the stratum corneum (iontophoresis).

[0197] For the above-mentioned pharmaceutical composition, the active ingredient can be used directly, or it can be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc. Examples of dosage forms include liquid preparations (such as injections), dispersions, suspensions, lozenges, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, tablets, inhalants, ointments, eye drops, nasal drops, ear drops, cataplasms, etc.

[0198] For formulation, for example, excipients, binders, disintegration adjuvants, lubricants, solubilizers, solubilization aids, coloring agents, flavoring and odor-correcting agents, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, preservatives, antioxidants, etc. can be appropriately used and carried out by conventional methods.

[0199] Examples of ingredients for formulation include, but are not limited to: distilled water, saline, phosphate buffer, glucose, glycerin, pharmaceutically acceptable organic solvents such as ethanol, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, anhydrous silicic acid, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum arabic, tragacanth gum, casein, agar, polyethylene glycol, diglycerol, glycerol, propylene glycol, petrolatum, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, human serum albumin, etc.

[0200] Since peptides are not easily absorbed through the mucosa, the above drug compositions may contain absorption promoters that improve the absorption of poorly absorbable drugs. As such absorption promoters, surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponins can be used; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylic acids; fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, and mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitin polysaccharides, nitric oxide donors, etc.

[0201] Pills or lozenges can also be coated with sugar coatings, gastric-soluble, and enteric-soluble substances.

[0202] Injections may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oils, alcohols, etc. Furthermore, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, solubilization aids, preservatives, etc. can be added.

[0203] The dosage of the drug composition of the present invention when administered to mammals (e.g., humans, mice, rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs, etc.), particularly to humans, varies depending on symptoms, the age, sex, weight, sensitivity differences of the patient, the administration method, the administration interval, the type of active ingredient, and the type of formulation, and is not particularly limited. For example, it can be administered once or in multiple doses of 30 μg to 100 g, 100 μg to 500 mg, 100 μg to 100 mg. When administered by injection, depending on the patient's weight, it can be administered once or in multiple doses of 1 μg / kg to 3000 μg / kg, 3 μg / kg to 1000 μg / kg.

[0204] The method for preventing or treating influenza using the peptide of the present invention can be implemented with reference to the description of the above drug composition.

[0205] Another embodiment disclosed in this specification relates to a virus detection drug, particularly to an influenza virus detection drug. This virus detection drug contains the above-mentioned hemagglutinin-binding peptide, its salt, or its solvate.

[0206] (Virus Detection Drug and Detection Kit)

[0207] The present invention also includes a virus detection drug containing the peptide of the present invention, particularly an influenza virus detection drug containing the peptide of the present invention. The peptide of the present invention specifically binds to hemagglutinin on the virus surface. Therefore, for example, the peptide of the present invention can be used to detect influenza virus in a sample instead of the anti-influenza antibody in immunoassays such as enzyme-linked immunosorbent assay.

[0208] When used as a detection drug, the peptide of the present invention can also be labeled to be detectable. The peptide can be labeled with known labeling substances. For example, antibodies labeled with enzymes such as peroxidase and alkaline phosphatase, radioactive substances such as 125I, 131I, 35S, and 3H, fluorescent substances such as fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine isothiocyanate, and near-infrared fluorescent materials, and luminescent substances such as luciferase, luciferin, and aequorin can be used. In addition, antibodies labeled with nanoparticles such as gold colloids and quantum dots can also be detected.

[0209] Furthermore, in immunoassays, the peptide of the present invention can also be labeled with biotin and bound to avidin or streptavidin labeled with an enzyme or the like for detection.

[0210] In immunoassays, the ELISA method using an enzyme label is preferably used as it can simply and rapidly measure the antigen. For example, an antibody that specifically recognizes a part other than hemagglutinin of the influenza virus is immobilized on a solid-phase carrier. After adding a sample and allowing it to react, the labeled peptide of the present invention is added and allowed to react. After washing, it is reacted with an enzyme substrate to develop color and measure the absorbance, thereby detecting the influenza virus. Alternatively, after allowing the antibody immobilized on the solid-phase carrier to react with the sample, the unlabeled peptide of the present invention is added, and then an antibody against the peptide of the present invention is labeled with an enzyme and further added. Also, the peptide of the present invention can be used as a capture substance and immobilized on the solid-phase carrier, and a labeled antibody that recognizes the influenza virus can be used as a detection substance. Furthermore, the peptide of the present invention can be used for both capture and detection.

[0211] When the enzyme of the enzyme substrate is peroxidase, 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), etc. can be used. When it is alkaline phosphatase, p-nitrophenyl phosphate (NPP), etc. can be used.

[0212] In this specification, the "solid phase carrier" is not particularly limited as long as it is a carrier capable of immobilizing an antibody, and examples thereof include microplates, substrates, beads, nitrocellulose membranes, nylon membranes, PVDF membranes, etc. made of glass, metal, resin, etc. The target substance can be immobilized on these solid phase carriers according to a known method.

[0213] The test kit described in the present invention includes the reagents and instruments required for the above detection (including the peptides, antibodies, solid phase carriers, buffers, enzyme reaction termination solutions, microplate analyzers, etc. of the present invention, but not limited thereto).

[0214] Another embodiment disclosed in this specification can also be considered as an influenza virus detection kit containing the above-mentioned influenza virus detection drug, and a tool for identifying the infection of influenza virus caused by hemagglutinin and various cellular functions or life phenomena associated therewith.

[0215] This specification also provides the use of hemagglutinin-binding peptides for manufacturing drugs for preventing or treating influenza. The hemagglutinin-binding peptides in this case can be any of the above.

[0216] This specification also provides a method for preventing or treating influenza, which includes the step of administering an effective amount of a hemagglutinin-binding peptide, a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient to a subject of human, non-human mammals or birds. The hemagglutinin-binding peptide can be appropriately used as the above-mentioned peptide. Examples of non-human mammals are primates other than humans, pigs, cows, dogs, cats, horses, sheep, rats and mice.

[0217] The abbreviations used in this specification, especially in the following representative examples, are well-known to those skilled in the art. Several abbreviations used are as follows: 9-fluorenylmethoxycarbonyl is Fmoc; 1-hydroxybenzotriazole is HOAt; O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate is HATU; acetonitrile is MeCN; 1,8-diazabicyclo "5.4.0"-7-undecene is DBU; N,N-diisopropylethylamine is DIPEA; 3,6-dioxa-1,8-octanedithiol is DODT; dimethyl sulfoxide is DMSO; N,N-dimethylformamide is DMF; milliliter (unit) is mL; molar concentration (unit) is M; monomethyldiphenylmethyl is Mtt; monomethoxytriphenylmethyl is Mmt; 2-nitrophenylsulfonyl is o-Ns; volume / volume is v / v; trifluoroacetic acid is TFA; triisopropylsilane is TIS; triphenylmethyl is Trt.

[0218] Examples and General Methods

[0219] All raw materials, structural units, reagents, acids, bases, solid-phase resins, and solvents used in the chemical synthesis of the present invention can be directly used as commercially available products or synthesized by those skilled in the art using organic chemistry methods. In addition, amino acids containing protecting groups are directly used as commercially available products unless otherwise specified.

[0220] In the present invention, the structure determination of the chemically synthesized peptide has confirmed the molecular weight by ESI-MS(+) in mass spectrometry analysis. This molecular weight is calculated considering the amino acids used according to the target sequence and the structural units used as required. In addition, "ESI-MS(+)" indicates electrospray ionization mass spectrometry analysis performed in the positive ion mode. The detected mass is reported in units of "m / z". In addition, compounds with a molecular weight of approximately more than 1000 have been detected at a high frequency as divalent or trivalent ions.

[0221] The purity of the peptide chemically synthesized in the present invention has been determined by any of the following analysis methods.

[0222] (Analysis conditions)

[0223] Analysis condition A

[0224] Column: CORTECS (registered trademark) UPLC (registered trademark) C18 column (NihonWaters Corporation), 1.6 μm, 2.1 x 100 mm

[0225] Mobile phase: MeCN / 0.025% TFA in H2

[0226] Temperature: 40 °C

[0227] Gradient: 5 - 95% MeCN / 0.025% TFA in H2 in 5.56 min; linear gradient

[0228] Flow rate: 0.4 mL / min

[0229] Detection method: UV 220 nm

[0230] Analysis condition B

[0231] Column: Kinetex EVO C18 2.6 μm, 2.1 ID x 150 mm, (Phenomenex Corporation)

[0232] Column temperature: 60 °C

[0233] Mobile phase A: 0.025% TFA in H2

[0234] Mobile phase B: 0.025% TFA in CH3CN

[0235] Gradient: as described in each example

[0236] Flow rate: 0.25 mL / min

[0237] Detection: PDA (225 nm)

[0238] The extension of the peptide chain in the solid-phase resin described in the present invention is carried out by using the resin described in each example as the starting material and using the commonly used peptide coupling reaction conditions and Fmoc removal reaction conditions. The reaction is carried out using Liberty Blue from CEM Corporation in accordance with the manufacturer's instructions. The general amino acids used are listed below, and the side-chain protecting groups are shown in parentheses.

[0239] Fmoc-Trp(Boc)-OH; Fmoc-Thr(tBu)-OH; Fmoc-N-Me-Gly-OH; Fmoc-Asp(OtBu)-OH; Fmoc-N-Me-Phe-OH; Fmoc-Ala-OH; Fmoc-N-Me-Ala-OH; Fmoc-His(Trt)-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Val-OH; Fmoc-HydPro(tBu)-OH; Fmoc-Cys(Trt)-OH; Fmoc-Lys(Mtt)-OH; Fmoc-Ser(tBu)-OH; Fmoc-N-Me-Ser(tBu)-OH.

[0240] The introduction of the chloroacetyl group is carried out as follows: for the solid-phase resin holding the Fmoc-protected peptide obtained in the previous step, after removing the Fmoc group of the α-amino group by the above method, chloroacetic acid (about 3 equivalents), a DMF solution (0.5 M) of about 3 equivalents of N,N'-diisopropylcarbodiimide, and a DMF solution (0.5 M) of about 3 equivalents of HOAt are added, and the mixture is shaken at room temperature for 40 minutes.

[0241] Deprotection of the side chain and cleavage from the solid-phase resin were carried out as follows: The resin obtained after the chloroacetyl group introduction step was washed 5 times each with DMF and dichloromethane and dried under reduced pressure. Subsequently, the reaction agent mixture - A (a mixture of TFA / H2 / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin, and it was shaken at room temperature for 150 minutes. The reaction solution was filtered and recovered from the frit. After the solid-phase resin remaining in the reaction vessel was shaken again with the cleavage mixture, the solution components were recovered from the frit and mixed with the above filtrate. When the filtrate was added to an excess of diethyl ether cooled to 0 °C, a turbid precipitate was formed. The mixture was centrifuged (9000 rpm, 3 min), and the solution was decanted. The obtained solid was washed with a small amount of diethyl ether cooled again to 0 °C, and the obtained solid was used for the next cyclization reaction.

[0242] In the present invention, the cyclization reaction of the peptide was carried out as follows: The final concentration of the peptide was adjusted to 5 mM based on the number of moles of the solid-phase resin, dissolved in DMSO, 6 equivalents of triethylamine was added, and it was stirred at room temperature for about 16 hours. The resulting reaction solution was made acidic with acetic acid and concentrated under reduced pressure using Biotage (registered trademark) V-10 (Biotage Japan Co., Ltd.).

[0243] As a method for purifying the obtained crude and refined peptide, reverse-phase separation HPLC was used in a Waters Corporation automated purification system - SQD2 single quadrupole mass spectrometer, and elution was carried out while monitoring the m / z ions from the target substance. It was confirmed that the mass spectrum obtained in the ESI-positive scan mode was consistent within the error range of the mass analyzer with the mass spectrum containing the polyvalent ions calculated from the molecular formula of the target substance. In addition, the purification conditions including the column used are shown in each example.

[0244] [Example 1]

[0245] Synthesis of HA119

[0246] [Chemical formula 32]

[0247]

[0248] Using Fmoc-NH-SAL-PEG resin (Watanabe Chemical, 0.15 mmol / g, 0.43 g), the target peptide was synthesized starting from the removal of Fmoc according to the above general method. Subsequently, the chloroacetyl group was introduced according to the general method.

[0249] The resulting crude product was purified under the following conditions (column: Waters Xbridge (registered trademark) C18 5μm OBD (registered trademark) 19x150 mm (Nihon Waters Corporation); mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (%B): 5 - 29% over 3 minutes, then 29 - 34% over 8 minutes; flow rate: 17 mL / min).

[0250] The purity of the target substance was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and it was 99.4%.

[0251] Analytical condition A: retention time = 3.57 minutes, ESI-MS(+) observed value m / z = 899.8 theoretical value 899.5 ((M / 2)+H)

[0252] Analytical condition B: retention time = 16.5 minutes; gradient (%B conc): 25 - 65% over 20 minutes, then 65 - 95% over 1 minute, then 95% over 5 minutes

[0253] [Example 2]

[0254] Synthesis of HA146

[0255] [Chemical formula 33]

[0256]

[0257] Fmoc-NH-SAL-PEG resin (Watanabe Chemical, 0.37 mmol / g, 1.35 g) was placed in a reaction vessel with a fritted glass and shaken with dichloromethane to swell it. According to the general method described above, after removing Fmoc and introducing Fmoc-Lys(Mtt)-OH by peptide coupling into the resulting solid-phase resin. The resulting solid-phase resin was swollen with dichloromethane, a reagent mixture - B (volume ratio of TFA / TIS / CH2Cl2 1:4:95) was added and shaken at room temperature for 30 minutes, and then the reaction solution was drained from the fritted glass. After repeating this operation 12 times, the color of the filtrate became colorless and transparent. This time point was regarded as the end of the reaction. To the resulting solid-phase resin, a DMF solution of capric acid (0.21 M, 12 mL), a DMF solution of HATU (0.5 M, 5 mL), and a DMF solution of DIPEA (1 M, 5 mL) were added, and shaken at 40 °C for 40 minutes. After draining the reaction solution from the fritted glass, the resulting solid-phase resin was washed in the order of DMF and then dichloromethane.

[0258] For the solid-phase resin obtained from the above operations, removal of Fmoc and introduction of Fmoc-amino acids were sequentially performed using an automatic synthesizer by the above general method. The amino acids and reagents used in the reaction were calculated in equivalents based on 0.5 mmol of the solid-phase resin. Peptide coupling was carried out using an automatic synthesizer, and then chloroacetyl groups were introduced according to the above general method.

[0259] Next, using the obtained solid-phase resin, deprotection of the side chain, cleavage from the solid-phase resin, and cyclization reaction were carried out according to the above general method.

[0260] The obtained crude product was purified under the following conditions (column: Waters Xbridge (registered trademark) C18 5μm OBD (registered trademark) 50x250mm (Nihon Waters Corporation); mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (%B): 16 - 41% over 3 minutes, then 47 - 52% over 7 minutes, then 47 - 80% over 1.5 minutes; flow rate: 120 mL / min).

[0261] The purity of the target substance was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analysis condition B, which was 99.3%.

[0262] Analysis condition A: retention time = 4.50 minutes; ESI-MS(+) observed m / z = 1040.4 theoretical 1040.2 ((M / 2)+H)

[0263] Analysis condition B: retention time = 18.8 minutes; gradient (%B conc): 25 - 65% over 20 minutes, then 65 - 95% over 1 minute, then 95% over 5 minutes

[0264] [Example 3]

[0265] Synthesis of HA145

[0266] [Chemical formula 34]

[0267]

[0268] HA145 was synthesized according to the synthesis method shown in Example 1 by using lauric acid instead of capric acid.

[0269] The obtained crude product was purified under the following conditions (column: Waters Xbridge (registered trademark) C18 5μm OBD (registered trademark) 50x250 mm (Nihon Waters Corporation); mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (%B): 21 - 46% over 3 minutes, then 46 - 51% over 7 minutes, then 51 - 80% over 1.5 minutes; flow rate: 120 mL / min).

[0270] The purity of the target substance was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under analytical condition B, and it was 99.3%.

[0271] Analytical condition A: retention time = 4.42 minutes, ESI - MS(+) observed value m / z = 1055.1 theoretical value 1054.3 ((M / 2)+H)

[0272] Analytical condition B: retention time = 18.8 minutes, gradient (%B conc): 25 - 65% over 20 minutes, then 65 - 95% over 1 minute, then 95% over 5 minutes

[0273] [Example 4]

[0274] Synthesis of HA152

[0275] [Chemical formula 35]

[0276]

[0277] Place PAL-PEG resin (Watanabe Chemical, 0.22 mmol / g, 1.16 g) in a reaction vessel equipped with a fritted disk, and shake it with dichloromethane to swell it. After discharging the dichloromethane from the fritted disk, add a dichloromethane solution (5 mL) of 2-nitrophenylsulfonyl chloride (4 equivalents) and a dichloromethane solution (4 mL) of DIPEA (4 equivalents), and stir at room temperature for 30 minutes. After discharging the reaction solution from the fritted disk, wash the solid-phase resin with dichloromethane. To the obtained solid-phase resin, add a THF solution (6 mL) of Fmoc-glycol (10 equivalents), a dichloromethane solution (5 mL) of triphenylphosphine (12 equivalents), and a dichloromethane solution (5 mL) of diisopropyl azodicarboxylate (10 equivalents), and shake at room temperature. The progress of the reaction can be confirmed by LCMS after taking out a small amount of the solid-phase resin and treating it with a cleavage mixture. After discharging the reaction solution from the fritted disk, wash the solid-phase resin with dichloromethane. To the obtained solid-phase resin, add a DMF solution (12 mL) of piperidine (20%), and shake at room temperature for 30 minutes. Drain the reaction solution from the fritted disk, add again a DMF solution (12 mL) of piperidine (20%), and shake at room temperature for 30 minutes. After discharging the reaction solution from the fritted disk, wash the obtained solid-phase resin in the order of DMF and then dichloromethane. To the obtained solid-phase resin, add a DMF solution (0.21 M, 5 mL) of capric acid, a DMF solution (0.5 M, 2 mL) of HATU, and a DMF solution (1 M, 2 mL) of DIPEA, and shake at 40 °C for 1 hour. After discharging the reaction solution from the fritted disk, wash the obtained solid-phase resin in the order of DMF and then dichloromethane. Immerse the obtained solid-phase resin in 10 mL of DMF, add DODT (0.4 mL, 10 eq) and DBU (0.37 mL, 10 eq), and stir at room temperature. After treating a small amount of the solid-phase resin, confirm the progress of the reaction and the disappearance of the starting materials by LCMS.

[0278] Sequentially introduce Fmoc-amino acids into the solid-phase resin obtained by the above operation using an automatic synthesizer according to the above general method. The amino acids and reagents used in the reaction are calculated in equivalents based on 0.25 mmol of the solid-phase resin. Perform peptide coupling using an automatic synthesizer, and then introduce a chloroacetyl group according to the above general method.

[0279] Using the obtained solid-phase resin, perform side-chain deprotection, cleavage from the solid-phase resin, and cyclization reaction according to the above general method.

[0280] The obtained crude product was purified under the following conditions (column: Waters Xbridge (registered trademark) C18 5μm OBD (registered trademark) 50x250 mm (Nihon Waters); mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (%B): 17 - 42% over 3 minutes, then 42 - 47% over 7 minutes, then 47 - 80% over 1.5 minutes; flow rate: 120 mL / min).

[0281] The purity of the target substance was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under Analytical Condition B, and it was 98.4%.

[0282] Analytical Condition A: retention time = 4.18 minutes, ESI-MS(+) observed value m / z = 998.6 theoretical value 998.2 ((M / 2)+H)

[0283] Analytical Condition B: retention time = 16.48 minutes, gradient (%B conc): 25 - 65% over 20 minutes, then 65 - 95% over 1 minute, then 95% over 5 minutes

[0284] [Example 5]

[0285] Synthesis of HA151

[0286] [Chemical Formula 36]

[0287]

[0288] HA151 can be synthesized according to the synthesis method shown in Example 4 by using lauric acid instead of capric acid.

[0289] The obtained crude product was purified under the following conditions (column: Waters Xbridge (registered trademark) C18 5μm OBD (registered trademark) 50x250 mm; mobile phase: A = 0.1% TFA in H2O, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (%B): 22 - 47% over 3 minutes, then 47 - 52% over 7 minutes, then 52 - 80% over 1.5 minutes; flow rate: 120 mL / min).

[0290] The purity of the target substance was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under Analytical Condition B, and it was 97.7%.

[0291] Analytical Condition A: retention time = 4.49 minutes, ESI-MS(+) observed value m / z = 1012.4 theoretical value 1012.2 ((M / 2)+H)

[0292] Analysis condition B: Holding time = 12.5 minutes; Gradient (%B conc): 40 - 80% over 20 minutes, then 80 - 95% over 1 minute, then 95% for 5 minutes

[0293] [Example 6]

[0294] [Evaluation of the antiviral activity of peptides against influenza virus]

[0295] To confirm the antiviral activity of the peptides against influenza virus in in vitro experiments, the test was conducted by the method shown below. The specific test method is shown below.

[0296] 1) Seed MDCK cells at 3×10 4 cells / well and culture for 24 hours at 37°C in the presence of 5% CO2 and MEM - 10% FBS.

[0297] 2) After culturing, add 100 μL of serum - free MEM to the well and wash the cell monolayer.

[0298] 3) Dissolve the test compound in the infection maintenance medium (serum - free MEM containing vitamins) and adjust to each measurement concentration.

[0299] 4) Add the test compound dissolved in serum - free MEM of the infection maintenance medium to each well.

[0300] 5) Dilute influenza virus A / Nagasaki / HA - 58 / 2009 (H1N1), A / Puerto Rico / 8 / 34 (H1N1) or A / Duck / Pennsylvania / 84 (H5N2) with the infection maintenance medium containing trypsin to prepare 1,000 TCID 50 / mL.

[0301] 6) Add the diluted virus solution at 100 μL / well and adjust the titer of each well to 100 TCID50.

[0302] 7) Culture at 37°C under 5% CO2 for 72 hours.

[0303] 8) After culturing is completed, remove the culture medium from each well.

[0304] 9) Add 70% aqueous ethanol solution at 200 ul / well and let stand at room temperature for 5 minutes.

[0305] 10) After removing the ethanol aqueous solution, add 0.5% aqueous crystal violet solution at 200 μL / well and let stand at room temperature for 5 minutes.

[0306] 11) Rinse with water and dry at room temperature.

[0307] 12) Using a TECAN infinite 200 (TECAN), measure the absorbance of each well at a measurement wavelength of λ = 560 nm.

[0308] 13) At each concentration, calculate the relative value (CV relative value, %) with the mock group (non-drug added and non-virus-infected group) set to 100%.

[0309] 14) Using GraphPad Prism 5.0 (GraphPad Software), determine the EC50 value for each sample.

[0310] To confirm the antiviral activity of the peptide in in vivo experiments against influenza virus, the test was conducted by the method shown below. The specific test method is shown below.

[0311] 1) As infected model mice, adjust BALB / cA Jcl [SPF] mice to 5 mice per group.

[0312] 2) Slowly thaw the virus stored at -80 °C on ice, centrifuge for a few seconds, and separately inject it into a tube containing PBS.

[0313] 3) Intranasally inoculate anesthetized mice with influenza virus A / Puerto Rico / 8 / 34 (H1N1) at 267 pfu per mouse, and set the above inoculation time point as "day 0".

[0314] 4) Dissolve HA152 in a 10% hydroxypropyl-β-cyclodextrin solution of the dissolution solvent.

[0315] 5) Dissolve peramivir in a PBS solution.

[0316] 6) Intravenously administer peramivir at a dose of 30 μmol / kg and HA152 at a dose of 15 μmol / kg via the tail vein. As a solvent control, intravenously administer a 10% hydroxypropyl-β-cyclodextrin solution in the same manner.

[0317] 7) After influenza virus infection and compound administration, observe the status, including daily survival observation.

[0318] 8) Convert the survival rate at 14 days with the infection day as day 0 and calculate the survival rate.

[0319] 3. Results

[0320] Results of the evaluation of the activity of HA152 of influenza viruses A / Nagasaki / HA-58 / 2009 (H1N1), A / Puerto Rico / 8 / 34 (H1N1), and A / Duck / Pennsylvania / 84 (H5N2)

[0321] Results of the evaluation of the antiviral activity of iHA100 and HA152 in in vitro experiments using influenza viruses A / Nagasaki / HA-58 / 2009 (H1N1), A / Puerto Rico / 8 / 34 (H1N1), and A / Duck / Pennsylvania / 84 (H5N2) are shown in Figure 1. iHA100 inhibited cell death caused by the virus in the high concentration range of 1 μm or more in the final concentration, but did not show significant inhibition of cell death in the low concentration range of the nM region. On the other hand, it was clearly shown that HA152 showed significant inhibition of cell death in the low concentration range of less than μm and also had significant antiviral activity in the low concentration range. Thus, it was confirmed that HA152 showed extremely high anti-influenza virus activity compared to iHA100.

[0322] Next, using influenza virus A / Duck / Pennsylvania / 84 (H5N2), the antiviral activity of the compounds containing iHA100 and HA152 in in vitro experiments was calculated as EC50. As Figure 2 shown, the compounds containing HA152 showed at least 10-fold higher antiviral activity compared to iHA100. Therefore, HA152 and Figure 2 each of the compounds shown showed significant high anti-influenza virus activity compared to iHA100.

[0323] Results of the evaluation of the activity of HA152 of influenza virus A / Puerto Rico / 8 / 34 (H1N1) in a mouse infection model

[0324] Results of the evaluation of the antiviral activity of peramivir and HA152 in in vivo experiments using influenza virus A / Puerto Rico / 8 / 34 (H1N1) are shown in Figure 3 .

[0325] The survival rate of mice infected with influenza virus was analyzed for 14 days. As a result, the survival rate of the group not administered with the agent was 0%. On the other hand, the survival rate of the group administered with peramivir at a single dose of 30 μmol / kg was 20%. In contrast, the survival rate of the group administered with HA152 at 15 μmol / kg was 60%. Thus, HA152 showed significant antiviral activity not only in in vitro experiments but also in in vivo experiments. Its antiviral activity was equal to or higher than that of the approved drug peramivir targeting neuraminidase.

[0326] Industrial Applicability

[0327] The present invention can be applied to the pharmaceutical industry and the medical device industry. Sequence Listing <110> PeptiDream INC. <120> Hemagglutinin-Binding Peptide <130> 110F0464-IE <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <220> <221> Post-translational modification of residues <222> (3)..(3) <223> N-Methylglycine <220> <221> Post-translational modification of residues <222> (5)..(5) <223> N-Methylphenylalanine <220> <221> Post-translational modification of residues <222> (6)..(6) <223> N-Methylphenylalanine <220> <221> Post-translational modification of residues <222> (8)..(8) <223> N-Methylalanine <220> <221> Post-translational modification of residues <222> (13)..(13) <223> 4-Hydroxy-L-proline <400> 1 Trp Thr Gly Asp Phe Phe Ala Ala His Tyr Thr Val Pro Ala Cys 1 5 10 15 <210> 2 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <220> <221> Post-translational modification of residues <222> (3)..(3) <223> N-Methylglycine <220> <221> Post-translational modification of residues <222> (5)..(5) <223> N-Methylphenylalanine <220> <221> Post-translational modification of residues <222> (6)..(6) <223> N-Methylphenylalanine <220> <221> Post-translational modification of residues <222> (8)..(8) <223> N-methylalanine <220> <221> Post-translational modification of residues <222> (13)..(13) <223> 4-hydroxy-L-proline <400> 2 Trp Thr Gly Asp Phe Phe Ala Ala His Tyr Thr Val Pro Ala Cys Lys 1 5 10 15 <210> 3 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <220> <221> Post-translational modification of residues <222> (1)..(1) <223> N-chloroacetyl-L-tryptophan <220> <221> Post-translational modification of residues <222> (3)..(3) <223> N-methylglycine <220> <221> Post-translational modification of residues <222> (5)..(5) <223> N-methylphenylalanine <220> <221> Post-translational modification of residues <222> (6)..(6) <223> N-methylphenylalanine <220> <221> Post-translational modification of residues <222> (8)..(8) <223> N-methylalanine <220> <221> Post-translational modification of residues <222> (13)..(13) <223> 4-hydroxy-L-proline <220> <221> Post-translational modification of residues <222> (15)..(15) <223> Usually located at the C-terminus of the mature active peptide <400> 3 Trp Thr Gly Asp Phe Phe Ala Ala His Tyr Thr Val Pro Ala Cys 1 5 10 15 <210> 4 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <220> <221> Post-translational modification of residues <222> (1)..(1) <223> N-chloroacetyl-L-tryptophan <220> <221> Post-translational modification of residues <222> (3)..(3) <223> N-methylglycine <220> <221> Post-translational modification of residues <222> (5)..(5) <223> N-methylphenylalanine <220> <221> Post-translational modification of residues <222> (6)..(6) <223> N-methylphenylalanine <220> <221> Post-translational modification of residues <222> (8)..(8) <223> N-methylalanine <220> <221> Post-translational modification of residues <222> (13)..(13) <223> 4-hydroxy-L-proline <220> <221> Post-translational modification of residues <222> (16)..(16) <223> Lysine [gamma-C(=O)n-C11H23]-NH2 <400> 4 Trp Thr Gly Asp Phe Phe Ala Ala His Tyr Thr Val Pro Ala Cys Lys 1 5 10 15 <210> 5 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <220> <221> Post-translational modification of residues <222> (1)..(1) <223> N-chloroacetyl-L-tryptophan <220> <221> Post-translational modification of residues <222> (3)..(3) <223> N-methylglycine <220> <221> Post-translational modification of residues <222> (5)..(5) <223> N-methylphenylalanine <220> <221> Post-translational modification of residues <222> (6)..(6) <223> N-methylphenylalanine <220> <221> Post-translational modification of residues <222> (8)..(8) <223> N-methylalanine <220> <221> Post-translational modification of residues <222> (13)..(13) <223> 4-hydroxy-L-proline <220> <221> Post-translational modification of residues <222> (16)..(16) <223> Lysine [gamma-C(=O)n-C9H19]-NH2 <400> 5 Trp Thr Gly Asp Phe Phe Ala Ala His Tyr Thr Val Pro Ala Cys Lys 1 5 10 15 <210> 6 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <220> <221> Post-translational modification of residues <222> (1)..(1) <223> N-chloroacetyl-L-tryptophan <220> <221> Post-translational modification of residues <222> (3)..(3) <223> N-methylglycine <220> <221> Post-translational modification of residues <222> (5)..(5) <223> N-methylphenylalanine <220> <221> Post-translational modification of residues <222> (6)..(6) <223> N-methylphenylalanine <220> <221> Post-translational modification of residues <222> (8)..(8) <223> N-methylalanine <220> <221> Post-translational modification of residues <222> (13)..(13) <223> 4-hydroxy-L-proline <220> <221> Post-translational modification of residues <222> (15)..(15) <223> Cysteine-[NHCH2CH2NHC(=O)n-C11H23] <400> 6 Trp Thr Gly Asp Phe Phe Ala Ala His Tyr Thr Val Pro Ala Cys 1 5 10 15 <210> 7 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <220> <221> Post - translational modification of residue <222> (1)..(1) <223> N - chloroacetyl - L - tryptophan <220> <221> Post - translational modification of residue <222> (3)..(3) <223> N - methylglycine <220> <221> Post - translational modification of residue <222> (5)..(5) <223> N - methylphenylalanine <220> <221> Post - translational modification of residue <222> (6)..(6) <223> N - methylphenylalanine <220> <221> Post - translational modification of residue <222> (8)..(8) <223> N - methylalanine <220> <221> Post - translational modification of residue <222> (13)..(13) <223> 4-Hydroxy-L-proline <220> <221> Post-translational modification of residues <222> (15)..(15) <223> Cysteine-[NHCH2CH2NHC(=O)n-C9H19] <400> 7 Trp Thr Gly Asp Phe Phe Ala Ala His Tyr Thr Val Pro Ala Cys 1 5 10 15

Claims

1. A hemagglutinin-binding peptide, a pharmaceutically acceptable salt thereof or a solvate thereof, characterized in that, The hemagglutinin-binding peptide is a cyclic peptide represented by any one of the following chemical formulas: ； ； ； ; or 。 2. A drug for treating viral infectious diseases, characterized in that, Comprising the hemagglutinin-binding peptide according to claim 1, a pharmaceutically acceptable salt thereof, or a solvate thereof.

3. A drug for treating influenza, characterized in that, Comprising the hemagglutinin-binding peptide according to claim 1, a pharmaceutically acceptable salt thereof, or a solvate thereof.

4. A virus detection drug, characterized in that, Comprising the hemagglutinin-binding peptide according to claim 1, a pharmaceutically acceptable salt thereof, or a solvate thereof.

5. An influenza virus detection drug, characterized in that, Comprising the hemagglutinin-binding peptide according to claim 1, a pharmaceutically acceptable salt thereof, or a solvate thereof.

6. A kit for virus detection, characterized in that, Comprising the virus detection drug according to claim 4.

7. A kit for detecting influenza virus, characterized in that, Comprising the influenza virus detection drug according to claim 5.

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