Peptide and peptide-containing composition

Abstract

The present invention pertains to a peptide and a composition containing the peptide. The peptide of the present invention comprises the following amino acid sequence: MeA-MeF-S-Cha-Y-S-Y-Y-R-R-Cha-C (SEQ ID NO: 2), or an amino acid sequence having a substitution, addition, deletion, or insertion at a quantity of 1-10 amino acid residues selected from the group consisting of amino acid residues at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 of the above amino acid sequence.

Description

Peptides and compositions containing peptides

[0001] The present invention relates to peptides and compositions comprising said peptides.

[0002] COVID-19 is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that became a global pandemic in early 2020. As of early December 2020, the number of deaths worldwide had exceeded 1.56 million, making countermeasures urgently needed worldwide.

[0003] SARS-CoV-2, like SARS-CoV and MERS-CoV, is a new species of virus belonging to the Betacoronavirus genus. Research is underway worldwide to identify effective therapeutic and preventative drugs. Currently available drugs for pharmacological therapy include antiviral drugs (remdesivir and favipiravir), anti-inflammatory drugs (dexamethasone and statins), and targeted immunomodulatory therapies (tocilizumab, sarilumab, etc.) (Non-Patent Document 1). However, these drugs were originally developed to treat other diseases, and many of them do not primarily target coronavirus infections. For example, remdesivir was developed as a treatment for Ebola hemorrhagic fever, which is caused by the Ebola virus, a filovirus. However, because it exhibits antiviral activity against single-stranded RNA viruses, including coronaviruses, it has received special approval as a treatment for COVID-19. As a result, some patients may not see any improvement in symptoms even after administration, or may experience side effects, which has become a problem. Furthermore, recently, mutant virus strains such as the European, Brazilian, and South African variants have been discovered that have mutated from the Wuhan variant of SARS-CoV-2 that triggered COVID-19, and research is awaited to determine whether these conventional drugs are also effective against these mutant viruses.

[0004] Additionally, the mRNA vaccine "COMIRNATY" (coronavirus-modified uridine RNA vaccine) was approved in Japan in December 2020 as a vaccine against SARS-CoV-2, and vaccination is being rushed. However, because it is a vaccine, it cannot be expected to be effective as a treatment after infection. Furthermore, it has been pointed out that its effectiveness is reduced against the South African variant virus (Non-Patent Document 2), so there is still a need for the development of preventive or therapeutic drugs against coronaviruses.

[0005] New England Journal of Medicine, Liu et al., Neutralizing Activity of BNT162b2-Elicited Serum Preliminary Report (DOI: 10.1056 / NEJMc2102017), New England Journal of Medicine, pp. 37-44 (2021, 4.2 edition), Ujiike M., and Taguchi T., Viruses. 2015 Apr 3; 7(4): 1700-1725. Yuan H. et al., Acta Pharmacol Sin. 2020 Sep; 41(9): 1141-1149.

[0006] The present inventors have devised the present invention as a result of extensive research and studies aimed at providing a preventive or therapeutic agent for coronavirus. The present invention provides peptides and compositions containing the peptides, particularly peptides having anti-SARS-CoV-2 virus activity, compositions for preventing or treating coronavirus infections, and compositions for diagnosing SARS-CoV-2 virus infections. The present invention also provides combination agents comprising the peptides of the present invention and other agents for preventing or treating coronavirus infections.

[0007] This application includes, but is not limited to, the following inventions. [1] A peptide comprising the following amino acid sequence: MeA-MeF-S-Cha-Y-S-Y-Y-R-R-Cha-C (SEQ ID NO: 2), or an amino acid sequence having a substitution, addition, deletion, or insertion at 1 to 10 amino acid residues selected from the group consisting of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, and 10th amino acid residues of the above amino acid sequence. [2] A peptide comprising the following amino acid sequence: MeA-MeF-S-Cha-Y-S-Y-Y-R-R-Cha-C (SEQ ID NO: 2), or an amino acid sequence having a substitution, addition, deletion, or insertion at 1 to 8 amino acid residues selected from the group consisting of the 1st, 2nd, 3rd, 5th, 6th, 8th, 9th, and 10th amino acid residues of the above amino acid sequence. [3] A peptide comprising the following amino acid sequence: X1-X2-X3-Cha-X4-X5-Y-X6-X7-X8-Cha-C (SEQ ID NO: 1), wherein X1 is A, E, MeA, or MeE; X2 is an N-methyl amino acid; X3 is S or Aib; X4 is an amino acid having an unsubstituted or substituted aromatic ring in the side chain; X5 is any amino acid; X6 is an amino acid having an unsubstituted or substituted aromatic ring in the side chain; X7 is any amino acid; and X8 is any basic amino acid. [4] The peptide of [3], wherein X1 is MeA or MeE. [5] The peptide of [3] or [4], wherein X2 is MeF or MeNle. [6] The peptide according to any one of [3] to [5], wherein X4 is unsubstituted or substituted Y, or unsubstituted or substituted F. [7] The peptide according to any one of [3] to [6], wherein X4 is Y or F4F. [8] The peptide according to any one of [3] to [7], wherein X5 is any hydrophilic amino acid or Aib, or any N-methylated hydrophilic amino acid or Aib. [9] The peptide according to any one of [3] to [8], wherein X5 is Aib, E, K, S, dd, ddap, ds, or MeE.

[10] The peptide according to any one of [3] to [9], wherein X6 is unsubstituted or substituted Y, or unsubstituted or substituted F.

[11] The peptide according to any one of [3] to

[10] , wherein X6 is any one of Y, Yae, 3Py, and Na1.

[12] The peptide according to any one of [3] to

[11] , wherein X7 is any one of A, Ahp, Cit, Dab, E, F4COO, K, and R.

[13] The peptide according to any one of [3] to

[12] , wherein X8 is any one of K, R, and A4p.

[14] The peptide according to any one of [1] to

[13] , further comprising D-glutamic acid at the C-terminus.

[15] The peptide according to any one of [1] to

[14] , comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 2-126, 132-234.

[16] The peptide according to any one of [1] to

[15] , which is a cyclic peptide.

[17] The peptide according to

[16] , which has a cyclic structure in which a chloroacetylated amino acid and a cysteine ​​residue contained in the peptide are bonded.

[18] The peptide according to any one of [1] to

[17] , which further contains an additional amino acid residue.

[19] A pharmaceutical composition comprising the peptide according to any one of [1] to

[17] .

[20] The pharmaceutical composition according to

[19] , which has anti-SARS-CoV-2 virus activity.

[21] The pharmaceutical composition according to

[19] or

[20] , for the prevention or treatment of coronavirus infection.

[22] The pharmaceutical composition according to any one of

[19] to

[21] , wherein the coronavirus infection is COVID-19.

[23] The pharmaceutical composition according to any one of

[19] to

[22] , which comprises a pharmaceutically acceptable salt of the peptide according to any one of [1] to

[17] , or a solvate thereof.

[24] A diagnostic composition for diagnosing SARS-CoV-2 virus infection, comprising the peptide according to any one of [1] to

[17] .

[25] The pharmaceutical composition according to any one of

[19] to

[23] , used in combination with other drugs for the prevention or treatment of coronavirus infection.

[26] The pharmaceutical composition according to

[25] , wherein the other drug for the prevention or treatment of coronavirus infection is selected from the group consisting of remdesivir, a combination of casirivimab and imdevimab, and molanupiravir or an active form thereof.

[27] The pharmaceutical composition according to

[24] or

[26] , which is administered simultaneously with another drug for the prevention or treatment of coronavirus infection.

[28] A combination comprising the peptide according to any one of [1] to

[17] and another drug for the prevention or treatment of coronavirus infection.

[29] Another drug for the prevention or treatment of coronavirus infection, which is administered in combination with a pharmaceutical composition comprising the peptide according to any one of [1] to

[17] .

[0008] The peptides of the present invention have anti-SARS-CoV-2 activity and are therefore useful as compounds for preventing or treating COVID-19. Furthermore, because the peptides of the present invention bind to SARS-CoV-2, they are also useful as diagnostic agents. Furthermore, the peptides of the present invention exhibit enhanced efficacy in preventing or treating coronavirus infections when used in combination with other drugs for preventing or treating coronavirus infections.

[0009] Figure 1-1 is a graph showing the results of measuring the antiviral activity of cyclic peptides against various mutant virus strains by measuring the amount of viral RNA in cell culture supernatants. Figure 1-2 is a graph showing the results of measuring the antiviral activity of cyclic peptides against various mutant virus strains by measuring the amount of viral RNA in cell culture supernatants. Figure 1-3 is a graph showing the results of measuring the antiviral activity of cyclic peptides against various mutant virus strains by measuring the amount of viral RNA in cell culture supernatants. Figure 1-4 is a graph showing the results of measuring the antiviral activity of cyclic peptides against various mutant virus strains by measuring the amount of viral RNA in cell culture supernatants. Figure 2-1 is a graph showing the effect of 5555_p020 and 55555_p028 on preventing the progression of SARS-CoV-2 infection in a lethal mouse model. Figure 2-2 is a graph showing the effect of 5555_p020 and 5555_p028 on improving mortality in a lethal mouse model of SARS-CoV-2 infection. Figure 3 is a photograph, instead of a drawing, showing lung tissue images at the end of observation in a mouse SARS-CoV-2 infection lethal model. The scale bar in the photograph indicates 200 μm. In the photograph, A1 indicates alveoli, Br indicates bronchioles, and V indicates blood vessels. Figure 4 is a photograph, instead of a drawing (histological image of the posterior lung lobe), showing the inhibitory effect of 5555_p028 on pneumonia caused by SARS-CoV-2 infection. The scale bars in the photographs a, c, e, and g indicate 2.0 mm, and b, d, f, and h indicate 100 μm. a and b indicate the results for the solvent control group, c and d indicate the 25 mg / kg group of 5555_p028, e and f indicate the 5 mg / kg group of 5555_p028, and g and h indicate the 1 mg / kg group of 5555_p028. Figure 5-1 is a graph showing the results of measuring the antiviral activity against the Wuhan strain when a cyclic peptide is combined with an existing compound having anti-SARS-CoV-2 activity, as measured by measuring the amount of viral RNA in a cell culture supernatant. Figure 5-2 is a graph showing the results of measuring the antiviral activity against the Wuhan strain when a cyclic peptide is combined with an existing compound having anti-SARS-CoV-2 activity, as measured by measuring the amount of viral RNA in a cell culture supernatant. Figure 5-3 is a graph showing the results of measuring the antiviral activity against the Wuhan strain when a cyclic peptide is combined with an existing compound having anti-SARS-CoV-2 activity, as measured by measuring the amount of viral RNA in a cell culture supernatant.Figure 5-4 is a graph showing the results of measuring the antiviral activity against the Delta strain when a cyclic peptide is combined with an existing compound having anti-SARS-CoV-2 activity, as measured by viral RNA levels in cell culture supernatants. Figure 5-5 is a graph showing the results of measuring the antiviral activity against the Delta strain when a cyclic peptide is combined with an existing compound having anti-SARS-CoV-2 activity, as measured by viral RNA levels in cell culture supernatants. Figure 5-6 is a graph showing the results of measuring the antiviral activity against the Delta strain when a cyclic peptide is combined with an existing compound having anti-SARS-CoV-2 activity, as measured by viral RNA levels in cell culture supernatants. Figure 5-7 is a graph showing the results of measuring the antiviral activity against the Omicron strain when a cyclic peptide is combined with an existing compound having anti-SARS-CoV-2 activity, as measured by viral RNA levels in cell culture supernatants. Figures 5-8 are graphs showing the results of measuring the antiviral activity against the Omicron strain when a cyclic peptide is combined with an existing compound having anti-SARS-CoV-2 activity, based on measurement of the amount of viral RNA in the cell culture supernatant.

[0010] 1. Abbreviations In this specification, the following abbreviations are used with the following meanings unless otherwise specified.

[0011] Abbreviations (general) Å: Angstrom (unit); AA: amino acid; Alloc: allyloxycarbonyl; ClAc: chloroacetyl; DCM: dichloromethane; DIC: N,N'-diisopropylcarbodiimide; DMSO: dimethyl sulfoxide; DMF: dimethylformamide; DIPEA or DIEA: N,N-diisopropylethylamine; DODT: 6-dioxa-1,8-octane-dithiol; FAM: fluorescein; Fmoc: 9-fluorenylmethyloxycarbonyl; Fmoc-Lys(Fmoc)-OH: N ２ , N ６-bis(((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysine; g: grams (unit); HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HOSu: N-hydroxysuccinimide; HPLC: high performance liquid chromatography; LC-MS or LC / MS: liquid chromatography mass spectrometry; MeCN: acetonitrile; mL: milliliters (unit); M: molar (unit); μL: microliters (unit); mM: millimolar (unit); μM: micromolar (unit); mg: milligrams (unit); MeCN: acetonitrile; mm: millimeters (unit); Mpe: 3-(3-methyl)pentyl; nm: nanometers (unit); nM: nanomolar (unit); Oxyma pure: ethyl cyanohydroxyiminoacetate; rpm: revolutions per minute (units); TAMRA: 5-carboxytetramethylrhodamine; tBu: tertiary butyl acetate; TFA: trifluoroacetic acid; TIS: triisopropylsilane; Trt or Tr: trityl group; MOI (multiplicity of infection): multiplicity of infection; RBD (receptor binding domain): receptor binding domain; (The "anti-SARS-CoV-2 antibody cocktail (casirivimab / imdevimab; REGN-COV2)" described in the Examples is a cocktail of two human IgG1 neutralizing antibodies (casirivimab and imdevimab) that target the receptor binding domain (RBD) of the SARS-CoV-2 spike protein.) RT-qPCR: Real-time quantitative PCR; HE staining: Hematoxylin and eosin staining; EDTA: Ethylenediaminetetraacetic acid; PBS: Phosphate-buffered saline; Pd(OAc)2: Palladium (II) acetate; PPh3: Triphenylphosphine; PhSiH3: Phenylsilane; FBS: Fetal bovine serum; CPE (cytopathic effect): Cytopathic effect; (morphological change of host cells caused by virus invasion) IC50 (half maximal (50%) inhibitory concentration): 50% inhibitory concentration CC50 (half cytotoxic concentration): 50% cytotoxic concentration WRCEVA: The World Reference Center for Emerging Viruses and Arboviruses.

[0012] Abbreviations (unnatural amino acids) 3Py: 3-pyridyl-L-alanine (CAS No. 64090-98-8); A4p: (S)-2-amino-3-(piperidin-4-yl)propanoic acid (CAS No. 342036-77-5); A4pipaa: 4-amino-1-(carboxymethyl)piperidine-4-carboxylic acid (Kishida Chemical); Ahp: (S)-2-aminoheptanoic acid (CAS No. 44902-02-5); Aib: α-methylalanine (CAS No. 62-57-7); Aind: (S)-2-amino-3-(2,3-dihydro-1H-inden-2-yl)propanoic acid (CAS No. 736122-13-7); aMeS: 2-methylserine; Cha: β-cyclohexyl-L-alanine (CAS No. 27527-05-5); Cit: (S)-2-amino-5-ureidopentanoic acid (CAS No. 372-75-8); Dab: L-α,γ-diaminobutanoic acid (CAS No. 1758-80-1); dd: D-aspartic acid; ddap: D-α,β-diaminopropionic acid (CAS No. 1915-96-4); de: D-glutamic acid; dk: D-lysine; dr: D-arginine; ds: D-serine; F4COO: 4-carboxy-L-phenylalanine (CAS No. 126109-42-0); F4F: 4-fluoro-L-phenylalanine (CAS No. 1132-68-9); Me: N-methyl; MeA: N-methylalanine; Meda: N-methyl-D-alanine; Medd: N-methyl-D-aspartic acid; Mede: N-methyl-D-glutamic acid; MeF: N-methylphenylalanine; MeE: N-methylglutamic acid; MeG: N-methylglycine; MeNle: N-methyl-L-norleucine (CAS No. 17343-27-0); Nal1: β-(1-naphthyl)L-alanine (CAS No. 55516-54-6); Yae: (S)-2-amino-3-(4-(2-aminoethoxy)phenyl)propanoic acid (CAS No. No. 1909283-20-0) Y3Me: (S)-2-amino-3-(4-hydroxy-3-methylphenyl)propanoic acid (CAS No.: 17028-03-4);cC10COO: 12-oxododecanoic acid; cC12COO: 14-oxotetradecanoic acid; cC14COO: 16-oxohexadecanoic acid; cC8COO: 10-oxodecanoic acid;

[0013] 2. Peptides (A) The present invention relates to peptides.

[0014] In one embodiment, the peptide of the present invention comprises the following amino acid sequence: MeA-MeF-S-Cha-Y-S-Y-Y-R-R-Cha-C (SEQ ID NO: 2) or an amino acid sequence having a substitution, addition, deletion or insertion at 1 to 10 amino acid residues selected from the group consisting of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th and 10th amino acid residues of the above amino acid sequence.

[0015] MeA-MeF-S-Cha-Y-S-Y-Y-R-R-Cha-C (SEQ ID NO: 2) is a peptide that was confirmed to bind to the spike protein of SARS-CoV-2 (GENE Accession No. YP_009724390) and have anti-SARS-CoV-2 activity in the Examples of this specification.

[0016] The amino acid sequence of SEQ ID NO: 2 may have a substitution, addition, deletion, or insertion of 1 to 10 amino acid residues selected from the group consisting of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, and tenth amino acid residues. Preferably, the substitution, deletion, addition, and / or insertion is of 1 to 8 amino acid residues selected from the group consisting of the first, second, third, fifth, sixth, eighth, ninth, and tenth amino acid residues of the amino acid sequence of SEQ ID NO: 2. The number of substituted, deleted, added, and / or inserted amino acids may be 1 to 10, with the lower limit being 1. The upper limit is 10, 9, 8, 7, 6, 5, 4, 3, or 2, with the minimum being 1. Preferably, the amino acid is "substituted." Such amino acid substitutions are suitably conservative amino acid substitutions.

[0017] A "conservative amino acid substitution" refers to a substitution with a functionally equivalent or similar amino acid. Conservative amino acid substitutions in a peptide result in a static change in the amino acid sequence of the peptide. For example, one or more amino acids with similar polarity act functionally equivalently, resulting in a static change in the amino acid sequence of the peptide. In general, substitutions within a group can be considered conservative in structure and function. However, as will be apparent to those skilled in the art, the role played by a particular amino acid residue can be determined by its context in the three-dimensional structure of the molecule containing that amino acid. For example, cysteine ​​residues can adopt an oxidized (disulfide) form that is less polar than its reduced (thiol) form. The long aliphatic portion of the arginine side chain can constitute a structurally and functionally important feature. Additionally, side chains containing aromatic rings (tryptophan, tyrosine, phenylalanine) can contribute to ion-aromatic or cation-pi interactions. In such cases, substitution of amino acids with these side chains with amino acids belonging to acidic or nonpolar groups can be structurally and functionally conservative. Residues such as proline, glycine, and cysteine ​​(in disulfide form) can have direct effects on the main-chain conformation and often cannot be substituted without structural distortion.

[0018] Conservative amino acid substitutions include specific substitutions based on side chain similarity (e.g., substitutions described in L. Lehninger, Biochemistry, 2nd edition, pp. 73-75, Worth Publisher, New York (1975)) and typical substitutions, as shown below.

[0019] Furthermore, conservative amino acid substitution is preferably, for example, substitution with an amino acid that belongs to the same group as a certain amino acid, in which natural amino acids are divided into groups based on the properties of their common side chains, as shown below.

[0020] Hydrophobic (also called non-polar) amino acids: Amino acids that exhibit hydrophobicity (non-polarity), including alanine (also written as "Ala" or simply "A"), glycine (also written as "Gly" or simply "G"), valine (also written as "Val" or simply "V"), leucine (also written as "Leu" or simply "L"), isoleucine (also written as "Ile" or simply "I"), proline (also written as "Pro" or simply "P"), phenylalanine (also written as "Phe" or simply "F"), tryptophan (also written as "Trp" or simply "W"), tyrosine (also written as "Tyr" or simply "Y"), and methionine (also written as "Met" or simply "M").

[0021] Hydrophobic amino acids can be further divided into the following groups:

[0022] Aliphatic Amino Acid: An amino acid having a fatty acid or hydrogen in the side chain, including Ala, Gly, Val, Ile, and Leu.

[0023] Aliphatic branched-chain amino acids: Amino acids having branched fatty acids in the side chain, including Val, Ile, and Leu.

[0024] Aromatic amino acids: Amino acids that have an aromatic ring in the side chain, including Trp, Tyr, and Phe.

[0025] Hydrophilic (also called polar) amino acids: Amino acids that exhibit hydrophilicity (polarity), including serine (also written as "Ser" or simply "S"), threonine (also written as "Thr" or simply "T"), cysteine ​​(also written as "Cys" or simply "C"), asparagine (also written as "Asn" or simply "N"), glutamine (also written as "Gln" or simply "Q"), aspartic acid (also written as "Asp" or simply "D"), glutamic acid (also written as "Glu" or simply "E"), lysine (also written as "lysine", "Lys" or simply "K"), arginine (also written as "Arg" or simply "R"), and histidine (also written as "His" or simply "H").

[0026] Hydrophilic amino acids can be further divided into the following groups:

[0027] Acidic amino acids: amino acids whose side chains are acidic, including Asp and Glu. Basic amino acids: amino acids whose side chains are basic, including Lys, Arg, and His.

[0028] Neutral amino acid: An amino acid whose side chain is neutral, including Ser, Thr, Asn, Gln, and Cys.

[0029] Furthermore, Gly and Pro can be classified as "amino acids that affect the direction of the main chain," and amino acids that contain sulfur molecules in their side chains, Cys and Met, can be classified as "sulfur-containing amino acids."

[0030] As used herein, the term "amino acid" includes not only natural amino acids but also unnatural amino acids. Unnatural amino acids include, for example, N-alkylamino acids in which the above-described natural amino acids are N-alkylated, and amino acids in which the nitrogen atom forming the peptide bond is modified with a branched or unbranched lower (e.g., C1-C5, preferably C1-C3, more preferably C1) alkyl group. N-Alkylamino acids are preferably N-ethylamino acids, N-butylamino acids, or N-methylamino acids, and more preferably N-methylamino acids. Unnatural amino acids also include chemically modified amino acids such as D-amino acids (also referred to as D-amino acids), β-amino acids, γ-amino acids, amino acid mutants, and amino acid derivatives, as well as amino acids that do not serve as building blocks of proteins in vivo, such as norleucine and ornithine. Furthermore, the amino acids include naturally occurring amino acids having a functional group added to the side chain or substituted with another functional group (for example, amino acids having a substitution or addition in an arylene group, alkylene group, or other portion of the side chain, amino acids having an increased C number in the arylene group, alkylene group, or alkyl group of the side chain, amino acids having a substitution in an aromatic ring of the side chain, and heterocyclized or condensed cyclized amino acids).

[0031] Furthermore, by adding or substituting a structure such as a functional group to the side chain of a natural amino acid, properties different from those of natural amino acids can be imparted. For example, A4p is an amino acid having a piperidyl group in the side chain of alanine. The addition of this piperidyl group gives A4p the properties of a polar amino acid, which is basic, unlike alanine, which belongs to the nonpolar amino acid group. In other words, unnatural amino acids with similar side chain properties can be included in the aforementioned groups, which are obtained by dividing natural amino acids based on the properties of their common side chains. For example, N-methylarginine (MeR), an amino acid in which the main chain nitrogen atom of arginine, which belongs to the basic amino acids, is methylated, is an unnatural amino acid, but can be classified as a basic amino acid because it exhibits basicity. In this way, unnatural amino acids with side chain properties similar to those of a certain amino acid can also be included as targets for conservative amino acid substitution.

[0032] Non-limiting examples of unnatural amino acids include N-methyl amino acids, 3Py, A4p, A4pipaa, Ahp, Aib, Aind, aMeS, Cha, Cit, Dab, dd, ddap, dk, dr, ds, F4COO, 4Py, F4F, MeNle, Meda, Medd, Mede, Na11, Yae, Y3Me, and the like. For example, Ahp, Aib, Aind, Cha, MeNle, F4COO, F4F, Nall, Yae, and Y3Me can be classified as hydrophobic amino acids, and 3Py, Cit, A4p, A4pipaa, Dab, ddap, dk, dr, dd, and ds can be classified as hydrophilic amino acids. Furthermore, Ahp, Aib, Cha, and MeNle can be classified as aliphatic amino acids, Cit as a polar amino acid, 3Py, A4p, A4pipaa, Dab, ddap, dk, and dr as basic amino acids, dd as an acidic amino acid, ds as a neutral amino acid, and 4Py, F4COO, F4F, Nall, Yae, and Y3Me as aromatic amino acids. D-amino acids such as dd and ds can be classified as D-amino acids, but can also be classified according to the properties of their side chains. N-methylamino acids can also be classified as N-alkylamino acids, or can be classified according to the properties of the side chains of the original non-N-methylated amino acids.

[0033] In one embodiment, the peptide of the present invention may be a peptide in which the fourth amino acid Cha is substituted in the amino acid sequence represented by SEQ ID NO: 2. In one embodiment, the fourth amino acid Cha may be substituted with Aind.

[0034] In one embodiment, the peptide of the present invention may be a peptide in which the seventh amino acid Y is substituted in the amino acid sequence represented by SEQ ID NO: 2. In one embodiment, the seventh amino acid Y may be substituted with a substituted Y, for example, Y3Me.

[0035] In one embodiment, the peptide may further comprise, but is not limited to, D-glutamic acid at the C-terminus. In this specification, the terms "but not limited to" and "in one embodiment" may be used interchangeably.

[0036] In one embodiment, the peptide is a cyclic peptide. "Cyclic peptides" will be described in detail in "3. Peptides (B)."

[0037] The peptide may contain additional amino acid residues in addition to SEQ ID NO: 2. The "additional amino acid residues" will be described in detail in "3. Peptides (B)."

[0038] The peptide preferably binds to the spike protein of SARS-CoV-2. The peptide has anti-SARS-CoV-2 activity. The peptide preferably binds to the spike protein of SARS-CoV-2 and has anti-SARS-CoV-2 activity. "Binds to the spike protein of SARS-CoV-2" and "has anti-SARS-CoV-2 activity" are described in detail in "3. Peptides (B)."

[0039] 3. Peptides (B) The present invention relates to peptides.

[0040] The peptide of the present invention comprises the following amino acid sequence: X1-X2-X3-Cha-X4-X5-Y-X6-X7-X8-Cha-C (SEQ ID NO: 1), wherein: X1 is A, E, MeA, or MeE; X2 is an N-methyl amino acid; X3 is S or Aib; X4 is an amino acid having an unsubstituted or substituted aromatic ring in the side chain; X5 is any amino acid; X6 is an amino acid having an unsubstituted or substituted aromatic ring in the side chain; X7 is any amino acid; and X8 is any basic amino acid.

[0041] Amino acids having an unsubstituted aromatic ring in their side chains are amino acids having an aromatic ring in their side chains, and include natural amino acids belonging to the aromatic amino acid group, unnatural amino acids such as N-acetylated aromatic amino acids, and amino acids in which an aromatic ring has been added or substituted to the side chain of a natural amino acid. Furthermore, amino acids having a substituted aromatic ring in their side chains are amino acids having an aromatic ring in their side chains, and include natural amino acids belonging to the aromatic amino acid group or unnatural amino acids such as N-acetylated aromatic amino acids, in which a portion of the aromatic ring has been substituted with another molecule or a functional group, or a heterocyclic ring, and amino acids having a fused ring. For example, amino acids having a substitution in the side chain hydroxy group of Tyr, amino acids having a substituent in the benzene ring of Phe, amino acids having a ring containing a heteroatom in the side chain indole ring of Trp, amino acids having a substituent, and amino acids having a functional group added to any of these.

[0042] The above options for X1 to X8 can be selected in any combination.

[0043] In one embodiment, X1 is MeA, MeE or 3Py. In one embodiment, X1 is MeA or MeE. In one embodiment, X1 is 3Py.

[0044] In one embodiment, X2 is MeF or MeNle.

[0045] In one embodiment, X4 is unsubstituted or substituted Y or unsubstituted or substituted F.

[0046] In one embodiment, X4 is Y or F4F.

[0047] In one embodiment, X5 is any hydrophilic amino acid or Aib, or any N-methylated hydrophilic amino acid or Aib. In one embodiment, X5 is any of Aib, A4pipaa, E, K, S, P, dd, ddap, ds, de, MeE, MeG, aMeS, Meda, Medd, and Med. In one embodiment, X5 is any of Aib, E, K, S, dd, ddap, ds, and MeE. In one embodiment, X5 is any of A4pipaa, P, de, MeG, aMeS, Meda, Medd, and Med.

[0048] In one embodiment, X6 is unsubstituted or substituted Y or unsubstituted or substituted F.

[0049] In one embodiment, X6 is any of Y, Yae, 3Py, Na1, and Y3Me. In one embodiment, X6 is any of Y, Yae, 3Py, and Na1. In one embodiment, X6 is Y3Me.

[0050] In one embodiment, X7 is A, Ahp, Cit, Dab, E, F4COO, K, or R.

[0051] In one embodiment, X8 is K, R, A4p, dk, or dr. In one embodiment, X8 is K, R, or A4p. In one embodiment, X8 is dk or dr.

[0052] In one embodiment, the peptide of the present invention may be a peptide in which the fourth amino acid Cha is substituted in the amino acid sequence represented by SEQ ID NO: 1. In one embodiment, the fourth amino acid Cha may be substituted with Aind.

[0053] In one embodiment, the peptide of the present invention may be a peptide in which the seventh amino acid Y is substituted in the amino acid sequence represented by SEQ ID NO: 1. In one embodiment, the seventh amino acid Y may be substituted with a substituted Y, for example, Y3Me.

[0054] The unsubstituted Y is tyrosine, a natural amino acid, and the substituted Y includes an amino acid derivative of Y in which the phenolic hydroxyl group in the side chain of Y is substituted, an amino acid derivative of Y having a heterocyclic ring, or an amino acid derivative of Y having a condensed polycyclic structure.

[0055] Furthermore, unsubstituted F is phenylalanine, a natural amino acid, and substituted F includes an amino acid having a substituent on the benzene ring that phenylalanine has in the side chain, an amino acid derivative of F having a heterocyclic ring, or an amino acid derivative of F having a condensed polycyclic structure.

[0056] The options of X1 to X8 in the above aspect can be selected in any combination.

[0057] In one embodiment, the peptide may further comprise, but is not limited to, D-glutamic acid at the C-terminus. In this specification, the terms "but not limited to" and "in one embodiment" may be used interchangeably.

[0058] In one embodiment, the peptide is a cyclic peptide. A "cyclic peptide" refers to a peptide in which two amino acids are bonded to form a ring structure, either wholly or partially. The peptide also includes peptides in which amino acids in the peptide form a cross-linked structure, peptides in which a ring structure is formed by lactam ring formation or macrocyclization, and peptides having a lasso peptide-like structure. In other words, the cyclic peptide may be any peptide in which a portion thereof forms a ring structure, and may also have a linear portion.

[0059] Peptides generally have poor metabolic stability in vivo and, due to their large size, have difficulty penetrating cell membranes. To address these issues, peptide cyclization has been used. Cyclization of peptides improves protease resistance and metabolic stability, and also restricts conformational changes, increasing their rigidity and suggesting improved membrane permeability and affinity for target proteins.

[0060] In one embodiment, the peptide has a cyclic structure in which a chloroacetylated amino acid is bonded to a cysteine ​​residue contained in the peptide. In one embodiment, the peptide has a cyclic structure in which an N-terminal amino acid (the first amino acid residue) is bonded to a cysteine ​​residue contained in the peptide. In one embodiment, the peptide has a cyclic structure in which an N-terminal amino acid (the first amino acid residue) is bonded to a twelfth cysteine ​​residue contained in the peptide. In one embodiment, the peptide has a cyclic structure in which a chloroacetylated N-terminal amino acid (the first amino acid residue) is bonded to a twelfth cysteine ​​residue contained in the peptide. "Chloroacetylation" may be "halogen acetylation" using another halogen. "Acetylation" may also be "acylation" using an acyl group other than an acetyl group.

[0061] In one embodiment, the peptide comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 2-126, 132-234. In one embodiment, the peptide is a cyclic peptide comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 2-126, 132-234.

[0062] The peptide may contain additional amino acid residues in addition to SEQ ID NO: 1. The additional amino acid residues may be contained in the peptide forming a cyclic structure, or further amino acid residues may be added to the cyclic peptide in the form of a linker. The number of amide bonds (number of amino acids / length) in the peptide or peptide portion is not particularly limited. The total number of amino acid residues (referring to the remaining number of amino acids contained in the peptide forming a cyclic structure; if further amino acid residues are added to the cyclic peptide in the form of a linker, these amino acids are not included) is preferably 20 or less. Preferably, the number of amino acids is 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more, and preferably 19 or less, 18 or less, 17 or less, 16 or less, or 15 or less. Most preferably, the number of amino acids is 12 to 13.

[0063] Furthermore, a linker may be further added to the cyclic peptide. Examples of linkers include the aforementioned amino acid linkers (peptide linkers), chemical linkers, fatty acid linkers, nucleic acid linkers, and sugar chain linkers. Alternatively, a complex of a chemical linker and a peptide linker may be used. An example of a chemical linker is a PEG (polyethyleneglycol) linker. For example, a PEG linker consisting of 1 to 24 ethylene glycol units may be used. The linker may also be a fatty acid linker containing a divalent chemical moiety derived from a fatty acid. The amino acid (peptide) linker is a linker containing at least one amino acid, and examples thereof include glycine-rich peptides such as peptides having the sequence [Gly-Gly-Gly-Gly-Ser]n (where n is 1, 2, 3, 4, 5, or 6) as described in U.S. Pat. No. 7,271,149, and serine-rich peptide linkers as described in U.S. Pat. No. 5,525,491.

[0064] The conjugate of a chemical linker and a peptide linker may be, for example, a conjugate of an amino acid and a PEG linker, a conjugate of an amino acid and a fatty acid linker, or a conjugate of an amino acid, a PEG, and a fatty acid linker. For example, among the peptides represented by SEQ ID NOS: 219 to 234, the structure from the 13th amino acid onwards may be used (e.g., in SEQ ID NOS: 219, the structure from the 13th amino acid onwards is the de-dk(cC10COO) structure, and in SEQ ID NOS: 225, the structure from the 13th amino acid onwards is the de-PEG2c-dk(cC10COO) structure). In addition, in peptide linkers, the bond between amino acids or between an amino acid and a chemical linker may be formed via the side chain of an amino acid. Addition of a linker may, without limitation, change the physical properties (e.g., solubility) of the peptide.

[0065] The linker may be added at any position. For example, it may be bound to Cys located on the N-terminus side, or to an amino acid contained in the cyclic peptide. Preferably, it is bound to Cys located on the N-terminus side, or to the side chain of an amino acid contained in the cyclic peptide.

[0066] Furthermore, the peptide may form a multimer via a linker or the like. For example, since the spike protein of SARS-CoV-2 is known to form a trimer, the peptide may be a trimer accordingly. In this case, the peptide may be a homomer consisting of peptides having the same sequence, or a heteromer consisting of peptides having different sequences.

[0067] The peptide preferably binds to the spike protein of SARS-CoV-2. The peptide has anti-SARS-CoV-2 activity. The peptide preferably binds to the spike protein of SARS-CoV-2 and has anti-SARS-CoV-2 activity.

[0068] "Binding to the spike protein of SARS-CoV-2" refers to binding to the spike protein of SARS-CoV-2 (GENE Accession No. YP_009724390). Binding to the spike protein can be measured by any known method of measuring intermolecular binding. It can be determined in any suitable manner known per se, including, but not limited to, surface plasmon resonance (SPR) assays, Scatchard analysis, and / or competitive binding assays such as radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays, as well as different variants thereof known per se in the art.

[0069] "Anti-coronavirus activity" refers to the activity of inhibiting coronavirus activity (also referred to as inactivation), and examples include inhibition of coronavirus infection, growth, maturation, reduction of virus count, reduction of infectivity, suppression of cytotoxicity, suppression of viral cytopathies, prevention of aggravation of viral infection, improvement of mortality rate, and suppression / amelioration of pneumonia symptoms. Inhibition of infection or growth is preferred. The peptide is determined to have "anti-coronavirus activity" when it inhibits coronavirus activity, for example, by 30%, 50%, 70%, or 90%. Alternatively, the peptide is determined to have "anti-coronavirus activity" when it exhibits an effect of inhibiting coronavirus activity equivalent to or greater than that of other known COVID drugs, such as remdesivir, REGN-COV2 (a combination drug of casirivimab and imdevimab), or EIDD-1931 as the active form of molanupiravir.

[0070] For example, and without limitation, a peptide is determined to have anti-coronavirus activity when its addition to a coronavirus cell culture supernatant reduces viral RNA in the supernatant, for example, by 30%, 50%, 70%, or 90%. "Anti-SARS-CoV-2 activity" refers to the activity of inhibiting the activity of SARS-CoV-2. A peptide is determined to have "anti-SARS-CoV-2 activity" when it inhibits SARS-CoV-2 activity by, for example, 30%, 50%, 70%, or 90% compared to when the peptide is not present. For example, in Example 1 of the present specification, it was determined that a peptide had anti-SARS-CoV-2 activity if the amount of SARS-CoV-2 RNA contained in the cell culture supernatant of SARS-CoV-2 (Wuhan strain) was reduced to 10% or less (a 90% reduction) when added at 1 μM. The amount of viral RNA can be measured by known methods, preferably by RT-qPCR. In Example 5, it was demonstrated that the peptide had anti-SARS-CoV-2 activity not only against the Wuhan strain but also against other mutant viruses such as alpha, beta, gamma, delta, and omicron viruses. That is, the addition of the peptide at 1 μM reduced the amount of SARS-CoV-2 RNA contained in the cell culture supernatant of SARS-CoV-2 to 10% or less (a 90% reduction).

[0071] In Examples 4 and 11, cells expressing SARS-CoV-2-derived S protein (pseudovirus) and luciferase subunits using an S protein expression vector into which a SARS-CoV-2-derived S protein gene has been inserted and an expression vector into which a luciferase subunit protein gene has been inserted were mixed with cells expressing human ACE2 protein using an expression vector into which a luciferase subunit protein gene has been inserted. When cells were fused via the binding of SARS-CoV-2-derived S protein and human ACE2 protein, the fluorescence intensity emitted as a result of the reconstitution of luciferase protein within the cells was measured, and the S protein inhibitory activity (%) and IC ５０The IC value was calculated. In a non-limiting example, an S protein inhibitory activity (%) of 50% or more, preferably 75% or more, and more preferably 90% or more is considered to have anti-SARS-CoV-2 activity. Alternatively, in a non-limiting example, an IC ５０ The value is 1 x 10 －６ mol / L or less, preferably 1x10 －８ mol / L or less, more preferably 5x10 －９ mol / L or less, it is determined to have anti-SARS-CoV-2 activity.

[0072] In Examples 6 and 11, the inhibitory effect on viral cytopathic effect (CPE) was investigated using cells expressing SARS-CoV-2 virus and human ACE2. The antiviral effect of the peptides added to a cell culture system was evaluated using the cell viability after a certain period of time after infection as an indicator of cytotoxicity. A peptide is considered to have anti-SARS-CoV-2 activity when the CPE reduction is 50% or more, preferably 75% or more, and more preferably 90% or more. Alternatively, the peptide is considered to have anti-SARS-CoV-2 activity when the IC ５０ The value is 1 x 10 －６ mol / L or less, preferably 1x10 －８ mol / L or less, it is determined to have anti-SARS-CoV-2 activity.

[0073] In Example 7, the peptide's effect of preventing the progression of SARS-CoV-2 infection in mice was evaluated using a mouse SARS-CoV-2 infection fatality model, and its effect of improving mortality rate and lung histology were evaluated. Anti-SARS-CoV-2 activity is determined when administration of the peptide prevents progression of SARS-CoV-2 infection compared to administration without the peptide, for example, when weight loss is prevented or when mortality rate is improved. "Weight loss is prevented" means, for example, that, 7 days after SARS-CoV-2 infection, when weight loss is 10% or more without administration of the peptide, weight loss is reduced to less than 10%, preferably less than 8%, and more preferably less than 5%, by administration of the peptide.

[0074] Alternatively, administration of the peptide suppresses and improves pneumonia symptoms compared to administration without the peptide. Pneumonia symptoms can be confirmed by, for example, cellular images of the proliferation phase of diffuse alveolar damage, such as perivascular cell clusters and inflammatory cell infiltration and alveolar epithelial regeneration in the alveolar field. Administration of the peptide can reduce these conditions. Furthermore, in Example 8, the efficacy of the peptide against pneumonia was investigated using a hamster SARS-CoV-2 infection model. Administration of the peptide can suppress and improve pneumonia symptoms, such as chronic active interstitial and pleural inflammation, compared to administration without the peptide. When the above-described effects against pneumonia are confirmed, the peptide is determined to have anti-SARS-CoV-2 activity.

[0075] The peptide is determined to have anti-SARS-CoV-2 activity when it exhibits at least one, and preferably a plurality of, the above-mentioned effects of reducing viral RNA, suppressing antiviral cytopathies, suppressing cytotoxicity, suppressing viral cytopathies, preventing viral infection from worsening, improving mortality, and suppressing / ameliorating pneumonia symptoms.

[0076] In one embodiment, the peptide has the structure:

[0077]

[0078] In this specification, unless otherwise specified, the term "peptide" includes both "2. Peptide (A)" and "3. Peptide (B)."

[0079] 4. Production of Peptides The peptides of the present invention can be produced by any known method for producing peptides, for example, as follows.

[0080] Chemical synthesis methods such as liquid phase methods, solid phase methods, and hybrid methods combining liquid phase and solid phase methods; genetic engineering methods, etc. The solid phase method involves, for example, an esterification reaction between the hydroxyl group of a resin having a hydroxyl group and the carboxyl group of a first amino acid (usually the C-terminal amino acid of the target peptide) whose α-amino group is protected with a protecting group. Known dehydration condensation agents such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIC) can be used as the esterification catalyst.

[0081] 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, and the carboxy group is 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 carboxy group of the main chain are protected is added, and the carboxy group is activated to bond the second and third amino acids. This process is repeated until a peptide of the desired length is synthesized, and then all functional groups are deprotected.

[0082] Examples of resins for solid-phase synthesis include Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, Amino-PEGA resin (Merck), HMPA-PEGA resin (Merck), etc. These resins can be used after washing with a solvent (dimethylformamide (DMF), 2-propanol, methylene chloride, etc.).

[0083] Examples of protecting groups for α-amino groups include benzyloxycarbonyl (Cbz or Z) group, tert-butoxycarbonyl (Boc) group, fluorenylmethoxycarbonyl (Fmoc) group, benzyl group, allyl group, allyloxycarbonyl (Alloc) group, etc. The Cbz group can be deprotected by hydrofluoric acid, hydrogenation, etc., the Boc group can be deprotected by trifluoroacetic acid (TFA), and the Fmoc group can be deprotected by treatment with piperidine or pyrrolidine.

[0084] For protection of the α-carboxy group, for example, methyl ester, ethyl ester, allyl ester, benzyl ester, tert-butyl ester, cyclohexyl ester, etc. can be used.

[0085] The activation of the carboxy group can be carried out using a condensing agent, such as dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), or 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU).

[0086] Cleavage of the peptide chain from the resin can be carried out by treating with an acid such as TFA or hydrogen fluoride (HF).

[0087] The production of peptides by recombinant DNA techniques (translation synthesis systems) can be carried out using nucleic acids that encode the peptides. The nucleic acids that encode the peptides may be DNA or RNA.

[0088] The nucleic acid encoding the peptide can be prepared by a known method or a method similar thereto. For example, it can be synthesized using an automated synthesizer. A restriction enzyme recognition site may be added to insert the obtained DNA into a vector. Alternatively, a base sequence encoding an amino acid sequence for excising the resulting peptide chain using an enzyme or the like may be incorporated.

[0089] As described above, when the peptide is fused with a membrane-permeable peptide or the like, the nucleic acid also includes a nucleic acid encoding the membrane-permeable peptide.

[0090] 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 encoding the peptide of interest and a peptide that binds to it.

[0091] Subsequently, an expression vector is prepared using a nucleic acid encoding the peptide. The nucleic acid can be inserted downstream of a promoter in the expression vector either directly or after digestion with a restriction enzyme or after addition of a linker. Examples of vectors include Escherichia 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 (retrovirus, vaccinia virus, adenovirus, adeno-associated virus (AAV), cauliflower mosaic virus, tobacco mosaic virus, baculovirus, etc.), and cosmids.

[0092] 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.

[0093] The expression vector may also incorporate, for example, 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.

[0094] Next, a suitable host cell is transformed with the expression vector. The host can be selected appropriately in relation to the vector. Examples of hosts that can be used include Escherichia coli, Bacillus subtilis, Bacillus sp., 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. Depending on the type of host, transformation can be performed according to known methods such as lipofection, calcium phosphate method, electroporation, microinjection, and particle gun method. The transformant is cultured according to standard methods to express the desired peptide.

[0095] To purify the peptide from the culture of the transformant, the cultured cells are harvested, suspended in an appropriate buffer, disrupted by sonication, freeze-thawing, or other methods, and then centrifuged or filtered to obtain a crude extract. If the peptide is secreted into the culture medium, the supernatant is collected.

[0096] 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, reversed-phase high performance liquid chromatography, etc.).

[0097] 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.

[0098] In one embodiment, the translation synthesis system may be a cell-free translation system. Cell-free translation systems generally allow expression products to be obtained in a highly pure form without purification. Cell-free translation systems contain, for example, ribosomal proteins, aminoacyl-tRNA synthetases (ARSs), ribosomal RNAs, amino acids, rRNAs, 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. Escherichia coli extract or wheat germ extract may be added to improve expression efficiency. Additionally, rabbit erythrocyte extract or insect cell extract may be added.

[0099] By continuously supplying energy to a system containing these using dialysis, it is possible to produce protein in amounts ranging from several hundred μg to several mg / mL, but this is not limited to this. 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 Escherichia coli-derived systems such as RTS-100 (registered trademark) from Roche Diagnostics, PURESYSTEM from GeneFrontier, and PURExpress In Vitro Protein Synthesis Kit from New England Biolabs, as well as systems using wheat germ extract from Zoigene and CellFree Sciences.

[0100] In a cellular 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 the tRNA may be used. Such an aminoacyl-tRNA can be synthesized using an artificial ribozyme.

[0101] Such ribozymes include flexizyme (H. Murakami, H. Saito, and H. Suga, (2003), Chemistry & Biology, Vol. 10, 655-662; and WO2007 / 066627, etc.). Flexizyme is also known by the name of the original flexizyme (Fx), and modified versions thereof, such as dinitrobenzyl flexizyme (dFx), enhanced flexizyme (eFx), and aminoflexizyme (aFx).

[0102] By using a 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 non-standard amino acid may also 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 linked peptide by this method.

[0103] The peptides can be chemically synthesized using various methods commonly used in the art, including, for example, stepwise solid-phase synthesis, semi-synthesis of peptide fragments via conformationally assisted religation, and chemical ligation. The peptides are synthesized using various solid-phase techniques, such as those described in K. J. Jensen, P. T. Shelton, and S. L. Pedersen, Peptide Synthesis and Applications, 2nd Edition, Springer, 2013. A preferred strategy is based on the combination of an Fmoc group, which temporarily protects the α-amino group and allows selective base removal, and a protecting group that temporarily protects side chain functional groups and is stable under Fmoc removal conditions. Selection of such general peptide side chains is described in the aforementioned Peptide Synthesis and Applications, 2nd Edition, and G. B. Fields and R. L. Noble, "Solid Phase Peptide Synthesis Utilizing 9-Fluorenylmethoxycarbonyl Amino Acids," Int. J. Peptide Protein Res. 35, 1990, 161-214, and the like. Preferred peptide side chain protecting groups include, for example, a benzyl group, a tert-butyl group, and a trityl (Trt) group for the hydroxy group of serine or threonine, a 2-bromobenzyloxycarbonyl group and a tert-butyl group for the hydroxy group of tyrosine, a Boc group, a methyltetrazolethiol (Mtt) group, an Alloc group, and an ivDde group for the amino group of the lysine side chain, and a methyltetrazolethiol (Mtt) group for the imidazole group of histidine. Trt and Boc groups for the guanidyl group of arginine; 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) group for the guanidyl group of arginine; tert-butyl, allyl, and 3-methylpentane (Mpe) groups for the carboxyl groups of glutamic acid and aspartic acid; Trt groups for the carboxamide groups of glutamine and asparagine; and Trt and monomethoxytrityl (Mmt) groups for the thiol group of cysteine.

[0104] The peptide 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 solid-phase resin described above is used, and the C-terminal carboxyl group of a peptide whose N-terminus is appropriately protected with Fmoc or the like, or the C-terminal carboxyl group of an amino acid protected with Fmoc, is converted into an activated ester with an appropriate reagent, followed by addition to the amino group on the solid-phase resin. Subsequent peptide chain elongation can be achieved by sequentially repeating the removal of the N-terminal protecting group (Fmoc group) and the condensation of a protected amino acid derivative according to the amino acid sequence of the target peptide. Note that this can liberate the target peptide at the final stage. For example, the conditions for liberation can be as described in Teixeira, W. E. Benckhuijsen, P. E. de Koning, A. R. P. M. Valentijn, J. W. The cleavage can be achieved by using a TFA solution containing water, silyl hydride, and thiol as scavengers in TFA, as described in 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).

[0105] Synthesis of the peptides 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 or their successors.

[0106] The activation of the carboxy group can be carried out using a condensing 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), or 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU).

[0107] Peptide cyclization can be carried out according to known methods. Non-limiting examples include designing a peptide to contain two or more cysteine ​​residues, which allows for the formation of a cyclic structure via disulfide bonds after translation. Cyclization can also be achieved by synthesizing a peptide with a chloroacetyl group at the N-terminus and placing a cysteine ​​residue containing a sulfur molecule in the peptide using genetic code reprogramming technology, according to the method of Goto et al. (Y. Goto, et al. ACS Chem. Biol. 3 120-129 (2008)). This allows spontaneous nucleophilic attack of the mercapto group on the chloroacetyl group after translation, resulting in cyclization of the peptide via a thioether bond. Cyclization can also be achieved by placing other amino acid combinations within the peptide that bond to form a ring using genetic code reprogramming technology. Alternatively, cyclization can be achieved by placing an L-2-aminoadipic acid residue in the peptide and bonding it to the N-terminal main chain amino group. As such, any known cyclization method can be used without particular limitation.

[0108] 5. Pharmaceutical Compositions The present invention also relates to pharmaceutical compositions comprising the peptides of the present invention. The diseases targeted by the pharmaceutical compositions are infectious diseases caused by coronaviruses, preferably infectious diseases caused by SARS-CoV-2, such as COVID-19. The peptides of the present invention are useful as active ingredients in pharmaceutical compositions for preventing or treating infectious diseases caused by coronaviruses.

[0109] Without limitation, the pharmaceutical composition has anti-SARS-CoV-2 virus activity. "Having anti-SARS-CoV-2 virus activity" is as described in "3. Peptide (B)."

[0110] In one embodiment, the pharmaceutical composition is for the prevention or treatment of a coronavirus infection. In one embodiment, the coronavirus infection is COVID-19.

[0111] "Coronaviruses" are a type of single-stranded RNA virus with a lipid bilayer membrane envelope. Taxonomically, they are classified in the Coronaviridae family of the Cornidovirales suborder of the Nidovirales order, and are further divided into the Letovirinae and Orthocoronavirinae subfamily. Inside the virus particle, there is a nucleocapsid consisting of genomic RNA and the N protein that binds to it. Furthermore, the surface of the envelope contains protein structures called spike (S) protein, envelope (E) protein, and membrane (M) protein. The spike protein in particular is known to play an important role during infection (Non-Patent Document 3).

[0112] Coronaviruses that infect humans are broadly divided into three types: coronaviruses that commonly infect humans (Human Coronavirus (HCoV)), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV). There are also coronaviruses that infect animals (such as porcine epidemic diarrhea virus (PEDV), transmissible porcine gastroenteritis virus (TGEV), infectious bronchitis virus (IBV), mouse hepatitis virus (MHV), and feline infectious peritonitis virus (FIPV)). It is known that coronaviruses that infect animals rarely infect other species of animals. Unless otherwise specified, the term "coronavirus" used herein refers to all of the above human- and animal-infecting coronaviruses.

[0113] "SARS-CoV-2" refers to a novel coronavirus discovered as a sister species of the severe acute respiratory syndrome coronavirus, which is a coronavirus that causes severe acute respiratory syndrome (SARS). It was first reported in Wuhan, China at the end of 2019 and has caused a global pandemic. SARS-CoV-2 has spike proteins on its surface, and is thought to infect hosts via these spike proteins (Non-Patent Document 4).

[0114] Like other viruses, SARS-CoV-2 mutates during repeated replication and infection, producing numerous mutant strains. Thousands of SARS-CoV-2 mutant strains exist, classified by lineage and phylogenetic group, with names such as the Pango lineage. However, in recent years, the World Health Organization (WHO) has introduced names using the Greek alphabet, leading to the use of names such as α and β strains (Non-Patent Document 5). Mutant strains that pose risks such as increased infectivity or reduced efficacy of vaccines and treatments are considered "Variants of Interest (VOI)." Once these risks are verified, they are renamed "Variants of Concern (VOC)" by national health authorities and other organizations, and are considered particularly dangerous and require vigilance. The α (alpha), β (beta), γ (gamma), δ (delta), and ο (omicron) strains are all considered to be VOCs. In particular, the delta strain is said to be more infectious than existing mutant strains and less sensitive to antibodies, so there is a need for a therapeutic agent that is also effective against the delta strain (Non-patent documents 5 and 6).

[0115] The mutation sites of the spike protein for the alpha, beta, gamma, and delta strains are as shown in Table 1 below. In this specification, "SARS-CoV-2" refers not only to the strain reported in Wuhan (Wuhan strain, wild strain), but also to the European lineage variants (alpha strain), South African lineage variants (beta strain), Brazilian lineage variants (gamma strain), Indian lineage variants (delta strain), US lineage variants (epsilon strain), US lineage variants (iota strain), Indian lineage variants (kappa strain), Philippine lineage variants (theta strain), Peruvian lineage variants (lambda strain), Colombian lineage variants (mu strain), Omicron strain, and multiply mutated virus strains such as the AY.4.2 lineage, which is a delta strain with additional mutations, and the BA.2 lineage, which is an Omicron strain with additional mutations, as well as other mutant strains.

[0116]

[0117] "COVID-19" refers to an infectious disease caused by SARS-CoV-2. It often presents with initial symptoms similar to those of influenza or the common cold, with acute respiratory illness being the primary symptom, but it may also be asymptomatic. In this specification, the term "COVID-19" refers to the various symptoms caused by SARS-CoV-2.

[0118] The pharmaceutical composition may contain the peptide itself, or may contain a pharmaceutically acceptable salt of the peptide or a solvate thereof. As used herein, "peptide" may include a pharmaceutically acceptable salt or a solvate thereof, unless otherwise specified. The pharmaceutical composition preferably contains an effective amount of the peptide as an active ingredient.

[0119] 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 such as intramuscular injection, intravenous injection, and subcutaneous injection, transdermal administration, and transmucosal administration (nasal, oral, ocular, pulmonary, vaginal, and rectal) administration.

[0120] The pharmaceutical composition can be modified in various ways, taking into account the tendency of polypeptides to be 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. Alternatively, the polypeptide can be encapsulated in a sustained-release base such as biodegradable polymer compounds such as polylactic acid glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, or nanospheres. For transdermal administration, a weak electric current can be applied to the skin surface to penetrate the stratum corneum (iontophoresis).

[0121] The pharmaceutical composition may contain the active ingredient as is, or may be formulated by adding pharmaceutically 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, and poultices. Formulation can be carried out by conventional methods using, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavorings, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, etc. as appropriate.

[0122] Examples of ingredients used in formulations include, but are not limited to, purified water, saline, phosphate buffer, dextrose, glycerol, ethanol and other pharmaceutically 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, and the like.

[0123] In view of the fact that peptides are generally poorly absorbed transmucosally, 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.

[0124] When the pharmaceutical composition is in the form of a pill or tablet, it may be coated with a sugar coating, gastric or enteric coating material.

[0125] When the pharmaceutical composition is an injection, it may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. Furthermore, it may also contain a wetting agent, an emulsifier, a dispersant, a stabilizer, a solubilizer, a solubilizing agent, a preservative, etc.

[0126] The pharmaceutical composition may be intended for not only humans but also non-human mammals or birds, including non-human primates (monkeys, chimpanzees, gorillas, etc.), livestock animals (pigs, cows, horses, sheep, etc.), dogs, cats, rats, mice, guinea pigs, rabbits, etc.

[0127] The dosage, particularly when administered to humans, 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 may be, but is not limited to, 30 μg to 100 g, 100 μg to 500 mg, or 100 μg to 100 mg, administered once or in divided doses. In the case of injection, 1 μg / kg to 3000 μg / kg, or 3 μg / kg to 1000 μg / kg may be administered once or in divided doses, depending on the patient's weight.

[0128] The present invention relates to a method for preventing or treating coronavirus infection by administering a peptide of the present invention.

[0129] The present invention relates to the use of the peptide of the present invention for the prevention or treatment of coronavirus infections.

[0130] The present invention relates to use of the peptide of the present invention for producing a pharmaceutical composition for the prevention or treatment of coronavirus infection.

[0131] The present invention relates to the use of the peptide of the present invention as a pharmaceutical composition for the prevention or treatment of coronavirus infections.

[0132] The present invention relates to a peptide of the present invention for use in a method for preventing or treating a coronavirus infection.

[0133] The present invention relates to the peptide of the present invention for use as a pharmaceutical composition for the prevention or treatment of coronavirus infections.

[0134] 6. Diagnostic Composition The present invention also relates to a diagnostic composition for diagnosing SARS-CoV-2 viral infection, comprising the peptide of the present invention.

[0135] Since the peptide of the present invention binds to SARS-CoV-2, it can also be used as a diagnostic composition for detecting SARS-CoV-2. The diagnostic agent may be a detection agent for diagnosing whether or not a person is infected with SARS-CoV-2, and when used as a detection agent, the peptide may be detectably labeled.

[0136] The peptide may be detectably labeled. Examples of peptide labels include antibodies labeled with enzymes such as peroxidase and alkaline phosphatase; radioactive substances such as 125I, 131I, 35S, and 3H; fluorescent substances such as fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine isothiocyanate, and near-infrared fluorescent materials; and luminescent substances such as luciferase, luciferin, and aequorin. Antibodies labeled with nanoparticles such as gold colloids and quantum dots can also be detected. For example, a complex can be prepared between the peptide and an antibody that binds to a specific target related to SARS-CoV-2 infection (e.g., the spike protein of SARS-CoV-2), and the antibody or peptide can be labeled to prepare a complex. The complex can then be administered and detected to detect the presence or severity of SARS-CoV-2 infection. In immunoassays, the peptides can be labeled with biotin and then detected by binding avidin or streptavidin labeled with an enzyme or the like.

[0137] Among immunoassays, ELISA using enzyme labeling is preferred because it allows for simple and rapid antigen measurement. For example, an antibody is immobilized on a solid support, a sample is added and reacted, and then the labeled peptide is added and reacted. After washing, the sample is reacted with an enzyme substrate to develop color, and the absorbance is measured, thereby enabling the presence or absence, severity, etc. of SARS-CoV-2 infection to be detected. After reacting the antibody immobilized on the solid support with the sample, the unlabeled peptide may be added, and an antibody against the peptide may be enzyme-labeled and then added. The antibody may be immobilized on the surface of the solid support or inside the solid support.

[0138] 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-nitrophenyl phosphate (pNPP), etc. can be used.

[0139] As used herein, the term "solid phase carrier" is not particularly limited as long as it is a carrier capable of immobilizing an antibody, and examples thereof include glass, metal, resin, etc. microtiter plates, substrates, beads, nitrocellulose membranes, nylon membranes, PVDF membranes, etc., and target substances can be immobilized on these solid phase carriers according to known methods.

[0140] The present invention relates to a diagnostic kit comprising the peptide of the present invention, which comprises reagents and instruments necessary for the above-mentioned detection (including, but not limited to, any or all of the peptide of the present invention, antibody, solid phase support, buffer, enzyme reaction stop solution, microplate reader, etc.).

[0141] The present invention relates to a tester comprising the peptide of the present invention.

[0142] The present invention relates to a method for diagnosing coronavirus infection by administering a peptide of the present invention.

[0143] The present invention relates to the use of the peptides of the present invention for the diagnosis of coronavirus infections.

[0144] The present invention relates to the use of the peptide of the present invention for the production of a diagnostic composition for the diagnosis of coronavirus infection.

[0145] The present invention relates to the use of the peptide of the present invention as a diagnostic composition for the diagnosis of coronavirus infections.

[0146] The present invention relates to a peptide of the present invention for use in a method for diagnosing a coronavirus infection.

[0147] The present invention relates to the peptide of the present invention for use as a diagnostic composition for diagnosing coronavirus infection.

[0148] 7. Combination Use The pharmaceutical composition of the present invention comprising the peptide of the present invention may be used in combination with other drugs for the prevention or treatment of coronavirus infections.

[0149] The present invention relates to a pharmaceutical composition comprising the peptide of the present invention, which is used in combination with other drugs for the prevention or treatment of coronavirus infections. The "pharmaceutical composition" is as explained in the section "5. Pharmaceutical Composition."

[0150] Drugs for the prevention or treatment of other coronavirus infections are drugs other than the peptides of the present invention that are effective in the prevention or treatment of coronavirus infections, and are not particularly limited. For example, they may be compounds such as RNA polymerase inhibitors such as remdesivir, favipiravir, and molanupiravir, steroids such as dexamethasone and ciclesonide, Janus kinase inhibitors such as baricitinib, and polymerase inhibitors such as nafamostat, or antibodies such as tocilizumab and sarilumab, or cocktails of multiple antibodies. They may also be mixtures thereof. Drugs for the prevention or treatment of other coronavirus infections are, but are not limited to, selected from the group consisting of remdesivir, a combination of casirivimab and imdevimab, and molanupiravir or its active form.

[0151] Remdesivir is a novel nucleotide analogue prodrug and antiviral drug. It has been found to exhibit antiviral activity against single-stranded RNA viruses (coronaviruses including respiratory syncytial virus, Junin virus, Lassa fever virus, Nipah virus, Hendra virus, MERS, and SARS virus). It has also demonstrated antiviral activity against multiple coronavirus infections, including SARS-CoV-2.

[0152] "Casirivimab-imdevimab" is sold under the trade name REGEN-COV®. It is an artificial "antibody cocktail" designed to create resistance to the SARS-CoV-2 coronavirus. It consists of two monoclonal antibodies (combined), casirivimab (REGN10933) and imdevimab (REGN10987), preferably used in combination.

[0153] "Molnupiravir" is an orally active antiviral drug developed as an anti-influenza drug. Molnupiravir is a prodrug of the synthetic nucleoside derivative N4-hydroxycytidine (also referred to as "EIDD-1931"). N4-hydroxycytidine is metabolized to β-D-N4-hydroxycytidine 5'-triphosphate (also referred to as "EIDD-1931 5'-triphosphate" or "NHC-TP"). Molnupiravir or its active forms include, but are not limited to, N4-hydroxycytidine, β-D-N4-hydroxycytidine 5'-triphosphate.

[0154] The timing of administration of the pharmaceutical composition comprising the peptide of the present invention and the drug for preventing or treating other coronavirus infections is not particularly limited, and the pharmaceutical composition comprising the peptide of the present invention may be administered before, simultaneously with, or after the administration of the drug for preventing or treating other coronavirus infections. In one embodiment, the pharmaceutical composition comprising the peptide of the present invention is administered simultaneously with the drug for preventing or treating other coronavirus infections.

[0155] The peptide of the present invention may be combined with another drug for preventing or treating coronavirus infections for simultaneous administration. In one aspect, the present invention relates to a combination comprising the peptide of the present invention and another drug for preventing or treating coronavirus infections.

[0156] The present invention also relates to a drug for preventing or treating other coronavirus infections, which is used in combination with a pharmaceutical composition comprising the peptide of the present invention.

[0157] The present invention relates to a method for preventing or treating a coronavirus infection by administering a peptide of the present invention and another drug for the prevention or treatment of a coronavirus infection. In one embodiment, the peptide of the present invention may be administered before, after, or simultaneously with the administration of another drug for the prevention or treatment of a coronavirus infection.

[0158] The present invention relates to the use of the peptide of the present invention and other agents for the prevention or treatment of coronavirus infections for the prevention or treatment of coronavirus infections.

[0159] The present invention relates to use of the peptide of the present invention for the manufacture of a pharmaceutical composition for the prevention or treatment of coronavirus infections, comprising the peptide of the present invention, in combination with other drugs for the prevention or treatment of coronavirus infections.

[0160] The present invention relates to the use of the peptide of the present invention as a pharmaceutical composition for the prevention or treatment of coronavirus infections in combination with other drugs for the prevention or treatment of coronavirus infections.

[0161] The present invention relates to the peptides of the present invention for use in methods for preventing or treating coronavirus infections and other agents for preventing or treating coronavirus infections.

[0162] The present invention relates to the peptide of the present invention for use as a pharmaceutical composition for the prevention or treatment of coronavirus infections in combination with other drugs for the prevention or treatment of coronavirus infections.

[0163] The present invention relates to the use of a drug for preventing or treating another coronavirus infection in combination with a pharmaceutical composition for preventing or treating a coronavirus infection, comprising the peptide of the present invention, for the prevention or treatment of coronavirus infection. In one aspect, the drug for preventing or treating another coronavirus infection suggests the prevention or treatment of coronavirus infection in combination with a pharmaceutical composition comprising the peptide of the present invention.

[0164] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Those skilled in the art can easily modify and alter the present invention based on the description in this specification, and such modifications and alterations are within the technical scope of the present invention.

[0165] All raw materials, building blocks, reagents, acids, bases, solid-phase resins, and solvents used in the chemical synthesis in the following examples were either commercially available or could be synthesized by those skilled in the art using organic chemistry techniques. Unless otherwise specified, commercially available amino acids containing protecting groups were used as they were.

[0166] Peptide chain elongation on the solid-phase resin 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 a CEM Liberty Blue automated peptide synthesizer according to the manufacturer's instructions.

[0167] The resin used was Sieber Amide resin, and the amount used ranged from 5 mg to 2 g depending on the peptide.

[0168] By way of example, some commonly used amino acids are listed below with the side chain protecting groups indicated in parentheses: Fmoc-N-Me-Phe-OH; Fmoc-Ala-OH; Fmoc-N-Me-Ala-OH; Fmoc-N-Me-Nle-OH; Fmoc-Aib-OH; Fmoc-Cha-OH; Fmoc-Cit-OH; Fmoc-Nal-OH; Fmoc-Tyr(tBu)-OH; Fmoc-Cys(Trt)-OH; Fmoc-Ser(Trt)-OH; Fmoc-dd(Mpe)-OH.

[0169] The resulting crude peptide was purified by reversed-phase preparative HPLC using a Waters AutoPurification System-SQD2 single quadruple mass spectrometer, and elution was performed while monitoring the m / z ions derived from the target substance. It was confirmed that the mass spectrum obtained in ESI-positive scan mode and the mass spectrum containing 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 column used, are shown in each example.

[0170] The structure of chemically synthesized peptides was determined by ESI-MS(+) mass spectrometry, where the molecular weight was calculated based on the amino acids used in the target sequence and the building blocks used as needed. "ESI-MS(+)" refers to electrospray ionization mass spectrometry performed in positive ion mode. Detected masses were reported in "m / z" units. Compounds with molecular weights greater than approximately 1000 were frequently detected as doubly or triply charged ions.

[0171] Example 1 Identification of Peptides with Anti-SARS-CoV-2 Activity In this example, several cyclic peptides with anti-SARS-CoV-2 activity were identified.

[0172] First, the spike protein of SARS-CoV-2 (GENE Accession No. YP_009724390) was targeted, and peptides that bind to the target protein were identified using the screening methods described in International Publication No. WO2014 / 119600, International Publication No. WO2012 / 033154, or International Publication No. WO2007 / 066627.

[0173] Using a SARS-CoV-2 infection experiment system, we investigated whether the peptide and its analogs actually have anti-SARS-CoV-2 activity. Specifically, an infection experiment using SARS-CoV-2 was performed in a BSL3 experimental section. VeroE6 / TMPRSS2 cells (a VeroE6 African green monkey cell line expressing transmembrane serine protease TMPRSS2, which is highly susceptible to Middle East respiratory syndrome coronavirus) were cultured in a 96-well plate at 3.0 x 10 ４ The cells were seeded and cultured overnight.

[0174] The cells were treated with a medium containing infectious SARS-CoV-2 (JPN / TY / WK-521 strain) at a multiplicity of infection (MOI) of 0.003 for 1 hour, washed, and then replaced with a new medium containing the cyclic peptides synthesized in Examples 2 and 3 described below, or remdesivir as a control, along with the compounds. The cyclic peptides were added to a final concentration of 1 μM or 50 nM. Remdesivir was added to a final concentration of 10 μM. The culture supernatant was collected after 24 hours, and viral RNA contained therein was extracted using a MagMax Viral / Pathogen II Nucleic Acid Isolation kit (Thermo Fisher Scientific).

[0175] Viral RNA contained in the extracts was quantified by real-time RT-qPCR using a one-step qRT-PCR kit (THUNDERBIRD® Probe One-step qRT-PCR kit; Toyobo Co., Ltd.).

[0176] Primers 5'-ACAGGTACGTTAATAGTTAATAGCGT-3' (SEQ ID NO: 127) 5'-ATATTGCAGCAGTACGCACACA-3' (SEQ ID NO: 128) Probe 5'-FAM-ACACTAGCCATCCTTACTGCGCTTCG-TAMRA-3' (SEQ ID NO: 129)

[0177] The results are shown in Table 2. 1-13 in the "Sequence" column of Table 2 indicate the amino acid residue numbers in the amino acid sequence of the peptide. All of the peptides in Table 2 are cyclic peptides in which the first amino acid residue is bonded to the 12th cysteine ​​residue (C). The actual synthesis method will be described later; after synthesizing an amino acid sequence consisting of 12 or 13 residues shown in the "Sequence" column of Table 2, a chloroacetyl group is introduced into the first amino acid residue, and cyclization is achieved by bonding the acetyl group contained in the chloroacetyl group with a sulfur molecule S contained in the cysteine ​​residue. Furthermore, peptides with "de" written as the 13th amino acid residue have D-glutamic acid at the C-terminus (outside the cyclic structure).

[0178] When the amount of viral RNA in the culture supernatant was 10% or less compared to before the addition of the peptide, it was determined that the peptide had anti-SARS-CoV-2 activity. In Table 2, samples that showed anti-SARS-CoV-2 activity when 50 nM of the peptide was added were marked with a "2," and samples that showed no activity when 50 nM of the peptide was added but showed activity when 1 μM of the peptide was added were marked with a "1." The ESI-MS (m / z) values ​​in Table 2 are the ESI-MS (+) observed values ​​([M+2H] ２＋ ) are shown. 125 cyclic peptides shown in Table 2 were shown to have anti-SARS-CoV-2 activity.

[0179] Remdesivir showed anti-SARS-CoV-2 activity when added at 10 μM.

[0180] Table 2: Peptide sequences and anti-SARS-CoV-2 activity

[0181]

[0182]

[0183]

[0184]

[0185]

[0186] Example 2 Synthesis of Cyclic Peptide 5555_p000 (SEQ ID NO: 2) This example describes the synthesis of cyclic peptide 5555_p000 (SEQ ID NO: 2).

[0187]

[0188] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical Industry Co., Ltd., 0.65 mmol / g) according to the general method described above. A CEM Liberty Blue solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. To introduce each residue, Fmoc-AA / HATU / DIEA (4.2 equivalents / 4 equivalents / 8 equivalents) was used per equivalent of resin. The reaction was carried out once for 10 minutes in DMF at 75°C. The first and second residues were reacted twice for 10 minutes at 75°C, the ninth and tenth residues were reacted twice for 20 minutes at 30°C, and the twelfth residue was reacted once for 20 minutes at 30°C.

[0189] The basic conditions for Fmoc removal were a 3-minute reaction with a 20% piperidine solution in DMF at 75°C. However, the first and second residues were reacted at 25°C for 5 minutes, followed by a 10-minute reaction. The introduction of a chloroacetyl group was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin carrying the Fmoc-protected peptide obtained in the previous step using the method described above, followed by adding a 0.2 M DMF solution of chloroacetic acid (5 equivalents), a 0.5 M DMF solution of HATU (5 equivalents), and a 0.5 M DIPEA DMF solution (5 equivalents) to the solid-phase resin and shaking at room temperature for 30 minutes.

[0190] Deprotection of the side chain and cleavage from the solid-phase resin were carried out by washing the resin obtained after the chloroacetyl group introduction step with DMF and methylene chloride and drying under reduced pressure, followed by adding reagent cocktail-A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) to the reaction vessel containing the solid-phase resin, followed by shaking at 25°C for 35 minutes.

[0191] 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 the frit and mixed with the filtrate. This filtrate was added to an excess of a diethyl ether / hexane (1 / 1) mixed solvent chilled to 0°C, resulting in a cloudy white precipitate. This mixture was centrifuged (9000 rpm, 1 minute), and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether / hexane chilled to 0°C again and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO / water (9 / 1) so that the final concentration of the peptide was 5 mM based on the molar number of the solid-phase resin, and then 6 equivalents of triethylamine were added and the mixture was shaken at 25°C for 18 hours. The resulting reaction solution was concentrated under reduced pressure using an EZ-2 Elite.

[0192] The resulting crude product was purified using the following conditions: column: Waters Xbridge® C18 5 μm® 30×150 mm; mobile phase: A=0.1% TFA(H ２ in 0), B = 0.1% TFA in MeCN; temperature: 40°C; gradient (% B): 9-34% over 3 min, then 34-39% over 8 min, then 39-60% over 1 min; flow rate: 45 mL / min).

[0193] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions: retention time = 3.86 min; column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm 100 Å; mobile phase: A = 0.025% TFA (H ２ in 0), B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% B conc): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min; Flow rate: 0.5 mL / min. ESI-MS (+) observed m / z = 909.85, (M+2H). ２＋

[0194] The cyclic peptides listed in Table 1 were synthesized in the same manner as 5555_p000 unless other synthesis examples are described in this specification.

[0195] Example 3: Synthesis of Cyclic Peptides This example describes the synthesis of cyclic peptides.

[0196] (1) Example 3-1 Synthesis of cyclic peptide 5555_p003 (SEQ ID NO: 5)

[0197]

[0198] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical Industry Co., Ltd., 0.65 mmol / g) according to the general method described above. A CEM Liberty Blue HT solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. To introduce each residue, Fmoc-AA / DIC / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used per equivalent of resin. The reaction was carried out once for 2 minutes at 105°C in DMF. The first residue was reacted twice for 30 minutes at 75°C, the second and third residues were reacted twice for 2 minutes at 105°C, the tenth residue was reacted twice for 15 minutes at 50°C, and the twelfth residue was reacted once for 15 minutes at 50°C.

[0199] The basic conditions for Fmoc removal were a 1-minute reaction with 83 mM Oxyma Pure in 4% pyrrolidine in DMF at 110°C. The second, third, fourth, fifth, and sixth residues were reacted with 10% pyrrolidine in DMF at 50°C for 90 seconds. The first residue was reacted twice with 10% pyrrolidine in DMF at 25°C for 1 minute. The introduction of a chloroacetyl group was performed by removing the Fmoc group from the α-amino group of the solid-phase resin carrying the Fmoc-protected peptide obtained in the previous step using the method described above. Then, a 0.2 M chloroacetic acid solution in DMF (5 equivalents), a 0.5 M HATU solution in DMF (5 equivalents), and a 1 M DIEA solution in DMF (10 equivalents) were added to the solid-phase resin and shaken at room temperature for 30 minutes.

[0200] For the deprotection of the side chain and the cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was washed with DMF and methylene chloride and dried under reduced pressure. Then, the reaction vessel containing the solid-phase resin was charged with reagent cocktail A (TFA / H ２A mixture of 0.25% O / TIS / DODT (volume ratio 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at 25°C for 30 minutes.

[0201] 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 the frit and mixed with the filtrate described above. When this filtrate was added to an excess of a diethyl ether / hexane (1 / 1) mixed solvent chilled to 0°C, a cloudy white precipitate formed. This mixture was centrifuged (10,000 rpm, 1 minute), and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether / hexane chilled to 0°C again and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO to a final concentration of 5 mM based on the molar number of the solid-phase resin, and then 5 equivalents of triethylamine were added and the mixture was shaken at 25°C for 16 hours. The resulting reaction solution was concentrated under reduced pressure using an EZ-2 Elite.

[0202] The resulting crude product was purified using the following conditions (Column: Waters Xbridge® C18 5 μm® 30×150 mm; Mobile phase: A=0.1% TFA(H ２ in 0), B = 0.1% TFA in MeCN; temperature: 40°C; gradient (% B): 18-43% over 3 min, then 43-48% over 8 min, then 48-60% over 1 min; flow rate: 45 mL / min).

[0203] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions: retention time = 5.70 min; column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; mobile phase: A = 0.025% TFA (H ２ in 0), B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% B conc): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min; Flow rate: 0.5 mL / min. ESI-MS(+) observed m / z = 888.87 (M+2H). ２＋

[0204] (2) Example 3-2 Synthesis of cyclic peptide 5555_p020 (SEQ ID NO: 22)

[0205]

[0206] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical Industry Co., Ltd., 0.65 mmol / g) according to the general method described above. A CEM Liberty Prime solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. To introduce each residue, Fmoc-AA / DIC / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used per equivalent of resin. The reaction was carried out once for 2 minutes at 105°C in DMF. The first residue was reacted twice for 30 minutes at 75°C; the second, third, fifth, and sixth residues were reacted twice for 2 minutes at 105°C; and the twelfth residue was reacted once for 15 minutes at 50°C.

[0207] The basic conditions for Fmoc removal were a 1-minute reaction with a 4% pyrrolidine solution of 83 mM Oxyma Pure in DMF at 110°C. However, the first residue was reacted twice for 1 minute at 25°C. The introduction of a chloroacetyl group was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin carrying the Fmoc-protected peptide obtained in the previous step using the method described above, followed by adding a 0.2 M chloroacetic acid solution in DMF (5 equivalents), a 0.5 M HATU solution in DMF (5 equivalents), and a 0.5 M DIEA solution in DMF (10 equivalents) to the solid-phase resin and shaking at room temperature for 30 minutes.

[0208] For the deprotection of the side chain and the cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was washed with DMF and methylene chloride and dried under reduced pressure. Then, the reaction vessel containing the solid-phase resin was charged with reagent cocktail A (TFA / H ２ A mixture of 0.25% O / TIS / DODT (volume ratio 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at 25°C for 5 minutes.

[0209] 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 the frit and mixed with the filtrate. This filtrate was added to an excess of a 1 / 1 diethyl ether / hexane mixed solvent chilled to 0°C, resulting in a cloudy white precipitate. This mixture was centrifuged (9000 rpm, 1 minute), and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether / hexane chilled to 0°C again and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO to a final concentration of 5 mM based on the molar amount of solid-phase resin, and then 10 equivalents of triethylamine were added and the mixture was shaken at 25°C for 16 hours. 20 equivalents of acetic acid were added to the resulting reaction solution, which was then concentrated under reduced pressure using an EZ-2 Elite.

[0210] The resulting crude product was purified using the following conditions: column: Waters Xbridge® C18 5 μm® 30×150 mm; mobile phase: A=0.1% TFA(H ２ in 0), B = 0.1% TFA in MeCN; temperature: 40°C; gradient (% B): 20-45% over 3 min, then 45-50% over 8 min, then 50-60% over 1 min; flow rate: 45 mL / min).

[0211] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions: retention time = 6.01 min; column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; mobile phase: A = 0.025% TFA (H ２ in 0), B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% B conc): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min; Flow rate: 0.5 mL / min. ESI-MS(+) observed m / z = 937.43 (M+2H). ２＋

[0212] (3) Example 3-3 Synthesis of cyclic peptide 5555_p028 (SEQ ID NO: 30)

[0213]

[0214] The target peptide was synthesized using HMPB-MBHA resin (Merck, 0.85 mmol / g) and Fmoc-de(OtBu)-HMPB-MBHA resin, which was synthesized by reacting 1 equivalent of resin with Fmoc-D-Glu(OtBu)-OH / DIC / DMAP (4 equivalents / 4 equivalents / 0.1-0.5 equivalents) in DMF / DCM (1 / 10) at room temperature for 30 minutes. A CEM Liberty Blue solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's instructions. Each residue was introduced by reacting 1 equivalent of resin with Fmoc-AA / DIC / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) in DMF at 90°C for 3 minutes. However, the first residue was reacted twice at 75°C for 30 minutes, the second residue was reacted twice at 75°C for 20 minutes, and the 12th residue was reacted once at 50°C for 20 minutes.

[0215] The basic conditions for Fmoc removal were a 3-minute reaction with a 20% piperidine solution in DMF at 75°C. However, the first, second, third, fourth, fifth, and sixth residues were reacted twice for 5 minutes at 25°C. The introduction of a chloroacetyl group was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin carrying the Fmoc-protected peptide obtained in the previous step using the method described above. Then, a 0.1 M DMF solution of chloroacetic acid (5 equivalents), a 0.1 M DMF solution of HATU (5 equivalents), and a 0.2 M DMF solution of DIEA (10 equivalents) were added to the solid-phase resin and shaken at room temperature for 30 minutes.

[0216] For the deprotection of the side chain and the cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was washed with DMF and methylene chloride and dried under reduced pressure. Then, the reaction vessel containing the solid-phase resin was charged with reagent cocktail A (TFA / H ２ A mixture of 0.25% O / TIS / DODT (volume ratio 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at 25°C for 30 minutes.

[0217] 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 the frit and mixed with the filtrate. This filtrate was added to an excess of diisopropyl ether solvent chilled to 0°C, resulting in a cloudy white precipitate. This mixture was centrifuged (9000 rpm, 1 minute), and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether chilled to 0°C again and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO / water (19 / 1) so that the final concentration of the peptide was 5 mM based on the molar number of the solid-phase resin, and then 15 equivalents of triethylamine were added and the mixture was shaken at 25°C for 4 hours. The resulting reaction solution was concentrated under reduced pressure using an EZ-2 Elite.

[0218] The resulting crude product was purified using the following conditions: column: Waters Xbridge® C18 5 μm® 50×250 mm; mobile phase: A=0.1% TFA(H ２ in 0), B = 0.1% TFA in MeCN; temperature: 50°C; gradient (% B): 14.4-38.8% over 3 min, then 38.8-43.9% over 15 min, then 43.9-60% over 3 min; flow rate: 120 mL / min).

[0219] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 81.62%. Analytical conditions: retention time = 5.40 min; column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; mobile phase: A = 0.025% TFA (H ２ in 0), B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% B conc): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min; Flow rate: 0.5 mL / min. ESI-MS(+) observed m / z = 922.92 (M+2H). ２＋

[0220] (4) Example 3-4 Synthesis of cyclic peptide 5555_p070 (SEQ ID NO: 72)

[0221]

[0222] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical Industry Co., Ltd., 0.65 mmol / g) according to the general method described above. A CEM Liberty Blue solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. To introduce each residue, Fmoc-AA / DIC / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used per equivalent of resin, and the reaction was carried out once for 3 minutes at 90°C in DMF. The first residue was reacted twice for 30 minutes at 75°C, the tenth residue was reacted twice for 20 minutes at 50°C, and the twelfth residue was reacted once for 20 minutes at 50°C.

[0223] The basic conditions for Fmoc removal were a 3-minute reaction with a 20% piperidine solution in DMF at 75° C. Introduction of a chloroacetyl group was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin carrying the Fmoc-protected peptide obtained in the previous step using the method described above, followed by adding a 0.2 M DMF solution of chloroacetic acid (4 equivalents), a 0.5 M DMF solution of HATU (4 equivalents), and a 1 M DMF solution of DIEA (8 equivalents) to the solid-phase resin and shaking at room temperature for 30 minutes.

[0224] For the deprotection of the side chain and the cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was washed with DMF and methylene chloride and dried under reduced pressure. Then, the reaction vessel containing the solid-phase resin was charged with reagent cocktail A (TFA / H ２ A mixture of 0.25% O / TIS / DODT (volume ratio 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at 25°C for 45 minutes.

[0225] 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 the frit and mixed with the filtrate. This filtrate was added to an excess of a diethyl ether / hexane (1 / 1) mixed solvent chilled to 0°C, resulting in a cloudy white precipitate. This mixture was centrifuged (9000 rpm, 1 minute), and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether / hexane chilled to 0°C again and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO / water (9 / 1) so that the final concentration of the peptide was 5 mM based on the molar number of the solid-phase resin, and then 6 equivalents of triethylamine were added and the mixture was shaken at 25°C for 18 hours. The resulting reaction solution was concentrated under reduced pressure using an EZ-2 Elite.

[0226] The resulting crude product was purified using the following conditions: column: Waters Xbridge® C18 5 μm® 30×150 mm; mobile phase: A=0.1% TFA(H ２ in 0), B = 0.1% TFA in MeCN; temperature: 40°C; gradient (% B): 14-39% over 3 min, then 39-44% over 8 min, then 44-60% over 1 min; flow rate: 45 mL / min).

[0227] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions: retention time = 4.91 min; column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; mobile phase: A = 0.025% TFA (H ２ in 0), B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% B conc): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min; Flow rate: 0.5 mL / min. ESI-MS(+) observed m / z = 940.37 (M+2H). ２＋

[0228] (5) Example 3-5 Synthesis of cyclic peptide 5555_p073 (SEQ ID NO: 75)

[0229]

[0230] The target peptide was synthesized using HMPB-MBHA resin (Merck, 0.85 mmol / g) and Fmoc-de(OtBu)-HMPB-MBHA resin, which was synthesized by reacting 1 equivalent of resin with Fmoc-D-Glu(OtBu)-OH / DIC / DMAP (4 equivalents / 4 equivalents / 0.1-0.5 equivalents) in DMF / DCM (1 / 10) at room temperature for 30 minutes. A CEM Liberty Prime solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's instructions. Each residue was introduced using Fmoc-AA / DIC / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) per equivalent of resin, and the reaction was carried out once for 2 minutes at 105°C in DMF. However, the 12th residue was reacted once for 15 minutes at 50° C. The 1st residue was reacted twice for 30 minutes in DMF at 75° C. using Fmoc-AA / HATU / DIEA (4.2 equivalents / 8 equivalents / 4 equivalents) per equivalent of resin.

[0231] The basic conditions for Fmoc removal were a 1-minute reaction with 83 mM Oxyma Pure in a 4% DMF solution of pyrrolidine at 110°C. However, for the first residue, a 5-minute reaction with a 20% DMF solution of piperidine at 25°C was followed by a 10-minute reaction. The introduction of a chloroacetyl group was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin carrying the Fmoc-protected peptide obtained in the previous step using the method described above. Then, a 0.1 M DMF solution of chloroacetic acid (4 equivalents), a 0.1 M DMF solution of HATU (4 equivalents), and a 0.2 M DMF solution of DIEA (8 equivalents) were added to the solid-phase resin and shaken at room temperature for 30 minutes.

[0232] For the deprotection of the side chain and the cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was washed with DMF and methylene chloride and dried under reduced pressure. Then, the reaction vessel containing the solid-phase resin was charged with reagent cocktail A (TFA / H ２A 92.5:2.5:2.5:2.5 volume ratio mixture of 0.01% PEG / TIS / DODT was added, and the mixture was shaken at 25°C for 10 minutes. The reaction solution was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate. This filtrate was added to an excess of a 1 / 1 diethyl ether / hexane mixture chilled to 0°C, resulting in a cloudy white precipitate. This mixture was centrifuged (9000 rpm, 2 minutes), and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether / hexane chilled to 0°C again and dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. The peptide cyclization reaction was carried out by dissolving the peptide in 1 / 1 MeCN / water to a final concentration of 5 mM based on the molar amount of solid-phase resin, adding 5 equivalents of triethylamine, and shaking at 25°C for 15 hours. The resulting reaction solution was concentrated under reduced pressure using EZ-2 Elite.

[0233] The resulting crude product was purified using the following conditions: column: Waters Xbridge® C18 5 μm® 30×150 mm; mobile phase: A=0.1% TFA(H ２ in 0), B = 0.1% TFA in MeCN; temperature: 40°C; gradient (% B): 14-39% over 3 min, then 39-44% over 8 min, then 44-60% over 1 min; flow rate: 45 mL / min).

[0234] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions: retention time = 5.01 min; column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; mobile phase: A = 0.025% TFA (H ２ in 0), B = 0.025% TFA in MeCN; Temperature: 60°C; Gradient (% B conc): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min; Flow rate: 0.5 mL / min. ESI-MS(+) observed m / z = 939.91 (M+2H). ２＋

[0235] Example 4 Measurement of antiviral activity of cyclic peptides against various mutant virus strains using cell fusion method In this example, the antiviral activity of cyclic peptides against various mutant virus strains was measured using the cell fusion method.

[0236] To generate VeroE6 / hTMPRSS2 / HiBiT cells stably expressing HiBiT in the VeroE6 / hTMPRSS2 cell line (JCRB1819 from the JCRB Cell Bank), the HaloTag gene was cloned into the pBiT3.1-N[CMV / HiBiT / Blast] vector (Promega N2361), and the vector was then introduced using an electroporator NEPA21 (Nepgene). Since the vector encodes a blasticidin resistance gene, blasticidin (Invitrogen) was added to the cells at a concentration of 10 μg / mL, and drug selection was performed until the vector-untransfected control cells died. The established cells were used for assays after three passages following drug selection. VeroE6 / hTMPRSS2 / HiBiT cells were cultured in DMEM medium (ThermoFisherScientific, 10569010) containing 10% heat-inactivated fetal bovine serum (ThermoFisherScientific, 10270-106) and 50 μg / mL gentamicin (Nacalai Tesque, 11980-14). The same medium was also used to culture HEK293T cells (European Collection of Authenticated Cell Cultures). Both VeroE6 / hTMPRSS2 / HiBiT cells (1:10 to 1:5) and HEK293T cells (1:10 to 1:5) were maintained by passage twice weekly and used in the study.

[0237] An S protein expression vector was prepared by inserting the SARS-CoV-2 S protein gene, which induces various mutant viral strains, into the pcDNA3.1(+) mammalian expression vector (V79020, Thermo Fisher Scientific) or the pcDNA3.4 vector (Thermo Fisher Scientific). Furthermore, an LgBiT expression vector was prepared by cloning the HaloTag gene into the pBiT1.1-N[TK / LgBiT] vector (N198A, Promega). Here, the various mutant viral strains refer to the alpha, beta, gamma, and delta strains mutated from the wild-type Wuhan strain.

[0238] VeroE6 / hTMPRSS / HiBiT cells (1.7x10 ４ The cells (cells / well) were seeded in a 96-well plate (Thermo Fisher Scientific 136101) and incubated at 37°C, 5% CO ２ HEK293T cells (2.0 × 10 ６ The cells (1.25 μg / well) were seeded in a 6-well plate (MS-80060, Sumitomo Bakelite Co., Ltd.), and co-transfected with the S protein expression vector or control vector (1.25 μg / well) and the LgBiT expression vector (1.25 μg / well) by reverse transfection using Lipofectamine® 3000 reagent (L3000-015, Thermo Fisher Scientific Co., Ltd.) and Opti-MEM® medium (11058-021, Thermo Fisher Scientific Co., Ltd.). The cells were then incubated at 37°C, 5% CO ２ The cells were cultured in the presence of S protein for 48 hours (preparation of S protein-expressing HEK293T cells and control cells).

[0239] To prepare a 2x concentrated solution of the cyclic peptide to be tested for the assay, a 100% DMSO solution of the peptide was diluted with assay solution (DMEM medium containing 0.5% heat-inactivated fetal bovine serum (10270-106, Thermo Fisher Scientific) and 50 μg / mL gentamicin (11980-14, Nacalai Tesque)), and seven 5-fold serial dilutions were prepared. These were then dispensed into a 96-well plate (MS-3296U, Sumitomo Bakelite). The final DMSO concentration was 0.1% or less. Following transfection, the medium was removed from HEK293T cells cultured for 48 hours, and the cells were incubated at room temperature for 10 minutes in 500 μL of 0.5 mM EDTA / PBS (13567-84, Nacalai Tesque) per well. The detached HEK293T cell suspension was added with a 4-fold volume of DMEM medium containing 10% heat-inactivated fetal bovine serum, centrifuged at 200 × g for 3 minutes, the supernatant was removed, and the cells were resuspended in the aforementioned assay solution (DMEM medium containing 0.5% heat-inactivated fetal bovine serum). The cell count and viability of the S protein-expressing HEK293T cells and control cells were measured using a cell counter (Bio-Rad TC20) and trypan blue (Bio-Rad 1450022). The viable cell count was 2 × 10 ６ The assay solution was added to adjust the concentration to 1 / mL. The medium of the VeroE6 / hTMPRSS2 / HiBiT cells described above, which had been seeded on a 96-well plate and cultured for 48 hours, was removed, and 50 μL / well of a 2x concentrated solution of the cyclic peptide was added. Immediately after, 50 μL / well of the suspension of the S protein-expressing HEK293T cells or control cells was added, and after pipetting, the cells were incubated at 37°C, 5% CO ２ After removing the cell medium, 75 μL / well of a detection solution prepared by diluting the substrate solution of the Nano-Glo® Live Cell Assay System (Promega N2012) 5-fold with Opti-MEM® medium containing 0.5% heat-inactivated fetal bovine serum was added, and the cells were incubated at room temperature for 5 minutes. After that, the luminescence intensity was measured using a SpectraMax® Paradigm® multimode microplate reader (Molecular Devices).

[0240] Based on the concentration-inhibition sigmoid curve of the compound in this system, Imax and IC were calculated using four-parameter logistic regression. ５０ Specifically, the S protein inhibitory activity (%) was calculated using the following formula based on the measured luminescence intensity values.

[0241] IC ５０ Values ​​were calculated using TIBCO Spotfire® software by four-parameter logistic regression using the following formula:

[0242] The calculated antiviral activity (IC ５０ ) are shown in Table 3.

[0243]

[0244] In Table 3, the activity of each peptide is expressed as an Imax value (maximum inhibitory effect). An Imax of 100% means that cell fusion and luminescence are below the detection limit due to the compound's inhibition of the binding between S protein and ACE2 protein. In Table 3, Imax shows a maximum effect of nearly 100% against five types of SARS-COV2 mutant strains, and it is determined that the cyclic peptide has an antiviral inhibitory effect against a wide range of viral mutations. Furthermore, the IC ５０ was detected at a very low concentration of several nM, indicating that the specific activity was high.

[0245] Table 3 shows that the cyclic peptides also have antiviral activity against various mutant virus strains of SARS-CoV-2.

[0246] Example 5 Measurement of Antiviral Activity Against Various Mutant Virus Strains In this example, the antiviral activity of cyclic peptides against various mutant virus strains was measured.

[0247] VeroE6 / TMPRSS2 cells were plated in a 96-well plate at 3.0 x 10 ４Cells were seeded and cultured overnight. Infectious SARS-CoV-2 (Wuhan strain: WK-521, alpha strain: QK002, gamma strain: TY7-501, beta strain: TY8-612, delta strain: TY11-927, or Omicron strain: TY38-873) was treated with the compounds at an MOI of 0.003 for 1 hour. The virus was then washed away, and the medium was replaced with fresh medium containing the cyclic peptides 5555_p000, 5555_p020, 5555_p013, or 5555_p028, or the anti-SARS-CoV-2 antibody cocktail (casirivimab-imdevimab; REGN-COV2) or remdesivir as a control compound. After 24 hours, the culture supernatant was collected and the viral RNA contained therein was extracted using a MagMax Viral / Pathogen II Nucleic Acid Isolation kit (Thermo Fisher Scientific). At this time, cytotoxicity was evaluated by microscopic observation.

[0248] The viral RNA contained in the extract was quantified by real-time RT-qPCR using a one-step RT-qPCR kit (THUNDERBIRD (registered trademark) Probe One-step RT-qPCR kit, Toyobo Co., Ltd.).

[0249] The following primer sequences were used: 5'-ACAGGTACGTTAATAGTTAATAGCGT-3' (SEQ ID NO: 127) 5'-ATATTGCAGCAGTACGCACACA-3' (SEQ ID NO: 128)

[0250] The following sequence was used as a probe: 5'-FAM-ACACTAGCCATCCTTACTGCGCTTCG-TAMRA-3' (SEQ ID NO: 129)

[0251] The results are shown in Figures 1-1, 1-2, 1-3, and 1-4. The vertical axis of Figure 1 shows the amount of viral RNA contained in the supernatant as a comparative value, with the value when no peptide or REGN-COV2 was added set to 1, and the horizontal axis shows the amount of peptide or REGN-COV2 added.

[0252] In Figure 1-1, open triangles indicate the effect on the Wuhan strain, and closed squares indicate the effect on the alpha strain. A shows the results for the test plots to which 5555_p000, B shows the results for the test plots to which REGN-COV2, C shows the results for the test plots to which 5555_p020, and D shows the results for the test plots to which 5555_p013 was added. In Figure 1-2, open triangles indicate the effect on the Wuhan strain, closed circles indicate the effect on the beta strain, and open squares indicate the effect on the gamma strain. A shows the results for the test plots to which REGN-COV2, B shows the results for the test plots to which 5555_p020, and C shows the results for the test plots to which 5555_p028 was added. In Figure 1-3, open triangles indicate the effect on the Wuhan strain, and closed diamonds indicate the effect on the delta strain. A shows the results for the test plots to which REGN-COV2 and B shows the results for the test plots to which 5555_p028 was added. In Figure 1-4, the white triangles indicate the effect on the Wuhan strain of virus, and the white circles indicate the effect on the Omicron strain. A shows the results of the test groups to which REGN-COV2, B shows remdesivir, and C shows the results of the test groups to which 5555_p028 was added.

[0253] Figures 1-1 to 1-4 show that the cyclic peptide has antiviral activity not only against the Wuhan strain, but also against other mutant viruses such as alpha, beta, gamma, delta, and omicron. Furthermore, no cytotoxicity was confirmed in any of the test groups.

[0254] Example 6 Antiviral Cytopathic Effect (CPE) Assay (1) Example 6-1 Cells with high ACE2 expression were selected from the VeroE6 cell line (American Type Culture Collection; CRL-1586) and used for the test. VeroE6 cells were cultured in MEM medium (Gibco 11095) containing 10% heat-inactivated fetal bovine serum (HI-FBS, Gibco 14000) and amphotericin B. The cells were passaged twice a week at a dilution ratio of 1:2 to 1:5, and the viability of 95% was confirmed using a Luna cell viability analyzer (Logos Biosystems) and trypan blue staining. After passage, the cells were suspended in MEM containing 2% FBS and 1% penicillin-streptomycin and used in infection experiments.

[0255] Cyclic peptides were prepared by dispensing 80 μL of 100% DMSO peptide solution into an empty 384-well ECHO plate (Labocyte P-05525), transferring 40 μL of the solution to an adjacent well containing 40 μL of DMSO, and repeatedly mixing to create eight two-fold dilutions (concentration ranges: 30-0.06 μM for the 10 mM stock solution, 1-0.002 μM for the 3.3 mM stock solution, and 100-0.2 nM for the 33.3 μM stock solution). The final DMSO concentration in the assay was 0.3%.

[0256] The assay for the antiviral CPE inhibitory effect was carried out as follows. 5 μL of the above cyclic peptide dilution series was dispensed into each well using an ECHO (registered trademark) 555 acoustic microautomatic dispenser (Beckman Coulter) to prepare an assay plate. VeroE6 cells were infected with SARS-CoV2 (WRCEVA USA_WA1 / 2020 strain) at an infectious titer (MOI of 0.002) that resulted in a 5% cell survival rate after 72 hours, and 25 μL of the prepared batch (4,000 VeroE6 cells / well) was dispensed into the assay plate. The plate was then incubated at 37°C and 5% CO ２ After incubation at 90% humidity for 72 hours, 30 μL of Cell Titer-Glo (registered trademark) (Promega) was added and incubated at room temperature for 10 minutes. After incubation, the fluorescence intensity was measured using a plate reader.

[0257] The cytotoxicity of the cyclic peptide was measured as follows: 25 μL of VeroE6 cells (4000 cells / well) were dispensed into the assay plate to which the cyclic peptide had been dispensed as described above, and the cells were incubated at 7° C., 5% CO ２ The plates were cultured at 90% humidity for 72 hours. 30 μL of Cell Titer-Glo® (Promega) was added, and after 10 minutes of incubation at room temperature, the fluorescence intensity was measured using a plate reader. As controls for high and low assay values, wells containing cells only and wells containing 100 μM Hyamine® 1622 (Sigma 1622) were also set up. The measured values ​​were converted to %CPE reduction values ​​using the following formula:

[0258]

[0259] IC ５０ The IC value was calculated by four-parameter logistic regression using the X1fit module of Activity Base (v.9.3; IDBS). Specifically, the antiviral CPE inhibitory effect was determined based on a sigmoid curve with the concentration of the cyclic peptide used on the horizontal axis and the %CPE reduction value calculated below on the vertical axis. The higher the %CPE reduction value on the vertical axis, the higher the antiviral effect. The concentration of the cyclic peptide at which the %CPE reduction value was 50% was defined as the IC ５０ The value was set as

[0260] Cytotoxicity was calculated as % cell viability using the following formula:

[0261]

[0262] CC ５０ Value, IC ５０ Similarly, the values ​​were calculated by four-parameter logistic regression using the X1fit module of Activity Base (v. 9.3.; IDBS).

[0263] The results are shown in Table 4.

[0264]

[0265] From Table 4, the IC of the antiviral inhibitory effect ５０ was detected at a very low concentration of a few nM, indicating high specific activity. On the other hand, if cells die due to the off-target cytotoxicity of the compound, it is difficult to distinguish between cell death caused by the virus and nonspecific cell death caused by the drug, making it difficult to correctly evaluate the activity of the compound. As shown in Table 4, there was no effect on cell viability (cytotoxic CC of >1 μM or more). ５０ If a compound is effective under these conditions, it has extremely low cytotoxicity and can be safely determined to be effective.

[0266] (2) Example 6-2: Cells with high ACE2 expression were selected from the VeroE6 cell line (American Type Culture Collection; CRL-1586) and used in the study. VeroE6 cells were cultured in MEM medium (Gibco, #11095) containing 10% heat-inactivated fetal bovine serum (HI-FBS, PEAK's PS-FB4) and 1% penicillin-streptomycin. The cells were passaged twice a week at a dilution ratio of 1:2 to 1:5, and the viability of 95% cells was confirmed using a Luna cell viability analyzer and trypan blue staining. The subcultured cells were suspended in MEM (assay media) containing 2% FBS, 1% penicillin-streptomycin (Corning 30-002-CL) and 1% HEPES (Corning 25-060-CL) and used in infection experiments.

[0267] Cyclic peptides were prepared by dispensing 80 μL of 100% DMSO peptide solution into an empty 384-well ECHO plate (Labocyte P-05525), transferring 40 μL of the solution to an adjacent well containing 40 μL of DMSO, and repeatedly mixing to create eight two-fold dilutions (concentration ranges: 100-2 nM for the 333 μM stock solution and 100-0.2 nM for the 33.3 μM stock solution). The final DMSO concentration in the assay was 0.3%.

[0268] The assay for the antiviral CPE inhibitory effect was performed as follows. 90 nL of the above cyclic peptide dilution series was dispensed into each well using an ECHO® 555 acoustic microautomatic dispenser (Beckman Coulter), and 5 μL of assay media was added to create an assay plate. VeroE6 cells were infected with SARS-CoV2 (WRCEVA, USA_WA1 / 2020 strain) at an infectious titer (MOI of 0.002) that resulted in a 5% cell survival rate after 72 hours, and 25 μL of the prepared batch (4,000 VeroE6 cells / well) was dispensed into the assay plate. The plate was then incubated at 37°C and 5% CO. ２After incubation at 90% humidity for 72 hours, 30 μL of Cell Titer-Glo (Promega) was added and incubated at room temperature for 10 minutes. After incubation, the fluorescence intensity was measured using a plate reader.

[0269] The cytotoxicity of the cyclic peptide was measured as follows: 25 μL of VeroE6 cells (4000 cells / well) were dispensed into the assay plate to which the cyclic peptide had been dispensed as described above, and the cells were incubated at 7° C., 5% CO ２ The plates were cultured at 100°C for 72 hours at 90% humidity. 30 μL of Cell Titer-Glo (Promega) was added, and after 10 minutes of incubation at room temperature, the fluorescence intensity was measured using a plate reader. As high and low assay controls, wells containing cells alone and 100 μM Hyamine® 1622 (Sigma 1622) were also set up.

[0270] As in Example 6-1, the antiviral CPE suppression effect (CPE reduction value), cytotoxicity (% cell viability), and IC ５０ Value and CC ５０ The values ​​were calculated, and the results are shown in Table 5.

[0271]

[0272] As can be seen from Table 5, the cyclic peptide tested in this example also had an IC value of antiviral inhibitory effect similar to that of the cyclic peptide tested in Example 6-1. ５０ was detected at a very low concentration of several nM, indicating high specific activity. In addition, there was no effect on cell viability (cytotoxic CC of >1 μM or more). ５０ The compound is effective in this condition, has extremely low cytotoxicity, and is safe and effective.

[0273] Example 7 Measurement of the effect of cyclic peptides to prevent progression to severe disease and improve mortality rate, and evaluation of lung tissue images using a mouse SARS-CoV-2 infection fatality model This example shows the results of measurement of the effect of cyclic peptides to prevent progression to severe disease and improve mortality rate, and evaluation of lung tissue images using a mouse SARS-CoV-2 infection fatality model.

[0274] Twenty-four 37-week-old BALB / c mice were inoculated with 3x10 QHmusX strain, a SARS-CoV-2 mouse passage strain. ３ T.C.I.D. ５０ (5LD ５０ Mice were then intranasally infected with 5555_p020 or 5555_p028 at 10 or 30 mg / kg (hereinafter referred to as the peptide administration group) intraperitoneally at 1, 7, 24, 48, 72, and 96 hours post-infection. The treatment control group received a single dose of 10 mg / kg of anti-RBD cocktail antibody (hereinafter referred to as the REGN-COV2 group) 1 hour after virus inoculation.

[0275] For each group, weight changes were monitored for 10 days after virus infection, and a humane endpoint or lethality assessment was performed. The observation period ended on day 10 after virus inoculation. Animals that died, were judged to have reached a humane endpoint, or were at the end of observation were euthanized, and after cardiac blood collection, the lungs were collected and immersed in 10% neutral buffered formalin to prepare tissue samples for pathological analysis.

[0276] The changes in body weight and survival rates in each group over 10 days after virus inoculation are shown in Figures 2-1 and 2-2.

[0277] The vertical axis of Figure 2-1 represents the percentage change in mouse weight, with the weight at the start of the test taken as 100, and the horizontal axis represents the number of days after the start of the test (virus infection). X represents the PBS group, black circles represent the 5555_p020 10 mg administration group, white circles represent the 5555_p020 30 mg administration group, black squares represent the 5555_p028 10 mg administration group, white squares represent the 5555_p028 30 mg administration group, and black triangles represent the REGN-COV2 group.

[0278] In Figure 2-2, the vertical axis represents the survival rate of mice as a percentage, with the survival rate at the start of the test taken as 100, and the horizontal axis represents the number of days after the start of the test. X represents the PBS group, black circles represent the 5555_p020 10 mg administration group, white circles represent the 5555_p020 30 mg administration group, black squares represent the 5555_p028 10 mg administration group, white squares represent the 5555_p028 30 mg administration group, and black triangles represent the REGN-COV2 group.

[0279] In the PBS group, weight loss was observed in all animals from day 2 onward, and by day 3 onward, 7 of 8 animals died or reached a humane endpoint. Only 1 of 8 animals gained weight after day 7 of inoculation, demonstrating a tendency toward recovery. In the REGN-COV2 group, weight loss was observed in all animals until day 2, but 7 of 8 animals gained weight after day 3 and recovered during the observation period. Only 1 of 8 animals died on day 5. In contrast, in the peptide-administered group, although some animals showed weight loss on day 2 of inoculation, all animals survived. Among the peptide-administered groups, no findings suggestive of weight loss or respiratory symptoms were observed during the observation period in the 5555_p020-administered group at any concentration. Although weight loss was observed on day 3 of inoculation in the 5555_p028-administered group, the weight loss rate was significantly improved (P<0.05) compared to the PBS-administered group from day 4 onward. Weight change was statistically analyzed using Dunnett's multiple comparisons test, and survival rate was analyzed using the Log-rank (Mantel-Cox) test. These results demonstrate that administration of the cyclic peptide suppresses weight loss after SARS-CoV-2 infection.

[0280] Representative lung tissue images from each group at the end of observation on day 10 after inoculation are shown in Figure 3 (hematoxylin-eosin stained, photographed at 10x magnification).

[0281] In (A), which shows an image of pneumonia in one animal in the PBS group that survived until day 10 after inoculation, histological findings corresponding to the proliferation phase of diffuse alveolar damage were observed, including perivascular cell clusters (*) and inflammatory cell infiltration and alveolar epithelial regeneration (#) in the alveolar fields. In (B), which shows lung tissue images from the REGN-COV2-treated group, only a mild increase in cells was observed in some alveolar fields. In (C), which shows lung tissue images from the 10 mg 5555_p020 group, mild lymphocyte clusters were observed around blood vessels near the bronchioles in some lung lobes (*). An increase in cells was observed in some alveolar fields. In (D), which shows lung tissue images from the 30 mg 5555_p028 group, mild perivascular cell clusters were observed in most lung lobes (*). The above lung tissue images showed that administration of the cyclic peptide suppressed pneumonia caused by SARS-CoV-2 infection.

[0282] Example 8 Evaluation of the efficacy of cyclic peptides on general condition and pneumonia using a hamster SARS-CoV-2 infection model This example demonstrates the evaluation of the efficacy of cyclic peptides on general condition (body weight) and pneumonia using a hamster SARS-CoV-2 infection model.

[0283] To evaluate the in vivo efficacy of 5555_p028 against general condition and pneumonia caused by SARS-CoV-2 infection, we conducted a study using a hamster infection model. This model has been reported to be effective with anti-SARS-CoV-2 antibody cocktails (Reference 7), and we therefore considered it a suitable model for evaluating in vivo efficacy.

[0284] Twenty-four female golden Syrian hamsters aged 8-9 weeks were given 8 x 10 doses of SARS-CoV-2 (USA_WA1 / 2020 strain). ５ T.C.I.D. ５０Hamsters were intranasally infected with 5555_p028. Hamsters were divided into groups of six per dose and intraperitoneally administered vehicle control or 5555_p028 at 1, 5, or 25 mg / kg at 2, 8, 24, 48, and 72 hours postinfection. Body weights were measured daily until 5 days postinfection. Five days postinfection, the hamsters were euthanized and their lungs were weighed. The left lobe was then sliced ​​and frozen at -70°C for quantification of viral RNA by RT-qPCR. The right anterior and posterior lobes were fixed in 10% neutral buffered formalin, and 5-μm-thick paraffin sections were prepared. They were stained with HE and used for histopathological examination.

[0285] Rodents, such as hamsters, are animals that continue to grow. Healthy animals continue to gain weight, but poor health can lead to weight gain inhibition and, in severe cases, weight loss. Therefore, weight is an important clinical parameter for assessing the physical condition of rodents. The weight trends are shown in Table 6. The vehicle control group showed a downward trend in mean values ​​from Day 1, while weight loss was similarly suppressed in the 5555_p028-treated groups at all doses. Weight loss in the vehicle control group indicates poor physical condition (general state) of the test hamsters, while reduced weight loss in the cyclic peptide-treated groups indicates improvement in physical condition (general state).

[0286]

[0287] Approximately 20 mg of the left lung was collected and weighed, and then buffer was added to a concentration of 25 mg / mL. The sample was then homogenized. RNA was extracted from 200 μL of the sample (equivalent to 5 mg of lung). The amount of SARS-CoV-2 N2 protein mRNA was quantified in triplicate by RT-qPCR using the N-2 primer set, Quant-iT Ribogreen RNA assay kit, TaqMan Fast Virus 4x Master Mix, and QuantStudio 6 (all from Thermo Fisher Scientific).

[0288] N-2 Primer Set Forward: 5'-TTA CAA ACA TTG GCC GCA AA-3' (SEQ ID NO: 130) Reverse: 5'-GCG CGA CAT TCC GAA GAA-3' (SEQ ID NO: 131)

[0289] As a result, a statistically significant decrease in the SARS-CoV-2 genome copy number was observed in the 5 mg / kg group of 5555_p0281, and a tendency toward a decrease was observed in the 25 mg / kg group (Table 7).

[0290]

[0291] Macroscopic findings of the lungs at necropsy revealed patchy discoloration in all groups, with no intergroup differences directly related to intraperitoneal administration of 5555_p028. At this time, no lung discoloration was observed in two animals in the 25 mg / kg 5555_p028 group (Table 8).

[0292]

[0293] Histopathologically, chronic active lung inflammation was observed in all groups. Interstitial inflammation was characterized by abundant macrophage infiltration, with less prominent neutrophils, lymphocytes, eosinophils, and occasional giant cells. In the affected areas, erythrocytes, eosinophilic fibrous material, inflammatory cells, and cellular debris were observed within the alveoli. Epithelial cells in the affected bronchioles and alveoli were hyperproliferating, and occasionally, alveolar epithelial cells became enlarged and showed a bizarre morphology (cellular atypia). Furthermore, multifocal pleural inflammation accompanied by mesothelial hyperplasia was observed.

[0294] The severity of lung inflammation was reduced by 5555_p028 administration compared with the vehicle control group. Lung inflammation was mild in the posterior lobe in two cases in the 5 mg / kg 5555_p028 group. In contrast, severe inflammation was observed in the anterior and posterior lobes in three of six cases in the vehicle control group (Table 9). Histopathological images are shown in Figure 4.

[0295]

[0296] These results suggest that SARS-CoV-2-infected golden Syrian hamsters develop chronic active interstitial and pleural inflammation, which may be ameliorated by administration of 5555_p028.

[0297] Example 9: Measurement of the effect of co-administration with anti-COVID drugs on the antiviral activity of a cyclic peptide In this example, to investigate the effect of co-administration with anti-COVID drugs on the antiviral activity of a cyclic peptide, known COVID drugs such as remdesivir, REGN-COV2 (a combination of casirivimab and imdevimab), or EIDD-1931, an active form of molanupiravir, were mixed with the cyclic peptide at various concentrations, administered to virus-infected cells, and the amount of viral RNA in the supernatant was measured. Details are provided below.

[0298] VeroE6 / TMPRSS2 cells were plated in a 96-well plate at 3.0 x 10 ４ Cells were seeded and cultured overnight. The cells were treated with a medium containing infectious SARS-CoV-2 (Wuhan strain: WK-521, Delta strain: TY11-927, or Omicron strain: TY38-873) at an MOI of 0.003 for 1 hour, along with a combination of a cyclic peptide and a compound with existing anti-SARS-CoV-2 activity (EIDD-1931 and 5555_p028, remdesivir and 5555_p028, or REGN-COV2 and 5555_p028). The virus was then washed away, and the medium was replaced with new compound-containing medium. After 24 hours, the culture supernatant was collected, and the viral RNA contained therein was extracted using a MagMax Viral / Pathogen II Nucleic Acid Isolation kit (Thermo Fisher Scientific). Cytotoxicity was evaluated by microscopic observation. The viral RNA contained in the extract was quantified in the same manner as in Example 5.

[0299] The results are shown in Figures 5-1 to 5-8. The vertical axis of Figure 5 shows the amount of viral RNA contained in the supernatant as a comparative value, with the value when no peptide or various existing compounds with anti-SARS-CoV-2 activity was added being set at 1, and the horizontal axis shows the amount of various existing compounds with anti-SARS-CoV-2 activity. Figures 5-1 to 5-3 show the results of measuring antiviral activity against the Wuhan strain, Figures 5-4 to 5-6 show the results of measuring antiviral activity against the Delta strain, and Figures 5-7 to 5-8 show the results of measuring antiviral activity against the Omicron strain.

[0300] Figures 5-1 to 5-8 show that the cyclic peptide not only has antiviral activity as a single agent, but also has even greater antiviral activity when used in combination with a compound known to have anti-SARS-CoV-2 activity.

[0301] No cytotoxicity was observed in any of the test groups.

[0302] Example 10 Synthesis of Cyclic Peptides Cyclic peptides were synthesized in the same manner as in Example 2. The sequences and structures of the synthesized peptides are shown in Tables 10 and 11.

[0303]

[0304]

[0305]

[0306]

[0307]

[0308] The peptides listed in Table 11 are peptides in which the first and twelfth amino acids form a cyclic structure, with the thirteenth and subsequent amino acids, PEG, or an alkyl chain structure added as a linker. Also, @R in the table indicates a structure in which the amino acid is bonded via a functional group contained in the side chain. K@R1 is bonded to the thirteenth amino acid, de, via the side chain amino group, and E@R2 is bonded to the lysine of K@R1 via the side chain carboxy group. For example, the cyclic peptide 5555_p233 (SEQ ID NO: 229) has the structure shown in [Chemical Formula 12] below, the cyclic peptide 5555_p234 (SEQ ID NO: 230) has the structure shown in [Chemical Formula 13] below, and the cyclic peptide 5555_p235 (SEQ ID NO: 231) has the structure shown in [Chemical Formula 14] below.

[0309]

[0310]

[0311]

[0312] Detailed examples of the synthesis of cyclic peptides are described below. (1) Example 10-1 Synthesis of cyclic peptide 5555_p173 (SEQ ID NO: 173)

[0313]

[0314] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical Industry Co., Ltd., 0.48 mmol / g) according to the general method described above. A CEM Liberty Blue solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. To introduce each residue, Fmoc-AA / DIC / Oxyma pure (5.25 equivalents / 10 equivalents / 5 equivalents) was used per equivalent of resin. The reaction was carried out once for 10 minutes in DMF at 75°C. The first residue was reacted three times for 30 minutes at 75°C; the second, third, fourth, fifth, and sixth residues were reacted once for 20 minutes at 75°C; and the twelfth residue was reacted once for 30 minutes at 40°C.

[0315] The basic conditions for Fmoc removal were a 3-minute reaction with a 10% pyrrolidine solution in DMF at 75°C. The first residue was reacted with a 10% pyrrolidine solution in DMF at room temperature for 5 minutes, followed by a 10-minute reaction. The introduction of a chloroacetyl group was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin carrying the Fmoc-protected peptide obtained in the previous step using the method described above, followed by adding a DMF / methylene chloride (1 / 1) solution (5 equivalents) of chloroacetic acid activated with DIC / HOSu to the solid-phase resin and shaking at room temperature for 60 minutes.

[0316] For the deprotection of the side chain and the cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was washed with DMF, methylene chloride, and diethyl ether and dried under reduced pressure. Then, the reaction vessel containing the solid-phase resin was charged with a reagent cocktail A (TFA / H ２A mixture of 92.5:2.5:2.5:2.5 (volume ratio of 0.0:0.0:0.0:0.0:0.0:0.0:0.0) of 1,000 sachets ...

[0317] The resulting crude product was purified using the following conditions (Column: Waters Xbridge® C18 5 μm® 50×250 mm; Mobile phase: A=0.1% TFA in water, B=0.1% TFA in MeCN; Temperature: 50° C.; Gradient (% B): 10-36% over 3 min, then 36-41% over 15 min, then 41-60% over 3 min; Flow rate: 118 mL / min).

[0318] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions: retention time = 5.03 min; column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; mobile phase: A = 0.025% TFA (in water), B = 0.025% TFA (in MeCN); temperature: 60 °C; gradient (% B conc): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min; flow rate: 0.5 mL / min. ESI-MS(+) observed value m / z = 986.93 (M + 2H). ２＋

[0319] (2) Example 10-2 Synthesis of cyclic peptide 5555_p176 (SEQ ID NO: 176)

[0320]

[0321] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical Industry Co., Ltd., 0.48 mmol / g) according to the general method described above. A CEM Liberty Blue solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. To introduce each residue, Fmoc-AA / DIC / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used per equivalent of resin. The reaction was carried out once for 10 minutes at 75°C in DMF. The first and third residues were reacted twice for 30 minutes at 75°C, the second residue twice for 10 minutes at 75°C, the fifth residue twice for 10 minutes at 90°C, and the twelfth residue once for 20 minutes at 50°C.

[0322] The basic conditions for Fmoc removal were a 3-minute reaction with a 20% piperidine solution in DMF at 75°C. The first, second, third, fourth, fifth, and sixth residues were reacted with a 20% piperidine solution in DMF at room temperature for 5 minutes, followed by a 10-minute reaction. The introduction of a chloroacetyl group was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin carrying the Fmoc-protected peptide obtained in the previous step using the method described above, followed by shaking in DMF at room temperature for 30 minutes using chloroacetic acid / HATU / DIPEA (5.2 equivalents / 5 equivalents / 5 equivalents).

[0323] For the deprotection of the side chain and the cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was washed with DMF, methylene chloride, and diethyl ether and dried under reduced pressure. Then, the reaction vessel containing the solid-phase resin was charged with a reagent cocktail A (TFA / H ２A mixture of 92.5:2.5:2.5:2.5 by volume of 0.01% HCl / TIS / DODT was added, and the mixture was shaken at room temperature for 5 minutes. The reaction solution was filtered and recovered through a frit. The solid phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate described above. When this filtrate was added to an excess of a mixed solvent of diethyl ether / hexane (1 / 1) cooled to 0°C, a cloudy white precipitate formed. This mixture was centrifuged, and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether cooled to 0°C again, and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. The peptide cyclization reaction was carried out in DMSO / MeCN / H2SO4 at a final peptide concentration of 5 mM based on the molar number of the solid phase resin. ２ After dissolving the residue in 100 ml of ethanol (1 / 1 / 1), 10 equivalents of triethylamine was added and the mixture was shaken at room temperature for 7 hours. The resulting reaction solution was concentrated under reduced pressure using HT-12.

[0324] The resulting crude product was purified using the following conditions (Column: Waters Xbridge® C18 5 μm® 50×250 mm; Mobile phase: A=0.1% TFA in water, B=0.1% TFA in MeCN; Temperature: 50° C.; Gradient (% B): 16-42% over 3 min, then 42-47% over 15 min, then 47-60% over 3 min; Flow rate: 118 mL / min).

[0325] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions: retention time = 5.82 min; column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; mobile phase: A = 0.025% TFA (in water), B = 0.025% TFA (in MeCN); temperature: 60 °C; gradient (% B conc): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min; flow rate: 0.5 mL / min. ESI-MS(+) observed value m / z = 959.55 (M + 2H). ２＋

[0326] (3) Example 10-3 Synthesis of cyclic peptide 5555_p195 (SEQ ID NO: 195)

[0327]

[0328] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical Industry Co., Ltd., 0.48 mmol / g) according to the general method described above. A CEM Liberty Prime solid-phase synthesizer was used, and synthesis was performed according to the manufacturer's instructions. To introduce each residue, Fmoc-AA / DIC / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used per equivalent of resin. The reaction was carried out once for 2 minutes in DMF at 105°C. The first residue was reacted twice for 10 minutes at 90°C, the 12th residue was reacted once for 15 minutes at 50°C, and the 13th residue was reacted once for 1 minute at 105°C.

[0329] The basic conditions for Fmoc removal were a 1-minute reaction with 83 mM Oxyma Pure in a 4% DMF solution of pyrrolidine at 110°C. The second, third, fourth, fifth, and sixth residues were reacted with a 10% DMF solution of pyrrolidine at 50°C for 90 seconds. The first residue was reacted twice with a 10% DMF solution of pyrrolidine at room temperature for 1 minute. The introduction of a chloroacetyl group was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin carrying the Fmoc-protected peptide obtained in the previous step using the method described above, followed by shaking in DMF at room temperature for 30 minutes using chloroacetic acid / HATU / DIPEA (5 equivalents / 5 equivalents / 10 equivalents).

[0330] For the deprotection of the side chain and the cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was washed with DMF, methylene chloride, and diethyl ether and dried under reduced pressure. Then, the reaction vessel containing the solid-phase resin was charged with a reagent cocktail A (TFA / H ２A mixture of 92.5:2.5:2.5:2.5 by volume of 0.01% HCl / TIS / DODT was added, and the mixture was shaken at room temperature for 90 minutes. The reaction solution was filtered and recovered through a frit. The solid phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate described above. When this filtrate was added to an excess of a mixed solvent of diethyl ether / hexane (1 / 1) cooled to 0°C, a cloudy white precipitate formed. This mixture was centrifuged, and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether cooled to 0°C again, and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. The peptide cyclization reaction was carried out in DMSO / isopropyl alcohol / H2SO4 such that the final peptide concentration was 5 mM based on the molar number of the solid phase resin. ２ After dissolving the residue in 0 (90 / 5 / 5), 10 equivalents of triethylamine was added and the mixture was shaken for 15 hours at 25° C. The resulting reaction solution was concentrated under reduced pressure using EZ-2 Elite.

[0331] The resulting crude product was purified using the following conditions (Column: Waters Xbridge® C18 5 μm® 50×150 mm; Mobile phase: A=0.1% TFA in water, B=0.1% TFA in MeCN; Temperature: 40° C.; Gradient (% B): 12-37% over 3 min, then 37-42% over 8 min, then 42-60% over 1 min; Flow rate: 120 mL / min).

[0332] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions: retention time = 4.51 min; column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; mobile phase: A = 0.025% TFA (in water), B = 0.025% TFA (in MeCN); temperature: 60 °C; gradient (% B conc): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min; flow rate: 0.5 mL / min. ESI-MS(+) observed value m / z = 954.90 (M+2H). ２＋

[0333] (4) Example 10-4 Synthesis of cyclic peptide 5555_p213 (SEQ ID NO: 212)

[0334]

[0335] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical Industry Co., Ltd., 0.48 mmol / g) according to the general method described above. A CEM Liberty Blue solid-phase synthesizer was used, and the synthesis was carried out according to the manufacturer's instructions. To introduce each residue, Fmoc-AA / DIC / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used per equivalent of resin, and the reaction was carried out once for 3 minutes at 90°C in DMF. However, the first residue was reacted twice for 30 minutes at 75°C, the second and third residues were reacted twice for 10 minutes at 90°C, the fourth residue was reacted once for 30 minutes at 75°C using Fmoc-AA / DIC / Oxyma pure (3 equivalents / 8 equivalents / 4 equivalents), the fifth residue was reacted twice for 3 minutes at 90°C, and the 12th residue was reacted once for 15 minutes at 50°C.

[0336] The basic conditions for Fmoc removal were a reaction with a 10% pyrrolidine solution in DMF at 90°C for 1 minute. The fifth residue was reacted with a 10% pyrrolidine solution in DMF at room temperature for 1 minute. The first residue was reacted twice with a 10% pyrrolidine solution in DMF at room temperature for 1 minute. The introduction of a chloroacetyl group was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin carrying the Fmoc-protected peptide obtained in the previous step using the method described above, followed by shaking in DMF at room temperature for 30 minutes using chloroacetic acid / HATU / DIPEA (5 equivalents / 5 equivalents / 10 equivalents).

[0337] For the deprotection of the side chain and the cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was washed with DMF, methylene chloride, and diethyl ether and dried under reduced pressure. Then, the reaction vessel containing the solid-phase resin was charged with a reagent cocktail A (TFA / H ２A 92.5:2.5:2.5:2.5 volume ratio mixture of 0.25% DISO / TIS / DODT was added, and the mixture was shaken at room temperature for 70 minutes. The reaction solution was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate. This filtrate was added to an excess of a 1 / 1 diisopropyl ether / hexane mixed solvent chilled to 0°C, resulting in a cloudy white precipitate. This mixture was centrifuged, and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether chilled to 0°C again and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in 90% aqueous DMSO to a final concentration of 6.3 mM based on the molar number of the solid-phase resin, and then 10 equivalents of triethylamine were added and the mixture was shaken at room temperature for 18 hours. The resulting reaction solution was concentrated under reduced pressure using an EZ-2 Elite.

[0338] The resulting crude product was purified using the following conditions (Column: Waters Xbridge® C18 5 μm® 50×150 mm; Mobile phase: A=0.1% TFA in water, B=0.1% TFA in MeCN; Temperature: 40° C.; Gradient (% B): 17-42% over 3 min, then 42-47% over 8 min, then 47-60% over 1 min; Flow rate: 120 mL / min).

[0339] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions: retention time = 5.53 min; column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; mobile phase: A = 0.025% TFA (in water), B = 0.025% TFA (in MeCN); temperature: 60 °C; gradient (% B conc): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min; flow rate: 0.5 mL / min. ESI-MS(+) observed value m / z = 917.38 (M + 2H). ２＋

[0340] (5) Example 10-5 Synthesis of cyclic peptide 5555_p216 (SEQ ID NO: 215)

[0341]

[0342] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical Industry Co., Ltd., 0.48 mmol / g) according to the general method described above. A CEM Liberty Blue solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. To introduce each residue, Fmoc-AA / DIC / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used per equivalent of resin, and the reaction was carried out once for 3 minutes at 90°C in DMF. The first residue was reacted twice for 30 minutes at 75°C, the second and third residues were reacted twice for 10 minutes at 90°C, the fourth residue was reacted once for 30 minutes at 75°C using Fmoc-AA / DIC / Oxyma pure (3 equivalents / 8 equivalents / 4 equivalents), and the 12th residue was reacted once for 15 minutes at 50°C.

[0343] The basic conditions for Fmoc removal were a reaction with a 10% pyrrolidine solution in DMF at 90°C for 1 minute. The first residue was reacted twice with a 10% pyrrolidine solution in DMF at room temperature for 1 minute. The introduction of a chloroacetyl group was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin carrying the Fmoc-protected peptide obtained in the previous step using the method described above, followed by shaking in DMF at room temperature for 30 minutes using chloroacetic acid / HATU / DIPEA (5 equivalents / 5 equivalents / 10 equivalents).

[0344] For the deprotection of the side chain and the cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was washed with DMF, methylene chloride, and diethyl ether and dried under reduced pressure. Then, the reaction vessel containing the solid-phase resin was charged with a reagent cocktail A (TFA / H ２A 92.5:2.5:2.5:2.5 volume ratio mixture of 0.25% DISO / TIS / DODT was added, and the mixture was shaken at room temperature for 70 minutes. The reaction solution was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate. This filtrate was added to an excess of a 1 / 1 diisopropyl ether / hexane mixed solvent chilled to 0°C, resulting in a cloudy white precipitate. This mixture was centrifuged, and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether chilled to 0°C again and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in 90% aqueous DMSO to a final concentration of 6.3 mM based on the molar number of the solid-phase resin, and then 10 equivalents of triethylamine were added and the mixture was shaken at room temperature for 18 hours. The resulting reaction solution was concentrated under reduced pressure using an EZ-2 Elite.

[0345] The resulting crude product was purified using the following conditions (Column: Waters Xbridge® C18 5 μm® 50×150 mm; Mobile phase: A=0.1% TFA in water, B=0.1% TFA in MeCN; Temperature: 40° C.; Gradient (% B): 17-42% over 3 min, then 42-47% over 8 min, then 47-60% over 1 min; Flow rate: 120 mL / min).

[0346] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions: retention time = 5.78 min; column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; mobile phase: A = 0.025% TFA (in water), B = 0.025% TFA (in MeCN); temperature: 60 °C; gradient (% B conc): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min; flow rate: 0.5 mL / min. ESI-MS(+) observed value m / z = 930.38 (M+2H). ２＋

[0347] (6) Example 10-6 Synthesis of cyclic peptide 5555_p221 (a structure in which dk(cC10COO) is added as a linker structure to the peptide represented by SEQ ID NO: 30 in the peptide portion, SEQ ID NO: 219)

[0348]

[0349] The target peptide was synthesized using Fmoc-NH-Sieber resin (Watanabe Chemical Industry Co., Ltd., 0.48 mmol / g) according to the general method described above. A CEM Liberty Blue solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. Fmoc removal was performed by reacting with a 10% DMF solution at 90°C for 1 minute. One equivalent of resin was reacted with Alloc-D-Lys(Fmoc)-OH / DIC / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) in DMF at 90°C for 3 minutes. After Fmoc removal under the same conditions, 12-(tert-butoxy)-12-oxododecanoic acid was used in one reaction in DMF at 90°C for 10 minutes. Alloc removal was performed with Pd(OAc) in one equivalent of resin. ２ / PPh ３ / PhSiH ３ The reaction was carried out in methylene chloride at room temperature for 60 minutes using Fmoc-AA / DIC / Oxyma pure (0.2 equivalents / 1 equivalent / 10 equivalents). To introduce each residue into the solid-phase resin obtained in the previous step, Fmoc-AA / DIC / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used per equivalent of resin. The reaction was carried out once for 3 minutes at 90°C in DMF. The first and second residues were reacted twice for 30 minutes at 75°C, the third residue twice for 10 minutes at 90°C, the fifth residue once for 10 minutes at 90°C, and the twelfth residue once for 15 minutes at 50°C.

[0350] The basic conditions for Fmoc removal were a 1-minute reaction with a 10% pyrrolidine solution in DMF at 90°C. The second, third, fourth, fifth, and sixth residues were reacted with a 10% pyrrolidine solution in DMF at 50°C for 90 seconds. The first residue was reacted twice with a 10% pyrrolidine solution in DMF at room temperature for 1 minute. The introduction of a chloroacetyl group was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin carrying the Fmoc-protected peptide obtained in the previous step using the method described above. Then, a 0.1 M chloroacetic acid solution in DMF (5 equivalents), a 0.1 M HATU solution in DMF (5 equivalents), and a 0.2 M DIEA solution in DMF (10 equivalents) were added to the solid-phase resin and shaken at room temperature for 30 minutes.

[0351] For the deprotection of the side chain and the cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was washed with DMF and methylene chloride and dried under reduced pressure. Then, the reaction vessel containing the solid-phase resin was charged with reagent cocktail A (TFA / H ２ A 92.5:2.5:2.5:2.5 volume ratio mixture of 0.01% HCl / TIS / DODT was added, and the mixture was shaken at room temperature for 5 minutes. The reaction solution was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate. This filtrate was added to an excess of a 1 / 1 diethyl ether / hexane mixture cooled to 0°C, resulting in a cloudy white precipitate. This mixture was centrifuged (9000 rpm, 2 minutes), and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether cooled to 0°C again and dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. The peptide cyclization reaction was performed by dissolving the peptide in 90% aqueous DMSO to a final concentration of 5 mM based on the molar amount of solid-phase resin, adding 10 equivalents of triethylamine, and shaking at room temperature for 6 hours. The resulting reaction solution was concentrated under reduced pressure.

[0352] The resulting crude product was purified using the following conditions (Column: Waters Xbridge® C18 5 μm® 50×150 mm; Mobile phase: A=0.1% TFA in water, B=0.1% TFA in MeCN; Temperature: 40° C.; Gradient (% B): 21-46% over 3 min, then 46-51% over 8 min, then 51-60% over 1 min; Flow rate: 120 mL / min).

[0353] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions: retention time = 6.01 min; column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; mobile phase: A = 0.025% TFA (in water), B = 0.025% TFA (in MeCN); temperature: 60 °C; gradient (% B conc): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min; flow rate: 0.5 mL / min. ESI-MS(+) observed value m / z = 1092.86 (M + 2H). ２＋

[0354] (7) Example 10-7 Synthesis of cyclic peptide 5555_p227 (a structure in which PEG2c-dk(cC10COO) is added as a linker structure to the peptide represented by SEQ ID NO: 30, SEQ ID NO: 225)

[0355]

[0356] The target peptide was synthesized using Fmoc-NH-Sieber resin (Watanabe Chemical Industry Co., Ltd., 0.48 mmol / g) according to the general method described above. A CEM Liberty Blue HT solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. Fmoc removal was performed by reaction with a 10% DMF solution at 90°C for 1 minute. One reaction was performed in DMF at 90°C for 3 minutes using Alloc-D-Lys(Fmoc)-OH / DIC / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) per equivalent of resin. After Fmoc removal under the same conditions, the product was reacted with 12-(tert-butoxy)-12-oxododecanoic acid in DMF at 90°C for 10 minutes. Alloc removal was performed using Pd(OAc) per equivalent of resin. ２ / PPh ３ / PhSiH ３ The reaction was carried out in methylene chloride at room temperature for 60 minutes using Fmoc-AA / DIC / Oxyma pure (0.2 equivalents / 1 equivalent / 10 equivalents). To introduce each residue into the solid-phase resin obtained in the previous step, Fmoc-AA / DIC / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used per equivalent of resin. The reaction was carried out once for 3 minutes at 90°C in DMF. The first and second residues were reacted twice for 30 minutes at 75°C, the third residue twice for 10 minutes at 90°C, the fifth residue once for 10 minutes at 90°C, and the twelfth residue once for 15 minutes at 50°C.

[0357] The basic conditions for Fmoc removal were a 1-minute reaction with a 10% pyrrolidine solution in DMF at 90°C. The second, third, fourth, fifth, and sixth residues were reacted with a 10% pyrrolidine solution in DMF at 50°C for 90 seconds. The first residue was reacted twice with a 10% pyrrolidine solution in DMF at room temperature for 1 minute. The introduction of a chloroacetyl group was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin carrying the Fmoc-protected peptide obtained in the previous step using the method described above. Then, a 0.1 M chloroacetic acid solution in DMF (5 equivalents), a 0.1 M HATU solution in DMF (5 equivalents), and a 0.2 M DIEA solution in DMF (10 equivalents) were added to the solid-phase resin and shaken at room temperature for 30 minutes.

[0358] For the deprotection of the side chain and the cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was washed with DMF and methylene chloride and dried under reduced pressure. Then, a reagent cocktail A (TFA / H ２ A 92.5:2.5:2.5:2.5 volume ratio mixture of 0.01% HCl / TIS / DODT was added, and the mixture was shaken at room temperature for 5 minutes. The reaction solution was filtered through a frit. The solid-phase resin remaining in the reaction vessel was shaken again with the cleavage cocktail, and the solution components were recovered through a frit and mixed with the filtrate. This filtrate was added to an excess of a 1 / 1 diethyl ether / hexane mixture cooled to 0°C, resulting in a cloudy white precipitate. This mixture was centrifuged (9000 rpm, 2 minutes), and the solution was decanted. The resulting solid was washed with a small amount of diethyl ether cooled to 0°C again and dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. The peptide cyclization reaction was performed by dissolving the peptide in 90% aqueous DMSO to a final concentration of 5 mM based on the molar amount of solid-phase resin, adding 10 equivalents of triethylamine, and shaking at room temperature for 6 hours. The resulting reaction solution was concentrated under reduced pressure.

[0359] The resulting crude product was purified using the following conditions (Column: Waters Xbridge® C18 5 μm® 50×150 mm; Mobile phase: A=0.1% TFA in water, B=0.1% TFA in MeCN; Temperature: 40° C.; Gradient (% B): 19-44% over 3 min, then 44-49% over 8 min, then 49-60% over 1 min; Flow rate: 120 mL / min).

[0360] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions: retention time = 5.94 min; column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; mobile phase: A = 0.025% TFA (in water), B = 0.025% TFA (in MeCN); temperature: 60 °C; gradient (% B conc): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min; flow rate: 0.5 mL / min. ESI-MS(+) observed value m / z = 1172.48 (M + 2H). ２＋

[0361] Example 11 Evaluation of anti-SARS-CoV-2 activity by various antiviral activity assays In this example, the antiviral activity of some of the peptides synthesized in Example 10 was measured and evaluated in the same manner as in Example 4. The Delta strain was used as the viral strain in this assay. The results are shown in Table 12.

[0362]

[0363] In addition, the antiviral activity of some of the peptides synthesized in Example 10 was measured and evaluated in the same manner as in Example 6. In this assay, the Wuhan strain and Delta strain were used as the virus strains. The results are shown in Table 13.

[0364]

[0365] The results in Tables 12 and 13 demonstrate that the cyclic peptides of the present invention have antiviral activity against SARS-CoV-2 (including various mutant virus strains) even when a linker is added.

[0366] In the unlikely event of any discrepancy between the tables in this specification and the sequence listing, the statements in the tables shall be interpreted as correct.

Claims

1. The amino acid sequence: MeA-MeF-S-Cha-Y-S-Y-Y-R-R-Cha-C (SEQ ID NO: 2) Alternatively, an amino acid sequence having a substitution, addition, deletion or insertion in 1 to 10 amino acid residues selected from the group consisting of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th and 10th amino acid residues of the above amino acid sequence. A peptide comprising:

2. The amino acid sequence: MeA-MeF-S-Cha-Y-S-Y-Y-R-R-Cha-C (SEQ ID NO: 2) Alternatively, an amino acid sequence having a substitution, addition, deletion or insertion in 1 to 8 amino acid residues selected from the group consisting of the 1st, 2nd, 3rd, 5th, 6th, 8th, 9th and 10th amino acid residues of the above amino acid sequence. A peptide comprising:

3. The amino acid sequence: X1-X2-X3-Cha-X4-X5-Y-X6-X7-X8-Cha-C (SEQ ID NO: 1) Here, X1 is A, E, MeA, or MeE; X2 is an N-methyl amino acid; X3 is S or Aib; X4 is an amino acid having an unsubstituted or substituted aromatic ring in the side chain; X5 is any amino acid; X6 is an amino acid having an unsubstituted or substituted aromatic ring in the side chain; X7 is any amino acid; X8 is any basic amino acid; A peptide comprising:

4. The peptide of claim 3, wherein X1 is MeA or MeE.

5. 5. The peptide of claim 3 or claim 4, wherein X2 is MeF or MeNle.

6. The peptide of any one of claims 3 to 5, wherein X4 is unsubstituted or substituted Y, or unsubstituted or substituted F.

7. The peptide according to any one of claims 3 to 6, wherein X4 is Y or F4F.

8. The peptide according to any one of claims 3 to 7, wherein X5 is any hydrophilic amino acid or Aib, or any N-methylated hydrophilic amino acid or Aib.

9. The peptide according to any one of claims 3 to 8, wherein X5 is any one of Aib, E, K, S, dd, ddap, ds, and MeE.

10. The peptide of any one of claims 3 to 9, wherein X6 is unsubstituted or substituted Y, or unsubstituted or substituted F.

11. The peptide according to any one of claims 3 to 10, wherein X6 is Y, Yae, 3Py, or Na1.

12. The peptide according to any one of claims 3 to 11, wherein X7 is any one of A, Ahp, Cit, Dab, E, F4COO, K, and R.

13. The peptide according to any one of claims 3 to 12, wherein X8 is K, R, or A4p.

14. The peptide of any one of claims 1 to 13, further comprising D-glutamic acid at the C-terminus.

15. A peptide according to any one of claims 1 to 14, comprising or consisting of an amino acid sequence according to any one of SEQ ID NOs: 2 to 126.

16. A peptide described in any one of claims 1-14, comprising or consisting of an amino acid sequence described in any one of SEQ ID NOs: 132-234.

17. 17. The peptide of any one of claims 1 to 16, which is a cyclic peptide.

18. The peptide according to claim 17, having a cyclic structure formed by binding a chloroacetylated amino acid and a cysteine ​​residue contained in the peptide.

19. 19. The peptide of any one of claims 1 to 18, further comprising additional amino acid residues.

20. A pharmaceutical composition comprising a peptide according to any one of claims 1 to 19.

21. The pharmaceutical composition of claim 20, having anti-SARS-CoV-2 virus activity.

22. 22. The pharmaceutical composition according to claim 20 or 21 for the prevention or treatment of a coronavirus infection.

23. The pharmaceutical composition of claim 22, wherein the coronavirus infection is COVID-19.

24. A pharmaceutical composition according to any one of claims 20 to 23, comprising a pharma- ceutically acceptable salt of the peptide according to any one of claims 1 to 19, or a solvate thereof.

25. A diagnostic composition for diagnosing SARS-CoV-2 viral infection, comprising a peptide described in any one of claims 1 to 19.

26. The pharmaceutical composition according to any one of claims 20 to 24, in combination with other drugs for the prevention or treatment of coronavirus infections.

27. 27. The pharmaceutical composition of claim 26, wherein the other drug for the prevention or treatment of a coronavirus infection is selected from the group consisting of remdesivir, a combination of casirivimab and imdevimab, and molanupiravir or an active form thereof.

28. 28. The pharmaceutical composition of claim 26 or 27, which is co-administered with other agents for the prevention or treatment of a coronavirus infection.

29. A combination comprising a peptide described in any one of claims 1 to 19 and another drug for the prevention or treatment of a coronavirus infection.

30. Another drug for the prevention or treatment of coronavirus infection, used in combination with a pharmaceutical composition comprising a peptide described in any one of claims 1 to 19.

Images