KR100832565B1 - Antibacterial furanone derivative and method of preventing a biofilm formation - Google Patents

Abstract

An antibacterial furanone derivative is provided to function as a quorum sensing antagonist, which interrupts communication among bacteria due to a chemical structure similar to a material used for the communication of the bacteria, thereby being used for preventing formation of a biofilm. A method for preventing formation of a biofilm comprises a step of contacting an antibacterial furanone derivative represented by a formula(1)(wherein R is C1-10 alkyl or C1-10 haloalkyl) with microorganism to inhibit the formation of the biofilm, wherein the antibacterial furanone derivative is represented by a formula(2), (3), (4), (5), (6), (7) or (8) and the microorganism includes Gram negative bacillus.

Description

Antibacterial furanone derivative and method for preventing biofilm formation using the same {ANTIBACTERIAL FURANONE DERIVATIVE AND METHOD OF PREVENTING A BIOFILM FORMATION}

1 to 8 are photographs showing the color change of the culture medium using the distilled water of Comparative Example and the furanone derivatives prepared in Examples 1 to 7, respectively.

The present invention relates to an antimicrobial furanone derivative and a method for preventing formation of a biofilm using the same. More specifically, the present invention relates to an antimicrobial furanone derivative having a function of interfering with communication between bacteria and a method for preventing formation of a biofilm using the same.

When a bacterium reaches a certain level of cell concentration, it uses its chemical language to transmit signals between cells and regulate the expression of specific genes. The mechanism by which gene expression depends on individual density is called quorum sensing (QS). The quorum sensing mechanism is characterized by a series of biological phenomena in which individual bacterial organisms accumulate small molecule signaling substances such as autoinducers or pheromones outside the cell to induce active proliferation and fill quorum. Refers to.

In many bacteria, gene regulation by quorum sensing is achieved by the interaction of autoinducer synthase and regulator protein. This increases the expression of specific genes as well as the expression of self-directed synthase genes and regulatory protein genes. Due to these characteristics, quorum sensing is also called autoinduction by magnetic induction.

For example, N-acyl homoserine lactone synthase among bacteria constituting proteins synthesizes a signaling material called normal acyl homomoserine Lactone (AHL). The synthesized normal acyl homoserine lactone freely diffuses through the cell membrane during bacterial growth and accumulates in the environment outside the cell. When the density of bacteria increases and the signaling material accumulated outside the cell reaches a certain concentration, the signaling material enters the cell again and binds to a transcriptional regulator to promote expression of a specific gene.

Bacteria control various physiological phenomena such as formation of biofilm, virulence, bioluminescence, production of antibiotics or delivery of tumor-induced plasmid by conjugation through the quorum sensing mechanism described above. do. For example, in a series of bacterial infections, bacteria first find their way into the host and settle in a habitat suitable for survival. It then survives while neutralizing the host's primary defense system. Finally, bacteria multiply and spread offspring to other hosts. In this process, bacteria express various virulence factors by quorum sensing mechanism.

The biofilm is a typical function of bacteria. Biofilm refers to a soft mucus layer on the surface formed by extracellular macromolecules secreted during the metabolism of microorganisms and attached microorganisms. The biofilm stays in human organs and causes a number of conditions. Therefore, there is a need for the development of antimicrobial agents that inhibit the formation of such biofilm.

Antimicrobial agents that kill bacteria directly have a problem that bacteria with strong resistance to antimicrobials such as superbacteria appear as the frequency of use increases, whereas quorum-sensing antagonists that interfere with communication between bacteria are used as antimicrobials. Antimicrobial resistance does not occur. Therefore, there is a need for the development of antimicrobial agents that interfere with communication between bacteria and inhibit bacteria from forming biofilms.

Accordingly, an object of the present invention is to provide an antimicrobial furanone derivative that can act as a quorum-sensing antagonist of bacteria to inhibit the onset and the formation of biofilm.

In addition, another object of the present invention to provide a method for preventing the formation of a biofilm using the antimicrobial furanone derivatives described above.

Antimicrobial furanone derivatives of the present invention according to an embodiment for achieving the above object of the present invention has a structure represented by the following formula (1).

...... (1)

...... (One)

In Structural Formula 1, R represents an alkyl group having 1 to 10 carbon atoms or a haloalkyl group having 1 to 10 carbon atoms.

In the method for preventing the formation of a biofilm according to another embodiment of the present invention, the antimicrobial furanone derivative represented by Structural Formula 1 is contacted with a microorganism to suppress the formation of the biofilm.

The antimicrobial furanone derivatives of the present invention as described above have a structure in which 4-hydroxymethylfuran-2-one and an alkanoic acid are ester-bonded. Antimicrobial furanone derivatives have a chemical structure similar to that used by bacteria to communicate, such as seaweed extracts, apples or fungal secretions, and act as a quorum sensing antagonist that interferes with communication between bacteria. Do it. Accordingly, the antimicrobial furanone derivatives can effectively block the growth of microorganisms and the formation of biofilms.

Hereinafter, an antimicrobial furanone derivative of the present invention and a method of preventing formation of a biofilm using the same will be described in detail.

Antimicrobial Puranone Derivatives Lactone Derivatives

The antimicrobial furanone derivative of the present invention has a structure represented by the following formula (1).

...... (1)

...... (One)

In Formula 1, R represents an alkyl group having 1 to 10 carbon atoms or a haloalkyl group having 1 to 10 carbon atoms. For example, R is methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, chloromethyl group, chloroethyl group, chlorobutyl group, bromomethyl group, bromo An ethyl group and the like.

In the development of quorum sensing antagonists, it mimics the quorum sensing antagonists that exist in nature. Halogenated furanone, patulin, and the like are known as quorum-sensing antagonists of Gram-negative bacteria existing in nature. Furanone is also called butenolide, and in the present invention, a furanone-based quorum sensing antagonist having a structure similar to patulin has been developed.

Specifically, the antimicrobial furanone derivatives of the present invention having the structure represented by Formula 1 have a structure in which 4-hydroxymethylfuran-2-one and alkanoic acid are ester-bonded. Antimicrobial furanone derivatives have a chemical structure similar to that used by bacteria to communicate, such as seaweed extracts, apples or fungal secretions, and act as a quorum sensing antagonist that interferes with communication between bacteria. Do it. Accordingly, the antimicrobial furanone derivatives can effectively block the growth of microorganisms and the formation of biofilms.

According to one embodiment of the present invention, the antimicrobial furanone derivative represented by Structural Formula 1 may be represented by the following Structural Formulas 2 to 8.

...... (2)

...... (2)

...... (3)

...... (3)

...... (4)

...... (4)

...... (5)

...... (5)

...... (6)

(6)

...... (7)

(7)

...... (8)

...... (8)

The antimicrobial furanone derivative represented by Structural Formula 1 is prepared by reacting alkanoyl chloride with 4-hydroxymethylfuran-2-one. For example, the antimicrobial furanone derivative represented by Formula 2 reacts with 4-hydroxymethylfuran-2-one and acetyl chloride to acetyl to the hydroxyl group of 4-hydroxymethylfuran-2-one. The carbonyl group of chloride is prepared by bonding.

The antimicrobial furanone derivatives of the present invention described above have excellent performance as quorum-sensing antagonists that interfere with communication between individuals of microorganisms. Accordingly, it is possible to effectively block the formation of the biofilm by the microorganisms, and to inhibit the growth of bacteria.

How to prevent the formation of biofilm

According to one embodiment of the present invention, it is possible to effectively prevent the formation of a biofilm using the antimicrobial furanone derivatives described above.

Antimicrobial furanone derivatives of the present invention have a structure represented by the following formula 1, it can interfere with the communication between bacteria to inhibit the reproduction and can effectively block the formation of biofilm (biofilm). Biofilm refers to a soft mucus layer on the surface formed by extracellular macromolecules secreted during the metabolism of microorganisms and attached microorganisms. The biofilm causes a number of diseases while staying in the human organs and living environment, the antimicrobial furanone derivatives of the present invention can effectively block the formation of such biofilm.

...... (1)

...... (One)

In Formula 1, R represents an alkyl group having 1 to 10 carbon atoms or a haloalkyl group having 1 to 10 carbon atoms.

The antimicrobial furanone derivative of the present invention having the structure represented by Formula 1 has a structure in which 4-hydroxymethylfuran-2-one and an alkanoic acid are ester-bonded. Accordingly, the quorum sensing antagonist has a chemical structure similar to that of a molecule that interferes with communication of bacteria in natural substances, such as seaweed extract, apple or fungal secretion, and prevents communication between bacteria. Function as. In particular, such antimicrobial furanone derivatives act as an excellent quorum sensing antagonist against microorganisms using an acyl homoserine lactone compound as a signaling material. For example, it functions as an excellent quorum-sensing antagonist against Gram-negative bacteria and can block the formation of biofilms.

As described above, the antimicrobial furanone derivative of the present invention is an excellent quorum-sensing antagonist, which prevents the growth of microorganisms by interfering with communication between microorganisms and effectively blocks the formation of biofilms. Therefore, the antimicrobial furanone derivative of the present invention may be applied to an apparatus or apparatus in which a biofilm is easily formed in contact with water, or an implant in a human body, thereby effectively blocking the formation of the biofilm.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples. However, the following examples are provided to illustrate the present invention, and the present invention is not limited to the following examples and may be variously modified and changed.

Preparation of Antimicrobial Puranone Derivatives

The antimicrobial furanone derivative represented by Structural Formula 1 was prepared according to the following Schemes (1) to (5).

Example  One

Using dipyridine as a solvent, dihydroxyacetone (1) and anhydrous acetic acid (anhydrous acetic acid) were reacted according to Scheme (1) to synthesize diacetoxyacetone (diacetoxyactone, 2). Dissolve 17.95 g (80 mmol) of triethyl phosphonoacetate in 50 mL of tetrahydrofuran (THF), and then add 1.92 g (80 mmol) of sodium hydride (NaH) in 150 mL of tetrahydrofuran (THF). The reaction was carried out for 1 hour. A solution of 13.92 g (80 mmol) of diacetoxyacetone dissolved in 40 mL of tetrahydrofuran (THF) was added to the solution obtained by the reaction, followed by reaction for 1 hour according to Scheme (2), followed by heating for 30 minutes. . Excessive water was added and precipitated, and extracted with ethyl ether (50 mL) about 6 times and dried to give ethyl 3,3-diacetoxymethylacrylate (3). .

Dissolve 4.0 g (16.4 mmol) of 3,3-diacetoxymethylacrylate (3) in 8 mL of a solvent containing methanol and 10% sulfuric acid in a volume ratio of 1: 1, and then, according to Scheme (3), for 3 hours. Reacted. Sodium bicarbonate (NaHCO ₃ ) was added to the resulting solution for neutralization, and the solid was filtered off and washed with methanol. The reaction product was extracted and dried using chloroform. As a result, 4-hydroxymethylfuran-2 (5H) -one (4-hydroxymethylfuran-2 (5H) -one, 4) was obtained.

0.5 mmol of 4-hydroxymethylfuran-2 (5H) -one (4), 2.5 equivalents of acetyl chloride, acylation catalyst and 2.5 equivalents of poly (4-vinylpyridine) Was added to 4 mL of methylene chloride and reacted according to Scheme (4) at room temperature for 6 hours. The filtrate obtained by filtration of the reaction product was placed in a container containing 1.15 g (1.9 mmol, 2.5 equivalents) of silica having an aminopropyl group attached to the end of an alkanoyl chloride scavenger, and stirred at room temperature for 2 hours. Residual acetyl chloride was removed. The remaining solution after filtration was washed with methylene chloride and the solvent was evaporated to give the final result (5).

The structure of the final product (5) was confirmed by hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectrum and mass spectrometry. Hydrogen nuclear magnetic resonance analysis was performed using a CDC1 ₃ as a solvent and using a 400 MHz nuclear magnetic resonance apparatus. Hydrogen nuclear magnetic resonance spectra showed chemical shifts (δ) at 6.12 (s, 1H), 4.82 (s, 2H), 4.65 (s, 2H), and 2.00 (s, 3H). In addition, a peak was obtained at 156.1381 corresponding to C ₇ H ₈ O ₄ (M ⁺ + 1, 156.1381) in mass spectra obtained using high resolution mass spectroscopy (HRMS). Therefore, the final result (5) is (5-oxo-2,5-dihydrofuran-3-yl) methyl acetate represented by the following structural formula 2 ((5-oxo-2,5-dihydrofuran-3-yl) methyl acetate ).

...... (2)

...... (2)

Example 2

The final result (5) was obtained in substantially the same manner as in Example 1 except that propionyl chloride was used instead of acetyl chloride.

The structure of the final product (5) was confirmed by hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectrum and mass spectrometry. In the hydrogen nuclear magnetic resonance spectrum, the chemical shifts (δ) were 6.12 (s, 1H), 4.82 (s, 2H), 4.65 (s, 2H), 2.37 (m, 2H), and 1.10 (t, 3H). In addition, in the mass spectrum, Peak appeared at 170.165 corresponding to C ₈ H ₁₀ O ₄ (M ⁺ + 1, 170.165). Thus, the final result (5) is (5-oxo-2,5-dihydrofuran-3-yl) methyl propionate ((5-oxo-2,5-dihydrofuran-3-yl) represented by the following structural formula (3) methyl propionate).

...... (3)

...... (3)

Example 3

The final product 5 was obtained in substantially the same manner as in Example 1 except for using butyryl chloride instead of acetyl chloride.

The structure of the final product (5) was confirmed by hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectrum and mass spectrometry. In the hydrogen nuclear magnetic resonance spectrum, the chemical shifts (δ) are 6.12 (s, 1H), 4.82 (s, 2H), 4.65 (s, 2H), 2.33 (t, 2H), 1.70 (m, 2H) and 0.92 ( t, 3H). In addition, in the mass spectrum, Peak appeared at 184.1919, which corresponds to C ₉ H ₁₂ O ₄ (M ⁺ + 1, 184.1919). Therefore, the final result (5) is (5-oxo-2,5-dihydrofuran-3-yl) methyl butyrate represented by the following structural formula 4 ((5-oxo-2,5-dihydrofuran-3-yl) methyl butyrate ).

...... (4)

...... (4)

Example 4

The final result (5) was obtained in substantially the same manner as in Example 1 except for using pentanoyl chloride instead of acetyl chloride.

The structure of the final product (5) was confirmed by hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectrum and mass spectrometry. In the hydrogen nuclear magnetic resonance spectrum, the chemical shifts (δ) are 6.12 (s, 1H), 4.82 (s, 2H), 4.65 (s, 2H), 2.33 (t, 2H), 1.66 (m, 2H), and 1.34 (m). , 2H) and 0.92 (t, 3H). In addition, in the mass spectrum, Peak appeared at 198.2187 corresponding to C ₁₀ H ₁₄ O ₄ (M ⁺ + 1, 198.2188). As a result, the final result (5) is 5-oxo-2,5-dihydrofuran-3-yl) methyl pentanoate represented by the following structural formula (5) ((5-oxo-2,5-dihydrofuran-3-yl methyl pentanoate).

...... (5)

...... (5)

Example 5

The final result (5) was obtained in substantially the same manner as in Example 1 except that hexanoyl chloride was used instead of acetyl chloride.

The structure of the final product (5) was confirmed by hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectrum and mass spectrometry. In the hydrogen nuclear magnetic resonance spectrum, the chemical shifts (δ) are 6.12 (s, 1H), 4.82 (s, 2H), 4.65 (s, 2H), 2.33 (t, 2H), 1.66 (m, 2H), 1.34-1.28 (m, 4H) and 0.92 (t, 3H). The mass spectrum showed a peak at 212.2457 corresponding to C ₁₁ H ₁₆ O ₄ (M ⁺ + 1, 212.2456). As a result, the final result (5) is 5-oxo-2,5-dihydrofuran-3-yl) methyl hexanoate ((5-oxo-2,5-dihydrofuran-3-yl) represented by the following structural formula (6). methyl hexanoate).

...... (6)

(6)

Example 6

The final result (5) was obtained in substantially the same manner as in Example 1 except that heptanoyl chloride was used instead of acetyl chloride.

The structure of the final product (5) was confirmed by hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectrum and mass spectrometry. In the hydrogen nuclear magnetic resonance spectrum, the chemical shifts (δ) are 6.12 (s, 1H), 4.82 (s, 2H), 4.65 (s, 2H), 2.33 (t, 2H), 1.66 (m, 2H), 1.34-1.28 (m, 6H) and 0.92 (t, 3H). The mass spectrum showed a peak at 226.2725, corresponding to C ₁₂ H ₁₈ O ₄ (M ⁺ + 1, 226.2725). As a result, the final result (5) is 5-oxo-2,5-dihydrofuran-3-yl) methyl heptanoate ((5-oxo-2,5-dihydrofuran-3-yl) represented by the following structural formula (7). methyl heptanoate).

...... (7)

(7)

Example 7

The final result (5) was obtained in substantially the same manner as in Example 1 except that 2-chloroacetyl chloride was used instead of acetyl chloride.

The structure of the final product (5) was confirmed by hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectrum and mass spectrometry. In the hydrogen nuclear magnetic resonance spectrum, the chemical shifts (δ) were 6.12 (s, 1H), 4.82 (s, 2H), 4.65 (s, 2H), and 4.33 (s, 2H). The mass spectrum showed a peak at 190.5829, corresponding to C ₇ H ₇ ClO ₄ (M ⁺ + 1, 190.5829). As a result, the final product (5) is 5-oxo-2,5-dihydrofuran-3-yl) methyl 2-chloroacetate represented by the following structural formula (8) ((5-oxo-2,5-dihydrofuran-3- yl) methyl 2-chloroacetate).

...... (8)

...... (8)

Functional Evaluation as a Quorum Sensing Antagonist

The furanone derivatives obtained in Examples 1 to 7 were evaluated as functioning as quorum sensing antagonists.

Agrobacterium tumefaciens A136 (Ti-) (pCF218) (pCF372) and Agrobacterium tumefaciens KYC6 were used as cultures. Agrobacterium tumefaciens A136 is a bacterium that is mutated to produce galactosidase by expressing the lac gene when exposed to acyl homoserine lactone (AHL). In addition, Agrobacterium tumefaciens KYC6 is a bacterium that has been modified to overproduce acyl homoserine lactone upon exposure to normal-3-oxoacyl homoserine lactone. In addition, Agrobacterium tumefaciens A136 is exposed to acyl homoserine lactone using X-gal (5-bromo-4-chloro-3-indolyl-D-galactopyranoside), which is green or blue when degraded by galactosidase. It was possible to visually observe whether galactosidase was produced.

Specifically, Agrobacterium tumefaciens KYC6 strain was placed in Luria-Bertani (LB) culture and incubated at about 30 ° C. overnight. 10uL of the KYC6 strain and 100uL of the furanone derivatives prepared in Examples 1 to 7 were respectively added to 5mL of the LB culture medium, and cultured at about 30 ° C. for about 24 hours. In addition, the Agrobacterium tumefaciens A136 strain was placed in an LB culture medium containing 50 ug / mL spectinomycin and 4.5 ug / mL tetracycline and incubated overnight at about 30 ° C. As a comparative example, Agrobacterium tumefaciens KYC6 strain was cultured by adding distilled water to the culture solution of Agrobacterium tumefaciens KYC6 strain instead of the furanone derivative.

In order to evaluate whether it functions as a quorum sensing antagonist, 16 μL of dimethylformamide solution and 50 μL of distilled water in which X-gal was dissolved at a concentration of 50 mg / mL and 50 μL of distilled water were plated to allow water to be absorbed. The A136 strain is largely drawn in the medium with platinum teeth (白 金 耳), and the KYC6 strain is drawn at intervals of about 1 cm. As a comparative example, incubation was carried out at about 30 ° C. for two days until green or blue color was observed in the cultured medium instead of the furanone derivative. Evaluation results are shown in FIGS. 1 to 8.

1 to 8 are photographs showing the color change of the culture medium using the distilled water of the comparative example and the furanone derivatives prepared in Examples 1 to 7, respectively.

As shown in Figures 1 to 8, the medium using distilled water as a reference (dark green) was observed in the portion where the Agrobacterium tumefaciens A136 strain is located. In contrast, it was observed that almost no turquoise color was observed in the media in which the Agrobacterium tumefaciens A136 strain was located in the culture medium using the furanone derivative according to the present invention. This since, by Pugh ranon derivatives of the present invention to compete with the acyl homoserine lactone magnetic inductance acting on the receiving protein (receptor protein) of Agrobacterium tumefaciens KYC6 strain prevent gene expression, producing a galactosidase claim is Agrobacterium tumefaciens A136 strain go It can be seen that suppression of doing. Therefore, it can be confirmed that the furanone derivative of the present invention has an excellent function as a quorum sensing antagonist.

Evaluation of inhibitory effect on biofilm formation

Inhibition of the biofilm formation of the furanone derivatives prepared in Examples 1 to 7 was evaluated. The specimen was placed in a nutrient medium containing P. aeruginosa , a Gram-negative indicator microorganism with strong surface adhesion properties, and the furanone derivatives prepared in Examples 1 to 7 at a concentration of about 10 μmol / L, and then after 4 hours The total number of bacteria of the microorganisms attached to it was measured. In Comparative Example 1, the specimen was placed in a nutrient medium containing distilled water instead of the furanone derivative, and after 4 hours, the total number of microorganisms attached to the specimen was measured. In Comparative Example 2, the specimen was placed in a nutrient medium containing N-3-oxooctanoyl homoserine lactone, which is known as an autoinducer, and microorganisms attached to the specimen after 4 hours. The total bacterial count of was measured. The ratio of the population when the total population of the measured microorganisms and the population of Comparative Example 1 is 100% is shown in Table 1 below.

Number of Pseudomonas aeruginosa attached [CFU / cm ² ] ratio[%] Example 1 2.2 × 10 ⁷ 29 Example 2 1.4 × 10 ⁷ 19 Example 3 3.6 × 10 ⁷ 47 Example 4 1.0 × 10 ⁷ 14 Example 5 2.5 × 10 ⁷ 34 Example 6 2.6 × 10 ⁷ 34 Example 7 2.3 × 10 ⁷ 30 Comparative Example 1 7.5 × 10 ⁷ 100 Comparative Example 2 1.0 × 10 ⁸ 140

Referring to Table 1, in the nutrient medium containing the furanone derivatives prepared in Examples 1 to 7 compared to the nutrient medium containing distilled water or self-inducing substance according to Comparative Example 1 or 2, the number of Pseudomonas aeruginosa was much less attached. Able to know. From this, it can be seen that the furanone derivative of the present invention has excellent ability to inhibit biofilm formation.

The antimicrobial furanone derivatives of the present invention described above have excellent performance as quorum-sensing antagonists that interfere with communication between individuals of microorganisms. Accordingly, by interfering with the gene expression of the microorganism, it is possible to effectively block the formation of the biofilm, and to inhibit the reproduction of bacteria. In particular, the antimicrobial furanone derivatives of the present invention have a mechanism of inhibiting the propagation of bacteria by interfering with communication between bacteria, and thus acting in an amount of 1 / 1,000 to 1 / 100,000 compared to an antimicrobial agent having a mechanism of killing bacteria. Even if it is effective to inhibit bacterial growth. Therefore, it can be effectively applied to various household goods and medical devices that need to block the onset caused by bacteria in advance or to prevent the formation of biofilms.

As described above with reference to a preferred embodiment of the present invention, those skilled in the art or those skilled in the art without departing from the spirit and scope of the present invention described in the claims below It will be appreciated that various modifications and variations can be made in the present invention.

Claims (5)

delete delete A method of preventing the formation of a biofilm comprising contacting an antimicrobial furanone derivative represented by the following Structural Formula 1 with a microorganism to inhibit the formation of the biofilm.

...... (One) (In Formula 1, R represents an alkyl group having 1 to 10 carbon atoms or a haloalkyl group having 1 to 10 carbon atoms.) The method of claim 3, wherein the microorganism comprises Gram-negative bacteria. The method of claim 3, wherein the antimicrobial furanone derivative is represented by the following Structural Formulas 2 to 8.

...... (2)

...... (3)

...... (4)

...... (5)

(6)

(7)

...... (8)

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