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 pharmaceutical product for the prevention or treatment of influenza, and a drug for detecting influenza. [Background technology]

[0002] Influenza viruses are highly infectious and pathogenic, and their outbreaks manifest as pandemics, exhibiting widespread pathogenicity in humans.

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

[0004] On the other hand, hemagglutinin is known to be an essential protein for influenza viruses to enter host cells, and antiviral molecules targeting this protein have been reported. Antiviral molecules targeting hemagglutinin are effective against viruses that have developed drug 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.

[0005] Antiviral molecules that bind to hemagglutinin have been reported (for example, Patent Documents 1-3). On the other hand, while antibody drugs have high activity, neutralizing antibodies 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 skeletons, such as N-methylamino acids and D-amino acids, have been reported as a new group of molecules. These molecules not only exhibit high binding affinity and biostability, but also have extremely small molecular weights compared to antibodies. For this reason, they are attracting attention as a group of molecules that can solve the problems faced by conventional antibody drugs (for example, Non-Patent Documents 1-4).

[0007] We have focused on peptide molecules with this special skeleton and have been searching for peptide molecules that target hemagglutinin. iHA100 is a peptide molecule that targets hemagglutinin, and unlike typical antibodies, it has a molecular weight and administration routes that differ significantly from conventional antibodies. It is an anti-influenza virus molecule that exhibits antiviral activity even when administered intranasally (Patent Document 4). On the other hand, there have been no reports of cases in which the anti-influenza virus activity of iHA100 has been significantly improved, nor has its structure been reported. Improving its anti-influenza virus activity could 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 Pamphlet [Non-Patent Document 1] Nature Reviews Drug Discovery 17,531-533(2018)

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0009] 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 medicament containing the compound having high activity as described above.

Means for Solving the Problems

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

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

[0012] This hemagglutinin-binding peptide is any one of the following peptides (1) to (7). (1) A polypeptide consisting of the amino acid sequence represented by 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 ID 2). (2) A polypeptide having an amino acid sequence in SEQ ID NO: 1 or 2 in which the N-terminal Trp is chloroacetyl-Trp. (3) A polypeptide comprising an amino acid sequence in which 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 the N-terminal Trp in SEQ ID NO: 2 is replaced with chloroacetyl-Trp and the C-terminal Lys is replaced with a lysine derivative modified as shown in formula (II). (5) A polypeptide consisting of an amino acid sequence in which the C-terminal Cys is modified via an amide bond as shown in formula (I) in sequence number 1. (6) A polypeptide comprising a sequence containing a lysine derivative represented by formula (II) in which the side chain of Lys in Sequence ID No. 2 is modified with an acyl group. (7) A peptide having an amino acid sequence in which one or two amino acids are deleted, added, substituted, or inserted in any of the amino acid sequences described in (1) to (6) above (excluding the one in SEQ ID NO: 1 in which the C-terminal Cys is deleted, and the one in SEQ ID NO: 2 in which the C-terminal Lys is deleted).

[0013] Equation (I) [ka] (In formula (I), * indicates the linkage portion of the C-terminal Cys to the carbonyl group, A 1 is, C8-C 12 (Indicates an alkyl group.)

[0014] Formula (II) [ka] (In formula (II), * indicates the linkage portion of the C-terminal Cys to the carbonyl group, A 1 is, C8-C12 (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 is modified via an amide bond as shown in formula (I) in sequence number 1. (6) A polypeptide consisting of a sequence in which the C-terminal Lys in Sequence ID No. 2 is replaced with a lysine derivative modified by formula (II); or (7) A peptide having an amino acid sequence in which one or two amino acids are deleted, added, substituted, or inserted in any of the amino acid sequences in (1), (5), and (6) above (excluding the one in SEQ ID NO: 1 in which the C-terminal Cys is deleted, and the one in SEQ ID NO: 2 in which the C-terminal Lys is deleted).

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

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

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

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

[0020] Another preferred example of a hemagglutinin-binding peptide is shown by formula (V) or (VI) below.

[0021]

Chem.

[0022]

Chem.

[0023]

Chem.

[0028] As shown in the examples, this application can provide a peptide with significantly higher anti-influenza virus activity than iHA100, a pharmaceutical for the prevention or treatment of influenza using the peptide, a detection agent for influenza viruses, and an intermediate for synthesizing the above peptide. [Brief explanation of the drawing]

[0029] [Figure 1-1] Figure 1-1 is a graph showing the in vitro anti-influenza virus activity evaluation results of iHA100 and HA152 using influenza virus A / Nagasaki / HA-58 / 2009 (H1N1). [Figure 1-2] Figure 1-2 is a graph showing the in vitro evaluation results of the anti-influenza virus activity of iHA100 and HA152 using influenza virus A / Puerto Rico / 8 / 34 (H1N1). [Figure 1-3] Figures 1-3 are graphs showing the in vitro anti-influenza virus activity evaluation results of iHA100 and HA152 using influenza virus A / Duck / Pennsylvania / 84 (H5N2). [Figure 2] Figure 2 shows the in vitro anti-influenza virus activity of influenza virus A / Duck / Pennsylvania / 84 (H5N2) against iHA100, HA119, HA145, HA146, HA151, and HA152, expressed as EC50 values. [Figure 3] Figure 3 is a graph showing the results of analyzing the anti-influenza virus activity of HA152 in vivo using an influenza A / Puerto Rico / 8 / 34 (H1N1) infection model. [Modes for carrying out the invention]

[0030] The following describes embodiments for carrying out the present invention. The present invention is not limited to the embodiments described below, but also includes modifications made to the embodiments described below to the extent that is obvious to those skilled in the art.

[0031] One embodiment disclosed herein relates to a hemagglutinin-conjugated peptide, a hemagglutinin-conjugated peptide, a pharmaceutically 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 the influenza virus, and is represented as "HA". Hemagglutinin is involved in the process of viral adhesion to host cells. Specifically, when hemagglutinin on the surface of the virus binds to sialic acid on the surface of the target host cell, the virus is enclosed in the cell membrane and taken into the cell in the form of an endosome containing the virus. Subsequently, the endosomal membrane and the viral membrane fuse, the viral genome is inserted into the cell, and replication begins. Influenza viruses are classified into three types: A, B, and C. Influenza A viruses, which are particularly prone to causing pandemics, have at least 16 subtypes of hemagglutinin, known as H1-H16. H1, 2, 5, 6, 8, 9, 11, 12, 13, 16, 17, and 18 are called Group I, while the other hemagglutinins (H3, 4, 7, 10, 14, and 15) are called Group II. The "H" in the influenza subtype name indicates hemagglutinin.

[0033] A hemagglutinin-binding peptide refers to a peptide that can bind to hemagglutinin. Whether or not a peptide can bind to hemagglutinin can be determined by methods known to those skilled in the art.

[0034] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of a 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 acids, 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 pharmaceutically acceptable solvates refer to pharmaceutically acceptable solvates of hemagglutinin-binding peptides or pharmaceutically acceptable solvates of salts of hemagglutinin-binding peptides. The solvent molecule may be coordinated to the compound or its salt, and examples of solvates are hydrates and alcoholic hydrates.

[0036] This hemagglutinin-binding peptide is one of the following peptides (1) through (7). (1) 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 (Sequence ID 1), Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala-Cys-Lys (Sequence ID 2). (2) A polypeptide having an amino acid sequence in SEQ ID NO: 1 or 2 in which the N-terminal Trp is chloroacetyl-Trp. (3) A polypeptide comprising an amino acid sequence in which 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 the N-terminal Trp in SEQ ID NO: 2 is replaced with chloroacetyl-Trp and the C-terminal Lys is replaced with a lysine derivative modified as shown in formula (II). (5) A polypeptide consisting of an amino acid sequence in which the C-terminal Cys is modified via an amide bond as shown in formula (I) in sequence number 1. (6) A polypeptide comprising a sequence containing a lysine derivative represented by formula (II) in which the side chain of Lys in Sequence ID No. 2 is modified with an acyl group. (7) A peptide having an amino acid sequence in which one or two amino acids are deleted, added, substituted, or inserted in any of the amino acid sequences described in (1) to (6) above (excluding the one in SEQ ID NO: 1 in which the C-terminal Cys is deleted, and the one in SEQ ID NO: 2 in which the C-terminal Lys is deleted).

[0037] Equation (I) [ka] (In formula (I), * indicates the linkage portion of the C-terminal Cys to the carbonyl group, A 1 is, C8-C 12 This indicates an alkyl group. )C8-C 12 Alkyl alkyl groups are alkyl groups with 8 to 12 carbon atoms. C8-C 12 The alkyl group may be a linear alkyl group or a branched alkyl group. C8-C 12 Alkyl groups include C8 alkyl groups, C9 alkyl groups, and C 10 Alkyl, C 11 Alkyl groups, and C 12 Any alkyl group may be used. A in each of the following groups 1 The same applies to this matter.

[0038] Formula (II) [ka] (In formula (II), * indicates the linkage portion of the C-terminal Cys to the carbonyl group, A 1 is, C8-C 12 (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 is modified via an amide bond as shown in formula (I) in sequence number 1. (6) A polypeptide consisting of a sequence in which the C-terminal Lys in Sequence ID No. 2 is replaced with a lysine derivative modified by formula (II); or (7) A peptide having an amino acid sequence in which one or two amino acids are deleted, added, substituted, or inserted in any of the amino acid sequences in (1), (5), and (6) above (excluding the one in SEQ ID NO: 1 in which the C-terminal Cys is deleted, and the one in SEQ ID NO: 2 in which the C-terminal Lys is deleted).

[0040] A specific example of the 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 (Sequence ID: 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 (Sequence ID: 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 (Sequence ID: 5) Chloroacetyl-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala-Cys-[NHCH2CH2NHC(=O)nC 11 H 23 ](Sequence ID: 6) Chloroacetyl-Trp-Thr-MeGly-Asp-MePhe-MePhe-Ala-MeAla-His-Tyr-Thr-Val-hydPro-Ala- Cys-[NHCH2CH2NHC(=O)n-C9H 19 ](Sequence ID: 7)

[0041] In this specification, the three-letter or one-letter notation accepted in the industry is used to represent amino acid residues (proteinogenic amino acids) that constitute the proteins comprising the peptides and polypeptides of the present invention.

[0042] Other amino acids disclosed herein include non-proteinogenic amino acids (also called non-natural amino acids or simply non-natural amino acids) that do not constitute proteins, or chemically synthesized compounds that possess the properties known in the industry that are characteristic of amino acids. Examples of non-natural 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, β-amino acids, and amino acids with different main chain structures from their natural forms (such as norleucine, homohistidine, and hydroxyproline).

[0043] Examples of non-natural amino acids disclosed herein include N-methylglycine, a type of N-methyl amino acid that is an N-alkyl-α-amino acid, which may be represented as MeGly, N-methylalanine as MeAla, and N-methylphenylalanine as MePhe. Additionally, as an example of an amino acid whose side chain structure differs from its natural form, 4R-hydroxyproline may be represented as hydPro.

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

[0045] In this specification, a polypeptide refers to a molecule in which two or more amino acids are linked by peptide bonds, for example, 8 to 30 amino acids linked by peptide bonds, and may be linear or cyclic. The polypeptide in this specification is preferably a cyclic amino acid molecule of 15 or 16 amino acids.

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

[0047] Circularization can be carried out by known methods, for example, according to the method described in the international publication WO2016 / 063969.

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

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

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

[0051] Another preferred example of a hemagglutinin-binding peptide is shown by formula (V) or (VI) below.

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

[0053] [ka] (In equation (IIa), * indicates a connecting part, A 1 is, C8-C 12 (Indicates an alkyl group.)

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

[0055] Another preferred example of a hemagglutinin-binding peptide, besides those mentioned above, is shown by formula (VII) below. [ka]

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

[0057] [ka]

[0058] [ka]

[0059] [ka]

[0060] [ka]

[0061] [ka]

[0062] The peptides of the present invention can be produced by chemical synthesis methods such as liquid-phase methods, solid-phase methods, and hybrid methods combining liquid-phase and solid-phase methods; or by known peptide production methods such as genetic engineering.

[0063] The solid-phase method involves, for example, esterifying the hydroxyl group of a resin containing a hydroxyl group with the carboxyl group of a first amino acid (usually the C-terminal amino acid of the target peptide) whose α-amino group is protected by a protecting group. Known dehydration condensation agents such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIPCDI) can be used as esterification catalysts. 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 carboxyl group of the main chain are protected, is added. This carboxyl group is then activated to bond the first and second amino acids. Furthermore, the α-amino group of the second amino acid is deprotected, and a third amino acid, in which all functional groups except the carboxyl group of the main chain are protected, is added. This carboxyl group is then activated to bond the second and third amino acids. This process is repeated until a peptide of the desired length is synthesized, at which point all functional groups are deprotected.

[0064] Examples of resins used in the solid-phase process include Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGA resin (Merck), and HMPA-PEGA resin (Merck). These resins can be used after being washed with a solvent (such as dimethylformamide (DMF), 2-propanol, or methylene chloride). The protecting group for the α-amino group is not particularly limited as long as it is a known protecting group, but examples include the benzyloxycarbonyl (Cbz or Z) group, tert-butoxycarbonyl (Boc) group, fluorenylmethoxycarbonyl (Fmoc) group, benzyl group, allyl group, and allyloxycarbonyl (Alloc) group. The Cbz group can be deprotected by hydrofluoric acid or hydrogenation, 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-carboxyl group is not particularly limited as long as it is a known protecting group, but examples include methyl esters, ethyl esters, benzyl esters, tert-butyl esters, cyclohexyl esters, and the like. Other functional groups of amino acids, though not limited to those mentioned above, include the hydroxyl groups of serine and threonine, which can be protected with benzyl or tert-butyl groups, and the hydroxyl group of tyrosine, which can be protected with 2-bromobenzyloxycarbonyl or tert-butyl groups. The amino groups of lysine side chains and the carboxyl groups of glutamic acid and aspartic acid can be protected in the same way as α-amino and α-carboxyl groups.

[0065] The carboxyl group can be activated using a condensing agent. Examples of condensing agents include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazole-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxidehexafluorophosphate (HBTU).

[0066] Peptide chains can be cleaved from resin by treatment with acids such as TFA and hydrogen fluoride (HF).

[0067] The production of peptides by genetic engineering (translation synthesis system) can be carried out using the nucleic acid encoding the peptide of the present invention. The nucleic acid encoding the peptide of the present invention may be DNA or RNA. The nucleic acids encoding the peptides of the present invention can be prepared by known methods or similar methods. For example, they can be synthesized by an automated synthesis apparatus. Restriction enzyme recognition sites may be added to insert the obtained DNA into a vector, or a base sequence encoding an amino acid sequence for cleaving the resulting peptide chain with an enzyme may be incorporated. As described above, when the peptide of the present invention is fused with a membrane-permeable peptide, etc., the nucleic acid includes the nucleic acid encoding the membrane-permeable peptide. To suppress degradation by host-derived proteases, a chimeric protein expression method can be used, in which the target peptide is expressed as a chimeric peptide with another peptide. In this case, the nucleic acids used are those encoding the target peptide and the 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, either as is, digested with restriction enzymes, or by adding a linker. Examples of vectors include E. coli plasmids (pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pUC118, pBluescript II, etc.), Bacillus subtilis plasmids (pUB110, pTP5, pC1912, pTP4, pE194, pC194, etc.), yeast plasmids (pSH19, pSH15, YEp, YRp, YIp, YAC, etc.), bacteriophages (e-phage, M13 phage, etc.), viruses (retroviruses, vaccinia virus, adenovirus, adeno-associated virus (AAV), cauliflower mosaic virus, tobacco mosaic virus, baculovirus, etc.), cosmids, etc.

[0069] The promoter can be appropriately selected depending on the type of host. If the host is an animal cell, for example, a promoter derived from SV40 (simian virus 40) or CMV (cytomegalovirus) can be used. If the host is E. coli, a promoter such as the trp promoter, T7 promoter, or lac promoter can be used. Expression vectors can also incorporate nucleic acids that encode DNA replication origins (oris), selection markers (such as antibiotic resistance and nutritional requirements), enhancers, splicing signals, polyalpha alpha addition signals, and tags (such as FLAG, HA, GST, and GFP).

[0070] Next, suitable host cells are transformed with the expression vector described above. The host can be appropriately selected in relation to the vector, and examples include Escherichia coli, Bacillus subtilis, Bacillus species, yeast, insects or insect cells, and animal cells. Examples of animal cells that can be used include HEK293T cells, CHO cells, COS cells, myeloma cells, HeLa cells, and Vero cells. Transformation can be carried out according to known methods such as lipofection, calcium phosphate, electroporation, microinjection, and particle gun, depending on the type of host. By culturing the transformed cells according to conventional methods, the target peptide is expressed.

[0071] Peptide purification from transformant cultures involves harvesting the cultured cells, suspending them in a suitable buffer, disrupting the cells by methods such as sonication or freeze-thaw cycles, and obtaining a crude extract by centrifugation or filtration. If peptides are 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 similar methods (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 known or equivalent methods.

[0072] The translation synthesis system may be a cell-free translation system. A cell-free translation system may include, 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 ribosomal regeneration factor (RRF), as well as other factors necessary for translation. E. coli extract or wheat germ extract may be added to increase expression efficiency. Rabbit red blood cell extract or insect cell extract may also be added. By continuously supplying energy to a system containing these components using dialysis, peptides can be produced in concentrations ranging from several hundred μg to several mg / mL. A system including RNA polymerase may also be used to facilitate transcription from gene DNA. Commercially available cell-free translation systems include Roche Diagnostics' RTS-100 (registered trademark), Gene Frontier's PURESYSTEM, and New England Biolabs' PURExpress In Vitro Protein Synthesis Kit, all derived from E. coli, and systems from Zoygene and CellFree Science, among others, which utilize wheat germ extract. Cell-free translation systems allow for the acquisition of highly purified expression products without the need for purification.

[0073] In cell-free translation systems, artificial aminoacyl-tRNAs may be used instead of aminoacyl-tRNAs synthesized by natural aminoacyl-tRNA synthetases, by cylating (acylating) a desired amino acid or hydroxy acid to the tRNA. Such aminoacyl-tRNAs can be synthesized using artificial ribozymes. 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 include "flexizyme that selectively charges amino acids activated by a water-friendly leaving group" (and WO2007 / 066627, etc.). Flexizyme is also known by the original flexizyme (Fx), and modified versions thereof such as dinitrobenzylflexizyme (dFx), enhanced flexizyme (eFx), and aminoflexizyme (aFx).

[0074] By using tRNA linked to a desired amino acid or hydroxy acid, generated by Flexizyme, a desired codon can be translated in association with the desired amino acid or hydroxy acid. Special amino acids may be used as the desired amino acid. For example, the non-natural amino acids necessary for the cyclization described above can also be introduced into hemagglutinin-binding peptides using this method.

[0075] The macrocyclic peptides and their analogues of the present invention can be synthesized using a variety of commonly used methods in the art, including stepwise solid-phase synthesis, semi-synthesis of peptide fragments via conformationally supported re-ligation, and chemical ligation. The synthesis of the peptides and their analogues described herein is a chemical synthesis using various solid-phase techniques, such as those described in KJ Jensen, PT Shelton, SL Pedersen, Peptide Synthesis and Applications, 2nd Edition, Springer, 2013. A preferred strategy is based on a combination of an Fmoc group that temporarily protects the α-amino group and allows for selective removal by a base, and a protecting group that temporarily protects the side-chain functional group and is stable under de-Fmoc conditions. Such general peptide side chain selections are 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, but preferred peptide side chain protecting groups include Boc and Mtt groups for amino groups such as lysine, tert-butyl groups for carboxyl groups of glutamic acid and aspartic acid, and Trt and Mmt groups for thiol groups of cysteine.

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

[0077] [ka]

[0078] Peptide synthesis in the present invention can also be carried out using commercially available resins as an alternative method. For example, as outlined in scheme-2, one preferred method for preparing a precursor resin is to use commercially available Rink Amide MBHA resin (Sigma-Aldrich), Fmoc-Rink Amide NovaPEG resin (Merck Millipour), or Fmoc-NH-SAL-PEG resin (Watanabe Chemical Co.) as a solid-phase resin (8). After removing Fmoc, an Fmoc amino acid 9 having a primary or secondary amino group in its side chain with a protecting group that can be removed under mild acidic conditions, such as Fmoc-Lys(Mtt)-OH(m=3, R 3 10 can be obtained by condensing (H, S-configuration). By treating the obtained solid phase resin with a solution of TFA / TIS / CH2Cl2 in a volume ratio of 1:4:95, it is possible to selectively remove the Mmt group to obtain 11. Subsequently, the aforementioned R 2 The compound can be modified to 12 by introducing the COOH group, and then to 13 by removing the Fmoc group, making it suitable for use in the synthesis of the target peptide.

[0079] [ka]

[0080] The peptides and their analogues described in the present invention can be synthesized stepwise on the solid-phase resin described above. The α-amino protecting group must be selectively removed from the C-terminal amino acid and all amino acids and peptides used in the synthesis process. Preferably, the synthesis is initiated by using the solid-phase resin described above, activating 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 appropriately protected with a protecting group such as Fmoc, using an appropriate reagent, and then adding it to an amino group on the solid-phase resin. Subsequent peptide chain extension can be achieved by sequentially removing the N-terminal protecting group (e.g., an Fmoc group) and then condensing the protected amino acid derivative, according to the amino acid sequence of the target peptide. In addition, the target peptide can be released in the final step. For example, as a condition for liberation, as mentioned in Teixeira, WE Benckhuijsen, PE de Koning, ARPM Valentijn, JW Drijfhout, Protein Pept. Lett., 2002, 9, 379-385, liberation can be achieved by using a TFA solution containing water / silyl hydride / thiol as a scavenger in TFA. A typical example is TFA / Water / TIS / DODT (volume ratio 92.5:2.5:2.5:2.5).

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

[0082] The carboxyl group can be activated using a condensing agent. Examples of condensing agents include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazole-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxidehexafluorophosphate (HBTU).

[0083] Another embodiment disclosed in this specification relates to a pharmaceutical product. This pharmaceutical product comprises the hemagglutinin-binding peptide described above, a pharmaceutically acceptable salt or solvate thereof (for simplicity, these will also be referred to below simply as hemagglutinin-binding peptide). Preferably, this pharmaceutical product contains an effective amount of the hemagglutinin-binding peptide described above as an active ingredient.

[0084] When hemagglutinin-binding peptides are used for pharmaceutical purposes, one or more amino acids of the peptide can be chemically modified with, for example, hydrocarbons, fatty acids, polyethylene glycol (PEG), etc., to impart function or to make various modifications. These chemical modifications can also be carried out using linkers. These modifications make it possible to obtain, for example, more chemically and metabolically stable peptides. A linker is a substructure that enables chemical modification for imparting function to a cyclic peptide exhibiting antiviral activity (for example, the basic structure shown in (II)). Specific examples of the basic structure in this 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 described later, 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 or other organisms via hemagglutinin or similar proteins. Examples of viruses include enveloped viruses, and more preferably, viruses having a class I fusion protein. Specific examples of viruses include seasonal influenza viruses (including human, avian, swine influenza viruses, or novel influenza viruses, also simply referred to as influenza viruses), highly pathogenic avian influenza viruses, swine epidemic diarrhea viruses, HIV-1, Ebola virus, yellow fever virus, and coronavirus. The target virus is preferably the influenza virus. Furthermore, the target influenza virus may be of type A, B, or C, but type A is preferred, and more preferably, an influenza virus having hemagglutinin belonging to group I is preferred. 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 a preventive agent for viral infections or as a therapeutic agent for viral infections, preferably as a preventive or therapeutic agent for influenza. Similarly, the peptide of the present invention is useful in a method for preventing viral infections or treating viral infections, preferably as a method for preventing or treating influenza.

[0086] In this specification, influenza refers to an acute infectious disease caused by the influenza virus. When infected with the influenza virus, humans develop cold-like symptoms such as 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" refers to either the process by which a virus enters a living organism through the skin or mucous membranes, or the process by which a virus enters a cell through membrane fusion. In this specification, "viral infection" refers to the state in which a virus has entered a living organism, regardless of whether or not symptoms are present. In this specification, "infectious disease" refers to the various symptoms caused by a 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 post-infection symptoms, preventing (delaying or stopping) the infection of cells by the virus in the body, preventing (delaying or stopping) the replication of the virus in the body, or reducing the number of viruses in the body. If at least one of these effects is observed, it is judged to be useful for the treatment or prevention of influenza. Furthermore, as shown in the examples described later, the peptide of the present invention has neutralizing activity against hemagglutinin, and is therefore understood to be able to obtain effects similar to those of an influenza vaccine.

[0088] In this specification, the form of administration of the pharmaceutical composition is not particularly limited and may be administered orally or parenterally. Examples of parenteral administration include injection (such as intramuscular injection, intravenous injection, or subcutaneous injection), transdermal administration, and transmucosal administration (through the nose, mouth, eye, lung, vagina, or rectum).

[0089] The above pharmaceutical composition can be modified in various ways, taking into account the easily metabolized and excreted nature of polypeptides. For example, polyethylene glycol (PEG) or sugar chains can be added to the polypeptide to lengthen its residence time in the blood and reduce its antigenicity. Alternatively, biodegradable polymer compounds such as polylactic acid glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, nanospheres, etc., may be used as sustained-release bases, and polypeptides may be encapsulated within them. When administered transdermally, a weak electric current can be passed through the stratum corneum by applying it to the skin surface (iontophoresis).

[0090] The above-mentioned pharmaceutical composition may use the active ingredient as is, or it may be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc. Examples of dosage forms include liquids (e.g., injections), dispersants, suspensions, tablets, pills, powders, suppositories, powders, granules, capsules, syrups, lozenges, inhalants, ointments, eye drops, nasal drops, ear drops, poultices, etc. Formulation can be carried out by conventional methods, for example, by using excipients, binders, disintegrants, lubricants, solvents, solubilizers, colorants, flavoring and odor-modifying agents, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, etc., as appropriate. Examples of ingredients used in formulation include, but are not limited to, purified water, saline solution, phosphate buffer, dextrose, glycerol, ethanol and other pharmaceutically acceptable organic solvents, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropylcellulose, starch, corn starch, anhydrous silicic acid, aluminum magnesium silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, acacia gum, 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 poorly absorbed through the mucous membrane, the above pharmaceutical composition may contain an absorption enhancer to improve the absorption of poorly absorbed drugs. Examples of such absorption enhancers include surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponins; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylic acids; fatty acids such as 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] Pills or tablets may also be coated with sugar, gastric-soluble, or enteric-soluble substances. The injectable preparation may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. Furthermore, humectants, emulsifiers, dispersants, stabilizers, solvents, solubilizers, preservatives, etc. may be added.

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

[0093] A 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 described above.

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

[0095] (Virus detection reagents and detection kits) The present invention also includes a virus detection agent containing the peptide of the present invention, particularly an influenza virus detection agent. 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 peptides of the present invention may be labeled in a detectable manner. While peptides can be labeled with known labeling substances, examples include 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, dancylkloride, phycoerythrin, tetramethylrhodamine isothiocyanate, and near-infrared fluorescent materials, and luminescent substances such as luciferase, luciferin, and aequorin. Furthermore, peptides labeled with nanoparticles such as gold colloid and quantum dots can also be detected. Furthermore, in immunoassays, the peptide of the present invention can be labeled with biotin and detected by binding it to avidin or streptavidin labeled with an enzyme or the like. Among immunoassays, the ELISA method using enzyme labeling is preferred because it allows for simple and rapid measurement of antigens. For example, an antibody that specifically recognizes the portion of the influenza virus other than hemagglutinin is immobilized on a solid support, the sample is added and reacted, and then the labeled peptide of the present invention is added and reacted. After washing, the mixture reacts with an enzyme substrate, color development occurs, and the absorbance is measured to detect the influenza virus. Alternatively, after reacting the antibody immobilized on the solid support with the sample, an unlabeled peptide of the present invention may be added, and an antibody against the peptide of the present invention, which has been enzyme-labeled, may be added further. Furthermore, the peptide of the present invention may be immobilized on a solid support as a capture substance, and a labeled antibody that recognizes the influenza virus may be used as a detection substance, or the peptide of the present invention may be used for both capture and detection. For enzyme substrates, if the enzyme is a peroxidase, 3,3'-diaminobenzidine (DAB), 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), etc. can be used, and if the enzyme is an alkaline phosphatase, p-nitropheny phosphate (NPP), etc. can be used.

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

[0097] The test kit according to the present invention includes reagents and equipment necessary for the above detection (including, but not limited to, the peptide, antibody, solid support, buffer, enzyme reaction stop solution, microplate reader, etc. of the present invention).

[0098] Another embodiment disclosed in this specification may be used as an influenza virus detection kit containing the influenza virus detection agent described above, or as a tool for elucidating influenza virus infection mediated by hemagglutinin, and the various cellular functions and life phenomena associated therewith.

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

[0100] This specification also provides a method for preventing or treating influenza, comprising 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 that is a human, a non-human mammal, or a bird. The hemagglutinin-binding peptide can be any of the peptides described above as appropriate. Examples of non-human mammals include primates other than humans, pigs, cattle, dogs, cats, horses, sheep, rats, and mice.

[0101] The abbreviations used in this specification, and especially in the following representative examples, will be well known to those skilled in the art. Some of the abbreviations used are as follows: Fmoc as 9-Fluorenylmethyloxycarbonyl; HOAt as 1-Hydroxybenzotriazole; HATU as O-(7-Azabenzotriazole-1-yl)-N,N,N',N'-Tetramethyluronium hexafluorophosphate; MeCN as acetonitrile; DBU as 1,8-Diazabicyclo"5.4.0"-7-Undecene; DIPEA as N,N-Diisopropylethylamine; DODT as 3,6-Dioxa-1,8-Octane-Dithiol; DMSO as dimethyl sulfoxide; DMF as N,N-Dimethylformamide; mL as milliliter; M as molar; Mtt as monomethyltrityl; Mmt as monomethoxytrityl; o-Ns as 2-Nitrobenzenesulfonyl; v / v as volume; TFA as trifluoroacetic acid; TIS as triisopropylsilane; Trt as 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 this invention were either commercially available or synthesized by those skilled in the art using organic chemical methods. Unless otherwise specified, amino acids containing protecting groups were commercially available.

[0103] The structure of the chemically synthesized peptides in this invention was determined by calculating the molecular weight, taking into account the amino acids used according to the target sequence and the building blocks used as needed, and confirming this by 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 molecular weights greater than approximately 1000 were frequently detected as divalent or trivalent ions.

[0104] The purity of the chemically synthesized peptides in this invention was determined by one of the following analytical methods. (Analysis conditions) Analysis conditions A Column: CORTECS® UPLC® C18 column (Waters Japan Co., Ltd.), 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: As described in each example. Flow rate: 0.25 mL / min Detection: PDA (225 nm)

[0105] The peptide chain elongation in the solid-phase resins described in this invention was carried out using the resins described in each example as starting materials, under commonly used peptide coupling reaction conditions and Fmoc removal reaction conditions. The reactions were performed using the CEM Liberty Blue automated peptide synthesizer, following the manufacturer's manual. The commonly used amino acids are listed below, with side-chain protecting groups indicated 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 in the solid-phase resin containing the Fmoc-protected peptide obtained in the previous step using the method described above, and then adding chloroacetic acid (approximately 3 equivalents) to approximately 3 equivalents of a DMF solution of N,N'-diiropropylcarbodiimide (0.5 M) and approximately 3 equivalents of a DMF solution of HOAt (0.5 M), and shaking at room temperature for 40 minutes. For side chain deprotection and cleavage from the solid phase resin, the resin obtained after the chloroacetyl group introduction step was first washed five times each with DMF and methylene chloride and dried under reduced pressure. Next, the reaction 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 the mixture was shaken at room temperature for 150 minutes. The reaction solution was filtered and recovered through the frit. The solid phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, the solution components were recovered through the frit, and mixed with the aforementioned filtrate. When this filtrate was added to excess diethyl ether cooled to 0°C, a turbid precipitate was formed. This mixture was centrifuged (9000 rpm, 3 min), and the solution was decanted. The obtained solid was washed again with a small amount of diethyl ether cooled to 0°C, and the obtained solid was used in the next cyclization reaction.

[0107] In this invention, the cyclization reaction of the peptide was carried out by dissolving the peptide in DMSO so that the final concentration of the peptide was 5 mM based on the number of moles of the solid phase resin, then 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® V-10 (Biotage Japan Co., Ltd.). For the purification of the obtained crude peptide, reverse-phase preparative HPLC was performed using a Waters AutoPurification System - SQD2 single quadruple mass spectrometer, and elution was carried out while monitoring the m / z ions derived from the target product. It was confirmed that the mass spectrum obtained in ESI-positive scan mode and the mass spectrum including polyvalent ions calculated from the molecular formula of the target product were in agreement within the error range of the mass spectrometer used. The purification conditions, including the column used, are shown in each example. [Examples]

[0108] Synthesis of HA119 [ka]

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

[0110] The obtained crude product was purified using the following conditions (Column: Waters Xbridge® C18 5 μm OBD® 19 x 150 mm (Waters Japan Co., Ltd.); 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).

[0111] 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 was found to be 99.4%. Analysis conditions A: Retention time = 3.57 minutes, ESI-MS(+). Observed value m / z = 899.8. Theoretical value 899.5 ((M / 2)+H). Analysis 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. [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 reaction vessel with frit and shaken with dichloromethane to expand it. Following the general method described above, Fmoc was removed and Fmoc-Lys(Mtt)-OH was introduced into the resulting solid resin by peptide coupling. The resulting solid resin was swollen with dichloromethane, and the reaction agent cocktail B (volume ratio of TFA / TIS / CH2Cl2 1:4:95) was added. The mixture was shaken at room temperature for 30 minutes, and the reaction solution was drained from the frit. This procedure was repeated 12 times until the filtrate became colorless and transparent. The reaction was considered complete at this point. Decanoic acid DMF solution (0.21 M, 12 mL), HATU DMF solution (0.5 M, 5 mL), and DIPEA DMF solution (1 M, 5 mL) were added to the resulting solid resin, and the mixture was shaken at 40 °C for 40 minutes. After the reaction mixture was drained from the frit, the resulting solid resin was washed with DMF, followed by dichloromethane.

[0114] Fmoc was removed from the solid-phase resin obtained by the above procedure, and Fmoc-amino acids were sequentially introduced using an automated synthesizer in the general manner described above. The amino acids and reagents used in the reaction were calculated in equal amounts, assuming a solid-phase resin volume of 0.5 mmol. Peptide coupling was performed using an automated synthesizer, and the subsequent introduction of chloroacetyl groups was carried out according to the general method described above. Subsequently, using the obtained solid-phase resin, the side chains were deprotected, cleaved from the solid-phase resin, and cyclized according to the general method described above. The obtained crude product was purified using the following conditions (Column: Waters Xbridge® C18 5 μm OBD® 50 x 250 mm (Waters Japan Co., Ltd.); 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).

[0115] 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 was found to be 99.3%. Analysis conditions A: Retention time = 4.50 minutes; ESI-MS(+) Observed value m / z = 1040.4 Theoretical value 1040.2 ((M / 2)+H) 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. [Examples]

[0116] Synthesis of HA145

[0117] [ka] HA145 was synthesized according to the synthesis method shown in Example 1, but using lauric acid instead of decanoic acid. The obtained crude product was purified using the following conditions (Column: Waters Xbridge® C18 5 m OBD® 50 x 250 mm (Waters Japan Co., Ltd.); 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). 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 was found to be 99.3%. Analysis conditions A: Retention time = 4.42 minutes, ESI-MS(+). Observed value m / z = 1055.1. Theoretical value 1054.3 ((M / 2)+H). 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. [Examples]

[0118] Synthesis of HA152 [ka]

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

[0120] Fmoc-amino acids were sequentially introduced into the solid-phase resin obtained by the above procedure using an automated synthesizer in accordance with the general method described above. The amino acids and reagents used in the reaction were calculated in equal amounts based on a solid-phase resin volume of 0.25 mmol. Peptide coupling using the automated synthesizer and subsequent introduction of chloroacetyl groups were carried out according to the general method described above.

[0121] Using the obtained solid-phase resin, deprotection of the side chains, cleavage from the solid-phase resin, and cyclization reactions were carried out according to the general method described above.

[0122] The obtained crude product was purified using the following conditions (Column: Waters Xbridge® C18 5 μm OBD® 50 x 250 mm (Waters Japan Co., Ltd.); 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). 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 was found to be 98.4%.

[0123] Analysis conditions A: Retention time = 4.18 minutes, ESI-MS(+). Observed value m / z = 998.6. Theoretical value 998.2 ((M / 2)+H). Analysis 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. [Examples]

[0124] Synthesis of HA151 [ka]

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

[0126] The obtained 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 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).

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

[0128] [Evaluation of the antiviral activity of peptides against influenza virus] To confirm the in vitro antiviral activity of the peptide against the influenza virus, tests were conducted using the method described below. The specific test method is as follows. MDCK cells 3 × 10 4 The cells were seeded in a cell / well and cultured for 24 hours at 37°C in 5% CO2 and MEM-10% FBS. After culturing, 100 μL of serum-free MEM was added to the well to wash the cell monolayer. The test compounds were dissolved in infection maintenance medium (vitamin-containing serum-free MEM) and adjusted to the respective measurement concentrations. The test compound, dissolved in serum-free MEM (Metabolic Emission Medium), an infection maintenance medium, was added to each well. Influenza virus A / Nagasaki / HA-58 / 2009 (H1N1), A / Puerto Rico / 8 / 34 (H1N1), or A / Duck / Pennsylvania / 84 (H5N2) was diluted in an infection maintenance medium containing trypsin and administered at 1,000 TCID. 50 Prepared in / mL Diluted virus solution was added at a rate of 100 μL / well, and the titer per well was set to 100 TCID. 50 I adjusted it to that. The cells were incubated at 37°C and 5% CO2 for 72 hours. After the culture was complete, the culture medium was removed from each well. A 70% ethanol aqueous solution was added at a rate of 200 ul / well, and the mixture was allowed to stand at room temperature for 5 minutes. After removing the ethanol aqueous solution, 200 μL / well of 0.5% crystal violet aqueous solution was added and allowed to stand at room temperature for 5 minutes. Rinse with water and dry at room temperature. The absorbance of each well at a measurement wavelength λ = 560 nm was measured using a TECAN infinite 200 (TECAN Corporation). For each concentration, the relative value (CV relative value, %) was calculated with the mock group (group without drug addition and without virus infection) set as 100%. The EC50 value for each sample was determined using GraphPad Prism 5.0 (GraphPad Software).

[0129] To confirm the in vivo antiviral activity of the peptide against the influenza virus, tests were conducted using the method described below. The specific test method is as follows. Five BALB / cA Jcl[SPF] mice were prepared per group to serve as an infection model mouse. The virus solution, stored at -80°C, was slowly 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 a dose of 267 pfu per mouse, and the inoculation time was designated as "day 0". HA152 was dissolved in a 10% hydroxypropyl-β-cyclodextrin solution, which was used as the solvent. Peramivir was dissolved in PBS. Peramivir was administered intravenously at a dose of 30 μmol / kg, and HA152 at a dose of 15 μmol / kg. A 10% hydroxypropyl-β-cyclodextrin solution was administered intravenously in the tail vein as a vehicle control. After influenza virus infection and administration of the compound, the patient's condition, including survival status, was monitored once a day. The survival rate was calculated by converting the 14-day survival rate, with the infection day being considered day 0.

[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). Figure 1 shows 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 / Peninsula / 84 (H5N2). iHA100 suppressed viral cell death in the high concentration range of 1 μM or higher, but did not show significant suppression of cell death in the low concentration range of nM. On the other hand, HA152 showed significant suppression of cell death even in the low concentration range of μM or lower, and it was revealed that it has significant antiviral activity against both human and avian influenza viruses, even at low concentrations. From this, it was confirmed that HA152 exhibits extremely high anti-influenza virus activity compared to iHA100.

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

[0132] Activity evaluation results of HA152 in a mouse infection model using influenza virus A / Puerto Rico / 8 / 34 (H1N1) Figure 3 shows the results of evaluating the in vivo antiviral activity of peramivir and HA152 using influenza virus A / Puerto Rico / 8 / 34 (H1N1). Analysis of the 14-day survival rate of mice infected with influenza virus revealed that the survival rate was 0% in the group that did not receive any drug. In contrast, the survival rate was 20% in the group that received a single dose of peramivir at 30 μmol / kg, while the survival rate was 60% in the group that received HA152 at 15 μmol / kg. This indicates that HA152 exhibited significant antiviral activity not only in vitro but also in vivo. Its antiviral activity was comparable to, or even greater than, that of peramivir, an approved drug that targets neuraminidase. [Industrial applicability]

[0133] This invention can be used in the pharmaceutical and medical device industries.

Claims

1. A hemagglutinin-binding peptide, a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein the hemagglutinin-binding peptide is represented by the following formula 1, formula 2, formula 3, or formula 4. 【Chemistry 1】 ...(Formula 1) 【Chemistry 2】 ...(Formula 2) 【Transformation 3】 ...Formula 3 【Chemistry 4】 ...Formula 4

2. A pharmaceutical agent for the prevention of viral infection or the treatment of a viral infection, comprising the hemagglutinin-binding peptide described in claim 1, a pharmaceutically acceptable salt thereof, or a solvate thereof.

3. A pharmaceutical agent for the prevention or treatment of influenza, comprising the hemagglutinin-binding peptide described in claim 1, a pharmaceutically acceptable salt thereof, or a solvate thereof.

4. A virus detection agent comprising the hemagglutinin-binding peptide described in claim 1, a pharmaceutically acceptable salt thereof, or a solvate thereof.

5. An influenza virus detection agent comprising the hemagglutinin-binding peptide described in claim 1, a pharmaceutically acceptable salt thereof, or a solvate thereof.

6. A virus detection kit comprising the virus detection agent described in claim 5.

7. A kit for detecting influenza viruses, comprising the influenza virus detection agent described in claim 6.

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