Antibacterial and antiviral agents

Abstract

To provide a novel antibacterial-antiviral agent.SOLUTION: An antibacterial-antiviral agent according to the present invention comprises a metal complex polymer having a constitutional unit derived from a metal complex monomer with an antibacterial and / or antiviral metal ion coordinated with a polymerizable monomer of the formula (1), where R1 is a hydrogen atom or a methyl group, Ar1 and Ar2 each denote a pyridyl group, a pyrrolyl group or the like, n1, n2, and n3 each denote an integer of 1-4.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] The present invention relates to an antibacterial and antiviral agent. [Background technology]

[0002] In recent years, from the viewpoint of infection prevention and hygiene, many products are required to have antibacterial and / or antiviral properties, and various antibacterial and antiviral agents are being used.

[0003] In general, inorganic antibacterial and antiviral agents that utilize metal ions or metal complexes are said to have a broader antimicrobial spectrum and superior durability compared to organic antibacterial and antiviral agents. For this reason, various metal complexes have been proposed as antibacterial and antiviral agents (see, for example, Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 10-7512 [Patent Document 2] Japanese Patent Application Laid-Open No. 2015-195826 Summary of the Invention [Problem to be solved by the invention]

[0005] However, after extensive investigations, the inventors have found that when a metal complex is added to a liquid, sufficient antibacterial and / or antiviral properties may not be exhibited at low concentrations that do not show toxicity to human cells.

[0006] An objective of the present invention is to provide a novel antibacterial and antiviral agent. [Means for solving the problem]

[0007] Specific means for solving the above problems include the following embodiments. <1> An antibacterial and antiviral agent comprising a metal complex polymer having a structural unit derived from a metal complex monomer in which a polymerizable monomer represented by the following formula (1) is coordinated to a metal ion having antibacterial and / or antiviral properties: [ka] [In the formula, R 1 represents a hydrogen atom or a methyl group, and Ar 1 and Ar 2 each independently represents a group represented by the following formula (2) or (3), and n 1 , n 2 , and n 3 each independently represents an integer of 1 to 4. [ka] [In the formula, R 2 represents a hydrogen atom or an alkyl group.

[0008] <2> the metal ions are ions of a metal selected from the group consisting of Cu, Ag, Au, Pt, Fe, Mn, Cr, Co, Ce, and Ru; <1> The antibacterial and antiviral agent according to claim 1.

[0009] <3> the proportion of structural units derived from the metal complex monomer in the metal complex polymer is 30 mol % or more; <1> or <2> The antibacterial and antiviral agent according to claim 1. [Effects of the Invention]

[0010] According to the present invention, a novel antibacterial and antiviral agent can be provided. [Brief explanation of the drawings]

[0011] [Figure 1A] FIG. 1 shows the growth curve of Escherichia coli after sterilization treatment with a copper complex polymer (pDPACu(II)MA). [Figure 1B] FIG. 1 shows the growth curve of Escherichia coli after sterilization treatment with a low molecular weight copper complex (DPACu(II)-OH). [Figure 2]FIG. 1 shows the amount of copper bound to E. coli incubated for 15 minutes in the presence of copper complexes (pDPACu(II)MA or DPACu(II)-OH). [Figure 3] FIG. 1 shows the cell viability of human dermal fibroblasts when cultured for 24 hours in the presence of copper complexes (pDPACu(II)MA or DPACu(II)-OH). [Figure 4] FIG. 1 is a graph showing the change over time in the residual rate of hydrogen peroxide when hydrogen peroxide is decomposed using a copper complex (pDPACu(II)MA or DPACu(II)-OH) as a catalyst. DETAILED DESCRIPTION OF THE INVENTION

[0012] <Antibacterial and antiviral agents> The antibacterial and antiviral agent according to this embodiment comprises a metal complex polymer having structural units derived from a metal complex monomer in which a polymerizable monomer, described below, is coordinated to a metal ion having antibacterial and / or antiviral properties (hereinafter also referred to as an "antibacterial and / or antiviral metal ion"). This metal complex polymer can exhibit excellent antibacterial and / or antiviral properties in liquid. While the reason for this is not clear, it is presumed that polymerizing the metal complex monomer limits the diffusion of the metal complex moieties in liquid, making it easier to form a polynuclear structure due to the local concentration effect.

[0013] In this specification, the term "antibacterial" is used to mean killing part or all of bacteria and inhibiting bacterial growth, and the term "antiviral" is used to mean inactivating viruses.

[0014] The antibacterial / antiviral metal ion is not particularly limited as long as it has antibacterial and / or antiviral properties, and specific examples of the antibacterial / antiviral metal ion include ions of Cu, Ag, Au, Pt, Fe, Mn, Cr, Co, Ce, Ru, etc.

[0015] The polymerizable monomer that coordinates with antibacterial and antiviral metal ions is represented by the following formula (1).

[0016] [ka]

[0017] In the above formula (1), R 1 represents a hydrogen atom or a methyl group, and Ar 1 and Ar 2 each independently represents a group represented by the following formula (2) or (3), and n 1 , n 2 , and n 3 each independently represents an integer of 1 to 4.

[0018] [ka]

[0019] In the above formulas (2) and (3), R 2 represents a hydrogen atom or an alkyl group. The group represented by the above formula (2) includes a pyridyl group and a substituted pyridyl group, and the group represented by the above formula (3) includes a pyrrolyl group and a substituted pyrrolyl group. R 2 The alkyl group in R is preferably an alkyl group having 1 to 4 carbon atoms, such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group, or an n-butyl group. 3 is preferably a hydrogen atom.

[0020] Ar 1 and Ar 2 may be the same or different, and preferably have the same structure. 1 and Ar 2 is preferably a group represented by the above formula (2), more preferably a pyridyl group.

[0021] n 1 is preferably 2 or 3, and more preferably 2. 2 and n 3are each independently preferably 1 or 2, and more preferably 1.

[0022] Specific examples of the polymerizable monomer represented by the above formula (1) include polymerizable monomers represented by the following formulae (1-1) to (1-3): 1 is synonymous with the above formula (1).

[0023] [ka]

[0024] A structural unit derived from a metal complex monomer in which a polymerizable monomer represented by the above formula (1) is coordinated to an antibacterial / antiviral metal ion is represented by the following formula (1a), in which M represents an antibacterial / antiviral metal ion.

[0025] [ka]

[0026] The metal complex polymer may contain a constituent unit derived from another polymerizable monomer in addition to the constituent unit represented by formula (1a), provided that the proportion of the constituent unit represented by formula (1a) is preferably 30 mol % or more, more preferably 50 mol % or more, even more preferably 70 mol % or more, and particularly preferably 90 mol % or more.

[0027] Other polymerizable monomers include unsaturated carboxylic acids such as (meth)acrylic acid, maleic acid and its anhydride, and itaconic acid and its anhydride; (meth)acrylic acid esters such as methyl (meth)acrylate, ethyl (meth)acrylate, chloroethyl (meth)acrylate, and polyethylene glycol (meth)acrylate; (meth)acrylamides such as (meth)acrylamide, N-methyl (meth)acrylamide, and N-phenyl (meth)acrylamide; allyl compounds such as allyl acetate, allyl caproate, and allyl caprylate; vinyl ethers such as hexyl vinyl ether, octyl vinyl ether, and methoxyethyl vinyl ether; vinyl esters such as vinyl butyrate, vinyl isobutyrate, and vinyl benzoate; and styrenes such as styrene, methylstyrene, and chlorostyrene. These polymerizable monomers may be used alone or in combination of two or more.

[0028] In this specification, the term "(meth)acrylic acid" means both "acrylic acid" and "methacrylic acid." The same applies to other terms such as "(meth)acrylate."

[0029] The metal complex polymer can be obtained, for example, by polymerizing the polymerizable monomer represented by formula (1) above (and other polymerizable monomers, if necessary) to obtain a polymer, and then coordinating the polymer with an antibacterial / antiviral metal ion. The method for producing the polymer is not particularly limited, and known production methods using a polymerization initiator or a chain transfer agent can be employed. Alternatively, the metal complex polymer can be obtained by coordinating the polymerizable monomer represented by formula (1) above with an antibacterial / antiviral metal ion to obtain a metal complex monomer, and then polymerizing the metal complex monomer (and other polymerizable monomers, if necessary).

[0030] The average degree of polymerization of the metal complex polymer is preferably 10 to 1,000, and more preferably 20 to 100, for example.

[0031] The antibacterial and antiviral agent according to this embodiment may contain other components, such as water, an organic solvent, a surfactant, a dispersant, etc. The antibacterial and antiviral agent according to this embodiment may further contain an antibacterial and antiviral substance other than the metal complex polymer.

[0032] <Antibacterial and antiviral treatment methods> An antibacterial and antiviral treatment method using an antibacterial and / or antiviral agent according to an embodiment includes contacting the antibacterial and / or antiviral agent with an object to which antibacterial and / or antiviral properties are to be imparted. Examples of the form of contacting the object include adding, coating, or spraying the antibacterial and / or antiviral agent to the object, or impregnating the object with the antibacterial and / or antiviral agent.

[0033] The object to which antibacterial and / or antiviral properties are imparted is not particularly limited, and examples thereof include detergents such as household detergents, laundry detergents, and dishwashing detergents; cosmetics such as lotions; filter water for water-type air purifiers; and the like.

[0034] The bacteria to be treated are not particularly limited as long as they are effective against the antibacterial / antiviral metal ions, and may be either gram-positive or gram-negative bacteria.The viruses to be treated are not particularly limited as long as they are effective against the antibacterial / antiviral metal ions, and may be either enveloped or non-enveloped viruses. [Example]

[0035] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0036] <Synthesis Example 1: Synthesis of copper complex polymer (pDPACu(II)MA)> (1) Synthesis of methacrylate monomer (DPAMA) having dipicolylamino group (DPA) as a ligand

[0037] [ka]

[0038] 2-(Chloromethyl)pyridine hydrochloride (40.11 g, 245 mmol), tetrabutylammonium bromide (TBAB; 1.31 g, 4.08 mmol), and potassium carbonate (112.6 g, 815 mmol) were dissolved in acetonitrile (500 mL) and stirred under a nitrogen atmosphere. 3-Amino-1-propanol (6.12 g, 81.5 mmol) was added to the mixture and refluxed at 95°C for 60 hours. The reaction solution was filtered through Celite (Celite No. 503, Fujifilm Wako Pure Chemical Industries, Ltd.), and the filtrate was concentrated using an evaporator. The concentrate was purified by silica gel column chromatography (eluent: ethyl acetate / methanol = 90 / 10), and the fraction containing the target product was dried under vacuum to obtain a viscous liquid compound (DPA-OH) (yield: 8.893 g, 42.0%). The structure of the obtained compound is 1 The results were confirmed by H-NMR (Bruker Avance 400 Hz).

[0039] Next, DPA-OH (4.06 g, 15.7 mmol) and triethylamine (TEA; 1.97 g, 19.5 mmol) were dissolved in dichloromethane (20 mL), and the resulting solution was ice-cooled. Separately, methacryloyl chloride (2.04 g, 19.5 mmol) was dissolved in dichloromethane (4 mL). The DPA-OH solution was added dropwise to the methacryloyl chloride solution while stirring in an ice bath, and the mixture was stirred at room temperature for 24 hours. The reaction solution was then filtered through Celite (Celite No. 503, Fujifilm Wako Pure Chemical Industries, Ltd.), and the filtrate was concentrated using an evaporator. To remove impurities, the concentrate was diluted with ethyl acetate (20 mL) and washed three times with saturated aqueous sodium bicarbonate and once with aqueous sodium chloride to recover the oil phase. The recovered oil phase was dehydrated with sodium sulfate, filtered through a Kiriyama funnel, and the filtrate was concentrated. The concentrate was purified by silica gel column chromatography (eluent: ethyl acetate / methanol = 90 / 10), and the fraction containing the target compound was dried under vacuum to obtain a viscous liquid compound (DPAMA) (yield: 1.582 g, 31.0%). The structure of the obtained compound is 1 The results were confirmed by H-NMR (Bruker Avance 400 Hz).

[0040] (2) Synthesis of polymer (pDPAMA) by RAFT polymerization of DPAMA

[0041] [ka]

[0042] DPAMA (955 mg, 2.94 mmol) and 2-phenyl-2-propyl-benzodithioate (8.41 mg, 30.9 μmol) as a RAFT agent were dissolved in N,N-dimethylformamide (DMF; 5 mL). The molar ratio of monomer to RAFT agent was determined by taking a portion of the solution. 1The reaction mixture was confirmed by H-NMR (Bruker Avance 400 Hz). A solution of 2,2'-azobisisobutyronitrile (AIBN; 1.01 mg, 6.3 μmol) dissolved in DMF (1 mL) was added to this solution to prepare a mixed solution. The mixed solution was degassed by repeating freeze-thaw cycles three times, and polymerization was carried out by stirring under a nitrogen atmosphere at 60°C for 48 hours. The monomer conversion rate was 1 The reaction mixture was confirmed by H-NMR. The reaction solution was then poured into diethyl ether (150 mL) to form a precipitate. The precipitate was dissolved in DMF (6 mL) and poured into diethyl ether (150 mL) to form a precipitate again, which was then dried in vacuo to obtain a powdery compound (pDPAMA) (yield: 604 mg, 62.7%). The structure of the obtained compound is 1 The chromatographic properties were determined by H-NMR and size-exclusion chromatography (HLC-8020 GPC system, Tosoh Corporation). The size-exclusion chromatography column used was TSKgel SuperHZM-H (Tosoh Corporation), and the eluent was DMF containing 10 mM lithium chloride.

[0043] Analysis revealed that the molar ratio of monomer to RAFT agent was 90. The monomer conversion was 73% and the degree of polymerization was 65. It was also confirmed that unreacted monomer had been completely removed by purification. 1 This was confirmed by H-NMR spectroscopy. The polyethylene glycol-equivalent number-average molecular weight (Mn) of the obtained pDPAMA was 6,980, the mass-average molecular weight (Mw) was 10,670, and the dispersity (Mw / Mn) was 1.528.

[0044] (3) Synthesis of copper complex polymer (pDPACu(II)MA) pDPAMA (52.89 mg; containing 160.5 μmol of DPA) was dissolved in methanol (2 mL). Separately, copper(II) chloride dihydrate (35.56 mg, 208.6 μmol) was dissolved in methanol (2 mL). The pDPAMA solution was added dropwise to the copper solution and stirred, followed by further stirring at room temperature for 12 hours. The solution was then dialyzed five times against water (MWCO: 3.5 kDa) and lyophilized to obtain the powdered copper complex polymer (pDPACu(II)MA) (yield: 79.40 mg, 98.8%).

[0045] <Synthesis Example 2: Synthesis of a comparative low molecular weight copper complex (DPACu(II)-OH)> DPA-OH (40 mg) obtained in Synthesis Example 1 (1) was dissolved in dimethyl sulfoxide (DMSO; 4.31 mL) to prepare a 36 mM DPA-OH solution. Copper (II) chloride dihydrate (33 mg) was dissolved in DMSO (64.5 mL) to prepare a 3 mM copper chloride solution. The copper chloride solution was then added to the DPA-OH solution to obtain a low molecular weight copper complex (DPACu(II)-OH).

[0046] <Experimental Example 1: Evaluation of antibacterial activity> Copper complexes (pDPACu(II)MA or DPACu(II)-OH) were mixed with deionized water to prepare sample solutions with various copper concentrations. 600 An E. coli suspension with an OD = 1.0 was prepared. 20 μL of the sample solution was added to 1 mL of the E. coli suspension and incubated at 37°C for 15 minutes. 500 μL of the incubated E. coli suspension was then added to 20 mL of LB medium and cultured to reach an OD 600 The growth curve of E. coli was obtained by monitoring the value of .

[0047] Figure 1A shows the growth curves of E. coli after sterilization with pDPACu(II)MA, and Figure 1B shows the growth curves of E. coli after sterilization with DPACu(II)-OH. As shown in Figures 1A and 1B, when sterilization was performed with pDPACu(II)MA, E. coli was almost completely killed even at a copper concentration as low as 25 μM. On the other hand, when sterilization was performed with DPACu(II)-OH, even when the copper concentration was increased to 400 μM, the bactericidal effect was inferior to that of pDPACu(II)MA at a copper concentration of 25 μM. In other words, by polymerizing the copper complex monomer, the bactericidal effect was improved by more than 15 times.

[0048] Furthermore, as described above, the amount of copper bound to E. coli after 15 minutes of incubation at 37°C in the presence of the copper complex was measured. First, the E. coli after incubation was centrifuged to recover the pellet. The pellet was then suspended in water, washed, and centrifuged twice, after which the pellet was dissolved in 0.1 M aqueous nitric acid. The amount of copper in the solution was then measured using an inductively coupled plasma optical emission spectrometer (SPECTRO ARCOS FHM22, SPECTRO Analytical Instruments). Note that yttrium was used as an internal standard during the measurements.

[0049] The amount of copper bound to E. coli is shown in Figure 2. As shown in Figure 2, E. coli after incubation in the presence of pDPACu(II)MA at a copper concentration of 25 μM had significantly more copper bound than E. coli after incubation in the presence of DPACu(II)-OH at the same concentration ( **** p<0.0001). This is presumably due to the multipoint interaction of pDPACu(II)MA with E. coli lipopolysaccharide. However, significantly more copper was bound to E. coli after incubation in the presence of DPACu(II)-OH at a copper concentration of 400 μM than to E. coli after incubation in the presence of pDPACu(II)MA at a copper concentration of 25 μM ( *****p<0.00001). These results show that the bactericidal effect per unit amount of copper was significantly improved by polymerizing the copper complex monomer.

[0050] <Experimental Example 2: Evaluation of toxicity to human fibroblasts> Human dermal fibroblasts (NHDF) were seeded at a cell density of 10,000 cells / well in a 24-well plate and cultured overnight in 400 μL of DMEM medium (containing 10% (v / v) FBS and 2% (w / v) penicillin / streptomycin). The cells were then cultured for 24 hours in 400 μL of DMEM medium containing various concentrations of copper complexes (pDPACu(II)MA or DPACu(II)-OH). After 24 hours, the medium was replaced with copper-complex-free DMEM, and cell viability was measured using Cell Counting Kit 8 (Dojindo Laboratories).

[0051] The results of measuring cell viability are shown in Figure 3. As shown in Figure 3, pDPACu(II)MA did not exhibit cytotoxicity to human skin fibroblasts, even when the copper concentration was set to 25 μM, at which E. coli was almost completely killed in Experimental Example 1. On the other hand, DPACu(II)-OH exhibited cytotoxicity to human skin fibroblasts when the copper concentration was increased to 100 μM or more, at which point it showed a bactericidal effect against E. coli in Experimental Example 1. ** p<0.01; ***** p<0.00001).

[0052] <Experimental Example 3: Evaluation of decomposition of hydrogen peroxide> The decomposition of hydrogen peroxide catalyzed by copper complexes was indirectly evaluated by detecting the generation of reactive oxygen radicals associated with the decomposition of hydrogen peroxide using 2,2-diphenyl-1-picrylhydrazyl (DPPH) as a probe. First, a methanol solution containing 10 mM DPPH, an aqueous solution containing 1 mM copper complex (pDPACu(II)MA or DPACu(II)-OH), an aqueous solution containing 10 mM hydrogen peroxide, and methanol were mixed to prepare sample solutions with DPPH, copper complex, and hydrogen peroxide concentrations of 250 μM, 25 μM, and 250 μM, respectively. The sample solutions were then shaken at 37°C, and the residual hydrogen peroxide was calculated from the change in absorbance at 512 nm after 10, 30, and 60 minutes.

[0053] Figure 4 shows the time course of the residual hydrogen peroxide. As shown in Figure 4, the addition of pDPACu(II)MA accelerated the decomposition of hydrogen peroxide (generation of reactive oxygen radicals) compared with the addition of DPACu(II)-OH. These results suggest that pDPACu(II)MA exerts its bactericidal effect by generating reactive oxygen radicals from hydrogen peroxide before it is eliminated by catalase or other enzymes. It is known that the formation of a dinuclear copper structure is important for the catalytic activity of copper catalysts against hydrogen peroxide (N. Oishi et al., Polyhedron, 1984, 3, 157). It is thought that the localized concentration of copper complex sites in pDPACu(II)MA facilitates the formation of a dinuclear copper structure.

Claims

[Claim 1] An antibacterial and antiviral agent comprising a copper complex polymer in which the average degree of polymerization of the constituent units represented by the following formula (1a) is 10 to 100: 【Chemistry 1】 [In the formula, R １ represents a hydrogen atom or a methyl group, and Ar １ and Ar ２ represents a group represented by the following formula (2), and n １ represents 3, n 2 and n 3 represent 1, and M represents copper.] 【Chemistry 2】 [In the formula, R ２ represents a hydrogen atom.

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