Method and agent for inactivating bacteria or viruses, and antiviral substrate using said agent

Abstract

[Problem] To provide an inexpensive means that can exert an inactivation effect on microorganisms without the presence of light while minimizing the burden on the environment. [Solution] A bacterium or a virus is inactivated by bringing a compound having a redox mechanism (in the formula, X-represents a counter anion, and the dashed line represents a bonding position with another atom) into contact with a subject at risk of contamination with the bacterium or the virus. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a method and an inactivating agent for inactivating bacteria or viruses, and an antiviral substrate using the inactivating agent. Background Art

[0002] Recent globalization has increased the risk of viral infections, and even after 1990, avian influenza (A / H5N1) in 1997, SARS (SARS-Cov) in 2002, new influenza (A / 4H1N1) in 2009, and MERS (MERS-Cov) in 2015 have spread worldwide as infectious diseases. This is a very high frequency of pandemics compared to before 1990. In addition, the emergence of a new coronavirus that caused a pandemic in 2019, COVID-19 (SARS-Cov-2) as an acute respiratory infectious disease, not only affects human lives, but also has a huge impact on the economy, posing a huge threat to the living environment that protects people's peace of mind and safety.

[0003] It is recommended to perform ventilation and external air introduction for removing viruses. However, ventilation and external air introduction disturb the temperature and humidity environment regulated by the air conditioner, so from the perspective of energy saving, it is hoped that the virus can be quickly inactivated by internal air circulation. It can be expected that bacteria and viruses in the internal air circulation can be inactivated by using filters and interior materials with antibacterial and antiviral properties. Here, as a previous technology for imparting antibacterial and antiviral properties, according to Japanese Patent Gazette No. 4584339 and Japanese Patent Gazette No. 2017-133137, a technology using metal ions to exert antibacterial and antiviral effects is proposed. In addition, inorganic oxides are also proposed as categories that function as photocatalysts. Summary of the invention

[0004] Technical problem solved by the invention

[0005] Inorganic metals such as those disclosed in Japanese Patent Gazette No. 4584339 and Japanese Patent Application No. 2017-133137 work by protein denaturation based on the sustained release of metal ions, which may have adverse effects on the environment. In addition, silver and copper, which are particularly effective, are not only useful as precious metals, but also relatively expensive. In addition, the sustained release of metal ions requires dissolution and diffusion of metal ions, and there is a problem that it takes time to obtain antibacterial and antiviral effects. In addition, inorganic oxides (photocatalysts) do not function in the absence of light, so there is a problem that light irradiation equipment is required when used under light such as sunlight or in the dark.

[0006] Therefore, an object of the present invention is to provide an inexpensive means which can minimize the burden on the environment and can exert an inactivation effect on microorganisms without requiring the presence of light.

[0007] Technical means of solving problems

[0008] The present inventors have conducted intensive studies to solve the above problems and have found that microorganisms can be inactivated by bringing a compound having a given redox mechanism into contact with an object that may be contaminated by microorganisms, thereby completing the present invention.

[0009] That is, one embodiment of the present invention is a method for inactivating bacteria or viruses, comprising: bringing a compound having the following redox mechanism into contact with an object at risk of being contaminated with bacteria or viruses,

[0010] [Chemical formula 1]

[0011]

[0012] Where, X - For counter anions, dashed lines indicate bonding sites to other atoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] [ Figure 1 ] Figure 1 The standard redox potential (E) between the nitroxyl radical form and the oxoammonium form was measured for four compounds: (a) TEMPO, (b) AZADO, (c) 1-methyl AZADO, and (d) 1,3-dimethyl AZADO by using cyclic voltammetry. 0 @25℃) and obtained cyclic voltammogram.

[0014] [ Figure 2 ] is a graph showing the results (cell survival rate) of evaluating the antiviral effect by the WST-8 method by treating a dilution series of the culture supernatant of CRFK cells infected with CoV with AZADO-Oxo or silver nitrate for the purpose of confirming the antiviral effect of AZADO.

[0015] [ Figure 3 ] is a figure showing a sample obtained by treating the culture supernatant of CRFK cells infected with CoV with AZADO-Oxo for the purpose of confirming the antiviral effect of AZADO, and the photographs obtained by observing the wells using a microscope are shown together with photographs of cells without added virus (no virus) and cells with added virus but not added AZADO-Oxo (with virus).

[0016] [ Figure 4] is a graph showing the results (cell survival rate) of evaluating the antiviral effect of AZADO by the WST-8 method for samples obtained by treating the culture supernatant of CRFK cells infected with CoV with AZADO-Oxo for the purpose of confirming the antiviral effect of AZADO.

[0017] [ Figure 5 ] is a graph showing the results of mixing S-RBD-His (wild type) or S-RBD-His (Omicron strain) with different final concentrations of AZADO-Oxo and evaluating them by flow cytometry for the purpose of confirming whether AZADO-Oxo exhibits antiviral activity by oxidizing the spike protein and denaturing it, thereby inhibiting the binding of the spike protein to ACE2. The results for the wild type (S-RBD-His (WT)) are as follows Figure 5 (a) shows the results for the Omicron strain (S-RBD-His (Omicron)). Figure 5 (b) as shown.

[0018] [ Figure 6 ] indicates that AZADO was used for the purpose of comparing the antiviral effects of the compounds. + BF4 - ,TEMPO + BF4 - and 4-AcNH-TEMPO + BF4 - , which is a graph showing the results of mixing S-RBD-His (wild type) with compounds at different final concentrations and evaluating them by flow cytometry. + BF4 - The results are as follows Figure 6 As shown in (a), for TEMPO + BF4 - The results are as follows Figure 6 As shown in (b), for 4-AcNH-TEMPO + BF4 - The results are as follows Figure 6 It should be noted that, in the present specification, "AcNH", "acetylamino", "acetamide" and "NHAc" represent the same functional group.

[0019] [ Figure 7 ] means to Figure 6 The graph shows the results of graphing the average fluorescence intensity (Mean Fluorescence Intensity) in each peak.

[0020] [ Figure 8 ] means for Figure 6 and Figure 7 AZADO+BF4 showed high antiviral effect in the experiment shown - and 4-AcNH-TEMPO + BF4 - , is a graph of MFI obtained by performing the same operation except that the treatment time when adding S-RBD-His (wild type) was extended from 30 minutes to 6 hours.

[0021] [ Fig. 9 ] is an electrophoresis photograph showing the results of CBB staining of the supernatant of samples obtained by treating ovalbumin (OVA) with AZADO / DB, AZADO-Cu(bpy) / DB, Cu(bpy) / DB and DENKABLACK(DB) prepared in the examples.

[0022] [ Fig.10 ] is an electrophoresis photograph showing the results of CBB staining of the supernatant and residue of samples obtained by treating ovalbumin (OVA) with AZADO / DB, AZADO-Cu(bpy) / DB, Cu(bpy) / DB and DENKA BLACK(DB) prepared in the examples.

[0023] [ Fig.11 ] is an electrophoresis photograph showing the result of CBB staining of the supernatant of a sample obtained by treating ovalbumin (OVA) with non-supported AZADO. Fig.11 Indicates the results of using AZADO-Oxo.

[0024] [ Fig. 12A ] represents the compound of the present invention (TEMPO + NO3 - AZADO + NO3 - and 4-AcNH-TEMPO + NO3 - ), a graph showing the results of calculating the viable bacterial rate by culturing a bacterial solution contacted with the compound on a plate for the purpose of evaluating the inactivation activity against Escherichia coli.

[0025] [ Fig. 12B ] represents the compound of the present invention (TEMPO + NO3 - AZADO + NO3 - and 4-AcNH-TEMPO + NO3 -), a graph showing the results of calculating the viable bacterial rate by culturing a bacterial solution contacted with the compound on a plate for the purpose of evaluating the inactivation activity against Staphylococcus aureus.

[0026] [ Fig. 12C ] represents the compound of the present invention (TEMPO + NO3 - AZADO + NO3 - and 4-AcNH-TEMPO + NO3 - ), a graph showing the results of calculating the viable bacterial rate by culturing a bacterial solution contacted with the compound on a plate for the purpose of evaluating the inactivation activity against Penicillium.

[0027] [ Fig.13 ] is for the compound of the present invention (AZADO + NO3 - ), when determining the minimum inhibitory concentration (MIC) against Escherichia coli, use AZADO + NO3 - A photograph of the agar medium after incubation of samples with a final concentration of 1 mM.

[0028] [ Fig.14 ] is an electrophoresis photograph showing the result of reacting the antiviral substrate (AZADO-Cu(bpy) / DB) of the present invention with S-RBD in a liquid phase before evaluating the inactivation effect on proteins in a mist state, and evaluating the amount of S-RBD after the reaction by SDS-PAGE and CBB staining.

[0029] [ Fig.15 ] is an electrophoresis photograph showing the result of spraying an aqueous solution containing S-RBD onto a filter containing the antiviral substrate (AZADO-Cu(bpy) / DB) of the present invention for the purpose of evaluating the inactivation effect on proteins in a mist state, and evaluating the amount of S-RBD remaining on the filter by SDS-PAGE and silver staining after incubation. DETAILED DESCRIPTION

[0030] Hereinafter, specific embodiments of the present invention will be described.

[0031] <<Methods for inactivating bacteria or viruses>>

[0032] One embodiment of the present invention is a method for inactivating bacteria or viruses, comprising: contacting a compound having the redox mechanism (hereinafter, also referred to as "the compound of the present embodiment") with an object at risk of being contaminated by bacteria or viruses. According to the present invention, an inexpensive means is provided, which minimizes the burden on the environment and can exert an inactivation effect on microorganisms without requiring the presence of light.

[0033] <Compounds of the present embodiment>

[0034] The compounds of this embodiment are known in the past as "organic nitroxyl free radicals". Nitroxyl free radicals are stable organic free radical molecules and are widely used as oxidation catalysts in the field of organic synthesis. In particular, organic nitroxyl free radicals exhibit reactivity in both oxidation and reduction directions. As shown in the reaction formula, a one-electron reduction reaction to produce hydroxylamine and a one-electron oxidation reaction to produce oxoammonium ions occur. The oxoammonium ions generated by the oxidation of nitroxyl free radicals have a strong oxidizing ability and obtain hydroxylamine after two-electron oxidation of the substrate. The compounds of this embodiment act as oxidants against bacterial and viral proteins and oxidize the proteins to reduce or eliminate their functions. Thereby, the bacteria and viruses are inactivated. Here, the substance movement involved in the redox mechanism is only H·, protons (H + ), electronic (e - ), a faster reaction occurs than the redox of metal ions such as Ag and Cu proposed in the past, thereby achieving more rapid inactivation of bacteria and viruses. In addition, the inactivation method of microorganisms of this mode is characterized in that it is based on an oxidation reaction, and bacteria and viruses are composed of proteins, so it has the advantage of being able to exert an inactivation effect regardless of the bacterial species, fungal (mold) species, and the presence or absence of the envelope of the virus species. It should be noted that in the oxidation reaction in which the oxoammonium ion acts as an oxidizing active substance, the oxoammonium ion can be regenerated by adding a reactant that promotes the reoxidation of the hydroxylamine to complete the cycle and act as a catalyst.

[0035] The compound of this embodiment has the redox mechanism described above, and there is no limitation on its specific structure. In addition, the compound of this embodiment can be in the hydroxylamine form, nitroxyl free radical form, or oxoammonium form in the redox mechanism described above. Among them, the hydroxylamine form or the nitroxyl free radical form is preferred, and the nitroxyl free radical form is particularly preferred.

[0036] In addition, from the viewpoint of exhibiting excellent inactivation activity against microorganisms, the standard redox potential (25° C.) between the nitroxyl radical form and the oxoammonium form of the compound of the present embodiment is preferably +100 mV to +1000 mV [Ag / Ag +], more preferably +130mV to +600mV [Ag / Ag + ]. It should be noted that the standard redox potential was measured by cyclic voltammetry (CV) at a temperature of 25°C. At this time, a glassy carbon electrode (inner diameter 3 mm) was used as the working electrode and a reference electrode (Ag / Ag + ) and a counter electrode (platinum wire) in a three-pole mode, and the potential scanning rate is 50mV / s. It should be noted that the hydroxylamine form of the compound of the present invention is generally unstable and is rapidly oxidized to a nitroxyl radical form. Therefore, the value of the standard redox potential measured by the method described is a value between the nitroxyl radical form and the oxoammonium form.

[0037] Here, among the compounds of the present embodiment, for four compounds, namely (a) TEMPO, (b) AZADO, (c) 1-methyl AZADO, and (d) 1,3-dimethyl AZADO, the standard redox potential (E) between the nitroxyl radical form and the oxoammonium form was measured by using the cyclic voltammetry method. 0 The cyclic voltammogram obtained by Figure 1 As shown. Figure 1 In this way, the E of these compounds 0 The values ​​are (d) 136mV < (c) 186mV < (b) 236mV < (a) 294mV. It should be noted that for Figure 1 As can be seen from the cyclic voltammogram shown, even when the measurement is repeated for more than 100 cycles, the stability can be maintained, and thus the compound of the present embodiment has very high durability as an oxidizing agent.

[0038] From the viewpoint of the inactivation activity against microorganisms, the compound of the present embodiment is preferably represented by the following compound formula 1 in the form of its nitroxyl radical.

[0039] [Chemical formula 2]

[0040] [Compound Formula 1]

[0041]

[0042] In compound formula 1, R1 to R4, Y1 and Y2 are each independent and are a hydrogen atom or an optionally substituted monovalent organic group. Here, the monovalent organic group is not particularly limited, and for example, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, etc. may be cited. It should be noted that R1 to R4, Y1 and Y2 may be the same or different.

[0043] The alkyl, alkenyl and alkynyl groups as monovalent organic groups may be any of linear, branched or cyclic, preferably linear. The number of carbon atoms in the alkyl group is not particularly limited, preferably 1 or more and 20 or less, more preferably 1 or more and 10 or less, and further preferably 1 or more and 4 or less. The number of carbon atoms in the alkenyl and alkynyl groups is not particularly limited, preferably 2 or more and 20 or less, more preferably 2 or more and 10 or less, and further preferably 2 or more and 4 or less.

[0044] The aryl group as a monovalent organic group is a group derived from a hydrocarbon ring having aromaticity as a part or as a whole. When the aryl group contains more than two hydrocarbon rings having aromaticity as a part or as a whole, these rings can be bonded or condensed to each other with a single bond. In addition, when the aryl group contains more than two hydrocarbon rings having aromaticity as a part or as a whole, one atom can serve as a ring-forming atom of any two of these rings to form a spiro ring. The number of carbon atoms of the aryl group is not particularly limited, preferably more than 6 and less than 30. The number of carbon atoms of the aryl group is more preferably more than 6 and less than 12, and more preferably 6. In particular, the aryl group is preferably a monovalent group derived from an aromatic hydrocarbon ring having more than 6 ring-forming atoms.

[0045] The heteroaryl group as a monovalent organic group is a group derived from a heterocyclic ring having aromaticity as a part or as a whole. When the heteroaryl group contains more than two heterocyclic rings having aromaticity as a part or as a whole, some or all of these rings can be bonded to each other with single bonds. When the heteroaryl group contains more than two heterocyclic rings or other rings having aromaticity as a part or as a whole, these rings can be fused to each other. In addition, when the heteroaryl group contains more than two heterocyclic rings or other rings having aromaticity as a part or as a whole, one atom can also serve as a ring-forming atom of these rings. The heteroatom possessed by the heteroaryl group is not particularly limited. For example, as a ring-forming atom, more than one heteroatom (for example, nitrogen atom (N), oxygen atom (O), phosphorus atom (P), sulfur atom (S), silicon atom (Si), selenium atom (Se), germanium atom (Ge)) etc. can be cited. The number of carbon atoms of the heteroaryl group is preferably more than 3 and less than 30. In addition, the number of ring-forming atoms of the heteroaryl group is not particularly limited, preferably more than 5 and less than 30. In addition, the number of ring atoms of the heteroaryl group is more preferably 5 or more and 14 or less, and further preferably 5 or more and 13 or less. The number of heteroatoms of the heteroaryl group is not particularly limited, but is preferably 1 or more and 3 or less. In addition, the number of heteroatoms of the heteroaryl group is more preferably 1 or more and 2 or less, and further preferably 1. In particular, the heteroaryl group is preferably a monovalent group derived from an aromatic heterocycle having 5 or more ring atoms.

[0046] The alkyl, alkenyl, alkynyl, aryl and heteroaryl groups as monovalent organic groups can be substituted groups. There is no particular limitation on the substituent, and for example, alkyl, alkoxy, alkenyl, alkynyl, halogen atom, hydroxyl, carboxyl, cyano, amino, carbamoyl and the like can be cited. Among them, as a substituent, preferably an alkyl or a halogen atom, more preferably a methyl, ethyl or fluorine atom. And, as a substituent, more preferably a methyl or fluorine atom. It should be noted that the substituent will not replace the same group. For example, the substituent of the substituted alkyl does not contain an alkyl.

[0047] In addition, in compound formula 1, from the viewpoint of excellent antimicrobial activity, it is preferred that Y1 and Y2 are bonded to each other and form a nitrogen-containing monocyclic ring containing a 5-membered ring or a 6-membered ring, which is optionally substituted, and R1 and R3 are bonded to each other and form a nitrogen-containing bicyclic ring containing a 5-membered ring or a 6-membered ring, which is optionally substituted, or the two rings constituting the nitrogen-containing bicyclic ring are further cross-linked to form a nitrogen-containing tricyclic ring containing a 5-membered ring or a 6-membered ring, which is optionally substituted. That is, in a preferred embodiment of the present embodiment, the nitroxyl radical form of the compound of the present embodiment is shown in the following compound formula 2.

[0048] [Chemical formula 3]

[0049] [Compound Formula 2]

[0050]

[0051] In compound formula 2, R1 to R4 are independently hydrogen atoms or monovalent organic groups that are optionally substituted, and Z is a nitrogen-containing monocyclic ring that is optionally substituted and contains a 5-membered ring or a 6-membered ring. In this case, R1 and R3 are further optionally bonded to each other to form a nitrogen-containing bicyclic ring that is optionally substituted and contains a 5-membered ring or a 6-membered ring, or the two rings constituting the nitrogen-containing bicyclic ring are further cross-linked to form a nitrogen-containing tricyclic ring that is optionally substituted and contains a 5-membered ring or a 6-membered ring.

[0052] As the nitrogen-containing monocyclic ring of Z, for example, aliphatic heterocyclic rings such as tetrahydropyrrole ring, piperidine ring, morpholine ring, pyrroline ring, aromatic heterocyclic rings such as pyrrole ring, imidazole ring, etc. can be cited. In addition, as a nitrogen-containing bicyclic ring formed by further bonding R1 and R3 to each other, for example, 9-azabicyclo [3.3.1] nonane ring and 9-aza-3-oxabicyclo [3.3.1] nonane ring can be cited. And, as a nitrogen-containing tricyclic ring formed by further cross-linking the two rings constituting the nitrogen-containing bicyclic ring, for example, 2-azaadamantane ring, 2,6-diazaadamantane ring, 6-aza-2-oxadamantane ring, 9-aza-6-noradamantane ring can be cited. Among them, from the viewpoint of excellent inactivation activity against microorganisms, the compound of this embodiment preferably has a nitrogen-containing monocyclic ring, a nitrogen-containing bicyclic ring or a nitrogen-containing tricyclic ring, more preferably has a nitrogen-containing monocyclic ring or a nitrogen-containing tricyclic ring, and particularly preferably has a nitrogen-containing tricyclic ring. The compound of this embodiment preferably has a nitrogen-containing monocyclic aliphatic heterocyclic ring or a nitrogen-containing tricyclic 2-azaadamantane ring or 9-aza-6-noradamantane ring, and more preferably has a nitrogen-containing monocyclic piperidine ring or a nitrogen-containing tricyclic 2-azaadamantane ring.

[0053] The nitrogen-containing monocyclic ring formed in the compound formula 2 or the nitrogen-containing bicyclic ring or nitrogen-containing tricyclic ring formed in the compound formula 2 is an optionally substituted ring structure. There is no particular limitation on the substituents that can replace these ring structures, and for example, alkyl, alkoxy, alkenyl, alkynyl, halogen atoms, hydroxyl, carboxyl, carbonyl, cyano, amino, carbamoyl, tosyl, (halogenated) acylamino, phosphoric acid, etc. can be cited. Among them, as a substituent, a methyl, ethyl, methoxy, ethoxy, fluorine atom, hydroxyl, carboxyl or tosyl group is more preferred.

[0054] Here, when the compound of the present embodiment does not contain the ring structure or contains a nitrogen-containing monocyclic ring, R1 to R4 are independently selected, preferably a hydrogen atom or an optionally substituted alkyl group, more preferably a hydrogen atom or an unsubstituted alkyl group, further preferably a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and particularly preferably a hydrogen atom, a methyl group or an ethyl group. In addition, from the viewpoint of antimicrobial activity, when the compound of the present embodiment contains the nitrogen-containing bicyclic ring or the nitrogen-containing tricyclic ring, R2 and R4 are independently selected, preferably a hydrogen atom or an optionally substituted alkyl group, more preferably a hydrogen atom or an unsubstituted alkyl group, further preferably a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, and particularly preferably a hydrogen atom, a methyl group or an ethyl group.

[0055] An example of the compound of the present embodiment is shown below (in the form of nitroxyl radical). It should be noted that for some compounds, the standard redox potential (E) between the nitroxyl radical form and the oxoammonium form is also described. 0In view of the mechanism by which the compound of the present embodiment exhibits excellent antimicrobial activity, it can be said that the compounds shown below all exhibit high antimicrobial activity.

[0056] [Chemical formula 4-1]

[0057]

[0058] [Chemical formula 4-2]

[0059]

[0060] [Chemical formula 4-3]

[0061]

[0062] Given the E 0 When heteroatoms are introduced into structures such as TEMPO, ABNO, and AZADO constituting the skeleton of the compound, the redox characteristics of the compound of this embodiment shift to the high potential side. It is believed that the oxidation ability of the potential corresponding to each oxoammonium form is improved.

[0063] Among the above compounds, TEMPO, 4-hydroxy TEMPO, 4-methoxy TEMPO, 4-acetylamino TEMPO, 4-oxo TEMPO, 4-carboxy TEMPO, 4-amino TEMPO, 4-phosphono TEMPO, 4-(2-bromoacetamide) TEMPO, AZADO, 1-methyl AZADO, 1,3-dimethyl AZADO, 1-fluoro AZADO, 5-fluoro AZADO, 5,7-difluoro AZADO, 1,3-dimethyl ... AZADO, 5-fluoro-1-methyl AZADO, 5,7-difluoro-1-methyl AZADO, 5-methoxy AZADO, 5-methoxy-1-methyl AZADO, 5,7-dimethoxy AZADO, oxa-AZADO, TsN-AZADO, diAZADO or Nor-AZADO, more preferably AZADO, 1-methyl AZADO, 1,3-dimethyl AZADO, 1-fluoro AZADO, 5-fluoro AZADO, 5,7-difluoro AZADO AZADO, 5-fluoro-1-methylAZADO, 5,7-difluoro-1-methylAZADO, 5-methoxyAZADO, 5-methoxy-1-methylAZADO, 5,7-dimethoxyAZADO, oxa-AZADO, TsN-AZADO, diAZADO, Nor-AZADO, TEMPO or 4-acetylamino TEMPO, and more preferably AZADO, 1-methylAZADO, 1,3-dimethylAZADO, 1-fluoroAZADO, 5-FluoroAZADO, 5,7-difluoroAZADO, 5-fluoro-1-methylAZADO, 5,7-difluoro-1-methylAZADO, 5-methoxyAZADO, 5-methoxy-1-methylAZADO, 5,7-dimethoxyAZADO, TEMPO or 4-acetylaminoTEMPO, particularly preferably 2-azaadamantane-N-oxyl (AZADO), 2,2,6,6-tetramethylpiperidinyl-N-oxyl (TEMPO) or 4-acetylaminoTEMPO.

[0064] <Co-catalyst>

[0065] With regard to the deactivation method of this mode, from the viewpoint of antimicrobial activity, it is preferred that the compound of this mode is contacted with the object in the presence of a co-catalyst comprising a transition metal. Here, the co-catalyst of this mode comprises an ion of a transition metal (i.e., a salt comprising a transition metal), or a monomer comprising a transition metal. As the transition metal that can be included in the co-catalyst of this mode, it is a metal element classified as a metal element from the 3rd group element to the 12th group element of the periodic table, and is not particularly limited. Among them, from the viewpoint of excellent antimicrobial activity, the transition metal preferably comprises one or more than two selected from Ag, Au, Pt, Pd, Ni, Mn, Fe, Ti, Al, Zn and Cu, and more preferably comprises its ion (salt). In addition, the transition metal further preferably comprises Ag or Cu, particularly preferably comprises an ion (salt) comprising Ag or Cu, and most preferably comprises an Ag ion or a divalent Cu ion (Cu (II) ion).

[0066] When the promoter includes an ion (salt) of a transition metal, the counter anion of the ion is not particularly limited. As a salt including a transition metal as a promoter, for example, sulfates, sulfites, hyposulfites, persulfates, thiosulfates, carbonates, phosphates, pyrophosphoric acids, hydrochlorides, nitrates, nitrites, halides (for example, fluorides, chlorides, bromides, iodides), trifluoromethanesulfonates, etc. can be cited.

[0067] In the inactivation method of this embodiment, when a co-catalyst is present, there is no particular limitation on the amount of the compound and the transition metal used. From the perspective of excellent microbial inactivation activity, the molar ratio of these components is preferably 1:2 to 4:1 (compound: transition metal), and more preferably 2:3 to 3:2.

[0068] <Target>

[0069] The method for inactivating bacteria or viruses of this embodiment includes: contacting the compound of this embodiment with an object at risk of being contaminated by bacteria or viruses. That is, according to other embodiments of the present invention, a bacteria or virus inactivator containing the compound is also provided. In terms of the inactivation of microorganisms using the compound and inactivator of this embodiment, for objects at risk of being contaminated by bacteria or viruses, for example, kitchens (kitchen spaces such as kitchen floors and walls, etc.), sinks, refrigerators and other food processing locations, knives, pots and other cooking utensils, plates and other tableware, refrigerators, toilets, bathrooms, washrooms, living rooms, bedrooms and other living spaces, sofas, cushions, bedding, curtains and other fiber products, etc., can be used by contacting these. Here, there is no particular limitation on the bacteria and viruses that are the objects of inactivation, and the inactivation effect on various bacteria, fungi and viruses is shown. Examples of the bacteria include Escherichia such as Escherichia coli; Pseudomonas such as Pseudomonas aeruginosa; Salmonella such as Salmonella choleraesuis; Moraxella such as Moraxella catarrhalis; Gram-negative bacteria such as Legionella such as Legionella pneumophila, Staphylococcus such as Staphylococcus aureus; and Gram-positive bacteria such as Clostridium such as Clostridium botulinum and Clostridium perfringens. Examples of the molds include Aspergillus species such as Aspergillus niger and Aspergillus sphaeroides; Paecilomyces species such as Paecilomyces variotii; Penicillium species such as Penicillium pinophilum and Penicillium citrinum; Trichoderma species such as Trichoderma virens; Chaetomium species such as Chaetomium globosum; Eurotium species such as Eurotium tonophilum; Rhizopus oryzae species such as Rhizopus oryzae; Cladosporium species such as Cladosporium cladosporioides; Aureobasidium pullulans species such as Aureobasidium pullulans; Myrothecium species such as Myrothecium verrucaria, and the like. In addition, as fungi other than molds, for example, yeasts such as Saccharomyces cerevisiae, Candida species such as Candida albicans, and Rhodotorula species such as Crimson Rhodotorula can be cited. In addition, as a virus, it can be an enveloped virus or a non-enveloped virus. As enveloped viruses, coronaviruses (including SARS-CoV-2), influenza viruses, herpes viruses, rubella viruses, etc. can be cited. As non-enveloped viruses, noroviruses, rotaviruses, etc. can be cited. The virus as the object is preferably an enveloped virus, more preferably a coronavirus, and particularly preferably SARS-CoV-2. Among them, in terms of being able to effectively inhibit the invasion of viruses into human cells, the inactivation method of the present method preferably inactivates the virus by causing oxidative damage to the spike protein constituting the virus.Furthermore, in another preferred embodiment, the object to be inactivated is bacteria, and examples of the bacteria include bacteria and fungi.

[0070] In addition, contact is not particularly limited, and the method of spraying the compound of this method or the composition containing it (for example, liquid composition) directly to the object or filling it into a spray device such as a trigger, sprayer, etc. can be cited. Through such a structure, the compound of this method can be distributed in a large range. The contact time between the compound of this method and the object is not limited, for example, it can be preferably placed for more than 5 seconds, more preferably more than 1 minute, and more preferably more than 30 minutes. There is no particular limitation on the upper limit of the contact time, preferably less than 24 hours, more preferably less than 12 hours, and more preferably less than 6 hours. In addition, the use method of the compound of this method when contacting the object is not particularly limited. For example, it can be liquid, gel or paste. It should be noted that, in the case of the contact in the solution, the oxidation reaction (for example, the denaturation of protein) can be monitored according to the color change of the solution.

[0071] In the case of liquid form, it can be used as a spray, lotion, etc. For example, it can be filled into a known spray container such as a trigger spray container (direct pressure or storage type), a dispenser pump spray container, etc., and the spray amount can be adjusted to spray the liquid at the location where bacteria or viruses exist or are thought to exist.

[0072] <<Antibacterial or antiviral substrate>>

[0073] According to another aspect of the present invention, there is provided an antibacterial or antiviral substrate in which the inactivator of one aspect of the present invention and a co-catalyst as required are supported or coated on a base substrate. The inactivator can be conveniently used for various purposes by using such an antimicrobial substrate.

[0074] As a base substrate constituting an antimicrobial substrate, as a material with a relatively low price and can easily carry / apply an inactivator, for example, woven fabrics, nonwoven fabrics, filters or urethane foams can be cited. There is no particular limitation on the method of carrying or applying the compound and the co-catalyst as required to these base substrates. Here, for the substrate of the described method, as an index of excellent antimicrobial activity, the antiviral activity value (Mv) for influenza virus or feline calicivirus measured based on JIS L 1922:2016 (antiviral test method for fiber products) is preferably 2.0 or more, more preferably 2.2 or more, and further preferably 2.5 or more. In addition, such an antimicrobial substrate can be suitable for use in, for example, masks, medical fiber products, air conditioners, air purifiers, freezers, temperature control machines, dehumidifiers, humidifiers or vacuum cleaner filters. Of course, it can also be used for other purposes.

[0075] It should be noted that the following embodiments are also included in the scope of the present invention: the inactivation method of claim 1 having the features of claim 2; the inactivation method of claim 1 having the features of claim 3; the inactivation method of any one of claims 1 to 3 having the features of claim 4; the inactivation method of any one of claims 1 to 4 having the features of claim 5; the inactivation method of any one of claims 1 to 5 having the features of claim 6; the inactivation method of claim 5 or 6 having the features of claim 7; the inactivation method of any one of claims 1 to 7 having the features of claim 8; the inactivation method of any one of claims 1 to 8 having the features of claim 9; An inactivation method according to any one of claims 1 to 9 having the features of claim 10; an inactivation method according to claim 10 having the features of claim 11; an inactivation method according to any one of claims 1 to 11 having the features of claim 12; an inactivating agent according to claim 13 having the features of claim 14; a substrate according to claim 15 using the inactivating agent according to claim 13 or 14; a substrate according to claim 15 having the features of claim 16; a substrate according to claim 15 or 16 having the features of claim 17; a substrate according to any one of claims 15 to 17 having the features of claim 18; a substrate according to any one of claims 15 to 18 having the features of claim 19.

[0076] Example

[0077] Hereinafter, although an embodiment of the present invention will be described in detail using examples, the technical scope of the present invention is not limited only to the following examples.

[0078] <<Study on the dilution multiples of the virus>>

[0079] First, the conditions of virus dilution multiples were studied for the purpose of studying the effect of feline coronavirus (FCov (VR-989)) on the viability of cat kidney-derived immortalized cells (CRFK).

[0080] Specifically, first, the supernatant obtained by diluting the culture supernatant of CRFK cells infected with FCoV for 3 days with PBS by 20 times was prepared as the stock solution of FCoV solution. In addition, the stock solution was diluted 10 times with PBS every 10 times. 10 The FCoV solution was prepared by diluting the solution in stages by multiple times.

[0081] On the other hand, CRFK cells suspended in DMEM medium containing 10% (v / v) FBS were inoculated into a 96-well plate at 100 μL / well and adjusted to 1×10 4 cells / well, and cultured for 4 days. After 4 days of culture, the medium was replaced with DMEM medium containing 2% (v / v) FBS at 100 μL / well. After adding each dilution of the FCoV solution prepared above at 100 μL / well, the cells were further cultured for 24 hours. Then, the medium was replaced with DMEM medium containing 2% (v / v) FBS at 200 μL / well, and the cells were further cultured for 2 or 3 days. And for the cells after the above culture, the cytopathic effect (CPE) was evaluated. For the cytopathic effect (CPE), each well was observed using an optical microscope, and the wells with more than 50% cytopathic cells were evaluated as CPE positive. Regarding the evaluation results of the CPE, for either case after 2 or 3 days of culture, a change in cell viability dependent on the dilution factor was confirmed until dilution to 10 5 times. Referring to the above results, the amount of virus to be used in the following experiments was determined.

[0082] <<Confirmation of the antiviral effect of AZADO (WST-8 method)>>

[0083] For the purpose of confirming whether AZADO, one kind of nitroxide radical, exhibits an antiviral effect, the following experiment was conducted to evaluate the antiviral effect by the WST-8 method.

[0084] Specifically, first, a culture supernatant obtained by diluting 20-fold with PBS the culture supernatant of CRFK cells infected with FCoV after 3 days of culture was prepared as the stock solution of the FCoV solution. In addition, FCoV solutions of various dilution factors were prepared by serially diluting the stock solution with PBS in a two-fold manner up to 160-fold dilution. And to each serially diluted FCoV solution, the oxoammonium form of AZADO (counter anion: nitrate ion; AZADO-Oxo) or silver nitrate (comparative example) was added to make the final concentration 100 mM, and the mixture was incubated at room temperature for 2 hours to prepare the sample solution.

[0085] On the other hand, CRFK cells suspended in DMEM medium containing 10% (v / v) FBS were inoculated into a 96-well plate at 100 μL / well and adjusted to 1×10 4Cells / well were cultured for 4 days. After 4 days of culture, the medium was replaced with DMEM medium containing 2% (v / v) FBS at 100 μL / well. After adding each sample solution at 100 μL / well, the cells were cultured for 2 days. Then, the cell viability in each sample was measured by the WST-8 method. It should be noted that the WST-8 method is a method for evaluating the presence of living cells by reducing a certain tetrazolium salt (WST-8) through an intracellular electron carrier in living cells to generate a highly water-soluble orange formazan dye. The results are as Figure 2 shown. As Figure 2 shown, by adding AZADO-Oxo, the recovery of the cell viability reduced by virus addition was observed. In contrast, such a phenomenon was not observed in the comparative example (addition of silver nitrate). These results indicate that AZADO-Oxo exhibits excellent antiviral effects.

[0086] <<Confirmation of the antiviral effect of AZADO (CPE method; incubation at 37 °C)>>

[0087] For the purpose of confirming whether AZADO, a kind of nitroxide radical, exhibits antiviral effects, the following experiment was conducted, and the antiviral effects were evaluated by the CPE method.

[0088] Specifically, first, a culture supernatant obtained by diluting 20-fold with PBS the culture supernatant of CRFK cells infected with FCoV after 3 days of culture was prepared and used as the stock solution of the FCoV solution. In addition, FCoV solutions with each dilution factor were prepared by successively diluting the stock solution 10-fold with PBS up to 10 5 times. And AZADO-Oxo was added to each of the FCoV solutions after stepwise dilution so that the final concentration became 2 mM, and the mixture was incubated at 37 °C for 2 hours, and then diluted 20-fold with PBS to prepare a sample solution. It should be noted that a sample solution of the comparative example without adding AZADO-Oxo was also prepared.

[0089] On the other hand, CRFK cells suspended in DMEM medium containing 10% (v / v) FBS were inoculated into a 96-well plate at 100 μL / well, and made to be 1×10 4Cells / well were cultured for 4 days. After 4 days of culture, the medium was replaced with DMEM medium containing 2% (v / v) FBS at 100 μL / well. After adding each of the above-prepared sample solutions at 100 μL / well, the cells were further cultured for 24 hours. Then, the medium was replaced with DMEM medium containing 2% (v / v) FBS at 200 μL / well, and the cells were further cultured for 2 days. And for the cultured cells, the cytopathic effect (CPE) was evaluated by the same method as above. The results are shown in Table 1 below. It should be noted that "TCID" 50 " in Table 1 refers to the abbreviation of 50% Tissue Culture Infectious Dose, which is the amount of virus that causes 50% of the host cells to die due to virus infection.

[0090] [Table 1]

[0091]

[0092] As shown in Table 1, it was confirmed that AZADO-Oxo exhibited excellent antiviral activity. In addition, the value of ΔlogTCID 50 as an index of antiviral activity was 2.8 (=5.2 - 2.4), and this value means that AZADO-Oxo reduced the virus amount to 1 / 10 2.8 (= approximately 1 / 1000). Therefore, it was confirmed here that AZADO-Oxo exhibited sufficient antiviral activity.

[0093] [[Confirmation of the antiviral effect of AZADO (CPE method; incubation at 25 °C)]]

[0094] For the purpose of meeting the conditions of JIS L 1922:2016 (Test method for antiviral properties of textile products), except that the incubation temperature was changed from 37 °C to 25 °C, and the number of days of culture after adding the sample solution and replacing the medium was changed from 2 days to 3 days, the antiviral effect based on the CPE method was evaluated by the same method as above. The results are shown in Table 2 below.

[0095] [Table 2]

[0096]

[0097] From the results shown in Table 2, it was confirmed that in this experimental system incubated at 25 °C, AZADO-Oxo exhibited sufficient antiviral activity.

[0098] In addition, in this experiment, 48 hours after the addition of the sample solution with (virus + AZADO-Oxo) or without (virus only) AZADO-Oxo, the wells were observed under a microscope, and the obtained photos were presented together with the photos of cells without virus addition (virus-free) in Figure 3 as shown. As Figure 3 shown, the addition of the virus changed the cell morphology to round, and the addition of AZADO-Oxo inhibited this morphological change, which also demonstrated the antiviral effect of AZADO-Oxo.

[0099] In addition, in this experiment, for the cells cultured for 3 days after the addition of the sample solution and medium replacement, the cell viability was measured using the WST-8 method. The results are as Figure 4 shown. As Figure 4 shown, the addition of the virus reduced the cell viability to approximately 50%, and the addition of (FCoV), AZADO-Oxo (FCoV + AZADO-Oxo) restored the cell viability to the same level as that of the blank (Non virus (virus-free)).

[0100] <<Confirmation of the Antiviral Effect of AZADO against SARS-Cov-2>>

[0101] In this experiment, it was confirmed whether AZADO-Oxo exhibited antiviral activity against the currently prevalent SARS-Cov-2. Here, SARS-CoV-2 invades human cells by binding the spike protein present on the surface of the virus particle to ACE2, which is an infection receptor on the surface of human cells. Therefore, it was confirmed whether AZADO-Oxo denatured the spike protein by oxidation, thereby hindering the binding of the spike protein to ACE2 and exhibiting antiviral activity. The virus spike proteins (two types: wild type and Omicron strain), cells, and fluorescently labeled antibodies for detection used in this experiment are as follows:

[0102] · Spike protein (wild type): SARS-CoV-2 Spike Protein (RBD) (aa319-541), His Tag Recombinant Protein (Thermo, RP-87678) (hereinafter also referred to as "S-RBD-His (WT)")

[0103] · Spike protein (Omicron strain): SARS-CoV-2 B.1.1.529 (Omicron) Spike RBD Protein (His Tag) (Sino Biological, 40592-V08H121) (hereinafter also referred to as "S-RBD-His (Omicron)")

[0104] Cells: ACE2-expressing HEK293T (hereinafter also referred to as "ACE2-HEK293T")

[0105] Fluorescently labeled antibody: 6x-His Tag Monoclonal Antibody (AD1.1.10), FITC (Thermo, MA1-81891) (hereinafter also referred to as “FITC-anti-6xHis-Tag antibody”).

[0106] In this experiment, first, 1 μg of S-RBD-His (WT) or S-RBD-His (Omicron) (2 μL of 0.5 mg / mL aqueous solution for WT and 4 μL of 0.25 mg / mL aqueous solution for Omicron) was mixed with AZADO-Oxo at a final concentration of 10 μM, 100 μM, or 1000 μM, respectively, and subjected to oxidation treatment at 25°C for 30 minutes to prepare a sample solution. In addition, a sample solution of a comparative example was prepared without adding AZADO-Oxo. Next, in a microtube containing 100 μL of PBS containing 1% BSA, 5×10 5 ACE2-HEK293T was suspended in the form of cells / mL. Then, 4 μL of the sample solution prepared above was added and allowed to stand at 4°C for 30 minutes to perform a binding reaction between the spike protein and the ACE2 receptor on the surface of ACE2-HEK293T cells.

[0107] To the cell suspension after the reaction, 800 μL of PBS containing 1% BSA was added, and the mixture was centrifuged at 4°C for 4 minutes and 400×g. After discarding the supernatant, 100 μL of PBS containing 1% BSA was added and resuspended. Therefore, 1 μg (10 μL) of FITC-anti-6xHis-Tag antibody was added, and the mixture was allowed to stand at 4°C for 30 minutes in the dark. Then, 1 mL of PBS containing 1% BSA was further added, and the mixture was centrifuged at 4°C for 4 minutes and 400×g. After discarding the supernatant, 300 μL of PBS containing 1% BSA was added and resuspended. The resulting solution was evaluated by flow cytometry. It should be noted that when this experiment was performed using HEK293T that does not express ACE2, the amount of S-RBD-His bound was significantly reduced, so it was determined that ACE2 specific binding could be detected. The results for the wild strain (S-RBD-His (WT)) are as follows Figure 5 (a) shows the results for the Omicron strain (S-RBD-His (Omicron)). Figure 5Here, the less the amount of spike protein bound to ACE2 (i.e., the more these bindings are inhibited by AZADO-Oxo), the more the peak shifts to the side with smaller fluorescence intensity (the left side of the figure).

[0108] Depend on Figure 5 The results shown in Figure 2 show that in the absence of AZADO-Oxo (S), the peak shifts to the right more (indicating a greater amount of binding to ACE2). In addition, it was confirmed that the peak shifted to the right shifts to the left depending on the amount of AZADO-Oxo added (the more the amount added, the greater the shift to the left). It should be noted that in the Omicron strain, the peak based on the addition of AZADO-Oxo shifts to the left more slowly than in the wild-type strain, and it is judged that a certain inactivation effect is also obtained for the Omicron strain.

[0109] <<Comparison of antiviral effects between compounds against SARS-Cov-2>>

[0110] For the purpose of comparing the antiviral effects of the compounds, the oxoammonium form of AZADO (with a counter anion of BF4 - ION;AZADO + BF4 - ), the oxoammonium form of TEMPO (the counter anion is BF4 - Ion; TEMPO + BF4 - ) and the oxoammonium form of 4-acetamidoTEMPO (the counter anion is BF4 - Ion; 4-AcNH-TEMPO + BF4 - ) and the antiviral effect was confirmed by the same method as above.

[0111] Specifically, first, 1 μg of S-RBD-His (WT) (2 μL of 0.5 mg / mL aqueous solution) was mixed with AZADO at a final concentration of 10 μM, 100 μM, or 1000 μM. + BF4 - TEMPO + BF4 - or 4-AcNH-TEMPO + BF4 - The mixture was mixed and subjected to oxidation treatment at 25°C for 30 minutes to prepare a sample solution. In addition, a sample solution of a comparative example was prepared without adding the compound. Next, in a microtube containing 100 μL of PBS (containing 2 mM EDTA) containing 1% BSA, 1×10 6ACE2-HEK293T was suspended in the form of cells / mL. Then, 4 μL of the sample solution prepared above was added and allowed to stand at 4°C for 30 minutes to perform a binding reaction between the spike protein and the ACE2 receptor on the surface of ACE2-HEK293T cells.

[0112] To the cell suspension after the reaction, 800 μL of PBS (containing 2 mM EDTA) containing 1% BSA was added, and the mixture was centrifuged at 400×g for 4 minutes at 4°C. After discarding the supernatant, 100 μL of PBS (containing 2 mM EDTA) containing 1% BSA was added for resuspending. Therefore, 1 μg (10 μL) of FITC-anti-6xHis-Tag antibody was added, and the mixture was left standing at 4°C for 30 minutes in the dark. Then, 1 mL of PBS (containing 2 mM EDTA) containing 1% BSA was further added, and the mixture was centrifuged at 400×g for 4 minutes at 4°C. After discarding the supernatant, 300 μL of PBS (containing 2 mM EDTA) containing 1% BSA was added for resuspending. The resulting solution was evaluated by flow cytometry. For AZADO + BF4 - The results (AZADO-Oxo) are as follows Figure 6 As shown in (a) (S is the spike protein, A is the compound), for TEMPO + BF4 - The results (TEMPO-Oxo) are as follows Figure 6 As shown in (b) (S is spike protein, T is compound), for 4-AcNH-TEMPO + BF4 - The results (Ac-TEMPO-Oxo) are as follows Figure 6 (c) (S is spike protein, AcT is compound). Figure 6 The results obtained by graphing the mean fluorescence intensity (Mean Fluorescence Intensity) in each peak are shown in the figure. Figure 7 shown.

[0113] according to Figure 6 The results shown in Figure 2 show that the peak shifted to the right in the absence of AZADO-Oxo (S). In addition, the peak shifted to the right shifted to the left depending on the amount of compound added. It should be noted that when comparing the activities of the compounds, AZADO + BF4 - and 4-AcNH-TEMPO + BF4 - The activity of TEMPO is roughly the same. + BF4 -The activity of Figure 7 The MFI chart also shows the same result.

[0114] In addition, AZADO, which showed a high antiviral effect in the above experiment + BF4 - and 4-AcNH-TEMPO + BF4 - (final concentration 10 μM), and the same operation was repeated except that the treatment time when adding S-RBD-His (WT) was extended from 30 minutes to 6 hours. The MFI graph thus obtained is shown in the figure below. Figure 8 As shown. Figure 8 By extending the processing time, AZADO + BF4 - and 4-AcNH-TEMPO + BF4 - Showed higher antiviral activity.

[0115] The results shown above show that according to the present invention, it is possible to provide an inexpensive means that can minimize the burden on the environment and can exert an inactivation effect on microorganisms without requiring the presence of light.

[0116] <<Evaluation of the Degradation Activity of AZADO on Ovalbumin (OVA)>>

[0117] (Preparation Example of Catalyst Support (AZADO / DB))

[0118] A catalyst support in which AZADO was supported on a catalyst support (carbon support) was prepared by the following method.

[0119] AZADO 5 mg (0.13 mmol) and dichloromethane 100 mL were added to a 300 mL eggplant flask and AZADO was dissolved by ultrasonication. DENKA BLACK (registered trademark) (Denka Black, DB) 100 mg as a carbon support was added and dispersed by ultrasonication for 30 minutes, and then stirred at room temperature for 30 minutes. Then, the mixture was evaporated to dryness at 40°C and 600 hPa using a rotary evaporator, and further vacuum dried for 10 hours to obtain a catalyst support (AZADO / DB).

[0120] (Preparation Example of Catalyst Support (AZADO-Cu(bpy) / DB))

[0121] Cu(OTf)2 0.03mmol (10.9mg), bpy 0.03mmol (4.7mg) and AZADO 0.03mmol (4.6mg) were added to a 5mL vial and dissolved in 3mL of acetonitrile, and added to a 300mL eggplant-shaped flask to which 90mL of acetonitrile was added. After adding each reagent, the wall of the vial was cleaned with acetonitrile and added to the flask. Next, 100mg of DENKA BLACK (DB) as a carbon carrier was added to the flask and dispersed with ultrasound for 30 minutes, and then stirred at room temperature for 30 minutes. Then, it was evaporated to dryness at 60°C and 250hPa using a rotary evaporator, and further vacuum dried for 10 hours to obtain a catalyst carrier (AZADO-Cu(bpy) / DB) simultaneously loaded with AZADO and a co-catalyst. It should be noted that by performing the same method without using AZADO, a catalyst carrier (Cu(bpy) / DB) loaded only with a co-catalyst was prepared.

[0122] (OVA treatment based on catalyst carrier (1))

[0123] 1 mg of each of the above-prepared AZADO / DB, AZADO-Cu(bpy) / DB, Cu(bpy) / DB, and DENKA BLACK(DB) was weighed into a microtube and mixed with 20 μL of a 10 mg / mL ovalbumin (OVA) aqueous solution and 980 μL of pure water to prepare a sample. In addition, 3 μL of an aqueous solution of AZADO-Oxo (concentration 100 mM) was used instead of the catalyst support to prepare a sample in the same manner (the amount of pure water was 977 μL). It should be noted that a control sample was prepared by mixing only OVA with pure water.

[0124] After each sample was allowed to stand at room temperature for 24 hours, it was centrifuged at room temperature for 3 minutes at 15000×g. Next, 80 μL of the supernatant of each sample was transferred to another tube, and 20 μL of 5× sample buffer (SB) was added. Then, it was allowed to stand at 95°C for 5 minutes, electrophoresed with molecular weight markers, and CBB stained. The results are as follows: Fig. 9 As shown. Fig. 9 As shown, the samples treated with AZADO-Oxo ( Fig. 9 In the sample (OVA+AZADO-Oxo), the band of OVA almost disappeared. This confirmed that the antiviral activity of AZADO-Oxo confirmed above is to act on the spike protein of the virus and oxidize it to decompose it. In addition, in the sample ( Fig. 9In the case of "OVA+AZADO-Cu(bpy) / DB"), the OVA band completely disappeared, showing a superior OVA decomposition activity compared to AZADO-Oxo. This is believed to be the result of a catalytic cycle of redox reaction occurring through the synergistic effect of AZADO and the co-catalyst, thereby oxidizing OVA more strongly. Fig. 9 In the sample (OVA+DB) to which the carbon carrier (DB) was added and the sample (OVA+Cu(bpy) / DB) to which Cu(bpy) / DB was added, the band of OVA almost did not disappear, and a band equivalent to that of the control (OVA) was confirmed.

[0125] (Ovalbumin (OVA) treatment based on catalyst carrier (2))

[0126] Each sample was prepared by the same method as above, wherein the amount of the aqueous solution of AZADO-Oxo (concentration 100 mM) added was 2.5 μL.

[0127] After each sample was allowed to stand at room temperature for 18 hours, it was centrifuged at room temperature at 15,000×g for 3 minutes. Next, 80 μL of the supernatant of each sample was transferred to another tube, 20 μL of 5×SB was added, and the mixture was allowed to stand at 95°C for 5 minutes to prepare a supernatant sample. In addition, for the sample containing the catalyst carrier (C), the remaining supernatant was discarded after the centrifugation, washed twice with 1 mL of pure water, and resuspended in 80 μL of pure water. Furthermore, 20 μL of 5×SB was added, and the mixture was allowed to stand at 95°C for 5 minutes to prepare a residue sample. The supernatant sample and residue sample thus obtained were subjected to electrophoresis together with molecular weight markers and CBB staining was performed. The results are as follows. Fig.10 As shown. Fig.10 As indicated, the supernatant samples were confirmed to be Fig. 9 The same results were obtained. On the other hand, in the residue samples, the samples with Cu(bpy) / DB added ( Fig.10 A very strong band of OVA was confirmed in "OVA+Cu(bpy) / DB"). It is believed that Cu(bpy) / DB strongly adsorbs OVA. In addition, in the sample ( Fig.10 In the case of "OVA+AZADO-Cu(bpy) / DB"), it was confirmed that the band of OVA adsorbed with Cu(bpy) / DB was significantly diffused (broadened). Fig. 9 and Fig.10The mechanism by which AZADO-Cu(bpy) / DB in the (supernatant sample) causes the OVA band to disappear (decomposes OVA) is that Cu(bpy) / DB adsorbs OVA and induces oxidative decomposition based on AZADO. Based on the mechanism, it is believed that the microbial inactivation method and inactivator of one embodiment of the present invention not only have high inactivation activity against viruses, but also against bacteria, molds, fungi, etc. that have proteins as essential components.

[0128] (OVA treatment with non-supported AZADO)

[0129] The following samples were prepared respectively:

[0130] (a1) Sample of 1000 μL pure water (None)

[0131] (a2) Sample obtained by mixing 980 μL of pure water and 20 μL of 10 mg / mL ovalbumin aqueous solution (OVA)

[0132] (a3) Sample obtained by mixing 980 μL of pure water, 20 μL of 10 mg / mL ovalbumin aqueous solution, and 2.5 μL of 100 mM AZADO-Oxo aqueous solution (OVA+AZADO-Oxo)

[0133] (a4) A sample obtained by mixing 980 μL of pure water, 20 μL of a 10 mg / mL ovalbumin aqueous solution, 2.5 μL of a 100 mM AZADO-Oxo aqueous solution, and 2.5 μL of a 100 mM Cu(NO3)2 aqueous solution (OVA+AZADO-Oxo+Cu(NO3)2)

[0134] (a5) A sample obtained by mixing 980 μL of pure water, 20 μL of a 10 mg / mL ovalbumin aqueous solution, 2.5 μL of a 100 mM AZADO-Oxo aqueous solution, and 2.5 μL of a 100 mM Cu(OTf)2 aqueous solution (OVA+AZADO-Oxo+Cu(OTf)2).

[0135] After each sample was allowed to stand at room temperature for 24 hours, it was centrifuged at room temperature for 3 minutes at 15000×g. Next, 80 μL of the supernatant of each sample was transferred to another tube, and 20 μL of 5×SB was added. Then, it was allowed to stand at 95°C for 5 minutes and electrophoresed with molecular weight markers, and CBB staining was performed. The results are as follows: Fig.11 As shown. Fig.11 As shown, in the sample in which AZADO-Oxo was added to OVA, the disappearance of the OVA band (protein decomposition activity) was confirmed similarly to the above, and the addition of the co-catalyst (Cu(NO3)2, Cu(OTf)2) did not enhance the effect.

[0136] <<Evaluation of inactivation activity against bacteria and fungi (molds)>>

[0137] For the oxoammonium form of TEMPO as the compound of the present invention (the counter anion is NO3 - Ion; TEMPO + NO3 - ), AZADO oxoammonium form (counter anion is NO3 - ION;AZADO + NO3 - ) and the oxoammonium form of 4-acetamidoTEMPO (the counter anion is NO3 - Ion; 4-AcNH-TEMPO + NO3 - ), and the inactivation activity against bacteria and fungi (molds) was evaluated. It should be noted that copper nitrate (II) was used in the comparative examples.

[0138] Specifically, first, as the objects of inactivation, Escherichia coli (Escherichia coli ATCC43895, serotype O157:H7, verotoxin toxin type I and II producing strains), Staphylococcus aureus (Staphylococcus aureus subsp. Aureus NBRC 12732) and Penicillium citrinum (Penicilliun citrinum NBRC 6352) were prepared. For Escherichia coli and Staphylococcus aureus, the pre-culture was carried out at 35°C ± 1°C for 18 to 24 hours in a normal agar medium (manufactured by Eiken Chemical Co., Ltd.). In addition, for Penicillium, it was carried out at 25°C ± 1°C for 7 to 10 days in a potato dextrose agar medium (manufactured by Difco). In addition, the bacterial liquid was prepared using purified water for Escherichia coli, physiological saline for Staphylococcus aureus, and 0.005% dioctyl sodium sulfosuccinate solution for Penicillium.

[0139] Next, to 5 mL of the aqueous solution containing the above compounds, the mixture was adjusted to 1×10 7 ~1×10 8 0.05 mL of a bacterial solution with a bacterial count of 100 μg / mL was added and gently stirred. Here, the concentration of the compound in the aqueous solution was 10 mM or 20 mM in the test for Escherichia coli, 0.1 mM or 1 mM in the test for Staphylococcus aureus, and 0.1 mM or 10 mM in the test for Penicillium.

[0140] After 1 minute, 5 minutes, and 15 minutes from the addition of the bacterial solution, the solution was sampled to the extent that the number of grown bacteria could be counted and diluted with SCDLP medium (manufactured by Shiota NI MS Co., Ltd.) to neutralize the compound. Then, the neutralized sample was applied to SCDLP agar medium (manufactured by Shiota NI MS Co., Ltd.) for Escherichia coli and Staphylococcus aureus, and the neutralized sample was applied to GPLP agar medium (manufactured by Shiota NI MS Co., Ltd.) for Penicillium, and plate culture was performed at 35°C±1°C for Escherichia coli and Staphylococcus aureus and at 25°C±1°C for Penicillium by the pour plate culture method, and the number of live bacteria on the culture medium was counted, and the ratio [%] of the number of live bacteria when the number of live bacteria in the comparative example was 100% was calculated as the live bacteria rate. It should be noted that, in addition, the plate culture period was 2 days for Escherichia coli and Staphylococcus aureus and 7 days for Penicillium. The results obtained by graphing the viable cell rates calculated for each compound for Escherichia coli are shown in the following table: Fig. 12A As shown, for Staphylococcus aureus the results are as follows Fig. 12B For Penicillium the results are as follows Fig. 12C shown.

[0141] For E. coli, Fig. 12A As shown in FIG. 1 , in the sample to which the compound of the present invention was added, the viable bacterial rate was approximately 0% 15 minutes after the addition, and the viable bacterial rate in the comparative example (copper nitrate II) did not drop to 0%. Fig. 12B As shown in Figure 1, the inactivation effect of the compound of the present invention is high, and the viable bacteria rate decreases rapidly after addition even at extremely low concentrations. On the other hand, in the comparative example (copper nitrate II), the viable bacteria rate did not decrease after 5 minutes from the addition. For Penicillium, Fig. 12C As shown, in the samples to which the compound of the present invention was added, a tendency was observed that the viable cell rate decreased with the passage of time from the addition, while in the comparative example (copper nitrate II), the viable cell rate increased after 5 minutes from the addition.

[0142] <<Determination of Minimum Inhibitory Concentration (MIC) against Bacteria (Escherichia coli)>>

[0143] For the oxoammonium form of AZADO as a compound of the present invention (the counter anion is NO3 - ION;AZADO + NO3 - ), the minimum inhibitory concentration (MIC) against Escherichia coli was determined by the following method.

[0144] First, as a pre-culture, Escherichia coli (DH5α strain; AMP+) was shake-cultured in LB medium for 20 hours, and the obtained culture solution was used as the Escherichia coli stock solution. Then, the Escherichia coli stock solution was diluted 1000 times using PBS to obtain a dilution. It should be noted that the dilution was used as a comparative example.

[0145] On the other hand, AZADO was added to the diluted solution obtained above so that the final concentration was 1 μM, 10 μM, 100 μM, 1 mM or 10 mM. + NO3 - , and let stand at room temperature for 15 minutes. + NO3 - For the purpose of inactivation, LB medium was used as a reaction stop solution and the diluent was further diluted 10 times. Finally, 25 μL of the reaction solution thus obtained was applied to LB agar medium and cultured at 37°C for 1 day. In addition, the number of colonies on the agar medium after culture was counted, and the colony forming unit (CFU / mL) per 1 mL was calculated. The results are shown in Table 3 below. It should be noted that the "CFU relative value" described in Table 3 is a relative value when the value of the colony forming unit of the comparative example is taken as 100. In addition, AZADO was used. + NO3 - The photo of the agar medium after culturing the sample with a final concentration of 1 mM is shown in FIG. Fig.13 shown.

[0146] [Table 3]

[0147]

[0148] From the results shown in Table 3, it can be seen that in AZADO + NO3 - Significant inactivation effects were observed above the final concentration of 100 μM. + NO3 - When the final concentration is 1mM or more, a high sterilization effect of 99% or more is confirmed. It should be noted that the concentration of 1mM is the same as the concentration confirmed to exert a significant antiviral effect in the FCoV infection experiment on CRFK cells. Therefore, it is indicated that the sterilization concentration of the compound of the present invention for Escherichia coli is the same as the concentration of the antiviral effect.

[0149] <<Evaluation of the Degradability of Protein in a Misty State>>

[0150] In order to simulate the state in which nitroxyl radicals exert their antiviral and sterilizing effects on filters present in the living environment, the antiviral effect of a carbon material (AZADO-Cu(bpy) / DB) loaded with nitroxyl radicals (AZADO) and a co-catalyst (Cu) was evaluated using the decomposition of S-RBD as an indicator by the following method using a sprayer.

[0151] (Confirmation of reactivity in liquid phase system)

[0152] First, 100 pmol of S-RBD and 0.1 mg of AZADO-Cu(bpy) / DB were mixed in 110 μL of an aqueous solvent (S-RBD:Cu:AZADO=1:250:250 (molar ratio)). Then, the mixture was incubated at room temperature for 2 or 6 hours. It should be noted that an experiment using Cu(bpy) / DB without AZADO was performed as a comparative example.

[0153] Then, 1×SB was added to the residue obtained by centrifugation, the protein in the residue was sampled, and the amount of S-RBD contained in the residue was evaluated by SDS-PAGE and CBB staining. Fig.14 shown.

[0154] Depend on Fig.14 The results shown confirmed that the band of S-RBD disappeared in a manner dependent on the incubation time in the system to which AZADO-Cu(bpy) / DB was added. Based on the results, the following experiment using a nebulizer was performed.

[0155] (Confirmation of the reactivity of proteins in a mist state)

[0156] An antiviral filter was prepared by passing 100 μL of an aqueous solution containing 0.1 mg of AZADO-Cu(bpy) / DB through a membrane filter (8 mm pores). The filter was placed at the opening of a 200 μL microchip and fixed with a sealing film. It should be noted that the filter of the comparative example was prepared using Cu(bpy) / DB.

[0157] For the filter prepared as above, 25 μL of an aqueous solution containing 125 pmol or 250 pmol of S-RBD was sprayed from the tip of the microchip using a micro sprayer (Micro FPS, manufactured by TORAY PRECISION Co., Ltd.) and incubated at room temperature for 24 hours. Then, the amount of S-RBD remaining on the filter was evaluated by SDS-PAGE and silver staining. The results (electrophoresis photos) are shown in FIG. Fig.15 shown.

[0158] Depend on Fig.15 The results shown confirmed that, regardless of the amount of S-RBD sprayed, the S-RBD band disappeared or decreased in the filter containing AZADO-Cu(bpy) / DB.

[0159] This application is based on Japanese Patent Application No. 2022-136098 filed on August 29, 2022, and the disclosed contents are cited as a whole by reference.

Claims

1. A method for inactivating bacteria or viruses, comprising: bringing a compound having the following redox mechanism into contact with an object at risk of being contaminated by bacteria or viruses, [Chemical formula 1] Where, X - For counter anions, dashed lines indicate bonding sites to other atoms.

2. The inactivation method according to claim 1, wherein: The virus is inactivated by causing oxidative damage to the spike protein that makes up the virus.

3. The inactivation method according to claim 1, which is a method for inactivating bacteria. The bacteria are bacteria or fungi.

4. The inactivation method according to claim 1 or 2, wherein: The standard redox potential (25°C) between the nitroxyl radical form and the oxoammonium form is +100 mV to +1000 mV [Ag / Ag + ].

5. The inactivation method according to claim 1, wherein: The nitroxyl radical form of the compound is shown in the following compound formula 1: [Chemical formula 2] [Compound Formula 1] In the formula, R1 to R4, Y1 and Y2 are each independently a hydrogen atom or an optionally substituted monovalent organic group, wherein: Y1 and Y2 are optionally bonded to each other to form a nitrogen-containing monocyclic ring containing a 5-membered ring or a 6-membered ring which is optionally substituted, and R1 and R3 are optionally bonded to each other to form an optionally substituted 5-membered or 6-membered nitrogen-containing bicyclic ring, or two rings constituting the nitrogen-containing bicyclic ring are optionally further cross-linked to form an optionally substituted 5-membered or 6-membered nitrogen-containing tricyclic ring.

6. The inactivation method according to claim 5, wherein: The nitroxyl radical form of the compound is shown in the following compound formula 2: [Chemical formula 3] [Compound Formula 2] In the formula, R1 to R4 are independently hydrogen atoms or monovalent organic groups which are optionally substituted, Z is a nitrogen-containing monocyclic ring which is optionally substituted and contains 5-membered rings or 6-membered rings, wherein further optionally R1 and R3 are bonded to each other to form a nitrogen-containing bicyclic ring containing a 5-membered ring or a 6-membered ring which is optionally substituted, or The two rings constituting the nitrogen-containing bicyclic ring are further cross-linked to form a nitrogen-containing tricyclic ring containing a 5-membered ring or a 6-membered ring which is optionally substituted.

7. The inactivation method according to claim 5 or 6, wherein: When the compound does not contain the monocyclic ring, the bicyclic ring or the tricyclic ring, or contains the nitrogen-containing monocyclic ring, R1 to R4 are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, When the compound includes the nitrogen-containing bicyclic ring or the nitrogen-containing tricyclic ring, R2 and R4 are each independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.

8. The inactivation method according to claim 1 or 2, wherein: The nitroxyl radical form of the compound is one or more selected from the following compounds: [Chemical formula 4-1] [Chemical formula 4-2] 9. The inactivation method according to claim 8, wherein: The nitroxyl free radical form of the catalyst is 2-azaadamantane-N-oxyl (AZADO), 2,2,6,6-tetramethylpiperidinyl-N-oxyl (TEMPO) or 4-acetylamino TEMPO.

10. The inactivation method according to claim 1 or 2, wherein: The compound is contacted with the object in the presence of a co-catalyst comprising a transition metal.

11. The inactivation method according to claim 10, wherein: The transition metal includes one or more selected from Ag, Au, Pt, Pd, Ni, Mn, Fe, Ti, Al, Zn and Cu.

12. The inactivation method according to claim 1 or 2, wherein: The compound is brought into contact with the object by spraying a solution containing the compound onto the object.

13. A bacteria or virus inactivator, comprising: a compound having the following redox mechanism, [Chemical formula 5] Where, X - For counter anions, dashed lines indicate bonding sites to other atoms.

14. The inactivator according to claim 13, which is a bacteria inactivator, The bacteria are bacteria or fungi.

15. An antibacterial or antiviral substrate obtained by supporting or coating the inactivating agent according to claim 13 or 14 on a base substrate.

16. The antibacterial or antiviral substrate according to claim 15, wherein The base substrate is further supported or coated with a promoter comprising a transition metal.

17. The antibacterial or antiviral substrate according to claim 15 or 16, wherein The base substrate is a woven fabric, a nonwoven fabric, a filter or a urethane foam.

18. The antibacterial or antiviral substrate according to claim 17, wherein The antiviral activity value (Mv) against influenza virus or feline calicivirus measured in accordance with JIS L 1922:2016 (Test method for antiviral activity of textile products) is 2.0 or more.

19. The antibacterial or antiviral substrate according to claim 15 or 16, which is used for masks, medical fiber products, air conditioners, air purifiers, refrigerators, temperature control machines, dehumidifiers, humidifiers or filters for vacuum cleaners.

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