Hemagglutinin-binding peptide

Abstract

To provide a compound having markedly higher antiviral activity than iHA 100, an intermediate for producing the compound, and a medical drug or the like containing the high-activity compound.SOLUTION: The invention relates to a hemagglutinin-binding peptide with a specific amino acid sequence, a pharmaceutically acceptable salt thereof, or a solvate thereof, exhibiting very high anti-influenza virus activity.SELECTED DRAWING: Figure 1-1

Description

[Technical field]

[0001] The present invention relates to a hemagglutinin-binding peptide having extremely high anti-influenza virus activity, a medicine for preventing or treating influenza, and an agent for detecting influenza. [Background technology]

[0002] Influenza viruses are highly infectious and pathogenic, and their epidemics, known as pandemics, show widespread pathogenicity to humans.

[0003] As medicines against influenza viruses, zanamivir (trade name: Relenza (trade name)), oseltamivir (trade name: Tamiflu (trade name)), peramivir (trade name: Rapiacta (trade name)), and laninamivir (trade name: Inavir (trade name)) that inhibit neuraminidase, which is necessary for influenza virus release, are widely used. In addition, amantadine (trade name: Symmetrel (trade name)) and flumadine (trade name: Rimantadine), which inhibit the process of viral enucleation, and baloxavir marboxil (trade name: Xofluza (trade name)), which inhibits cap-dependent endonuclease, are also known as medicines. However, although the above medicines, especially anti-influenza drugs against neuraminidase, are widely used as medicines, drug resistance due to viral mutations is a problem.

[0004] On the other hand, hemagglutinin is known to be an essential protein for influenza viruses when they invade host cells, and antiviral molecules targeting this protein have been reported. Antiviral molecules targeting hemagglutinin are effective against viruses that have resistance to existing antiviral drugs. Therefore, antiviral molecules targeting molecules different from those of existing drugs are highly useful from the perspective of drug resistance as well.

[0005] Antibody molecules that bind to hemagglutinin have been reported as antiviral molecules against hemagglutinin (e.g., Patent Documents 1 to 3). On the other hand, while antibody drugs have high activity, neutralizing antibodies that are generated when antibody drugs are recognized as foreign substances in the body can significantly reduce the potency of the drug.

[0006] In recent years, peptide molecules with special backbones such as N-methylamino acids and D-amino acids have been reported as a new group of molecules. Not only do they exhibit high binding strength and biostability, but their molecular weights are also extremely small compared to antibodies. For this reason, they have attracted attention as a group of molecules that can solve the problems faced by conventional antibody drugs (e.g., Non-Patent Documents 1-4).

[0007] We have focused on peptide molecules with this unique backbone and have been searching for peptide molecules against hemagglutinin. iHA100 is a peptide molecule against hemagglutinin, and its molecular weight and administration route are significantly different from those of general antibodies, making it an anti-influenza virus molecule that exhibits anti-viral activity even when administered intranasally (Patent Document 4). On the other hand, there have been no reported cases in which the anti-influenza virus activity of iHA100 has been significantly improved, and no reported structure has been reported, and improving the anti-influenza virus activity of this molecule can greatly contribute to improving the efficacy of the drug. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2018 / 108086 Brochure [Patent Document 2] International Publication No. 2018 / 015012 Brochure [Patent Document 3] International Publication No. 2017 / 122087 Brochure [Patent Document 4] International Publication No. 2013 / 071904 Brochure [Non-Patent Document 1] Nature Reviews Drug Discovery 17,531-533(2018) [Non-Patent Document 2] Current opinion in chemical biology, 34, 44-52 (2016) [Non-Patent Document 3] Annual Review of Biochemistry, 83, 727-752 (2014) [Non-Patent Document 4] Chemistry, 19, 6530-6536 (2013) Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention aims to provide a compound having significantly higher antiviral activity than iHA100, an intermediate for producing the compound, and a medicine containing the highly active compound. [Means for solving the problem]

[0010] The present invention is based on the finding that a newly synthesized hemagglutinin-binding peptide has significant activity compared to known hemagglutinin-binding peptides having anti-influenza virus activity.

[0011] One embodiment disclosed in the present specification relates to a hemagglutinin-binding peptide, a pharma- ceutically acceptable salt thereof, or a solvate thereof (also referred to as the peptide of the present invention).

[0012] The hemagglutinin-binding peptide is any one of the following peptides (1) to (7). (1) A polypeptide consisting 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 (sequence number 2). (2) A polypeptide consisting of an amino acid sequence in which the N-terminal Trp in SEQ ID NO: 1 or 2 is chloroacetyl-Trp. (3) A polypeptide consisting of an amino acid sequence in which, in SEQ ID NO: 1, the N-terminal Trp is chloroacetyl-Trp and the C-terminal Cys is modified via an amide bond as shown in formula (I). (4) A polypeptide having a sequence in which, in SEQ ID NO: 2, the N-terminal Trp is chloroacetyl-Trp and the C-terminal Lys is replaced with a modified lysine derivative shown in formula (II). (5) A polypeptide consisting of an amino acid sequence in which the C-terminal Cys in SEQ ID NO:1 has been modified via an amide bond as shown in formula (I). (6) A polypeptide consisting of a sequence in SEQ ID NO: 2, which contains a lysine derivative represented by formula (II) in which the side chain of Lys is modified with an acyl group. (7) A peptide having an amino acid sequence selected from any of the above (1) to (6) in which one or two amino acids have been deleted, added, substituted or inserted (excluding, however, those in which Cys at the C-terminus in SEQ ID NO: 1 has been deleted and those in which Lys at the C-terminus in SEQ ID NO: 2 has been deleted).

[0013] Formula (I) [ka] (In formula (I), * represents the linking portion to the carbonyl group of the C-terminal Cys, and A 1 is, C8-C 12 It indicates an alkyl group.

[0014] Formula (II) [ka] (In formula (II), * indicates the linking portion to the carbonyl group of the C-terminal Cys, and A 1 is, C8-C12 It indicates an alkyl group.

[0015] Preferred examples of hemagglutinin-binding peptides are as follows: (1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2. (5) A polypeptide consisting of an amino acid sequence in which the C-terminal Cys in SEQ ID NO:1 has been modified via an amide bond as shown in formula (I). (6) A polypeptide consisting of a sequence in which the C-terminal Lys in SEQ ID NO:2 is replaced with a modified lysine derivative represented by formula (II); or (7) A peptide having an amino acid sequence in any one of the above (1), (5), and (6) in which one or two amino acids have been deleted, added, substituted, or inserted (excluding, however, those in which Cys at the C-terminus in SEQ ID NO: 1 has been deleted, and those in which Lys at the C-terminus in SEQ ID NO: 2 has been deleted).

[0016] A preferred example of the hemagglutinin-binding peptide is one in which the hemagglutinin-binding peptide is cyclic.

[0017] A preferred example of the hemagglutinin-binding peptide is represented by the following formula (III) or (IV).

[0018] [ka] (In formula (III), formula A 2 represents a group represented by -NH2 or a group represented by formula (I).

[0019] [ka] (In formula (IV), formula A 3 represents a group represented by -NH2 or a group represented by formula (II).

[0020] Another preferred example of the hemagglutinin-binding peptide is represented by the following formula (V) or (VI).

[0021] [ka] (In formula (V), formula A 4 represents a group represented by -NH2 or a group represented by formula (IIa).

[0022] [ka] (IIa) (In formula (IIa), * represents a linking portion, 1 is, C8-C 12 It indicates an alkyl group.

[0023] [ka] In formula (VI), formula A 5 represents a group represented by -NH2 or a group represented by formula (I).

[0024] Another preferred example of the hemagglutinin-binding peptide is represented by the following formula (VII). [ka]

[0025] Another embodiment of this specification is a pharmaceutical for preventing viral infection or treating viral infection, comprising any of the above-mentioned hemagglutinin-binding peptides, a pharma- ceutical acceptable salt thereof, or a solvate thereof. Yet another embodiment is a pharmaceutical for preventing or treating influenza (an influenza prophylactic or therapeutic agent, pharmaceutical product), comprising any of the above-mentioned hemagglutinin-binding peptides, a pharma- ceutical acceptable salt thereof, or a solvate thereof.

[0026] Another embodiment disclosed in this specification relates to a virus detection drug. Yet another embodiment relates to an influenza virus detection drug. This virus detection drug contains the above-mentioned hemagglutinin-binding peptide.

[0027] Another embodiment disclosed in this specification relates to a kit for virus detection comprising the above-mentioned virus detection agent. Effect of the Invention

[0028] As shown in the examples, this application can provide peptides having significantly higher anti-influenza virus activity than iHA100, medicines for the prevention or treatment of influenza using such peptides, and drugs for detecting influenza viruses, as well as intermediates for synthesizing the above peptides. [Brief description of the drawings]

[0029] [Figure 1-1] Figure 1-1 is a graph showing the results of in vitro evaluation of the anti-influenza virus activity of iHA100 and HA152 using influenza virus A / Nagasaki / HA-58 / 2009 (H1N1). [Figure 1-2] Figure 1-2 is a graph showing the results of in vitro evaluation of the anti-influenza virus activity of iHA100 and HA152 using influenza virus A / Puerto Rico / 8 / 34 (H1N1). [Figure 1-3] FIG. 1-3 is a graph showing the results of evaluating the in vitro anti-influenza virus activity of iHA100 and HA152 using influenza virus A / Duck / Pennsylvania / 84 (H5N2). [Diagram 2] Figure 2 shows the in vitro anti-influenza virus activity against iHA100, HA119, HA145, HA146, HA151, and HA152 using influenza virus A / Duck / Pennsylvania / 84 (H5N2), expressed as EC50 values. [Diagram 3] FIG. 3 is a graph showing the results of analyzing the in vivo anti-influenza virus activity of HA152 using an influenza virus A / Puerto Rico / 8 / 34 (H1N1) infection model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The following describes the embodiments of the present invention. The present invention is not limited to the embodiments described below, and includes appropriate modifications of the embodiments described below within the scope obvious to those skilled in the art.

[0031] One embodiment disclosed herein relates to a hemagglutinin-binding peptide, a hemagglutinin-binding peptide, a pharma- ceutically acceptable salt thereof, or a solvate thereof.

[0032] In this specification, hemagglutinin refers to an antigenic glycoprotein present on the surface of many bacteria and viruses, including influenza viruses, and is abbreviated as "HA." Hemagglutinin is involved in the process of virus attachment to host cells. Specifically, when hemagglutinin on the viral surface binds to sialic acid on the surface of the target host cell, the virus is enveloped in the cell membrane and taken into the cell in the form of an endosome containing the virus. Next, the endosomal membrane and the viral membrane fuse, the viral genome is inserted into the cell, and proliferation begins. Influenza viruses are classified into three types: A, B, and C. Influenza virus type A, which is particularly prone to causing pandemics, has at least 16 subtypes of hemagglutinin, called H1 to H16. H1, 2, 5, 6, 8, 9, 11, 12, 13, 16, 17, and 18 are called group I, and the other hemagglutinins (H3, 4, 7, 10, 14, and 15) are called group II. The H in the influenza subtype name stands for hemagglutinin.

[0033] The hemagglutinin-binding peptide refers to a peptide capable of binding to hemagglutinin. Whether or not a peptide can bind to hemagglutinin can be confirmed by those skilled in the art according to known methods.

[0034] The pharma- ceutically acceptable salt means a pharma- ceutically 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-bromophenylsulfonic acid, carboxylic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc.), addition salts of inorganic bases (ammonium hydroxide or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, etc.), and addition salts of amino acids.

[0035] The pharma- ceutically acceptable solvate means a pharma- ceutically acceptable solvate of a hemagglutinin-binding peptide or a pharma- ceutically acceptable solvate of a salt of a hemagglutinin-binding peptide. The solvent molecule may be coordinated to the compound or its salt, and examples of the solvate include a hydrate and an alcohol solvate.

[0036] The hemagglutinin-binding peptide is any one of the following peptides (1) to (7). (1) A polypeptide consisting 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 (sequence number 2). (2) A polypeptide consisting of an amino acid sequence in which the N-terminal Trp in SEQ ID NO: 1 or 2 is chloroacetyl-Trp. (3) A polypeptide consisting of an amino acid sequence in which, in SEQ ID NO: 1, the N-terminal Trp is chloroacetyl-Trp and the C-terminal Cys is modified via an amide bond as shown in formula (I). (4) A polypeptide having a sequence in which, in SEQ ID NO: 2, the N-terminal Trp is chloroacetyl-Trp and the C-terminal Lys is replaced with a modified lysine derivative shown in formula (II). (5) A polypeptide consisting of an amino acid sequence in which the C-terminal Cys in SEQ ID NO:1 has been modified via an amide bond as shown in formula (I). (6) A polypeptide consisting of a sequence in SEQ ID NO: 2, which contains a lysine derivative represented by formula (II) in which the side chain of Lys is modified with an acyl group. (7) A peptide having an amino acid sequence selected from any of the above (1) to (6) in which one or two amino acids have been deleted, added, substituted or inserted (excluding, however, those in which Cys at the C-terminus in SEQ ID NO: 1 has been deleted and those in which Lys at the C-terminus in SEQ ID NO: 2 has been deleted).

[0037] Formula (I) [ka] (In formula (I), * represents the linking portion to the carbonyl group of the C-terminal Cys, and A 1 is, C8-C 12 Indicates an alkyl group.) C8-C 12 The alkyl group is an alkyl group having 8 to 12 carbon atoms. C8-C 12 The alkyl group may be a straight-chain alkyl group or a branched alkyl group. 12 The alkyl group is C8 alkyl group, C9 alkyl group, C 10 Alkyl group, C 11 Alkyl groups, and C 12 In each of the following groups, A may be any of the alkyl groups. 1 The same applies to.

[0038] Formula (II) [ka] (In formula (II), * indicates the linking portion to the carbonyl group of the C-terminal Cys, and A 1 is, C8-C 12 It indicates an alkyl group.

[0039] Preferred examples of hemagglutinin-binding peptides are as follows: (1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2. (5) A polypeptide consisting of an amino acid sequence in which the C-terminal Cys in SEQ ID NO:1 has been modified via an amide bond as shown in formula (I). (6) A polypeptide consisting of a sequence in which the C-terminal Lys in SEQ ID NO:2 is replaced with a modified lysine derivative represented by formula (II); or (7) A peptide having an amino acid sequence in any one of the above (1), (5), and (6) in which one or two amino acids have been deleted, added, substituted, or inserted (excluding, however, those in which Cys at the C-terminus in SEQ ID NO: 1 has been deleted, and those in which Lys at the C-terminus in SEQ ID NO: 2 has been deleted).

[0040] An example of a specific amino acid sequence of this peptide is as follows: Chloroacetyl-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala-Cys-NH2 (SEQ ID NO: 3) Chloroacetyl-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala-Cys-Lys[gamma-C(=O)nC 11 H 23 ]-NH2 (SEQ ID NO:4) Chloroacetyl-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala-Cys-Lys[gamma-C(=O)n-C9H 19 ]-NH2 (SEQ ID NO:5) Chloroacetyl-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala-Cys-[NHCH2CH2NHC(=O)nC 11 H 23 ] (SEQ ID NO: 6) 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)

[0041] In this specification, the proteinogenic amino acid residues constituting the peptides and polypeptides of the present invention are represented by three-letter or one-letter symbols accepted in the art.

[0042] Other amino acids disclosed herein include non-proteinogenic amino acids, or simply unnatural amino acids, or chemically synthesized compounds having properties known in the art that are characteristic of amino acids. Examples of unnatural amino acids include, but are not limited to, α,α-disubstituted amino acids (such as α-methylalanine), N-alkyl-α-amino acids, N-alkyl-α-D-amino acids, and β-amino acids whose main chain structure differs from that of natural amino acids, and amino acids whose side chain structure differs from that of natural amino acids (such as norleucine, homohistidine, and hydroxyproline).

[0043] As an example of the unnatural amino acid disclosed herein, N-methylglycine, which is a type of N-methylamino acid that is an N-alkyl-α-amino acid, may be represented as MeGly, N-methylalanine as MeAla, and N-methylphenylalanine as MePhe. As an example of an amino acid whose side chain structure differs from that of the natural type, 4R-hydroxyproline may be represented as hydPro.

[0044] Chloroacetyl- means chloroacetylation, and Chloroacetyl-Trp refers to chloroacetyl-Trp.

[0045] As used herein, a polypeptide refers to a compound in which two or more amino acids are bound by peptide bonds, for example, 8-30 amino acids bound by peptide bonds, and may be linear or cyclic. A polypeptide as used herein is preferably a cyclic compound of 15 or 16 amino acids.

[0046] Furthermore, the hemagglutinin-binding peptide of the present invention may be cyclized (macrocyclized). In this specification, cyclization means that two amino acids separated by one or more amino acids in one peptide are directly or indirectly bound via a linker or the like to form a cyclic structure within the molecule.

[0047] Cyclization can be carried out by known methods, for example, according to the method described in International Publication WO2016 / 063969.

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

[0049] [ka] (In formula (III), formula A 2 represents a group represented by -NH2 or a group represented by formula (I).

[0050] [ka] (In formula (IV), formula A 3 represents a group represented by -NH2 or a group represented by formula (II).

[0051] Another preferred example of the hemagglutinin-binding peptide is represented by the following formula (V) or (VI).

[0052] [ka] (In formula (V), formula A 4represents a group represented by -NH2 or a group represented by formula (IIa).

[0053] [ka] (In formula (IIa), * represents a linking portion, 1 is, C8-C 12 It indicates an alkyl group.

[0054] [ka] In formula (VI), formula A 5 represents a group represented by -NH2 or a group represented by formula (I).

[0055] Another preferred example of the hemagglutinin-binding peptide is represented by the following formula (VII). [ka]

[0056] The specific structure of the cyclic peptide is as follows:

[0057] [ka]

[0058] [ka]

[0059] [ka]

[0060] [ka]

[0061] [ka]

[0062] The peptide of the present invention can be produced by known peptide production methods, such as chemical synthesis methods, such as the liquid phase method, the solid phase method, and a hybrid method combining the liquid phase method and the solid phase method, and gene recombination methods.

[0063] In the solid phase method, for example, the hydroxyl group of a resin having a hydroxyl group is esterified with the carboxyl group of the first amino acid (usually the C-terminal amino acid of the target peptide) whose α-amino group is protected with a protecting group. As an esterification catalyst, a known dehydration condensation agent such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), or diisopropylcarbodiimide (DIPCDI) can be used. Next, the protecting group of the α-amino group of the first amino acid is removed, and a second amino acid in which all functional groups except the carboxy group of the main chain are protected is added, the carboxy group is activated, and the first and second amino acids are bonded. Furthermore, the α-amino group of the second amino acid is deprotected, and a third amino acid in which all functional groups except the carboxy group of the main chain are protected is added, the carboxy group is activated, and the second and third amino acids are bonded. This process is repeated until a peptide of the desired length is synthesized, and then all functional groups are deprotected.

[0064] Resins used in the solid-phase method include Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGA resin (Merck), HMPA-PEGA resin (Merck), etc. These resins can be used after washing with a solvent (dimethylformamide (DMF), 2-propanol, methylene chloride, etc.). The protecting group for the α-amino group is not particularly limited as long as it is a known protecting group, and examples thereof include a benzyloxycarbonyl (Cbz or Z) group, a tert-butoxycarbonyl (Boc) group, a fluorenylmethoxycarbonyl (Fmoc) group, a benzyl group, an allyl group, an allyloxycarbonyl (Alloc) group, etc. The Cbz group can be deprotected by hydrofluoric acid, hydrogenation, etc., the Boc group can be deprotected by trifluoroacetic acid (TFA), and the Fmoc group can be deprotected by treatment with piperidine. The protecting group for the α-carboxy group is not particularly limited as long as it is a known protecting group, and examples thereof include methyl ester, ethyl ester, benzyl ester, tert-butyl ester, cyclohexyl ester, and the like. Other functional groups of amino acids are not particularly limited, but for example, the hydroxyl groups of serine and threonine can be protected with benzyl groups or tert-butyl groups, and the hydroxyl group of tyrosine can be protected with 2-bromobenzyloxycarbonyl groups or tert-butyl groups. The amino groups of the lysine side chains and the carboxyl groups of glutamic acid and aspartic acid can be protected in the same manner as the α-amino and α-carboxyl groups.

[0065] The activation of the carboxyl group can be carried out using a condensation agent, such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU), etc.

[0066] The peptide chain can be cleaved from the resin by treatment with an acid such as TFA or hydrogen fluoride (HF).

[0067] The production of peptides by gene recombination (translation synthesis) can be carried out using a nucleic acid that encodes the peptide of the present invention. The nucleic acid that encodes the peptide of the present invention may be either DNA or RNA. The nucleic acid encoding the peptide of the present invention can be prepared by a known method or a method equivalent thereto. For example, it can be synthesized by an automatic synthesizer. A restriction enzyme recognition site may be added to insert the obtained DNA into a vector, or a base sequence encoding an amino acid sequence for excising the resulting peptide chain with an enzyme or the like may be incorporated. As described above, when the peptide of the present invention is fused with a membrane-permeable peptide or the like, the above-mentioned nucleic acid also includes a nucleic acid encoding the membrane-permeable peptide. In order to suppress degradation by proteases derived from the host, a chimeric protein expression method can be used in which the peptide of interest is expressed as a chimeric peptide with another peptide. In this case, the nucleic acid used is a nucleic acid that encodes the peptide of interest and a peptide that binds to it.

[0068] Next, an expression vector is prepared using the nucleic acid encoding the peptide of the present invention. The nucleic acid can be inserted downstream of the promoter of the expression vector as it is, or after digestion with a restriction enzyme or addition of a linker. Examples of vectors include E. coli-derived plasmids (pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pUC118, pBluescript II, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, pC1912, pTP4, pE194, pC194, etc.), yeast-derived plasmids (pSH19, pSH15, YEp, YRp, YIp, YAC, etc.), bacteriophages (e phage, M13 phage, etc.), viruses (retroviruses, vaccinia viruses, adenoviruses, adeno-associated viruses (AAV), cauliflower mosaic viruses, tobacco mosaic viruses, baculoviruses, etc.), cosmids, etc.

[0069] The promoter can be appropriately selected depending on the type of host. When the host is an animal cell, for example, a promoter derived from SV40 (simian virus 40) or a promoter derived from CMV (cytomegalovirus) can be used. When the host is Escherichia coli, a trp promoter, a T7 promoter, a lac promoter, etc. can be used. An expression vector can also incorporate a DNA replication origin (ori), a selection marker (antibiotic resistance, auxotrophy, etc.), an enhancer, a splicing signal, a polyA addition signal, a nucleic acid encoding a tag (FLAG, HA, GST, GFP, etc.), etc.

[0070] Next, a suitable host cell is transformed with the expression vector. The host can be appropriately selected in relation to the vector, and 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, and Vero cells can be used. Transformation can be performed according to known methods such as lipofection, calcium phosphate, electroporation, microinjection, and particle gun methods depending on the type of host. The target peptide is expressed by culturing the transformant according to a conventional method.

[0071] To purify peptides from the culture of the transformant, the cultured cells are harvested and suspended in an appropriate buffer, then disrupted by ultrasonication or freezing and thawing, and a crude extract is obtained by centrifugation or filtration. If the peptide is secreted into the culture medium, the supernatant is collected. Purification from the crude extract or culture supernatant can also be carried out by known methods or methods equivalent thereto (e.g., salting out, dialysis, ultrafiltration, gel filtration, SDS-PAGE, ion exchange chromatography, affinity chromatography, reverse phase high performance liquid chromatography, etc.). The obtained peptide may be converted from a free form to a salt, or from a salt to a free form, by a known method or a method similar thereto.

[0072] The translation synthesis system may be a cell-free translation system. The cell-free translation system includes, for example, ribosomal proteins, aminoacyl-tRNA synthetases (ARS), ribosomal RNA, amino acids, rRNA, GTP, ATP, translation initiation factors (IFs), elongation factors (EFs), release factors (RFs), and ribosome recycling factors (RRFs), as well as other factors necessary for translation. In order to increase the expression efficiency, Escherichia coli extract or wheat germ extract may be added. In addition, rabbit red blood cell extract or insect cell extract may be added. By continuously supplying energy to a system containing these by dialysis, peptides can be produced in the amount of several hundred μg to several mg / mL. A system containing RNA polymerase may also be used to perform transcription from gene DNA. Commercially available cell-free translation systems that can be used include systems derived from Escherichia coli such as RTS-100 (registered trademark) from Roche Diagnostics, PURESYSTEM from Gene Frontier, and PURExpress In Vitro Protein Synthesis Kit from NEW ENGLAND Biolabs, and systems using wheat germ extract such as those from Zoigene and Cell Free Sciences. Using a cell-free translation system, expression products can be obtained in a highly pure form without purification.

[0073] In a cell-free translation system, instead of the aminoacyl-tRNA synthesized by a natural aminoacyl-tRNA synthetase, an artificial aminoacyl-tRNA in which a desired amino acid or hydroxy acid is linked (acylated) to a tRNA may be used. Such an aminoacyl-tRNA can be synthesized using an artificial ribozyme. Such ribozymes 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; Murakami, A. Ohta, H. Ashigai, H. Suga (2006) Nature Methods 3, 357-359 ``The flexizyme system: a flexible tRNA aminoacylation tool for the synthesis of highly nonnatural peptides'';N. Niwa, Y. Yamagishi, H. Murakami, H. Suga (2009) Bioorganic & Medicinal Chemistry Letters 19, 3892-3894”A Examples of such compounds include "flexizyme that selectively charges amino acids activated by a water-friendly leaving group"; and WO2007 / 066627. Flexizyme is also known as the original flexizyme (Fx), and modified forms such as dinitrobenzyl flexizyme (dFx), enhanced flexizyme (eFx), and amino flexizyme (aFx).

[0074] By using tRNA produced by Flexizyme to which a desired amino acid or hydroxy acid is linked, a desired codon can be translated in association with the desired amino acid or hydroxy acid. A special amino acid may be used as the desired amino acid. For example, the unnatural amino acid required for the above-mentioned cyclization can also be introduced into the hemagglutinin-binding peptide by this method.

[0075] The macrocyclic peptides of the present invention and their analogs can be chemically synthesized using various methods commonly used in the art, including stepwise solid-phase synthesis, semi-synthesis of peptide fragments via conformationally assisted religation, and chemical ligation. The peptides and their analogs described herein are chemically synthesized using various solid-phase techniques, such as those described in KJ Jensen, PT Shelton, SL Pedersen, Peptide Synthesis and Applications, 2nd Edition, Springer, 2013. A preferred strategy is based on the combination of the Fmoc group, which temporarily protects the α-amino group and allows selective removal by base, and a protecting group that temporarily protects side chain functional groups and is stable to Fmoc deprotection conditions. Such general peptide side chain selection is known from the aforementioned Peptide Synthesis and Applications, 2nd edition, and GB Fields, RL Noble, Solid Phase Peptide Synthesis Utilizing 9-Fluorenylmethoxycarbonyl Amino Acids, Int. J. Peptide Protein Res. 35, 1990, 161-214, among others. Preferred peptide side chain protecting groups include the Boc group or Mtt group for amino groups such as lysine, the tert-butyl group for carboxyl groups of glutamic acid and aspartic acid, and the Trt and Mmt groups for the thiol group of cysteine.

[0076] The synthesis of resin (or simply resin) as a precursor for peptide synthesis in the present invention can be carried out by using, for example, commercially available PAL-PEG-resin or PAL-PEG-PS, with reference to reports such as Biopolymers 2011;96(6):715-22. For example, an outline is shown in Scheme 1, where n represents the length of the carbon number; R1 represents hydrogen or an alkyl group having 1-4 carbon atoms; and R2 represents various alkyl chains or alkyl chains having a substituent. The amino group of resin 1 is sulfonylated with o-Ns-Cl in the presence of an appropriate base to prepare 2, and solid-phase resin 4 can be obtained by reacting it with amino primary alcohol 3 whose N-terminus is protected with Fmoc, such as Fmoc-glycinol, in the presence of a reagent used in combination in the Mitsunobu reaction, such as triphenylphosphine and diisopropyl azodicarboxylate. Deprotection of Fmoc can be achieved by the combination of an appropriate secondary amine and a solvent, such as piperidine in DMF, to give 5, and various acyl groups can be introduced to the regenerated amine using known peptide coupling techniques to give 6. 2 For example, decanoic acid (composition formula CH3(CH2)8COOH) with a long-chain alkyl group can be introduced by reacting it with the condensing agent HATU in the presence of DIPEA. Finally, the o-Ns group can be removed by treating it with a suitable base and thiol, for example, a combination of DBU and DODT, to prepare resin 7, a precursor for peptide synthesis.

[0077] [ka]

[0078] In the peptide synthesis of the present invention, commercially available resins can also be used as other methods. For example, as outlined in scheme 2, another method for preparing a preferred precursor resin is to use commercially available Rink Amide MBHA resin (Sigma-Aldrich), Fmoc-Rink Amide NovaPEG resin (Merck Millipore) or Fmoc-NH-SAL-PEG resin (Watanabe Chemicals) as a solid-phase resin (8), and after Fmoc removal, an Fmoc amino acid 9 having a primary or secondary amino group in the side chain with a protecting group removable under mild acidic conditions, such as Fmoc-Lys(Mtt)-OH (m=3, R 3 =H, S-configuration) can be condensed to obtain 10. The resulting solid phase resin can be treated with a solution of TFA / TIS / CH2Cl2 in a volume ratio of 1:4:95 to selectively remove the Mmt group to obtain 11. 2 After the introduction of COOH, 12 is obtained, and by removing the Fmoc group, 13 is obtained, which can be used for the synthesis of the desired peptide.

[0079] [ka]

[0080] The peptides and their analogs described in the present invention can be synthesized in a stepwise manner on the solid-phase resin described above. The α-amino protecting group of the C-terminal amino acid used and all amino acids and peptides used in the synthesis must be selectively removed during the synthesis process. Preferably, the above-mentioned solid-phase resin is used, and the C-terminal carboxyl group of a peptide whose N-terminus is appropriately protected with a protecting group such as Fmoc or the C-terminal carboxyl group of an amino acid whose N-terminus is appropriately protected with a protecting group such as Fmoc is converted into an activated ester with an appropriate reagent, and then added to the amino group on the solid-phase resin. Subsequent elongation of the peptide chain can be achieved by sequentially repeating the removal of the N-terminal protecting group (e.g., Fmoc group) and then the condensation of the protected amino acid derivative according to the amino acid sequence of the desired peptide. These can liberate the desired peptide at the final stage. For example, the release can be achieved by using a TFA solution containing water, silyl hydride, and thiol as trapping agents in TFA, as described in Teixeira, WE Benckhuijsen, PE de Koning, ARPM Valentijn, and JW Drijfhout, Protein Pept. Lett., 2002, 9, 379-385, etc. A typical example is TFA / Water / TIS / DODT (volume ratio 92.5: 2.5: 2.5: 2.5).

[0081] Synthesis of the peptide analogs described herein can be carried out using a single or multi-channel peptide synthesizer, such as a CEM Liberty Blue synthesizer or a Biotage Syro I synthesizer.

[0082] The activation of the carboxyl group can be carried out using a condensation agent, such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU), etc.

[0083] Another embodiment disclosed in this specification relates to a medicine. This medicine contains the above-mentioned hemagglutinin-binding peptide, or a pharma- ceutical acceptable salt or solvate thereof (for simplicity, hereinafter, these are also simply referred to as hemagglutinin-binding peptide). This medicine preferably contains an effective amount of the above-mentioned hemagglutinin-binding peptide as an active ingredient.

[0084] When hemagglutinin-binding peptides are used for medical purposes, one or more of the amino acids of the peptide can be chemically modified with, for example, hydrocarbons, fatty acids, polyethylene glycol (PEG), etc., to impart functionality or perform various modifications. These chemical modifications can also be performed using a linker. These modifications can, for example, make it possible to obtain peptides that are more chemically and metabolically stable. A linker is a partial structure that allows for chemical modification to impart functionality to a cyclic peptide that exhibits antiviral activity (for example, the basic structure shown in (II)). Specific examples of the basic structure in the present invention are the 1,2-ethylenediamine structure shown in (I) and the lysine amide structure with an NH2 C-terminus shown in (II).

[0085] As shown in the examples below, the peptide of the present invention exhibits antiviral activity by binding to hemagglutinin on the surface of a virus. The type of virus is not particularly limited as long as it exhibits antiviral activity against viruses that infect humans and the like via hemagglutinin or similar proteins. The type of virus is preferably, for example, an enveloped virus, and further a virus having a class I fusion protein. Specific examples of viruses include seasonal influenza viruses (including human, avian, and swine influenza viruses, or new influenza viruses, and also simply referred to as influenza viruses), highly pathogenic avian influenza viruses, porcine epidemic diarrhea viruses, HIV-1, Ebola viruses, yellow fever viruses, and coronaviruses. The target virus is preferably influenza viruses. The target influenza viruses may be any of types A, B, and C, but are preferably type A, and more preferably influenza viruses having hemagglutinin belonging to group I. In this specification, anti-influenza virus activity is also referred to as antiviral activity. A composition containing the peptide of the present invention is useful as an agent for preventing viral infection or treating viral infectious diseases, preferably an agent for preventing or treating influenza, and similarly, the peptide of the present invention is useful in a method for preventing viral infection or treating viral infectious diseases, preferably a method for preventing or treating influenza.

[0086] In this specification, influenza refers to acute infectious disease caused by the influenza virus. When infected with the influenza virus, humans experience cold-like symptoms accompanied by high fever and muscle pain. Gastrointestinal symptoms such as abdominal pain, vomiting, and diarrhea may also occur, and complications include pneumonia and influenza encephalopathy. In this specification, infection is used as a term to refer to either the process in which a virus invades a living body through the skin or mucous membrane, or the process in which a virus invades a cell by membrane fusion. In addition, in this specification, viral infection refers to a state in which a virus invades a living body, regardless of the presence or absence of symptoms. In addition, in this specification, infectious disease refers to various symptoms caused by viral infection.

[0087] In this specification, the treatment or prevention of influenza is used in its broadest sense, and refers to, for example, alleviating or preventing the worsening of one or more symptoms associated with influenza virus infection, suppressing the onset of symptoms after infection, preventing (delaying or stopping) the infection of viral cells in the body, preventing (delaying or stopping) the proliferation of viral cells in the body, reducing the number of viruses in the body, etc. When at least one of these effects is exhibited, it is determined to be useful for the treatment or prevention of influenza. Furthermore, as shown in the examples below, the peptide of the present invention has neutralizing activity against hemagglutinin, and therefore it is understood that it can provide the same effect as an influenza vaccine.

[0088] In the present specification, the administration form of the pharmaceutical composition is not particularly limited, and may be oral or parenteral. Examples of parenteral administration include injection administration such as intramuscular injection, intravenous injection, and subcutaneous injection, transdermal administration, and transmucosal administration (nasal, oral, ocular, pulmonary, vaginal, and rectal) administration.

[0089] The above pharmaceutical composition can be modified in various ways in consideration of the fact that the polypeptide is easily metabolized and excreted. For example, polyethylene glycol (PEG) or sugar chains can be added to the polypeptide to increase its blood residence time and reduce its antigenicity. Furthermore, the polypeptide can be encapsulated in biodegradable polymer compounds such as polylactic acid glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, nanospheres, etc., which are used as sustained-release bases. When administering transdermally, a weak electric current can be passed through the skin surface to penetrate the stratum corneum (iontophoresis method).

[0090] The above pharmaceutical compositions may be formulated by using the active ingredient as it is, or by adding pharma- ceutically acceptable carriers, excipients, additives, etc. Dosage forms include, for example, liquids (e.g., injections), dispersions, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, lozenges, inhalants, ointments, eye drops, nasal drops, ear drops, poultices, etc. The formulation can be carried out in a conventional manner using, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilization aids, colorants, flavorings, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, etc. as appropriate. Examples of ingredients used in formulations include, but are not limited to, purified water, saline, phosphate buffer, dextrose, glycerol, ethanol and other pharma- ceutically acceptable organic solvents, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, silicic anhydride, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, tragacanth, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, human serum albumin, etc. In view of the fact that peptides are difficult to absorb through mucous membranes, the pharmaceutical composition may contain an absorption enhancer that improves the absorption of poorly absorbed drugs. Examples of such absorption enhancers include surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponin; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylic acids; fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, and mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, and nitric oxide donors.

[0091] The pills or tablets may also be coated with sugar, gastric or enteric substances. The injection preparation may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. In addition, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, solubilizing agents, preservatives, etc. may be added.

[0092] The dosage when the pharmaceutical composition of the present invention is administered to mammals (e.g., humans, mice, rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs, etc.), particularly humans, varies depending on symptoms, the patient's age, sex, weight, sensitivity difference, administration method, administration interval, type of active ingredient, and type of formulation, and is not particularly limited, but may be, for example, 30μg-100g, 100μg-500mg, or 100μg-100mg administered once or in several divided doses. In the case of administration by injection, 1μg / kg-3000μg / kg, or 3μg / kg-1000μg / kg may be administered once or in several divided doses depending on the patient's weight.

[0093] The method for preventing or treating influenza using the peptide of the present invention can be carried out with reference to the description of the pharmaceutical composition above.

[0094] Another embodiment disclosed in this specification relates to a virus detection agent, particularly an influenza virus detection agent, which comprises the above-mentioned hemagglutinin-binding peptide, a salt thereof, or a solvate thereof.

[0095] (Virus detection drugs and detection kits) The present invention also includes a virus detection drug, 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 surface of the virus. Therefore, for example, the peptide of the present invention can be used to detect influenza viruses in a sample instead of anti-influenza antibodies in immunoassays such as ELISA. When used as a detection agent, the peptide of the present invention may be detectably labeled. The peptide can be labeled with a known labeling substance, for example, peptides 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 are used. In addition, peptides labeled with nanoparticles such as gold colloids and quantum dots can also be detected. In addition, in immunoassays, the peptide of the present invention can be labeled with biotin and then detected by binding avidin or streptavidin labeled with an enzyme or the like. Among immunoassays, the ELISA method using enzyme labeling is preferred because it can measure antigens simply and quickly. For example, an antibody that specifically recognizes a part of influenza virus other than hemagglutinin is fixed to a solid phase carrier, a sample is added and reacted, and then a labeled peptide of the present invention is added and reacted. After washing, the sample is reacted with an enzyme substrate to develop color and absorbance is measured, thereby enabling influenza virus to be detected. After reacting the antibody fixed to the solid phase carrier with the sample, an unlabeled peptide of the present invention is added, and an antibody against the peptide of the present invention may be enzyme-labeled and further added. Alternatively, the peptide of the present invention may be fixed to a solid phase carrier as a capture substance, and a labeled antibody that recognizes influenza virus may be used as a detection substance, or the peptide of the present invention may be used for both capture and detection. When the enzyme is peroxidase, 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), etc. can be used as the enzyme substrate, and when the enzyme is alkaline phosphatase, p-nitropheny phosphate (NPP), etc. can be used.

[0096] In this specification, the term "solid phase carrier" is not particularly limited as long as it is a carrier capable of immobilizing peptides, and examples thereof include glass, metallic, or resin microtiter plates, substrates, beads, nitrocellulose membranes, nylon membranes, and PVDF membranes, and the target substance can be immobilized on these solid phase carriers according to known methods.

[0097] The test kit according to the present invention contains reagents and instruments necessary for the above detection (including, but not limited to, the peptide of the present invention, an antibody, a solid phase carrier, a buffer solution, an enzyme reaction stopping solution, a microplate reader, etc.).

[0098] Other embodiments disclosed in this specification may also be considered for use as influenza virus detection kits containing the above-mentioned influenza virus detection drugs, and as tools for elucidating influenza virus infection that occurs via hemagglutinin, and the various cellular functions and life phenomena associated therewith.

[0099] This specification also provides use of a hemagglutinin-binding peptide for the manufacture of a medicament for the prevention or treatment of influenza. In this case, the hemagglutinin-binding peptide may be any of those described above.

[0100] The present specification also provides a method for preventing or treating influenza, comprising the step of administering an effective amount of a hemagglutinin-binding peptide, a pharma- ceutically acceptable salt thereof, or a solvate thereof as an active ingredient to a subject that is a human, non-human mammal, or bird. The hemagglutinin-binding peptide may be any of the peptides described above. Examples of non-human mammals include non-human primates, pigs, cows, dogs, cats, horses, sheep, rats, and mice.

[0101] The abbreviations used herein, and particularly in the representative examples below, are well known to those of skill in the art. Some abbreviations used are: Fmoc for 9-fluorenylmethyloxycarbonyl; HOAt for 1-hydroxybenzotriazole; HATU for O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; MeCN for acetonitrile; DBU for 1,8-diazabicyclo[5.4.0]-7-undecene; DIPEA for N,N-diisopropylethylamine; DODT for 3,6-dioxa-1,8-octane-dithiol; DMSO for dimethylsulfoxide; DMF for N,N-dimethylformamide; mL for milliliters; M for molar; Mtt for monomethyltrityl; Mmt for monomethoxytrityl; o-Ns for 2-nitrobenzenesulfonyl; v / v for volume / volume; TFA for trifluoroacetic acid; TIS for triisopropylsilane; Trt for trityl.

[0102] Examples and General Methods All raw materials, building blocks, reagents, acids, bases, solid-phase resins, and solvents used in the chemical synthesis of the present invention were either commercially available or could be synthesized by those skilled in the art using organic chemistry techniques. Unless otherwise specified, amino acids containing protecting groups were commercially available and used as is.

[0103] In the present invention, the structure of the chemically synthesized peptide was determined by confirming the molecular weight calculated by taking into account the amino acids used according to the target sequence and the building blocks used as necessary, using ESI-MS(+) in mass spectrometry. "ESI-MS(+)" refers to electrospray ionization mass spectrometry performed in positive ion mode. The detected mass was reported in "m / z" units. Compounds with a molecular weight of more than approximately 1000 were frequently detected as doubly or triply charged ions.

[0104] The purity of the chemically synthesized peptides in the present invention was determined by any of the following analytical methods. (Analysis conditions) Analysis conditions A Column: CORTECS® UPLC® C18 column (Nihon Waters), 90 Å, 1.6 μm, 2.1 x 100 mm Mobile phase: MeCN / 0.025% TFA in H2 Temperature: 40℃ Gradient: 5 - 95% MeCN / 0.025% TFA in H2in 5.56 min; linear gradient Flow rate: 0.4 mL / min Detection method: UV 220nm Analysis conditions B Column: Kinetex EVO C18 2.6 μm, 2.1 ID x 150 mm, 100 Å (Phenomenex) Column temperature: 60 °C Mobile phase A: 0.025% TFA in H2 Mobile phase B:0.025% TFA in CH3CN Gradient: described in each example Flow rate: 0.25 mL / min Detection: PDA (225 nm)

[0105] The peptide chain elongation on the solid-phase resin described in this invention was carried out using the resin described in each example as the starting material, under commonly used peptide coupling reaction conditions and Fmoc removal reaction conditions. The reaction was carried out using an automatic peptide synthesizer, CEM Liberty Blue, according to the manufacturer's manual. The commonly used amino acids are listed below, with the side chain protecting groups shown in parentheses. 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.

[0106] The chloroacetyl group was introduced by removing the Fmoc group from the α-amino group of the solid-phase resin retaining the Fmoc-protected peptide obtained in the previous step using the method described above, then adding chloroacetic acid (approximately 3 equivalents) to a DMF solution (0.5 M) of approximately 3 equivalents of N,N'-diisopropylcarbodiimide and a DMF solution (0.5 M) of approximately 3 equivalents of HOAt, and shaking at room temperature for 40 minutes. For the deprotection of the side chain and cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times with DMF and methylene chloride, and then dried under reduced pressure. Then, the reaction vessel containing the solid-phase resin was added with reagent cocktail A (a mixture of TFA / H2 / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) and shaken at room temperature for 150 minutes. The reaction solution was filtered and collected through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were collected through a frit and mixed with the filtrate mentioned above. When this filtrate was added to an excess of diethyl ether cooled to 0°C, a cloudy precipitate was formed. This mixture was centrifuged (9000 rpm, 3 min), and the solution was decanted. The solid obtained was washed again with a small amount of diethyl ether cooled to 0°C, and the solid obtained was used in the next cyclization reaction.

[0107] In the present invention, the peptide cyclization reaction was carried out by dissolving the peptide in DMSO so that the final concentration was 5 mM based on the molar number of the solid-phase resin, adding 6 equivalents of triethylamine, and stirring at room temperature for about 16 hours. The resulting reaction solution was acidified with acetic acid and concentrated under reduced pressure using Biotage (registered trademark) V-10 (Biotage Japan, Inc.). The resulting crude peptide was purified using reversed-phase preparative HPLC with a Waters AutoPurification System - SQD2 single quadruple mass spectrometer, and the m / z ions derived from the target substance were monitored while eluting. It was confirmed that the mass spectrum obtained in ESI-positive scan mode and the mass spectrum including multiply charged ions calculated from the molecular formula of the target substance matched within the error range of the mass analyzer used. The purification conditions, including the columns used, were shown in each example. EXAMPLES

[0108] Synthesis of HA119 [ka]

[0109] Using Fmoc-NH-SAL-PEG resin (Watanabe Chemical Co., Ltd., 0.15 mmol / g, 0.43 g), the target peptide was synthesized starting from the removal of Fmoc according to the general method described above. Subsequently, the chloroacetyl group was introduced according to the general method.

[0110] The resulting crude product was purified using the following conditions (column: Waters Xbridge® C18 5 μm OBD® 19 x 150 mm (Japan Waters); mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (%B): 5 - 29% over 3 min, then 29 - 34% over 8 min; flow rate: 17 mL / min).

[0111] 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 and was found to be 99.4%. Analysis condition A: Retention time = 3.57 min, ESI-MS (+) observed value m / z = 899.8 theoretical value 899.5 ((M / 2)+H) Analysis condition B: Retention time = 16.5 min; Gradient (% B conc): 25 - 65% over 20 min, then 65 - 95% over 1 min, then 95% over 5 min EXAMPLES

[0112] Synthesis of HA146 [ka]

[0113] Fmoc-NH-SAL-PEG resin (Watanabe Chemical, 0.37 mmol / g, 1.35 g) was placed in a fritted reaction vessel and swelled by shaking with dichloromethane. Following the general method described above, Fmoc-Lys(Mtt)-OH was introduced into the resulting solid-phase resin by peptide coupling after removal of Fmoc. The resulting solid-phase resin was swollen with dichloromethane, and reactant cocktail-B (TFA / TIS / CH2Cl2 in a volume ratio of 1:4:95) was added and the mixture was shaken at room temperature for 30 min, after which the reaction solution was discharged from the frit. After repeating this procedure 12 times, the filtrate became colorless and transparent. At this point, the reaction was considered complete. To the resulting solid-phase resin, a DMF solution of decanoic 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 the mixture was shaken at 40 °C for 40 min. After the reaction solution was discharged through the frit, the resulting solid phase resin was washed with DMF and then with dichloromethane.

[0114] The solid-phase resin obtained by the above procedure was subjected to removal of Fmoc and sequential introduction of Fmoc-amino acids using an automatic synthesizer according to the general method described above. The amino acids and reagents used in the reaction were calculated in equivalent amounts, assuming 0.5 mmol of solid-phase resin. Peptide coupling using the automatic synthesizer and subsequent introduction of chloroacetyl groups were performed according to the general method described above. Subsequently, the obtained solid phase resin was used for deprotection of the side chain, cleavage from the solid phase resin, and cyclization reaction according to the general method described above. The resulting crude product was purified using the following conditions (column: Waters Xbridge® C18 5 μm OBD® 50 x 250 mm (Japan Waters); mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (% B): 16-41% over 3 min, then 47-52% over 7 min, then 47-80% over 1.5 min; flow rate: 120 mL / min).

[0115] 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 and was found to be 99.3%. Analysis condition A: Retention time = 4.50 min; ESI-MS (+) observed value m / z = 1040.4, theoretical value 1040.2 ((M / 2)+H) Analysis condition B: Retention time = 18.8 min; Gradient (%B conc): 25-65% over 20 min, then 65-95% over 1 min, then 95% over 5 min EXAMPLES

[0116] Synthesis of HA145

[0117] [ka] HA145 was synthesized according to the synthesis method shown in Example 1, except that lauric acid was used instead of decanoic acid. The resulting crude product was purified using the following conditions (column: Waters Xbridge® C18 5 m OBD® 50 x 250 mm (Japan Waters); mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (% B): 21-46% over 3 min, then 46-51% over 7 min, then 51-80% over 1.5 min; flow rate: 120 mL / min). 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 and was found to be 99.3%. Analysis condition A: Retention time = 4.42 min, ESI-MS (+) observed value m / z = 1055.1, theoretical value 1054.3 ((M / 2)+H) Analysis condition B: Retention time = 18.8 min, Gradient (%B conc): 25-65% over 20 min, then 65-95% over 1 min, then 95% over 5 min EXAMPLES

[0118] Synthesis of HA152 [ka]

[0119] PAL-PEG resin (Watanabe Chemical, 0.22 mmol / g, 1.16 g) was placed in a fritted reaction vessel and swelled by shaking with dichloromethane. After the dichloromethane was drained from the frit, a solution of 2-nitrobenzenesulfonyl chloride (4 equivalents) in dichloromethane (5 mL) and a solution of DIPEA (4 equivalents) in dichloromethane (4 mL) were added and stirred at room temperature for 30 minutes. After the reaction solution was drained from the frit, the solid phase resin was washed with dichloromethane. To the resulting solid phase resin, a solution of Fmoc-glycinol (10 equivalents) in THF (6 mL), a solution of triphenylphosphine (12 equivalents) in dichloromethane (5 mL), and a solution of diisopropyl azodicarboxylate (10 equivalents) in dichloromethane (5 mL) were added and shaken at room temperature. The progress of the reaction was confirmed by LCMS after taking out a small amount of the solid phase resin and treating it with a cleavage cocktail. After the reaction solution was discharged from the frit, the solid phase resin was washed with dichloromethane. A DMF solution (12 mL) of piperidine (20%) was added to the obtained solid phase resin and the mixture was shaken at room temperature for 30 minutes. The reaction solution was discharged from the slit, and a DMF solution (12 mL) of piperidine (20%) was added again and the mixture was shaken at room temperature for 30 minutes. After the reaction solution was discharged from the slit, the obtained solid phase resin was washed with DMF and then with dichloromethane in that order. A DMF solution of decanoic acid (0.21 M, 5 mL), a DMF solution of HATU (0.5 M, 2 mL) and a DMF solution of DIPEA (1 M, 2 mL) were added to the obtained solid phase resin and the mixture was shaken at 40 °C for 1 hour. After the reaction solution was discharged from the frit, the obtained solid phase resin was washed with DMF and then with dichloromethane in that order. The solid phase resin obtained was immersed in 10 mL of DMF, and DODT (0.4 mL, 10 eq) and DBU (0.37 mL, 10 eq) were added and stirred at room temperature. The progress of the reaction and disappearance of the raw materials were confirmed by LCMS after treating a small amount of the solid phase resin.

[0120] Fmoc-amino acids were sequentially introduced to the solid-phase resin obtained by the above operation using an automatic synthesizer according to the general method described above. The amino acids and reagents used in the reaction were calculated in equivalent amounts based on 0.25 mmol of solid-phase resin. Peptide coupling using the automatic synthesizer and subsequent introduction of chloroacetyl groups were performed according to the general method described above.

[0121] The resulting solid-phase resin was used for deprotection of the side chain, cleavage from the solid-phase resin, and cyclization, according to the general method described above.

[0122] The resulting crude product was purified using the following conditions (column: Waters Xbridge® C18 5 μm OBD® 50 x 250 mm (Japan Waters); mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; temperature: 40 °C; gradient (% B): 17-42% over 3 min, then 42-47% over 7 min, then 47-80% over 1.5 min; flow rate: 120 mL / min). 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 and was found to be 98.4%.

[0123] Analysis condition A: Retention time = 4.18 min, ESI-MS (+) observed value m / z = 998.6 theoretical value 998.2 ((M / 2)+H) Analysis condition B: Retention time = 16.48 min, Gradient (% B conc): 25-65% over 20 min, then 65-95% over 1 min, then 95% over 5 min EXAMPLES

[0124] Synthesis of HA151 [ka]

[0125] HA151 was synthesized according to the synthesis method shown in Example 4, except that lauric acid was used instead of decanoic acid.

[0126] The resulting crude product was purified using the following conditions (Column: Waters Xbridge® C18 5 μm OBD® 50 x 250 mm; Mobile phase: A = 0.1% TFA in HO, B = 0.1% TFA in MeCN; Temperature: 40 °C; Gradient (%B): 22-47% over 3 min, then 47-52% over 7 min, then 52-80% over 1.5 min; Flow rate: 120 mL / min).

[0127] 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 and was found to be 97.7%. Analysis condition A: Retention time = 4.49 min, ESI-MS (+) observed value m / z = 1012.4 theoretical value 1012.2 ((M / 2)+H) Analysis condition B: Retention time = 12.5 min; Gradient (% B conc): 40-80% over 20 min, then 80-95% over 1 min, then 95% over 5 min EXAMPLES

[0128] [Antiviral activity of peptides against influenza virus] In order to confirm the in vitro antiviral activity of the peptide against influenza virus, tests were carried out using the following methods. The specific test methods are shown below. 3 x 10 MDCK cells 4 The cells were seeded at 100 cells / well and cultured at 37°C, 5% CO2, in the presence of MEM-10% FBS for 24 hours. After incubation, 100 μL of serum-free MEM was added to the wells to wash the cell monolayer. The test compounds were dissolved in an infection maintenance medium (vitamin-containing serum-free MEM) and adjusted to each measurement concentration. A test compound dissolved in serum-free MEM, which is an infection maintenance medium, was added to each well. Influenza viruses A / Nagasaki / HA-58 / 2009 (H1N1), A / Puerto Rico / 8 / 34 (H1N1), or A / Duck / Pennsylvania / 84 (H5N2) were diluted in infection maintenance medium containing trypsin and incubated at 1,000 TCID 50 / mL. The diluted virus solution was added at 100 μL / well to obtain a titer of 100 TCID 50 was adjusted to. The cells were cultured at 37°C and 5% CO2 for 72 hours. After the incubation was completed, the culture medium was removed from each well. A 70% aqueous ethanol solution was added at 200 ul / well, and the plate was left to stand at room temperature for 5 minutes. After removing the aqueous ethanol solution, 200 μL / well of 0.5% aqueous crystal violet solution was added and the plate was left to stand at room temperature for 5 minutes. It was rinsed with water and dried at room temperature. The absorbance of each well was measured at a measurement wavelength λ = 560 nm using a TECAN infinite 200 (TECAN). For each concentration, the CV relative value (%) was calculated with the mock group (group without drug addition and non-virus infection) set at 100%. The EC50 value of each sample was calculated using GraphPad Prism 5.0 (GraphPad Software).

[0129] In order to confirm the in vivo antiviral activity of the peptide against influenza virus, tests were carried out using the following methods. Specific test methods are shown below. As an infection model mouse, BALB / cA Jcl[SPF] mice were prepared, five per group. The virus solution stored at -80°C was gently thawed on ice, centrifuged for a few seconds, and then dispensed into tubes containing PBS. Anesthetized mice were intranasally inoculated with influenza virus A / Puerto Rico / 8 / 34 (H1N1) at 267 pfu per mouse, and the time point of inoculation was designated as "day 0." HA152 was dissolved in a 10% hydroxypropyl-β-cyclodextrin solution as a dissolution solvent. Peramivir was dissolved in PBS. Peramivir was administered at a dose of 30 μmol / kg, and HA152 was administered at a dose of 15 μmol / kg, via the tail vein. As a vehicle control, 10% hydroxypropyl-β-cyclodextrin solution was administered via the tail vein in the same manner. After influenza virus infection and compound administration, the animals were observed once a day, including whether they were alive or dead. The day of infection was set as day 0, and the survival rate over 14 days was converted to calculate the survival rate.

[0130] 3.Results Activity evaluation results of HA152 using influenza viruses A / Nagasaki / HA-58 / 2009 (H1N1), A / Puerto Rico / 8 / 34 (H1N1), and A / Duck / Pennsylvania / 84 (H5N2) The results of in vitro antiviral activity evaluation of iHA100 and HA152 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 virus-induced cell death at high final concentrations of 1 μM or higher, but did not show significant cell death inhibition at low concentrations in the nM range. On the other hand, HA152 showed significant cell death inhibition even at low concentrations below μM, demonstrating that it has significant antiviral activity at low concentrations against both human and avian influenza viruses. This confirmed that HA152 exhibits extremely high anti-influenza virus activity compared to iHA100.

[0131] Next, the in vitro antiviral activity of compounds including iHA100 and HA152 was calculated as EC50 using influenza virus A / Duck / Pennsylvania / 84 (H5N2). As shown in Figure 2, compounds including HA152 showed antiviral activity at least 10 times higher than that of iHA100. Therefore, it was shown that HA152 and each compound shown in Figure 2 have significantly higher antiviral activity than iHA100.

[0132] Activity evaluation results of HA152 in a mouse infection model using influenza virus A / Puerto Rico / 8 / 34 (H1N1) The results of evaluating the in vivo antiviral activity of peramivir and HA152 using influenza virus A / Puerto Rico / 8 / 34 (H1N1) are shown in Figure 3. Analysis of the survival rate of mice infected with influenza virus over a 14-day period showed that the survival rate of the group not administered any drug was 0%. On the other hand, the survival rate of the group administered a single dose of peramivir at 30 μmol / kg was 20%, while the survival rate of the group administered 15 μmol / kg of HA152 was 60%. This shows that HA152 exhibited significant antiviral activity not only in vitro but also in vivo. The antiviral activity was equal to or greater than that of peramivir, an approved drug that targets neuraminidase. [Industrial Applicability]

[0133] The present invention can be used in the pharmaceutical industry and the medical device industry.

Claims

[Claim 1] A hemagglutinin-binding peptide, a pharma- ceutically acceptable salt thereof, or a solvate thereof.

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