Inactivation method and inactivator for bacteria or viruses, and antiviral base material using same

Abstract

[Problem] To provide an inexpensive means by which an inactivation effect on microorganisms can be exhibited without the need for light to be present, while minimizing the burden on the environment. [Solution] A compound having the following redox mechanism is brought into contact with an object, which is likely to be contaminated due to bacteria or viruses, to inactivate the bacteria or viruses. In the formula, X- is a counter anion, and the broken line represents a bonding position to another atom.

Description

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

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

[0002] Recent globalization has increased the risk of viral infection outbreaks, and since 1990, there have been global outbreaks of infectious diseases such as avian influenza (A / H5N1) in 1997, SARS (SARS-CoV) in 2002, novel influenza (A / 4H1N1) in 2009, and MERS (MERS-CoV) in 2015. These outbreaks are far more frequent than the frequency of pandemics prior to 1990. Furthermore, the emergence of the novel coronavirus that caused the pandemic in 2019, COVID-19 (SARS-CoV-2), an acute respiratory infection, not only endangers human lives but also inflicts a major blow on the economy, posing a serious threat to the safety and security of human living environments.

[0003] Ventilation and the introduction of fresh air are recommended to remove viruses. However, because ventilation and the introduction of fresh air disrupt the temperature and humidity environment regulated by air conditioning, it is desirable to be able to quickly inactivate viruses through internal air circulation from the perspective of energy conservation. The use of antibacterial and antiviral filters and interior materials can be expected to inactivate bacteria and viruses through internal air circulation. Conventional techniques for imparting antibacterial and antiviral properties have been proposed in Japanese Patent No. 4584339 and Japanese Patent Laid-Open No. 2017-133137, which use metal ions to exert antibacterial and antiviral effects. Furthermore, inorganic oxide systems that function as photocatalysts have also been proposed.

[0004] The inorganic metal systems disclosed in Japanese Patent No. 4584339 and Japanese Patent Laid-Open No. 2017-133137 exert their effects through protein denaturation based on the sustained release of metal ions, but there are concerns about adverse environmental effects. Furthermore, silver and copper, which are considered to be particularly effective, are not only useful as precious metals but also expensive. Furthermore, sustained release of metal ions requires dissolution and diffusion of the metal ions, which poses the issue of time required for antibacterial and antiviral effects to be achieved. Furthermore, inorganic oxide systems (photocatalysts) do not function without light, so they must be used in situations where they are exposed to light such as sunlight, or a separate light irradiation device is required when used in dark places.

[0005] Therefore, an object of the present invention is to provide an inexpensive means that can exert an inactivating effect on microorganisms without requiring the presence of light, while minimizing the burden on the environment.

[0006] The present inventors have conducted extensive research to solve the above problems, and as a result have found that microorganisms can be inactivated by contacting a compound having a specific oxidation-reduction mechanism with an object that is at risk of microbial contamination, thereby completing the present invention.

[0007] That is, one aspect of the present invention is a method for inactivating bacteria or viruses, which comprises contacting a target suspected of being contaminated with bacteria or viruses with a compound having the following oxidation-reduction mechanism:

[0008]

[0009] In the formula, X － is a counter anion, and the dashed lines represent the bond positions to other atoms.

[0010] Figure 1 shows the standard redox potential (E ) between the nitroxyl radical form and the oxoammonium form for four compounds: (a) TEMPO, (b) AZADO, (c) 1-methyl AZADO, and (d) 1,3-dimethyl AZADO, measured by cyclic voltammetry. ０This figure shows cyclic voltammograms obtained by measuring the cyclic voltammograms (at 25°C). This figure shows graphs illustrating the results of evaluating the antiviral effect (cell viability) of serially diluted culture supernatants of CRFK cells infected with CoV, treated with AZADO-Oxo or silver nitrate, using the WST-8 method, in order to confirm the antiviral effect of AZADO. This figure shows microscopic photographs of wells of samples of culture supernatants of CRFK cells infected with CoV, treated with AZADO-Oxo, in order to confirm the antiviral effect of AZADO, along with photographs of cells to which no virus had been added (virus-free) and cells to which virus had been added but no AZADO-Oxo had been added (virus-present). This figure shows graphs illustrating the results of evaluating the antiviral effect (cell viability) of samples of culture supernatants of CRFK cells infected with CoV, treated with AZADO-Oxo, in order to confirm the antiviral effect of AZADO, using the WST-8 method. 5(a) and 5(b) show graphs showing the results of flow cytometry analysis of S-RBD-His (wild-type) or S-RBD-His (Omicron strain) mixed with different final concentrations of AZADO-Oxo to confirm whether AZADO-Oxo exhibits antiviral activity by inhibiting the binding of spike protein to ACE2 through oxidative denaturation of the spike protein. The results for the wild-type strain (S-RBD-His (WT)) are shown in FIG. 5(a), and the results for the Omicron strain (S-RBD-His (Omicron)) are shown in FIG. 5(b). To compare the antiviral effects of the compounds, AZADO-Oxo was used. ＋ BF ４ － , TEMPO ＋ BF ４ － and 4-AcNH-TEMPO ＋ BF ４ － 1 is a graph showing the results of flow cytometry evaluation of S-RBD-His (wild type) mixed with compounds at different final concentrations using AZADO. ＋ BF ４ － The results for TEMPO are shown in FIG. ＋ BF ４－ The results for 4-AcNH-TEMPO are shown in FIG. ＋ BF ４ － The results for (c) of Figure 6 are shown. In this specification, "AcNH", "acetylamino", "acetamide", and "NHAc" refer to the same functional group. This figure shows the results of graphing the mean fluorescence intensity at each peak shown in Figure 6. AZADO+BF showed high antiviral effects in the experiments shown in Figures 6 and 7. ４ － and 4-AcNH-TEMPO ＋ BF ４ － 1 is a graph showing the MFI obtained by performing the same procedure when S-RBD-His (wild-type) was added to AZADO / DB, AZADO-Cu(bpy) / DB, Cu(bpy) / DB, and Denka Black (DB) were treated with ovalbumin (OVA) using AZADO / DB, AZADO-Cu(bpy) / DB, Cu(bpy) / DB, and Denka Black (DB) prepared in the Examples. 2 is an electrophoretic photograph showing the results of CBB staining of the supernatant and residue of the sample prepared in the Examples where ovalbumin (OVA) was treated with AZADO / DB, AZADO-Cu(bpy) / DB, Cu(bpy) / DB, and Denka Black (DB). 11 shows the results of CBB staining of the supernatant of a sample in which ovalbumin (OVA) was treated with unloaded AZADO. FIG. 12 shows the results using AZADO-Oxo. ＋ NO ３ － , AZADO ＋ NO ３ － and 4-AcNH-TEMPO ＋ NO ３ － 2 is a graph showing the results of calculating the viability of a bacterial solution contacted with the compound (TEMPO) according to the present invention, which was cultured on a plate in order to evaluate the inactivation activity of the compound against Escherichia coli. ＋ NO ３－ , AZADO ＋ NO ３ － and 4-AcNH-TEMPO ＋ NO ３ － 1 is a graph showing the results of calculating the viability of a bacterial solution contacted with the compound (TEMPO) according to the present invention, which was cultured on a plate in order to evaluate the inactivation activity of the compound against Staphylococcus aureus. ＋ NO ３ － , AZADO ＋ NO ３ － and 4-AcNH-TEMPO ＋ NO ３ － 1 is a graph showing the results of calculating the viability of a bacterial solution contacted with the compound (AZADO) according to the present invention, which was cultured on a plate in order to evaluate the inactivation activity against Penicillium. ＋ NO ３ － ) when measuring the minimum inhibitory concentration (MIC) against Escherichia coli, AZADO ＋ NO ３ － 1 is a photograph of an agar medium after incubation with a sample having a final concentration of 1 mM. This is an electrophoretic photograph showing the results of reacting the antiviral substrate (AZADO-Cu(bpy) / DB) of the present invention with S-RBD in a liquid phase prior to evaluating the inactivation effect of the substrate on proteins in a mist state, and evaluating the amount of S-RBD after the reaction by SDS-PAGE and CBB staining. This is an electrophoretic photograph showing the results of spraying an aqueous solution containing S-RBD onto a filter containing the substrate and evaluating the amount of S-RBD remaining on the filter after incubation by SDS-PAGE and silver staining, for the purpose of evaluating the inactivation effect of the antiviral substrate (AZADO-Cu(bpy) / DB) of the present invention on proteins in a mist state.

[0011] Hereinafter, embodiments of the present invention will be described.

[0012] <<Method for Inactivating Bacteria or Viruses>> One aspect of the present invention is a method for inactivating bacteria or viruses, which comprises contacting a compound having the above-described oxidation-reduction mechanism (hereinafter also referred to as "the compound of this aspect") with an object that is at risk of bacterial or viral contamination. The present invention provides an inexpensive means that can exert an inactivating effect on microorganisms without requiring the presence of light, while minimizing the burden on the environment.

[0013] <Compound of the Present Form> The compound of the present form has been known as an "organic nitroxyl radical." Nitroxyl radicals are stable organic free radical molecules and are widely used as oxidation catalysts in the field of organic synthesis. In particular, organic nitroxyl radicals exhibit two-way reactivity, oxidation and reduction, and as shown in the above reaction formula, they undergo a one-electron reduction to produce hydroxyamine, and a one-electron oxidation to produce oxoammonium ions. The oxoammonium ions produced by the oxidation of nitroxyl radicals have strong oxidizing power and give hydroxyamine after two-electron oxidation of the substrate. The compound of the present form acts as an oxidizing agent on proteins of bacteria and viruses, oxidizing the proteins and reducing or eliminating their function. This exerts the effect of inactivating bacteria and viruses. Here, the mass transfer involved in the above oxidation-reduction mechanism is H, protons (H ＋ ), electron (e － ), which produces a faster reaction than the oxidation-reduction reactions of metal ions such as Ag and Cu that have been proposed in the past, thereby achieving more rapid inactivation of bacteria and viruses. Furthermore, the microorganism inactivation method according to this embodiment is characterized by being based on an oxidation reaction, and because bacteria and viruses are composed of proteins, it has the advantage of being able to inactivate bacteria and fungi (mold) regardless of the species, and even whether the virus is enveloped or not. In addition, in the oxidation reaction in which oxoammonium ions act as the active oxidizing species, a reactant that promotes the reoxidation of this hydroxylamine can be added to regenerate the oxoammonium ions, completing the cycle and allowing them to function like a catalyst.

[0014] The compound of this embodiment may have the above-described oxidation-reduction mechanism, and there is no limitation on its specific structure. Furthermore, the compound of this embodiment may be in the hydroxylamine form, the nitroxyl radical form, or the oxoammonium form in the above-described oxidation-reduction mechanism. Among these, the hydroxylamine form or the nitroxyl radical form is preferred, and the nitroxyl radical form is particularly preferred.

[0015] In addition, from the viewpoint of exhibiting excellent inactivation activity against microorganisms, the standard oxidation-reduction potential (25°C) between the nitroxyl radical form and the oxoammonium form of the compound of this form is preferably +100 mV to +1000 mV [Ag / Ag ＋ ], and more preferably +130 mV to +600 mV [Ag / Ag ＋ The standard oxidation-reduction potential is measured by cyclic voltammetry (CV) at a temperature of 25°C. In this case, a glassy carbon electrode (inner diameter 3 mm) is used as a working electrode, and a reference electrode (Ag / Ag ＋ The measurement is performed in a three-electrode system using a first electrode (anode) and a counter electrode (platinum wire), and the potential sweep rate is 50 mV / s. Note that the hydroxylamine form of the compound of the present invention is generally unstable and is rapidly oxidized to the nitroxyl radical form. Therefore, the standard redox potential measured by the above method is a value between the nitroxyl radical form and the oxoammonium form.

[0016] Here, for the four compounds of this embodiment, (a) TEMPO, (b) AZADO, (c) 1-methyl AZADO, and (d) 1,3-dimethyl AZADO, the standard oxidation-reduction potential (E ０ The cyclic voltammograms obtained by measuring the E of these compounds are shown in Figure 1. ０The values ​​of (d) 136 mV < (c) 186 mV < (b) 236 mV < (a) 294 mV, respectively. The cyclic voltammogram shown in Figure 1 remained stable even after 100 or more cycles of measurement, which indicates that the compound of this embodiment has extremely high durability as an oxidizing agent.

[0017] From the viewpoint of inactivating activity against microorganisms, the compound of this embodiment preferably has a nitroxyl radical form represented by the following chemical formula 1.

[0018]

[0019] In Chemical Formula 1, R １ ~R ４ , Y １ and Y ２ are each independently a hydrogen atom or an optionally substituted monovalent organic group. The monovalent organic group is not particularly limited, but examples thereof include a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a substituted or unsubstituted aryl group, and a substituted or unsubstituted heteroaryl group. １ ~R ４ , Y １ and Y ２ may be the same or different.

[0020] The alkyl group, alkenyl group, and alkynyl group as the monovalent organic group may each be linear, branched, or cyclic, but are preferably linear. The number of carbon atoms in the alkyl group is not particularly limited, but is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 4. The number of carbon atoms in the alkenyl group and alkynyl group is not particularly limited, but is preferably 2 to 20, more preferably 2 to 10, and even more preferably 2 to 4.

[0021] The aryl group as a monovalent organic group is a group derived from a hydrocarbon ring having aromaticity in part or as a whole. When the aryl group contains two or more hydrocarbon rings having aromaticity in part or as a whole, these rings may be bonded to each other by a single bond or fused together. Furthermore, when the aryl group contains two or more hydrocarbon rings having aromaticity in part or as a whole, one atom may serve as a ring-forming atom of any two of these rings to form a spiro ring. The number of carbon atoms in the aryl group is not particularly limited, but is preferably 6 to 30. The number of carbon atoms in the aryl group is more preferably 6 to 12, and even more preferably 6. In particular, the aryl group is preferably a monovalent group derived from an aromatic hydrocarbon ring having 6 or more ring-forming atoms.

[0022] The heteroaryl group as a monovalent organic group is a group derived from a heterocycle having aromaticity in part or as a whole. When the heteroaryl group contains two or more heterocycles having aromaticity in part or as a whole, some or all of these rings may be bonded to each other via single bonds. When the heteroaryl group contains two or more heterocycles having aromaticity in part or as a whole, or other rings, these rings may be fused to each other. Furthermore, when the heteroaryl group contains two or more heterocycles having aromaticity in part or as a whole, or other rings, one atom may also serve as a ring-forming atom of these rings. The heteroatom contained in the heteroaryl group is not particularly limited, and examples thereof include one or more heteroatoms (e.g., nitrogen atom (N), oxygen atom (O), phosphorus atom (P), sulfur atom (S), silicon atom (Si), selenium atom (Se), germanium atom (Ge)) as ring-forming atoms. The number of carbon atoms in the heteroaryl group is preferably 3 to 30. Furthermore, the number of ring-forming atoms in the heteroaryl group is not particularly limited, but is preferably 5 to 30. Furthermore, the number of ring atoms in the heteroaryl group is more preferably 5 to 14, and even more preferably 5 to 13. The number of heteroatoms in the heteroaryl group is not particularly limited, but is preferably 1 to 3. The number of heteroatoms in the heteroaryl group is more preferably 1 to 2, and even more preferably 1. In particular, the heteroaryl group is preferably a monovalent group derived from an aromatic heterocycle having 5 or more ring atoms.

[0023] The alkyl group, alkenyl group, alkynyl group, aryl group, and heteroaryl group as monovalent organic groups may each be a substituted group. The substituent is not particularly limited, but examples thereof include alkyl groups, alkoxy groups, alkenyl groups, alkynyl groups, halogen atoms, hydroxy groups, carboxy groups, cyano groups, amino groups, and carbamoyl groups. Among these, alkyl groups or halogen atoms are preferred as the substituent, and methyl groups, ethyl groups, or fluorine atoms are more preferred. Furthermore, methyl groups or fluorine atoms are even more preferred as the substituent. The above-mentioned substituents do not substitute the same type of group. For example, the substituents substituting alkyl groups do not include alkyl groups.

[0024] In addition, in Chemical Formula 1, from the viewpoint of excellent antimicrobial activity, Y １ and Y ２ are bonded to each other to form an optionally substituted nitrogen-containing monocyclo ring including a 5-membered or 6-membered ring, or further R １ and R ３ are bonded to each other to form a nitrogen-containing bicyclo ring containing a 5- or 6-membered ring which may be substituted, or two rings constituting the nitrogen-containing bicyclo ring are further crosslinked to form a nitrogen-containing tricyclo ring containing a 5- or 6-membered ring which may be substituted. That is, in a preferred embodiment of this aspect, the nitroxyl radical form of the compound of this aspect is represented by the following chemical formula 2.

[0025]

[0026] In Chemical Formula 2, R １ ~R ４ are each independently a hydrogen atom or an optionally substituted monovalent organic group, Z is an optionally substituted nitrogen-containing monocyclo ring containing a 5-membered or 6-membered ring, and in this case, R １ and R ３may be bonded to each other to form an optionally substituted nitrogen-containing bicyclo ring containing a 5- or 6-membered ring, or two rings constituting the nitrogen-containing bicyclo ring may be further bridged to form an optionally substituted nitrogen-containing tricyclo ring containing a 5- or 6-membered ring.

[0027] Examples of nitrogen-containing monocyclo rings as Z include aliphatic heterocycles such as a pyrrolidine ring, a piperidine ring, a morpholine ring, and a pyrroline ring, and aromatic heterocycles such as a pyrrole ring and an imidazole ring. １ and R ３ Examples of nitrogen-containing bicyclo rings formed by bonding together include a 9-azabicyclo[3.3.1]nonane ring and a 9-aza-3-oxabicyclo[3.3.1]nonane ring. Examples of nitrogen-containing tricyclo rings formed by further crosslinking two rings constituting the nitrogen-containing bicyclo ring include a 2-azaadamantane ring, a 2,6-diazaadamantane ring, a 6-aza-2-oxaadamantane ring, and a 9-aza-6-noradamantane ring. Among these, from the viewpoint of excellent inactivating activity against microorganisms, the compound of this embodiment preferably has a nitrogen-containing monocyclo ring, a nitrogen-containing bicyclo ring, or a nitrogen-containing tricyclo ring, more preferably a nitrogen-containing monocyclo ring or a nitrogen-containing tricyclo ring, and particularly preferably a nitrogen-containing tricyclo ring. Furthermore, the compound of this embodiment preferably has an aliphatic heterocycle which is a nitrogen-containing monocyclo ring or a 2-azaadamantane ring or a 9-aza-6-noradamantane ring which is a nitrogen-containing tricyclo ring, and more preferably has a piperidine ring which is a nitrogen-containing monocyclo ring or a 2-azaadamantane ring which is a nitrogen-containing tricyclo ring.

[0028] The nitrogen-containing monocyclo ring formed in Chemical Formula 2, or the nitrogen-containing bicyclo ring or nitrogen-containing tricyclo ring that can be formed in Chemical Formula 2, is an optionally substituted ring structure. Substituents that can substitute these ring structures are not particularly limited, and examples include alkyl groups, alkoxy groups, alkenyl groups, alkynyl groups, halogen atoms, hydroxy groups, carboxy groups, carbonyl groups, cyano groups, amino groups, carbamoyl groups, tosyl groups, (halogenated) acylamino groups, and phosphate groups. Among these, methyl groups, ethyl groups, methoxy groups, ethoxy groups, fluorine atoms, hydroxy groups, carboxy groups, and tosyl groups are more preferred as the substituent.

[0029] Here, when the compound of this embodiment does not contain the above ring structure or contains a nitrogen-containing monocyclo ring, R １ ~R ４ are each independently preferably a hydrogen atom or an optionally substituted alkyl group, more preferably a hydrogen atom or an unsubstituted alkyl group, even more 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. From the viewpoint of antimicrobial activity, when the compound of this embodiment contains the above-mentioned nitrogen-containing bicyclo ring or nitrogen-containing tricyclo ring, R ２ and R ４ are each independently preferably a hydrogen atom or an optionally substituted alkyl group, more preferably a hydrogen atom or an unsubstituted alkyl group, even more 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.

[0030] Examples of compounds of this type are as follows (shown in nitroxyl radical form): Note that for some compounds, the standard oxidation-reduction potential (E ０ In view of the mechanism by which the compounds according to the present invention exhibit excellent antimicrobial activity, it can be said that all of the compounds shown below exhibit high antimicrobial activity.

[0031]

[0032]

[0033]

[0034] The above-mentioned E ０ In view of the value of , when a heteroatom is introduced into the structure of TEMPO, ABNO, AZADO, etc. that constitutes the skeleton of the compound, the redox characteristics of the compound in this form shift to the higher potential side. From this, it is thought that the oxidizing power as the potential of the corresponding oxoammonium form increases.

[0035] Among the above compounds, from the viewpoint of antimicrobial activity, preferred are TEMPO, 4-hydroxy TEMPO, 4-methoxy TEMPO, 4-acetylamino TEMPO, 4-oxo TEMPO, 4-carboxy TEMPO, 4-amino TEMPO, 4-phosphono TEMPO, 4-(2-bromoacetamido) TEMPO, AZADO, 1-methyl AZADO, 1,3-dimethyl AZADO, 1-fluoro AZADO, 5-fluoro AZADO, and 5,7-difluoro AZADO. 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, 5-fluoro AZADO Preferred are 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, Nor-AZADO, TEMPO, and 4-acetylamino TEMPO, and more preferred are AZADO, 1-methyl AZADO, 1,3-dimethyl AZADO, 1-fluoro AZADO, 5-fluoro AZADO, 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, and particularly preferred are 2-azaadamantane-N-oxyl (AZADO), 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), or 4-acetylaminoTEMPO.

[0036] <Co-catalyst> From the viewpoint of antimicrobial activity, the inactivation method according to this embodiment preferably involves contacting the compound of this embodiment with a target in the presence of a co-catalyst containing a transition metal. Here, the co-catalyst of this embodiment contains a transition metal ion (i.e., a salt containing a transition metal) or a simple transition metal. The transition metal that can be contained in the co-catalyst of this embodiment is not particularly limited as long as it is a metal element classified as an element of Groups 3 to 12 of the periodic table. In particular, from the viewpoint of excellent antimicrobial activity, the transition metal preferably contains one or more selected from the group consisting of Ag, Au, Pt, Pd, Ni, Mn, Fe, Ti, Al, Zn, and Cu, and more preferably contains these ions (salts). Furthermore, the transition metal more preferably contains Ag or Cu, particularly preferably contains Ag or Cu ions (salts), and most preferably contains Ag ions or divalent Cu ions (Cu(II) ions).

[0037] When the co-catalyst contains a transition metal ion (salt), the counter anion of the ion is not particularly limited. Examples of the salt containing a transition metal as the co-catalyst include sulfate, sulfite, hyposulfite, persulfate, thiosulfate, carbonate, phosphate, pyrophosphate, hydrochloride, nitrate, nitrite, halide (e.g., fluoride, chloride, bromide, iodide), and trifluoromethanesulfonate.

[0038] In the inactivation method of this embodiment, when a co-catalyst is also present, there are no particular restrictions on the amounts of the compound and the transition metal used. However, from the viewpoint of achieving excellent microbial inactivation activity, the molar ratio of the amounts of these components used is preferably 1:2 to 4:1 (compound:transition metal), and more preferably 2:3 to 3:2.

[0039] <Subject> The method for inactivating bacteria or viruses according to this embodiment includes contacting the compound of this embodiment with an object that is suspected of being contaminated by bacteria or viruses. That is, according to another embodiment of the present invention, a bacterial or viral inactivator comprising the compound is also provided. The compound or inactivator of this embodiment can be used to inactivate microorganisms by contacting it with an object that is suspected of being contaminated by bacteria or viruses, such as a kitchen (kitchen space such as kitchen floors and walls), food handling areas such as sinks and refrigerators, cooking utensils such as knives and pots, tableware such as plates, living spaces such as refrigerators, toilets, baths, washrooms, living rooms, and bedrooms, and textile products such as sofas, cushions, bedding, and curtains. Here, the bacteria or viruses to be inactivated are not particularly limited, and the compound exhibits an inactivating effect against various bacteria, fungi, and viruses. Examples of bacteria include gram-negative bacteria such as Escherichia genus such as Escherichia coli; Pseudomonas genus such as Pseudomonas aeruginosa; Salmonella genus such as Salmonella cholerae suis; Moraxella genus such as Moraxella catarrhalis; Legionella genus such as Legionella pneumophila; Staphylococcus genus such as Staphylococcus aureus; and gram-positive bacteria such as Clostridium genus such as Clostridium botulinum and Clostridium perfringens. Examples of molds include the genus Aspergillus, such as Aspergillus niger and Aspergillus penicilloides; the genus Paecilomyces, such as Paecilomyces variotii; the genus Penicillium, such as Penicillium pinophyllum and Penicillium citrinum; the genus Trichoderma, such as Trichoderma virens; the genus Chaetomium, such as Chaetomium globosum; the genus Eurotium, such as Eurotium tonohilum; the genus Rhizopus, such as Rhizopus oryzae; the genus Cladosporium, such as Cladosporium cladosporioides; the genus Aureobasidium, such as Aureobasidium pullulans; and the genus Myrothecium, such as Myrothecium verrucaria. Examples of fungi other than molds include the genus Saccharomyces, such as Saccharomyces cerevisiae; the genus Candida, such as Candida albicans; and the genus Rhodotorula, such as Rhodotorula rubra. Furthermore, the virus may be an enveloped virus or a non-enveloped virus.Examples of enveloped viruses include coronaviruses (including SARS-CoV-2), influenza viruses, herpes viruses, rubella viruses, etc. Examples of non-enveloped viruses include noroviruses and rotaviruses. The target viruses are preferably enveloped viruses, more preferably coronaviruses, and particularly preferably SARS-CoV-2. In particular, the inactivation method according to this embodiment is preferably one that inactivates a virus by inflicting oxidative damage on the spike protein that constitutes the virus, in view of being able to effectively inhibit virus entry into human cells. In another preferred embodiment, the target of inactivation is a fungus, and examples of the fungus include bacteria and fungi.

[0040] The contact is not particularly limited, and examples include spraying the compound of the present invention or a composition containing the compound (e.g., a liquid composition) directly onto the target, or spraying the compound by filling a spray device such as a trigger or aerosol sprayer. This configuration allows the compound of the present invention to be distributed over a wide area. The contact time between the compound of the present invention and the target is not particularly limited, but is preferably at least 5 seconds, more preferably at least 1 minute, and even more preferably at least 30 minutes. The upper limit of the contact time is also not particularly limited, but is preferably 24 hours or less, more preferably 12 hours or less, and even more preferably 6 hours or less. The form of use for contacting the compound of the present invention with the target is also not particularly limited. For example, the compound can be in liquid, gel, or paste form. When the contact is performed in a solution, it may be possible to monitor the progress of the oxidation reaction (e.g., protein denaturation) from the color change of the solution.

[0041] When in liquid form, it can be used particularly 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 pressure accumulation type) or a dispenser-type pump spray container, and the spray amount can be adjusted so that it can be sprayed onto a location where bacteria or viruses are present or thought to be present.

[0042]

[0023] According to yet another aspect of the present invention, there is also provided an antibacterial or antiviral substrate comprising a base substrate supported or coated with the inactivator according to one aspect of the present invention described above and, if necessary, a co-catalyst. By using such an antimicrobial substrate, the inactivator can be easily used for various purposes.

[0043] Examples of the base substrate constituting the antimicrobial substrate include inexpensive materials that can easily support or coat an inactivating agent, such as woven fabrics, nonwoven fabrics, filters, and urethane foams. There are no particular limitations on the method for supporting or coating these base substrates with the compound and, if necessary, the co-catalyst. Here, for the substrate according to this embodiment, as an indicator of excellent antimicrobial activity, the antiviral activity value (Mv) against influenza virus or feline calicivirus measured according to JIS L 1922:2016 (Testing Method for Antiviral Activity of Textile Products) is preferably 2.0 or higher, more preferably 2.2 or higher, and even more preferably 2.5 or higher. Such antimicrobial substrates can be suitably used for applications such as masks, medical textiles, air conditioners, air purifiers, freezers, temperature controllers, dehumidifiers, humidifiers, and suction vacuum cleaner filters. Of course, they may also be used for other applications.

[0044] The following embodiments are also included within the scope of the present invention: an inactivation method according to claim 1 having the features of claim 2; an inactivation method according to claim 1 having the features of claim 3; an inactivation method according to any one of claims 1 to 3 having the features of claim 4; an inactivation method according to any one of claims 1 to 4 having the features of claim 5; an inactivation method according to any one of claims 1 to 5 having the features of claim 6; an inactivation method according to claim 5 or 6 having the features of claim 7; an inactivation method according to any one of claims 1 to 7 having the features of claim 8; an inactivation method according to 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 characteristic of claim 0; an inactivation method according to claim 10 having the characteristic of claim 11; an inactivation method according to any one of claims 1 to 11 having the characteristic of claim 12; an inactivation agent according to claim 13 having the characteristic of claim 14; a substrate according to claim 15 using an inactivation agent according to claim 13 or 14; a substrate according to claim 15 having the characteristic of claim 16; a substrate according to claim 15 or 16 having the characteristic of claim 17; a substrate according to any one of claims 15 to 17 having the characteristic of claim 18; a substrate according to any one of claims 15 to 18 having the characteristic of claim 19.

[0045] Hereinafter, the embodiments of the present invention will be described in detail using examples, but the technical scope of the present invention is not limited to the following examples.

[0046] <<Investigation of Virus Dilution Rate>> First, conditions for the virus dilution rate were investigated in order to investigate the effect of feline coronavirus (FCov (VR-989)) on the viability of feline kidney-derived immortalized cells (CRFK).

[0047] Specifically, CRFK cells infected with FCoV were cultured for 3 days, and the culture supernatant was diluted 20-fold with PBS to prepare a stock solution of FCoV. １０ Serial dilutions were carried out up to 2 times dilution to prepare FCoV solutions at each dilution factor.

[0048] On the other hand, 1 × 10 CRFK cells were suspended in 10% (v / v) FBS-containing DMEM medium. ４The cells were seeded into a 96-well plate at 100 μL / well to provide 10 cells / well and cultured for 4 days. After 4 days of culture, the medium was replaced with 100 μL / well of 2% (v / v) FBS-containing DMEM medium, and the FCoV solution prepared above at each dilution was added at 100 μL / well, followed by further culture for 24 hours. The medium was then replaced with 200 μL / well of 2% (v / v) FBS-containing DMEM medium, and the cells were cultured for another 2 or 3 days. The cells were then evaluated for cytopathic effect (CPE). For cytopathic effect (CPE), each well was observed using an optical microscope, and wells in which 50% or more of the cells had degenerated were evaluated as CPE-positive. As a result of this CPE evaluation, after both 2 and 3 days of culture, 10 ５ A dilution-dependent change in cell viability was observed up to 2x dilution. Based on this result, the amount of virus to be added in the following experiments was determined.

[0049] <Confirmation of Antiviral Effect of AZADO (WST-8 Method)> In order to confirm whether AZADO, a type of nitroxyl radical, exhibits antiviral effect, the following experiment was carried out and the antiviral effect was evaluated by the WST-8 method.

[0050] Specifically, CRFK cells infected with FCoV were cultured for 3 days, and the culture supernatant was diluted 20-fold with PBS to prepare a stock FCoV solution. This stock solution was then serially diluted 2-fold with PBS up to 160-fold to prepare FCoV solutions at each dilution. Then, AZADO oxoammonium form (counter anion: nitrate ion; AZADO-Oxo) or silver nitrate (comparison) was added to each serially diluted FCoV solution to a final concentration of 100 mM, and the mixture was incubated at room temperature for 2 hours to prepare a sample solution.

[0051] On the other hand, 1 × 10 CRFK cells were suspended in 10% (v / v) FBS-containing DMEM medium. ４The cells were seeded into a 96-well plate at 100 μL / well to achieve a cell / well ratio and cultured for 4 days. After 4 days of culture, the medium was replaced with 2% (v / v) FBS-containing DMEM medium at 100 μL / well, and each sample solution was added at 100 μL / well. Cultures were then cultured for 2 days. The cell viability of each sample was then measured using the WST-8 assay. The WST-8 assay assesses the presence of viable cells by utilizing the reduction of a certain tetrazolium salt (WST-8) by intracellular electron carriers to produce a highly water-soluble orange formazan dye. The results are shown in Figure 2. As shown in Figure 2, the addition of AZADO-Oxo restored the cell viability that had decreased with virus addition. In contrast, this phenomenon was not observed in the comparative sample (silver nitrate addition). These results demonstrate that AZADO-Oxo exerts excellent antiviral effects.

[0052] <Confirmation of Antiviral Effect of AZADO (CPE Method; Incubation at 37°C)> The following experiment was carried out to confirm whether AZADO, a type of nitroxyl radical, exhibits antiviral effect, and the antiviral effect was evaluated by the CPE method.

[0053] Specifically, CRFK cells infected with FCoV were cultured for 3 days, and the culture supernatant was diluted 20-fold with PBS to prepare a stock solution of FCoV. ５ Serial dilutions were performed up to 1:1000 dilutions to prepare FCoV solutions at each dilution ratio. AZADO-Oxo was then added to each serially diluted FCoV solution to a final concentration of 2 mM, and the solution was incubated at 37°C for 2 hours. The solution was then further diluted 20-fold with PBS to prepare a sample solution. A comparative sample solution was also prepared without adding AZADO-Oxo.

[0054] On the other hand, 1 × 10 CRFK cells were suspended in 10% (v / v) FBS-containing DMEM medium. ４The cells were seeded into a 96-well plate at 100 μL / well so that there were 10 cells / well, and cultured for 4 days. After 4 days of culture, the medium was replaced with 100 μL / well of DMEM medium containing 2% (v / v) FBS, and each sample solution prepared above was added at 100 μL / well, followed by further culture for 24 hours. Next, the medium was replaced with 200 μL / well of DMEM medium containing 2% (v / v) FBS, and further cultured for 2 days. After this culture, the cells were evaluated for cytopathic effect (CPE) using the same method as above. The results are shown in Table 1 below. Note that the "TCID" listed in Table 1 indicates the percentage of cells that were cultured. ５０ " is an abbreviation for 50% Tissue Culture Infectious Dose, which is the amount of virus that kills 50% of host cells due to viral infection.

[0055]

[0056] As shown in Table 1, it was confirmed that AZADO-Oxo exhibited excellent antiviral activity. In addition, the ΔlogTCID ５０ The value of 2.8 (= 5.2 - 2.4) was obtained, which means that AZADO-Oxo reduced the viral load to 1 / 10. ２．８ (= approximately 1 / 1000). Therefore, it was confirmed that AZADO-Oxo exhibited sufficient antiviral activity.

[0057] <Confirmation of the antiviral effect of AZADO (CPE method; incubation at 25°C)> The antiviral effect was evaluated by the CPE method using the same method as above, except that the incubation temperature was changed from 37°C to 25°C and the number of days of incubation after addition of the sample solution and medium change was changed from 2 days to 3 days in order to conform to the conditions of JIS L 1922:2016 (Testing methods for antiviral activity of textile products). The results are shown in Table 2 below.

[0058]

[0059] The results shown in Table 2 confirm that AZADO-Oxo exhibits sufficient antiviral activity even in this experimental system where incubation was carried out at 25°C.

[0060] Furthermore, in this experiment, wells were observed under a microscope 48 hours after the addition of sample solutions with or without AZADO-Oxo (virus + AZADO-Oxo) or without (virus present). Photographs of wells were taken together with photographs of cells without virus (no virus) and are shown in Figure 3. As shown in Figure 3, the addition of virus caused the cells to change morphology to a round shape, but this morphological change was suppressed by the addition of AZADO-Oxo, further demonstrating the antiviral effect of AZADO-Oxo.

[0061] Furthermore, in this experiment, the cells were cultured for three days after the addition of the sample solution and the medium change, and then the cell viability was measured using the WST-8 method. The results are shown in Figure 4. As shown in Figure 4, the addition of virus reduced the cell viability to approximately 50% (FCoV), but the addition of AZADO-Oxo (FCoV + AZADO-Oxo) restored the cell viability to the same level as the blank (non-virus) control.

[0062] <Confirmation of the antiviral effect of AZADO against SARS-CoV-2> This experiment examined whether AZADO-Oxo exhibits antiviral activity against the currently circulating SARS-CoV-2. SARS-CoV-2 enters human cells by binding the spike protein present on the surface of the virus particle to ACE2, an infection receptor on the surface of human cells. Therefore, we examined whether AZADO-Oxo exhibits antiviral activity by denaturing the spike protein through oxidation, thereby inhibiting the binding of the spike protein to ACE2. The viral spike proteins (two types, wild-type and Omicron strain), cells, and fluorescently labeled antibodies for detection used in this experiment are as follows: Spike protein (wild-type): SARS-CoV-2 Spike Protein (RBD) (aa319-541), His Tag Recombinant Protein (Thermo, RP-87678) (hereinafter referred to as "S-RBD-His (WT)") Spike protein (Omicron strain): SARS-CoV-2 B. 1.1.529 (Omicron) Spike RBD Protein (His Tag) (Sino Biological, 40592-V08H121) (hereinafter referred to as "S-RBD-His (Omicron)") Cells: ACE2-expressing HEK293T (hereinafter referred to as "ACE2-HEK293T") Fluorescently labeled antibody: 6x-His Tag Monoclonal Antibody (AD1.1.10), FITC (Thermo, MA1-81891) (hereinafter referred to as "FITC-anti-6xHis-Tag antibody").

[0063] In this experiment, first, 1 μg of S-RBD-His (WT) or S-RBD-His (Omicron) (2 μL of a 0.5 mg / mL aqueous solution for WT, and 4 μL of a 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, and the mixture was oxidized at 25°C for 30 minutes to prepare a sample solution. A comparative sample solution was also prepared without adding AZADO-Oxo. Next, 5 × 10 ACE2-HEK293T cells were added to a microtube containing 100 μL of PBS containing 1% BSA. ５ Then, 4 μL of the sample solution prepared above was added, and the mixture was allowed to stand at 4°C for 30 minutes to allow a binding reaction between the spike protein and the ACE2 receptor on the surface of the ACE2-HEK293T cells.

[0064] To the cell suspension after the reaction, 800 μL of 1% BSA-containing PBS was added and centrifuged at 400 × g for 4 minutes at 4°C. After discarding the supernatant, 100 μL of 1% BSA-containing PBS was added and the cells were resuspended. Then, 1 μg (10 μL) of FITC-anti-6xHis-Tag antibody was added and the cells were left standing at 4°C for 30 minutes in the dark. An additional 1 mL of 1% BSA-containing PBS was then added and the cells were centrifuged at 400 × g for 4 minutes at 4°C. After discarding the supernatant, 300 μL of 1% BSA-containing PBS was added and the cells were resuspended. The resulting solution was evaluated by flow cytometry. When this experiment was performed using HEK293T cells that do not express ACE2, the amount of S-RBD-His binding was significantly reduced, indicating that ACE2-specific binding had been detected. The results for the wild-type strain (S-RBD-His(WT)) are shown in Figure 5(a), and the results for the Omicron strain (S-RBD-His(Omicron)) are shown in Figure 5(b). Here, the lower the binding between the spike protein and ACE2 (i.e., the more their binding is inhibited by AZADO-Oxo), the more the peak shifts to the side with lower fluorescence intensity (to the left of the figure).

[0065] The results shown in Figure 5 indicate that in the absence of AZADO-Oxo (S), the peak shifted significantly to the right (indicating a high level of binding to ACE2). Furthermore, it was confirmed that this right-shifted peak shifted to the left depending on the amount of AZADO-Oxo added (the greater the amount added, the greater the left shift). Although the left shift of the peak with the addition of AZADO-Oxo was more gradual in the Omicron strain than in the wild-type strain, it was determined that a certain level of inactivation effect was also achieved in the Omicron strain.

[0066] Comparison of antiviral effects between compounds against SARS-CoV-2: In order to compare the antiviral effects between compounds, the oxoammonium form of AZADO (counter anion is BF ４ － ION; AZADO ＋ BF ４ － ), TEMPO oxoammonium form (counter anion is BF ４ － Ion; TEMPO ＋ BF ４ － ) and the oxoammonium form of 4-acetylamino TEMPO (counter anion is BF ４ － Ion: 4-AcNH-TEMPO ＋ BF ４ － ) and the antiviral effect was confirmed by the same method as above.

[0067] Specifically, 1 μg of S-RBD-His (WT) (2 μL of an aqueous solution with a concentration of 0.5 mg / mL) was first added to a final concentration of 10 μM, 100 μM, or 1000 μM of AZADO. ＋ BF ４ － , TEMPO ＋ BF ４ － or 4-AcNH-TEMPO ＋ BF ４ －The mixture was mixed with the above and subjected to oxidation treatment at 25°C for 30 minutes to prepare a sample solution. A comparative sample solution was also prepared without adding the above compound. Next, 1 x 10 ACE2-HEK293T was added to a microtube containing 100 µL of 1% BSA-containing PBS (containing 2 mM EDTA). ６ Then, 4 μL of the sample solution prepared above was added, and the mixture was allowed to stand at 4°C for 30 minutes to allow a binding reaction between the spike protein and the ACE2 receptor on the surface of the ACE2-HEK293T cells.

[0068] To the cell suspension after the reaction, 800 μL of 1% BSA-containing PBS (containing 2 mM EDTA) was added and centrifuged at 400 × g for 4 minutes at 4 °C. After discarding the supernatant, 100 μL of 1% BSA-containing PBS (containing 2 mM EDTA) was added and resuspended. Then, 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 1% BSA-containing PBS (containing 2 mM EDTA) was added and centrifuged at 4 °C for 4 minutes at 400 × g. After discarding the supernatant, 300 μL of 1% BSA-containing PBS (containing 2 mM EDTA) was added and the mixture was resuspended. The solution thus obtained was evaluated by flow cytometry. AZADO ＋ BF ４ － The results for TEMPO (AZADO-Oxo) are shown in Figure 6(a) (S is spike protein, A is compound). ＋ BF ４ － The results for 4-AcNH-TEMPO (TEMPO-Oxo) are shown in Figure 6(b) (S is spike protein, T is compound). ＋ BF ４ － The results (Ac-TEMPO-Oxo) are shown in Figure 6(c) (S is the spike protein, and AcT is the compound). Figure 7 shows a graph of the mean fluorescence intensity at each peak shown in Figure 6.

[0069] From the results shown in Figure 6, in the absence of AZADO-Oxo (S), the peak shifted significantly to the right. Furthermore, this right-shifted peak shifted to the left depending on the amount of compound added. When comparing the activities of the compounds, AZADO ＋ BF ４ － and 4-AcNH-TEMPO ＋ BF ４ － The activity of TEMPO is almost as strong as that of TEMPO. ＋ BF ４ － The activity of was slightly weaker than this. The MFI graph in Figure 7 also shows similar results.

[0070] Furthermore, AZADO, which showed a high antiviral effect in the above experiment, ＋ BF ４ － and 4-AcNH-TEMPO ＋ BF ４ － The same procedure was performed on S-RBD-His (WT) (final concentration 10 μM) except that the treatment time was extended from 30 minutes to 6 hours. The MFI graph obtained in this manner is shown in Figure 8. As shown in Figure 8, by extending the treatment time, AZADO ＋ BF ４ － and 4-AcNH-TEMPO ＋ BF ４ － showed higher antiviral activity.

[0071] The results shown above demonstrate that the present invention can provide an inexpensive means for exerting an inactivation effect on microorganisms without requiring the presence of light, while minimizing the burden on the environment.

[0072] <<Evaluation of AZADO Decomposition Activity Against Ovalbumin (OVA)>> (Preparation Example of Catalyst Support (AZADO / DB)) A catalyst support in which AZADO was supported on a catalyst support (carbon support) was prepared by the following method.

[0073] 5 mg (0.13 mmol) of AZADO and 100 mL of dichloromethane were added to a 300 mL eggplant-shaped flask, and the AZADO was dissolved using ultrasonic waves. 100 mg of a carbon support, Denka Black (registered trademark) (DB), was added, and the mixture was dispersed using ultrasonic waves for 30 minutes, followed by stirring at room temperature for 30 minutes. The mixture was then evaporated to dryness using a rotary evaporator at 40°C and 600 hPa, and further dried in vacuum for 10 hours to obtain a catalyst support (AZADO / DB).

[0074] (Preparation Example of Catalyst Support (AZADO-Cu(bpy) / DB)) Cu(OTf) ２ 0.03 mmol (10.9 mg), 0.03 mmol (4.7 mg) of bpy, and 0.03 mmol (4.6 mg) of AZADO were each added to a 5 mL vial, dissolved in 3 mL of acetonitrile, and added to a 300 mL eggplant-shaped flask containing 90 mL of acetonitrile. After adding each reagent, the vial walls were washed with acetonitrile and added to the flask. Next, 100 mg of Denka Black (DB), a carbon support, was added to the flask, dispersed ultrasonically for 30 minutes, and then stirred at room temperature for 30 minutes. The mixture was then evaporated to dryness using a rotary evaporator at 60 °C and 250 hPa, and further dried in vacuo for 10 hours to obtain a catalyst support (AZADO-Cu(bpy) / DB) in which both AZADO and the cocatalyst were supported. A catalyst support (Cu(bpy) / DB) in which only the cocatalyst was supported was also prepared by performing the same method without AZADO.

[0075] (Treatment of ovalbumin (OVA) with catalyst carrier (1)) 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 samples. Similarly, a sample was prepared using 3 μL of an aqueous solution of AZADO-Oxo (concentration: 100 mM) instead of the catalyst carrier (the amount of pure water was 977 μL). A control sample was also prepared by mixing only OVA and pure water.

[0076] Each sample was allowed to stand at room temperature for 24 hours and then centrifuged at 15,000 × g for 3 minutes at room temperature. Next, 80 μL of the supernatant from each sample was transferred to a separate tube, and 20 μL of 5x sample buffer (SB) was added. The samples were then allowed to stand at 95°C for 5 minutes, subjected to electrophoresis with molecular weight markers, and then stained with CBB. The results are shown in Figure 9. As shown in Figure 9, the OVA band was almost completely eliminated in the sample treated with AZADO-Oxo ("OVA + AZADO-Oxo" in Figure 9). This confirmed that the antiviral activity of AZADO-Oxo confirmed above is due to its action on the viral spike protein, which is then oxidized and degraded. Furthermore, in a sample in which OVA was treated with AZADO supported on a catalyst carrier together with a co-catalyst ("OVA + AZADO-Cu(bpy) / DB" in Figure 9), the OVA band completely disappeared, demonstrating superior OVA decomposition activity compared to AZADO-Oxo. This is thought to be the result of a catalytic cycle of oxidation-reduction reactions occurring due to the cooperative action of AZADO and the co-catalyst, resulting in stronger OVA oxidation. Note that in Figure 9, the OVA band barely disappeared in the sample to which a carbon carrier (DB) was added (OVA + DB) and the sample to which Cu(bpy) / DB was added (OVA + Cu(bpy) / DB), and a band equivalent to that of the control (OVA) was confirmed.

[0077] (Treatment of ovalbumin (OVA) with catalyst carrier (2)) Each sample was prepared in the same manner as above, except that the amount of AZADO-Oxo aqueous solution (concentration: 100 mM) added was 2.5 μL.

[0078] Each sample was allowed to stand at room temperature for 18 hours and then centrifuged at 15,000 × g for 3 minutes at room temperature. Next, 80 μL of the supernatant from each sample was transferred to a separate tube, and 20 μL of 5×SB was added. The tube was then allowed to stand at 95°C for 5 minutes to prepare a supernatant sample. For the sample containing catalyst support (C), the supernatant remaining after the centrifugation was discarded, washed twice with 1 mL of pure water, and resuspended in 80 μL of pure water. 20 μL of 5×SB was added, and the tube was allowed to stand at 95°C for 5 minutes to prepare a residue sample. The supernatant and residue samples thus obtained were subjected to electrophoresis with molecular weight markers and CBB staining. The results are shown in Figure 10. As shown in Figure 10, the supernatant sample showed the same results as Figure 9. Among the residue samples, a very strong OVA band was observed in the sample to which Cu(bpy) / DB had been added ("OVA + Cu(bpy) / DB" in Figure 10). From this, it is believed that Cu(bpy) / DB strongly adsorbed OVA. Furthermore, in a sample in which OVA was treated with AZADO supported on a catalyst carrier together with a co-catalyst ("OVA + AZADO-Cu(bpy) / DB" in Figure 10), it was confirmed that the OVA band adsorbed by Cu(bpy) / DB was significantly smeared (broadened). From this, it was suggested that the mechanism by which AZADO-Cu(bpy) / DB eliminates the OVA band (decomposes OVA) in Figures 9 and 10 (supernatant sample) is that Cu(bpy) / DB adsorbs OVA, thereby inducing oxidative degradation by AZADO. Based on this mechanism, it is believed that the microorganism inactivation method and inactivating agent according to one embodiment of the present invention exhibit high inactivation activity not only against viruses but also against bacteria, molds, fungi, and other organisms that contain proteins as essential components.

[0079] (Treatment of ovalbumin (OVA) with unsupported AZADO) The following samples were prepared: (a1) a sample of 1000 μL of pure water (None) (a2) a sample of 980 μL of pure water mixed with 20 μL of a 10 mg / mL aqueous solution of ovalbumin (OVA) (a3) ​​a sample of 980 μL of pure water mixed with 20 μL of a 10 mg / mL aqueous solution of ovalbumin and 2.5 μL of a 100 mM aqueous solution of AZADO-Oxo (OVA+AZADO-Oxo) (a4) a sample of 980 μL of pure water mixed with 20 μL of a 10 mg / mL aqueous solution of ovalbumin, 2.5 μL of a 100 mM aqueous solution of AZADO-Oxo, and 100 mM Cu(NO) ３ ) ２ A sample mixed with 2.5 μL of aqueous solution (OVA + AZADO-Oxo + Cu(NO ３ ) ２ (a5) 20 μL of 10 mg / mL egg albumin aqueous solution, 2.5 μL of 100 mM AZADO-Oxo aqueous solution, and 100 mM Cu(OTf) were added to 980 μL of pure water. ２ A sample mixed with 2.5 μL of aqueous solution (OVA + AZADO-Oxo + Cu(OTf) ２ ).

[0080] Each sample was allowed to stand at room temperature for 24 hours, and then centrifuged at 15,000 x g for 3 minutes at room temperature. Next, 80 µL of the supernatant from each sample was transferred to a separate tube, and 20 µL of 5xSB was added. The samples were then allowed to stand at 95°C for 5 minutes, and electrophoresis was performed together with molecular weight markers, followed by CBB staining. The results are shown in Figure 11. As shown in Figure 11, in the sample to which AZADO-Oxo was added to OVA, disappearance of the OVA band (proteolytic activity) was confirmed as above, but the promoter (Cu(NO ３ ) ２ and Cu(OTf) ２ This effect was not enhanced by the addition of

[0081] <Evaluation of inactivation activity against bacteria and fungi (molds)> The oxoammonium form of TEMPO (counter anion is NO), which is the compound according to the present invention, was used. ３ － Ion; TEMPO ＋ NO ３－ ), oxoammonium form of AZADO (counter anion is NO ３ － ION; AZADO ＋ NO ３ － ), and the oxoammonium form of 4-acetylamino TEMPO (counter anion is NO ３ － Ion: 4-AcNH-TEMPO ＋ NO ３ － The inactivation activity of each of these compounds against bacteria and fungi (mold) was evaluated. Copper (II) nitrate was used as a comparative example.

[0082] Specifically, Escherichia coli (ATCC 43895, serotype O157:H7, verotoxin type I and II-producing strain), Staphylococcus aureus (Staphylococcus aureus subsp. Aureus NBRC 12732), and Penicilliun citrinum (Penicilliun citrinum NBRC 6352) were prepared as inactivation targets. These targets were pre-cultured in nutrient agar medium (Eiken Chemical Co., Ltd.) at 35°C ± 1°C for 18 to 24 hours for E. coli and Staphylococcus aureus. Penicilliun citrinum was pre-cultured in potato dextrose agar (Difco) at 25°C ± 1°C for 7 to 10 days. The bacterial solutions were prepared using purified water for E. coli, saline for Staphylococcus aureus, and 0.005% dioctyl sodium sulfosuccinate solution for Penicilliun citrinum.

[0083] Next, 5 mL of an aqueous solution containing each of the above compounds was added with 1 × 10 ７ ~1 x 10 ８ 0.05 mL of a bacterial solution adjusted to a bacterial count of 1 / mL was added and gently stirred. The concentrations of the compounds in the aqueous solutions were 10 mM or 20 mM for the Escherichia coli test, 0.1 mM or 1 mM for the Staphylococcus aureus test, and 0.1 mM or 10 mM for the Penicillium test.

[0084] One minute, five minutes, and 15 minutes after the addition of the bacterial solution, the solution was sampled to an extent that the number of grown colonies could be counted, and the compound was neutralized by diluting with SCDLP medium (Shiotani MS Co., Ltd.). The neutralized samples were then spread on SCDLP agar medium (Shiotani MS Co., Ltd.) for E. coli and Staphylococcus aureus, and GPLP agar medium (Shiotani MS Co., Ltd.) for Penicillium spp. The plates were cultured by the pour plate culture method at 35°C ± 1°C for E. coli and Staphylococcus aureus, and at 25°C ± 1°C for Penicillium spp. The number of viable bacteria on the medium was counted, and the percentage of viable bacteria relative to the viable bacteria count in the comparative example (100%) was calculated as the viable bacteria rate. The plate culture period was 2 days for E. coli and Staphylococcus aureus, and 7 days for Penicillium spp. The viable cell rates calculated for each compound in this manner were graphed and the results are shown in FIG. 12A for Escherichia coli, FIG. 12B for Staphylococcus aureus, and FIG. 12C for Penicillium.

[0085] As shown in Figure 12A, in the case of Escherichia coli, the viability of the sample containing the compound of the present invention was nearly 0% 15 minutes after addition, whereas in the case of the comparative example (copper nitrate II), the viability did not decrease to 0%. As shown in Figure 12B, in the case of Staphylococcus aureus, the compound of the present invention had a high inactivating effect, and the viability decreased rapidly after addition even at very low concentrations. On the other hand, in the comparative example (copper nitrate II), the viability did not decrease 5 minutes after addition. As shown in Figure 12C, in the case of Penicillium, the viability of the sample containing the compound of the present invention tended to decrease over time after addition, whereas in the comparative example (copper nitrate II), the viability began to increase 5 minutes after addition.

[0086] <<Measurement of minimum inhibitory concentration (MIC) against bacteria (Escherichia coli)>> The oxoammonium form of AZADO, the compound of the present invention (counter anion is NO), ３ － ION; AZADO ＋ NO ３ － The minimum inhibitory concentration (MIC) of each of the above compounds against Escherichia coli was measured by the following method.

[0087] First, as a preculture, Escherichia coli (DH5α strain; AMP+) was cultured with shaking in LB medium for 20 hours, and the resulting culture solution was used as an Escherichia coli stock solution. Next, this Escherichia coli stock solution was diluted 1000-fold with PBS to obtain a diluted solution. This diluted solution was used as a comparative example.

[0088] On the other hand, the diluted solution obtained above was added with AZADO ＋ NO ３ － was added to the solution to a final concentration of 1 μM, 10 μM, 100 μM, 1 mM, or 10 mM, and the solution was left to stand at room temperature for 15 minutes. ＋ NO ３ － To inactivate the β-amyloid ... ＋ NO ３ － A photograph of the agar medium after incubation with a sample having a final concentration of 1 mM is shown in FIG.

[0089]

[0090] From the results shown in Table 3, AZADO ＋ NO ３ － A significant inactivation effect was observed when the final concentration of AZADO was 100 μM or higher. ＋ NO ３ － A high disinfecting effect of 99% or more was confirmed when the final concentration of the compound was 1 mM or more. This concentration of 1 mM is similar to the concentration at which a significant antiviral effect was confirmed in an FCoV infection experiment on CRFK cells. This suggests that the disinfecting effect of the compound of the present invention on E. coli is comparable to that of the compound's antiviral effect.

[0091] <<Evaluation of Protein Degradation in a Mist State>> In order to simulate the conditions in which nitroxyl radicals exert their antiviral and disinfecting effects on filters present in the living environment, the antiviral activity of a carbon material (AZADO-Cu(bpy) / DB) carrying nitroxyl radicals (AZADO) and a co-catalyst (Cu) was evaluated using the decomposition activity of S-RBD as an index by the following method using a sprayer.

[0092] (Confirmation of reactivity in a liquid phase system) First, 100 pmol of S-RBD and 0.1 mg of AZADO-Cu(bpy) / DB were mixed in 110 μL of water solvent (S-RBD:Cu:AZADO=1:250:250 (molar ratio)). The mixture was then incubated at room temperature for 2 or 6 hours. An experiment using Cu(bpy) / DB without AZADO was also conducted as a comparative example.

[0093] Thereafter, 1xSB was added to the residue obtained by centrifugation, and 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. The results (electrophoresis photograph) are shown in Figure 14.

[0094] The results shown in Figure 14 confirmed that in the system to which AZADO-Cu(bpy) / DB was added, the S-RBD band disappeared in an incubation time-dependent manner. Based on these results, the following experiment using a nebulizer was carried out.

[0095] (Confirmation of reactivity to proteins in a mist state) An antiviral filter was prepared by impregnating a membrane filter (8 mm punch) with 100 μL of an aqueous solution containing 0.1 mg of AZADO-Cu(bpy) / DB. This filter was placed in the opening of a 200 μL microchip and fixed with parafilm. A comparative filter was also prepared using Cu(bpy) / DB.

[0096] 25 μL of an aqueous solution containing 125 pmol or 250 pmol of S-RBD was sprayed onto the filter prepared above from the tip of the microchip using a microsprayer (Micro FPS, manufactured by Toray Precision Co., Ltd.), and the filter was incubated at room temperature for 24 hours. The amount of S-RBD remaining on the filter was then evaluated by SDS-PAGE and silver staining. The results (electrophoresis photograph) are shown in Figure 15.

[0097] The results shown in FIG. 15 confirmed that the S-RBD band disappeared or was reduced in the filter containing AZADO-Cu(bpy) / DB, regardless of the amount of S-RBD sprayed.

[0098] This application is based on Japanese Patent Application No. 2022-136098, filed on August 29, 2022, the disclosure of which is incorporated herein by reference in its entirety.

Claims

**Claim 1** A method for inactivating bacteria or viruses, comprising contacting a compound having the following redox mechanism with a subject at risk of contamination by bacteria or viruses in the presence of a cocatalyst containing a transition metal, wherein: 【Chemical 1】 wherein X － is a counter anion, and the dashed line represents the bonding position with other atoms The method, wherein the nitroxyl radical form of the compound is one or more selected from the following group: 【Chemical Formula 2-1】 ​ **Claim 2** The inactivation method according to claim 1, wherein the virus is inactivated by imparting oxidative damage to the spike protein constituting the virus. **Claim 3** The inactivation method according to claim 1, which is a method for inactivating bacteria, wherein the bacteria are bacteria or fungi. **Claim 4** The standard redox potential (25 °C) between the nitroxyl radical form and the oxoammonium form is +100 mV to +1000 mV [Ag / Ag ＋ , and the inactivation method according to claim 1 or 2. **Claim 5** The inactivation method according to claim 1 or 2, wherein the nitroxyl radical form of the compound is 2-azabicyclo[2.2.2]octane-N-oxyl (AZADO), 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), or 4-acetylamino TEMPO. **Claim 6** The inactivation method according to claim 1 or 2, wherein the transition metal contains one or more selected from the group consisting of Ag, Au, Pt, Pd, Ni, Mn, Fe, Ti, Al, Zn, and Cu. **Claim 7** The inactivation method according to claim 1 or 2, wherein the compound-containing solution is sprayed onto the subject to contact the compound with the subject. **Claim 8** An antibacterial or antiviral substrate, wherein an inactivator for bacteria or viruses comprising a compound having the following redox mechanism is supported or coated on a base substrate, wherein: [Chemical Formula 3] In the formula, X － is a counter anion, and the dashed line represents the bonding position with other atoms. A cocatalyst containing a transition metal is further supported or coated on the base substrate, and The substrate, wherein the nitroxyl radical form of the compound is one or more selected from the following group: 【Chemical Formula 4-1】 【Chemical Formula 4-2】 **Claim 9** The antibacterial or antiviral substrate according to claim 8, wherein the bacteria are bacteria or fungi. **Claim 10** The antibacterial or antiviral substrate according to claim 8 or 9, wherein the base substrate is a woven fabric, non-woven fabric, filter, or urethane foam. **Claim 11** The antibacterial or antiviral substrate according to claim 8 or 9, wherein the antiviral activity value (Mv) against influenza virus or feline calicivirus measured based on JIS L 1922:2016 (Test method for antiviral properties of textile products) is 2.0 or more. **Claim 12** The antibacterial or antiviral substrate according to claim 8 or 9, which is used for a mask, a medical fiber product, an air conditioner, an air cleaner, a refrigerator, a temperature controller, a dehumidifier, a humidifier, or a filter for a suction cleaner. **Claim 13**: A method for imparting oxidative damage to a spike protein constituting a virus, the method comprising contacting the virus with a compound having the following redox mechanism: 【Chemical Formula 5】 wherein X− is a counter anion, and the dashed line represents the bonding position with other atoms, the method, wherein the nitroxyl radical form of the compound is one or more selected from the following group: ​ ​ **Claim 14**: The method according to claim 13, wherein the standard redox potential (25° C.) between the nitroxyl radical form and the oxoammonium form is +100 mV to +1000 mV [Ag / Ag+].

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