JP3162871B2 - Antibacterial antifungal antiviral agent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antibacterial and antifungal agent, and more particularly, it has a broad antibacterial spectrum, is excellent in durability and residual effect, and has reduced skin irritation and mucous membrane irritation. And a highly safe antibacterial and antifungal agent.

[0002]

2. Description of the Related Art In recent years,
The development and use of functional materials are becoming active. Above all, many attempts have been made to apply functional materials having antibacterial and antifungal activity to living environments such as fibers, sanitary products, tableware, packaging materials and the like. These functional materials are provided with new functions by adding or kneading agents having antibacterial and antifungal activity to the materials.

[0003] In order to apply to these fields, it is required that the drug has a safety and a broad spectrum of antibacterial and antifungal properties, and that the drug does not affect the quality of the material and that the durability is high. , Residual effect, economy, etc. are required.

[0004] Agents used so far as antibacterial and antifungal agents include benzimidazoles, nitriles, isothiazolines, haloallylsulfones, iodopropargyls, benzothiazoles, phenols, organotins, and pyridines. System, diphenyl ether system, chlorhexidine system and the like.

[0005] However, many of these antibacterial and antifungal agents cannot exhibit a sufficient antibacterial and antifungal effect with only one kind of agent. In addition, even if the antimicrobial mold activity of the drug itself is excellent, there is a problem in compatibility with the material, and when applied to the material, it does not necessarily show a sufficient antibacterial and antifungal effect. . On the other hand, regarding the safety of these antibacterial and antifungal agents, many of them show acute oral toxicity, skin irritation, mucous membrane irritation, etc., and their effects are generally stronger with stronger antibacterial and antifungal activity. There have been problems in applying various antibacterial and antifungal agents to the living environment.

Therefore, it has a broad antibacterial and antifungal spectrum, is excellent in durability and residual effect without affecting the materials used, and has acute oral toxicity, skin irritation and mucous membrane irritation. It has been desired to develop a reduced antibacterial antifungal agent.

[0007]

Means for Solving the Problems As a result of intensive studies in view of the above-mentioned circumstances, the present inventors have found that a complex obtained by coordinating a thiol group-containing compound described below with a silver ion can be obtained in addition to a broad antibacterial antifungal spectrum. The present inventors have found that they exhibit antiviral activity, do not adversely affect applied materials, and have significantly reduced toxicity, skin irritation, mucous membrane irritation, and the like, and have completed the present invention.

[0008]

That is, the present invention relates to thiosalicylic acid, p
-Toluenethiol, 2-naphthalentiol, α-thiolactic acid, cysteine, thioglycerol, 5-mercapto- (1H) tetrazolylacetic acid, 2-mercaptoethanol, 2,3-dimercaptosuccinic acid, thiophenol, 2-mercaptobenz An antibacterial and antifungal agent comprising as an active ingredient a complex in which a thiol group-containing compound selected from imidazole, 2-mercaptobenzothiazole, thiourea, mercaptonicotinic acid, 2-thiouracil and 2-mercaptohypoxanthine is coordinated to silver ions. It provides a viral agent.

Further, the present invention provides a method for preparing a solid carrier comprising thiosalicylic acid, p-toluenethiol, 2-naphthalene thiol, α-thiolactic acid, cysteine, thioglycerol,
-Mercapto- (1H) tetrazolylacetic acid, 2-mercaptoethanol, 2,3-dimercaptosuccinic acid, thiophenol, 2-mercaptobenzimidazole, 2-mercaptobenzimidazole
An antibacterial antifungal antiviral material obtained by immobilizing a complex in which a thiol group-containing compound selected from mercaptobenzothiazole, thiourea, mercaptonicotinic acid, 2-thiouracil and 2-mercaptohypoxanthine is coordinated with silver ions, And use of the complex for producing the same.

The complex of the present invention is produced, for example, by reacting a thiol group-containing compound with silver ions in an aqueous solution, and adding an alcohol or the like to precipitate the complex.

As the thiol group-containing compound used in the present invention, for example, the compound may have a functional group such as a carboxyl group, a hydroxyl group and an amino group.
Thiosalicylic acid and its salts, p-toluenethiol, 2
-Naphthalene thiol, thiomalic acid, thioglycolic acid, α-thiolactic acid, β-thiolactic acid, cysteine, thioglycerol, 5-mercapto- (1H) tetrazolylacetic acid sodium salt, 2-mercaptoethanol, 2,3-
Dimercaptosuccinic acid, thiophenol, 2-mercaptobenzimidazole, 2-mercaptohenzothiazole, thiourea, mercaptonicotinic acid, 2-thiouracil, 2-mercaptohypoxanthine and the like.

The silver ion source used in the present invention is preferably a water-soluble silver salt.

The complex of the present invention is used in an amount of 0.5 to 0.5 mol per mol of silver.
Preferably, 2 mol of the ligand is reacted, and the pH during the reaction is preferably 0.5 to 13.5.

The counter cation added to the complex is preferably K ⁺ , Na ⁺ , Ca ⁺⁺ , Ba ⁺⁺ or the like.

The complex of the present invention thus obtained has excellent antibacterial, antifungal and antiviral activities and can be used as it is as an antibacterial, antifungal or antiviral agent. Further, the complex of the present invention can be used as an antibacterial antifungal material by immobilizing the complex on various solid carriers.

The carrier which can immobilize the complex of the present invention includes:
For example, silica, hydroxyapatite, zeolite, titanium oxide and the like can be mentioned.

In order to immobilize the complex on the carrier, it is preferable to use, for example, a heat treatment, a chemical bonding method or the like.

In the present invention, by forming a thiol-silver complex using silver as an anionic silver complex as described above, for example, the existence of a salt which is a drawback of existing silver-containing antibacterial agents represented by zeolite-silver is considered. It is possible to improve a decrease in antibacterial activity due to a silver substitution reaction below, discoloration of silver ions by light, and the like.

[0020]

The antibacterial and antifungal agent of the present invention has a broad antibacterial and antifungal spectrum and antiviral properties, does not affect the quality of the material, and is effective for a long period of time. In addition, since acute oral toxicity, skin irritation, mucous membrane irritation, etc. are low, the following various uses,
For example, it can be applied to fibers, sanitary products, foods, green products, clean films, packaging materials, bactericidal materials, paints, glass filters for inorganic rooms, and the like. On the other hand, it is also effective in preventing bacterial infection due to blue wounds, bedsores, etc., and can be applied to the treatment thereof. At present, the emergence of antibiotic-resistant bacteria as a result of heavy use of antibiotics has become a major social problem. The antibacterial agent of the present invention solves such a problem at once.

[0021]

EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 6.17 g of thiosalicylic acid was weighed and dissolved in 200 ml of ethanol. 6.79 g of silver nitrate was weighed and dissolved in 10 ml of ion-exchanged water, and then gradually added to an ethanol solution of thiosalicylic acid with stirring. After stirring for about 1 hour, the resulting complex was centrifuged (3,500 rpm, 5
Min). The recovered complex was further washed with water (centrifugation conditions: 3,500 rpm, 5 minutes) four times, and then washed with ethanol and acetone three times each (centrifugation conditions: 10,000 rpm, 10 minutes).
Air-dried to obtain a complex (code No. # 1L-8H). The yield was about 9.7 g of orange powder.

The physical properties data of the obtained complex are shown. (1) IR spectrum Absorption of SH-derived absorption around 2550 cm ^-1 . The broad absorption of OH of 1700 cm ^-1 vicinity of C = 0 absorption and 3300~2200cm ^-1, the carboxyl group remains -COOH. In addition, there is absorption near 3400 cm ^-1 which is considered to be caused by water of crystallization. (2) Thermal analysis Decomposition point: about 317 ° C. (3) Elemental analysis values (% by weight, calculated values in parentheses) C; 30.79 (32.65), H; 1.92 (1.94), S; 11.5 (12.4), O; 11.9
(12.4), N; 0.2 or less (-), Na; 0.1 or less (-), Ag; 43.0 (41.
9). From this formula seems to _{(C 7 H 5 O 2 SAg} ) 2 · 0~1H 2 O. (4) ESCA (Electron Spectroscopy for Chemical)
Analysis) S2p spectrum peak is from 164ev (SH) to 162.8ev (SM)
Shift to. (5) NMR Chemical shifts of ¹ H and ¹³ C-NMR are shown in Table 1.

[0024]

[Table 1]

The structural formula expected from the above data is shown.

[0026]

Embedded image

Example 2 6.17 g of thiosalicylic acid was weighed out and 200 ml of 0.5
Dissolved in N NaOH solution. 6.79 g of silver nitrate was weighed and dissolved in 10 ml of ion-exchanged water, and then gradually added to an alkaline solution of thiosalicylic acid with stirring. After stirring for about 1 hour, centrifugation (10,000 rpm, 10 rpm)
Min) to remove insoluble substances, and centrifuged supernatant (216 ml)
An equal volume of ethanol was added to the mixture with stirring to precipitate and recover the complex (centrifugation conditions: 3,500 rpm, 5 minutes).
The recovered complex is dissolved in water and centrifuged (10,000 rp).
m, 10 minutes) to obtain a centrifugal supernatant. The centrifuged supernatant (27
An equal volume of ethanol was added to 8 ml) with stirring to precipitate and recover the complex (centrifugation conditions: 3,500 rpm, 5 minutes).
did. The recovered complex was dissolved again in water and centrifuged (1
(000 rpm, 10 minutes) to obtain a centrifugal supernatant. An equal volume of ethanol was added to the centrifuged supernatant (310 ml) with stirring to precipitate and recover the complex (centrifugation conditions: 3,500
rpm, 5 minutes). The recovered complex was then washed three times each with ethanol and acetone (centrifugation conditions: 3,500 rpm, 5 minutes) and air-dried to obtain a complex (code No. # 1L-8J). The yield was about 10 g of a yellow powder.

The physical properties data of the obtained complex are shown below. (1) Absorption of SH-derived absorption around 2550 cm ^{-1 in} IR spectrum. 1700cm ^-1
Carboxylic acid absorption around has changed to 1550 and 1400 cm ^-1, COOH is COO ^- indicating that the change to. The absorption of OH derived from COOH at 3300 to 2200 cm ^-1 has disappeared. At around 3400 cm ^-1 , absorption of OH which is considered to be water of crystallization is observed. (2) Thermal analysis Decomposition point: about 302 ° C. Weight loss that is considered as dehydration of crystallization water is 1
It was observed around 00 ° C. (3) Elemental analysis values (% by weight, calculated values in parentheses) C; 27.32 (27.91), H; 2.15 (1.99), S; 10.2 (10.7), O; 16.4
(15.9), N; 0.2 or less (-), Na; 6.92 (7.64), Ag; 36.4 (35.
9). From this formula seems to _{(C 7 H 4 O 2 SAgNa} ) 2 · 2~3H 2 O. (4) ESCA Shift similar to that of the complex of Example 1. (5) NMR Chemical shifts of ¹ H and ¹³ C-NMR are shown in Table 2.

[0029]

[Table 2]

The structural formula expected from the above data is shown.

[0031]

Embedded image

6.17 g of thiosalicylic acid was weighed, and 14
Dissolved in 0 ml ethanol. 6.79 g of silver nitrate was weighed, dissolved in 20 ml of ion-exchanged water, and gradually added to an ethanol solution of thiosalicylic acid with stirring. About 1
After stirring for an hour, the resulting complex was centrifuged (3,5
00 rpm, 5 minutes). A barium hydroxide aqueous solution was added to the collected complex to adjust the pH to 12.5. The complex was then collected by centrifugation (3,500 rpm, 5 minutes). The recovered complex is washed with water (centrifugation conditions: 6,000).
(rpm, 10 minutes) six times, followed by washing twice each with ethanol and acetone (centrifugation conditions: 6,000).
(0 rpm, 10 minutes), and air-dried to obtain a complex (code No. # 1L-8P). The yield was about 15 g of a yellow powder.

Example 4 4.51 g of thiomalic acid was weighed and dissolved in 60 ml of water. 3.40 g of silver nitrate was weighed and dissolved in 40 ml of water, and then gradually added to an aqueous thiomalic acid solution with stirring. 200 ml of ethanol was added to this solution, and further 2N NaO was added.
H was added until a yellow precipitate formed. The precipitate formed at this time was collected (centrifugation conditions: 3,500 rpm, 5 minutes), washed three times each with ethanol and acetone (centrifugation conditions: 3,500 rpm, 5 minutes), air-dried, and the complex (code No. # 4) was obtained. The yield is about 4.2 of yellow powder.
g.

The physical properties data of the obtained complex are shown below. (1) IR spectrum SH absorption around 2550 cm ^-1 has disappeared. 170
Absorption is observed at ⁰ , 1550 and 1400 cm ^-1 , respectively. 3400 cm ^-1 water of crystallization or OH of carboxyl group
(Monomer) is observed. (2) Thermal analysis Decomposition point: around 188 ° C. (3) Elemental analysis values (% by weight, calculated values in parentheses) C; 16.67 (17.06), H; 1.74 (1.78), S; 11.1 (10.8), O; 25.0
(24.4), N; 0.1 or less (-), Na;. 8.0 (7.33), Ag; 37.5 (38.3) than this, the composition formula is expected to _{(C 4 H 4 O 4 SAgNa} ) 2 · 0~2H 2 O . (4) NMR Chemical shifts of ¹ H and ¹³ C-NMR are shown in Table 3.

[0035]

[Table 3]

The structural formula expected from the above data is shown.

[0037]

Embedded image

Example 5 (Synthesis of Various Compounds) As shown in Tables 4 and 5, various ligands were reacted with silver ions under various synthesis conditions to obtain complexes.

[0039]

[Table 4]

[0040]

[Table 5]

The IR spectra of all the synthesized compounds were measured. As a result, it was confirmed that the absorption derived from SH at around 2550 cm ^{-1 which} was observed in the raw materials of most of the compounds disappeared in the synthesized product. This means that SH
Is changed to a bond such as S-Ag-. Further, those having a carboxyl group may have CO 2 depending on the synthesis conditions.
OH (1700 cm ^-1) or COO ^- (1550 and 140
(Around 0 cm ^-1 ).

Test Example 1 (Measurement of antibacterial activity) The antibacterial activities of the complexes shown in Table 5 were measured by the following measuring methods (a) and (b).

(A) Method of measuring antibacterial activity by surface smearing method Potato dextrose (PD) agar medium (15 ml) was 90 mm
Put in a Petri dish and solidify. The preparation of the spore suspension is carried out in 0.1 ml.
Aqueous solution 5 of 017% surfactant (Ravisol B-30)
In 3 ml, 3 to 5 loops of spores were suspended from the slant medium that had been cultured in advance, and filtered using gauze to obtain a spore suspension. Sample (about 10mg) and spore suspension (0.1m
l) was mixed and spread evenly on the agar surface with a conradi bar.
The cells were cultured at 25 ° C. for 4 days. After the cultivation, the antimicrobial activity was evaluated in comparison with the degree of proliferation of a control containing no specimen. Antimicrobial activity was measured at 80% of the control growth level (100%,-).
+ Up to about 40%, ++ up to about 40%,
The case where it was almost completely suppressed was defined as +++.

(B) Method for measuring antibacterial activity using minimum inhibitory concentration (MIC) Bacteria: Soybean casein digest (SCD)
5 ml of a liquid medium was inoculated, pre-cultured at 35 ° C. for 24 hours, and 0.1 ml of a 100-fold dilution of the pre-cultured bacterial liquid was inoculated into 2 ml of an SCD liquid medium. After shaking culture at 35 ° C. for 48 hours, the presence or absence of proliferation was determined. Yeast: Inoculated in 5 ml of glucose / peptone (GP) liquid medium, pre-cultured at 35 ° C. for 24 hours, and one of the pre-cultured bacterial liquids
0.1 ml of the 1:00 dilution was inoculated into a GP liquid medium containing 2 ml of the specimen. After shaking culture at 35 ° C. for 48 hours, the presence or absence of proliferation was determined. The antibacterial activity measurement results of each test complex are also shown in Tables 6 and 7.

[0045]

[Table 6]

[0046]

[Table 7]

As is clear from the results shown in Table 7, the complexes of the present invention show excellent antibacterial and antifungal activities against bacteria and fungi.

Test Example 2 (Light Stability Test) 1 ml of ethanol was added to 10 mg of each test complex shown in Table 8, and the particles were dispersed by ultrasonic treatment. The dispersed particles were uniformly suction-fixed on Kiriyama filter paper (No. 3, diameter 21 mm), and the test piece obtained after drying was sandwiched between colorless and transparent plastic films (0.1 mm thick), and the periphery was sealed and fixed.
The sun exposure was performed outdoors on a sunny day, and the test piece was placed on the surface of the test piece so that direct sunlight had an incident angle of about 90 °. The measurement was carried out four times for each test specimen 6 hours after sun exposure using a color difference meter CR-300 manufactured by Minolta Co. (using _D65 light), and the average value was obtained. The results are shown in the L * a * b color system according to CIF 1976. Also shown in Table 8.

[0049]

[Table 8]

As is clear from the results shown in Table 8, it can be seen that some of the complexes of the present invention have good photostability and also have excellent antibacterial and antifungal activities.

Test Example 3 (Safety test) The complexes shown in Table 4 were examined for toxicity (single dose, ICR, male) by oral administration. Table 9 also shows the results on day 14 after administration.

[0052]

[Table 9]

As can be seen from the results shown in Table 9, the complexes of the present invention were shown to be highly safe. In addition, $ 1L-8
LD ₅₀ for J is 2.5~5.0g / kg, whereas, # 1
The LD- _{50 of} L-8H was 3.9 g / kg or more.

Test Example 4 [Measurement of anti-human cytomegalovirus (HCMV) activity] Complexes 4, ♯1L-8H, and ♯1L-8J were added to MEM (2
% Fetal bovine serum; hereinafter 2% MEM)
/ Ml was used as a starting material, and was measured under the following conditions. Reaction solution: HCMV was 1 × 10 ³ p in Experiment 1.
fu / ml, and in Experiment 2, 1 × 10 ⁴ pfu / ml. In 1 ml of 2% MEM, HCMV, compound 50,
10, 1 μg were added. Cells: Human fetal lung cells (HEL) 10-14 generations
1 × 10 ⁴ ce for “Lab-Tek chamber”
11 seeds. Method: The above reaction mixture was incubated at room temperature with occasional stirring, incubated for 1 hour, and 0.5 ml thereof was infected with HEL.
The cells were cultured at 37 ° C. in an O ₂ incubator. Experiment 1: Culture for 72 hours without medium exchange Experiment 2: (1) Culture for 72 hours without medium exchange and (2) 1 hour after adsorption, washing twice with 2% MEM, and adding 0.5 ml of the same medium 72 Cultivation of CO ₂ for hours. Evaluation: Antiviral activity was performed by a fluorescent antibody staining method. That is, fluorescent staining was performed using a “Microtrak” kit (including an anti-early early protein antibody and an anti-early protein antibody) manufactured by Syva. The fewer cells that are fluorescently stained,
Strong antiviral activity. Approximately 400 cells were seen in one field of the fluorescence microscope. After the completion of Experiment 1, the number of cells positive for fluorescence staining in one field of the fluorescence microscope was measured under the above conditions. Table 10 shows the results.

[0055]

[Table 10] Complex concentration (μg / ml) complex 0 50 10 1 control 30 − − − Δ4 − 20 30 20 Δ1L-8H − −5 − Δ1L-8J − − 10 10

On the other hand, in each of Experiment 2 (1) and (2) after completion, the number of fluorescently stained positive cells in one visual field of the fluorescent microscope was measured. The results are shown in Tables 11 and 12, respectively.

[0057]

[Table 11] Compound concentration (μg / ml) complex 0 50 10 1 Control 390 − − − Δ4 − 220 300 − Δ1L-8H − 100 240 280 Δ1L-8J − 150 130 170

[0058]

[Table 12] Complex concentration (μg / ml) complex 0 50 10 1 control 120 − − − ♯4-70 80 80 ♯1L-8H -20 30 30 ♯1L-8J -20 20 30

As is clear from the results shown in Tables 10 to 12, the complexes of the present invention have remarkable antiviral activity.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification code FI // C07F 1/10 C07F 1/10 (72) Inventor Sakuma Sadatoshi 540 Narita, Odawara-shi, Kanagawa Prefecture Meiji Dairy Cell Engineering Center ( 72) Inventor Kenji Nomiya 390-6 Nagogi, Hadano-shi, Kanagawa (56) Reference Pharm. Weekbl. , Sc i. Ed. 1981 Vol. 3 No. 2, p. 79-81 Agents Actions, 1978, Vol. 8 No. 1-2, p. 85-90 Anal. Chem. , 1975, Vol. 47 No. 13, p. 2316-2319 Curr. Sci. 1989, Vol. 58 No. 21, p. 1201-1203 Curr. Sci. , 1972, Vol. 41 No. 16, p. 601-603 Chemical Abstracts, 1971, Vol. 74, 69726m (58) Field surveyed (Int. Cl. ⁷ , DB name) A01N 59/16 A61K 31/28 A61K 33/38 A61P 31/04 A61P 31/12 C07F 1/10 CA (STN) REGISTRY (STN )

Claims (5)

(57) [Claims] 1. thiosalicylic acid, p-toluenethiol, 2-naphthalentiol, α-thiolactic acid, cysteine, thioglycerol, 5-mercapto- (1H) tetrazolylacetic acid, 2-mercaptoethanol, 2,3-dimercaptosuccinate A complex in which a thiol group-containing compound selected from an acid, thiophenol, 2-mercaptobenzimidazole, 2-mercaptobenzothiazole, thiourea, mercaptonicotinic acid, 2-thiouracil and 2-mercaptohypoxanthine is coordinated with silver ions. An antibacterial, antifungal and antiviral agent comprising as an active ingredient. 2. The antibacterial and antifungal agent according to claim 1, wherein the thiol group-containing compound is thiosalicylic acid. 3. The counter cation added to the complex is K ⁺ , N
3. The antibacterial antifungal and antiviral agent according to claim 1, which is selected from a ⁺ , Ca ⁺⁺ and Ba ⁺⁺ . 4. A solid carrier comprising thiosalicylic acid, p-toluenethiol, 2-naphthalene thiol, α-thiolactic acid, cysteine, thioglycerol, 5-mercapto-
(1H) tetrazolylacetic acid, 2-mercaptoethanol, 2,3-dimercaptosuccinic acid, thiophenol,
2-mercaptobenzimidazole, 2-mercaptobenzothiazole, thiourea, mercaptonicotinic acid, 2
-An antibacterial antifungal material obtained by immobilizing a complex in which a thiol group-containing compound selected from thiouracil and 2-mercaptohypoxanthine is coordinated to silver ions. 5. A thiosalicylic acid, p-toluenethiol, 2-naphthalene thiol, α-thiolactic acid, cysteine, thioglycerol, or thiosalicylic acid for producing an antibacterial, antifungal, or antiviral material by immobilizing it on a fixed carrier.
-Mercapto- (1H) tetrazolylacetic acid, 2-mercaptoethanol, 2,3-dimercaptosuccinic acid, thiophenol, 2-mercaptobenzimidazole, 2-mercaptobenzimidazole
Use of a complex in which a thiol group-containing compound selected from mercaptobenzothiazole, thiourea, mercaptonicotinic acid, 2-thiouracil and 2-mercaptohypoxanthine is coordinated to silver ions.