KR20080060777A - Antibacterial amide derivative and method of preventing a biofilm formation using the same - Google Patents

Abstract

An antibacterial amide derivative is provided to interrupt communication between bacteria as a quorum sensing antagonist, so that the compound is useful for preventing the biofilm formation by bacteria and disease occurrence. An antibacterial amide derivative is represented by the formula(1) or formula(2), wherein R1 and R2 are each independently aromatic hydrocarbon group or aromatic hydrocarbon group having hetero atom. The biofilm formation is prevented by contacting the antibacterial amide derivative represented by the formula(1) or formula(2) with bacteria including gram negative bacteria.

Description

ANTIBACTERIAL AMIDE DERIVATIVE AND METHOD OF PREVENTING A BIOFILM FORMATION USING THE SAME}

1 to 5 are photographs showing the color change of the culture medium using the distilled water of Comparative Example and the antimicrobial amide derivatives prepared in Examples 1, 5, 9 and 13, respectively.

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

Bacteria are largely divided into planktonic cells and sessile cells. Specifically, the floating cells exist in the form of floating alone near the root of the plant and in the human or animal body. In contrast, adherent cells exist in the form of a biofilm attached to a surface in a microcolonies state surrounded by a polysaccharide matrix.

Until a few years ago, the presence of bacteria in the form of biofilms was unknown, and only recently were studies of fixed cells. Recent studies have shown that bacteria present in the form of biofilms have the ability to protect themselves very strongly from external attacks. In particular, bacteria in the form of biofilms have a strong resistance to antibacterial and disinfectants. In addition, new planktonic daughter cells are separated from the biofilm to form new colonies on the surface. As this proliferation is repeated, the area of the biofilm increases, and the bacterial population increases beyond the control of the population of bacteria.

As a bacterium having the characteristics of forming a biofilm as described above, Pseudomonas aeruginosa, which is one of Gram-negative bacteria, may be mentioned. Pseudomonas aeruginosa communicates with its neighbors through quorum sensing. The quorum sensing is a series in which each bacterial entity accumulates small molecule signaling materials such as autoinducer, pheromone or quomone outside the cell to induce active proliferation and fill the quorum. Refers to the biological phenomenon of.

As a signaling material used by the Pseudomonas aeruginosa for quorum sensing, butanoyl homoserine lactone, 3-oxododecanoyl homoserine lactone, 2-heptyl-3-hydroxy-4 2-heptyl-3-hydroxy-4-quinolone and the like are known. Pseudomonas aeruginosa does not form a biofilm without these signaling materials. Therefore, if a quorum-sensing antagonist having a chemical structure similar to that of the signaling material can be developed, it can interfere with communication between bacteria and inhibit reproduction and block the formation of a biofilm.

As a method of inhibiting the formation of a biofilm, a method of directly killing bacteria may be considered, but an antimicrobial agent that directly kills bacteria has a problem in that bacteria with strong resistance to antimicrobial agents such as superbacteria appear as the frequency of use increases. In contrast, when a quorum-sensing antagonist, which interferes with communication between bacteria, is used as an antimicrobial agent, the resistance of the antimicrobial agent 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 amide derivative capable of acting as a quorum sensing antagonist of bacteria to inhibit the onset and the formation of a biofilm.

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

The antimicrobial amide derivative 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) or (2).

...... (1)

...... (One)

...... (2)

...... (2)

In the formulas 1 and 2, R ₁ and R ₂ each independently represent an aromatic hydrocarbon group or an aromatic hydrocarbon group containing a hetero atom.

In the method for preventing the formation of a biofilm according to another embodiment of the present invention, the antimicrobial amide derivative represented by Formula 1 or 2 is contacted with a microorganism to suppress the formation of the biofilm.

As described above, the antimicrobial amide derivative of the present invention has a structure in which butyric acid or 3-oxododecanoic acid is amide-bonded with an aromatic compound having an amine group. Antimicrobial amide derivatives have a chemical structure similar to that used by bacteria to communicate with them, acting as a quorum sensing antagonist that interferes with the communication between them. Accordingly, the antimicrobial amide derivatives can effectively block the growth of microorganisms and the formation of biofilms.

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

Antimicrobial Amide Derivatives

The antimicrobial amide derivative of the present invention has a structure represented by the following formula (1) or (2).

...... (1)

...... (One)

...... (2)

...... (2)

In the formulas 1 and 2, R ₁ and R ₂ each independently represent an aromatic hydrocarbon group or an aromatic hydrocarbon group containing a hetero atom. As a specific example of R <_1> and R <_2> , the phenyl group, pyrimidinyl group, etc. which contain an alkyl group, an alkoxy group, a halogen atom, a thiol group, a hydroxyl group, a hydroxyalkyl group, an alkylthio group, etc. are mentioned.

The compound represented by Formula 1 is a butylamide derivative, which is similar to butanoyl homoserine lactone, a low molecular weight signaling material produced by bacteria for gene expression, that is, an autoinducer. Has a chemical structure That is, the butylamide group corresponding to a tail part is the same, and the substituent of a head part differs from each other. In addition, the compound represented by Structural Formula 2 is a 3-oxododecanamide derivative, a chemical similar to 3-oxododecanoyl homoserine lactone, which is a self-inducer of bacteria. It has a structure. That is, the 3-oxododecaneamide groups corresponding to the tail portion are identical to each other, and the substituents in the head portion are different from each other.

As described above, the antimicrobial amide derivative of the present invention represented by Structural Formulas 1 or 2 has a chemical structure similar to that of bacteria inducing bacteria, and thus competitively acts with a self-inducing agent on a regulatory protein that induces bacterial growth or formation of a biofilm. Thereby inhibiting the expression of the gene. Accordingly, the antimicrobial amide derivative of the present invention may function as a quorum sensing antagonist that interferes with communication of bacteria.

According to one embodiment of the present invention, the antimicrobial butylamide derivative represented by Structural Formula 1 may be represented by the following Structural Formulas 3 to 10.

...... (3)

...... (3)

...... (4)

...... (4)

...... (5)

...... (5)

...... (6)

(6)

...... (7)

(7)

...... (8)

...... (8)

...... (9)

(9)

...... (10)

...... (10)

In addition, the antimicrobial 3-oxododecaneamide derivative represented by Formula 2 may be represented by the following Formula 11 or 18.

...... (11)

...... (11)

...... (12)

...... (12)

...... (13)

...... (13)

...... (14)

...... (14)

...... (15)

...... (15)

...... (16)

...... (16)

...... (17)

...... (17)

...... (18)

...... (18)

The antimicrobial amide derivative represented by Formula 1 or 2 is prepared by combining an aromatic compound having an amine group with butyric acid or 3-oxododecanoic acid. For example, the antimicrobial amide derivative represented by Formula 3 is prepared by reacting butyric acid with 2-amino-5-chlorophenol (2-amino-5-chlorophenol).

The antimicrobial amide derivatives of the present invention described above have a chemical structure similar to that used by bacteria to communicate, and thus function as a quorum sensing antagonist that interferes with communication between bacteria. Accordingly, the antimicrobial amide derivatives can effectively block the growth of microorganisms and the formation of biofilms.

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 above-described antimicrobial amide derivatives.

Antimicrobial amide derivatives of the present invention has a structure represented by the following formula 1 or 2, it can interfere with the communication between bacteria to inhibit the reproduction and 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 numerous diseases while staying in the human organs and living environment, the antimicrobial amide derivative of the present invention can effectively block the formation of such biofilm in contact with microorganisms.

...... (1)

...... (One)

...... (2)

...... (2)

In the formulas 1 and 2, R ₁ and R ₂ each independently represent an aromatic hydrocarbon group or an aromatic hydrocarbon group containing a hetero atom.

The compound represented by the formula 1 is a butylamide derivative (butyramide derivative), the compound represented by the formula 2 is a 3-oxododecanamide derivative (3-oxododecanamide derivative), a signal that bacteria produce for gene expression It has a chemical structure similar to a substance.

For example, the antimicrobial amide derivative represented by Structural Formulas 1 or 2 may be a butanoyl homoserine lactone or 3-oxododecanoyl homoserine lactone, which Gram-negative bacteria such as Pseudomonas aeruginosa are used as a signaling material. It has a chemical structure similar to that of oxododecanoyl homoserine lactone. That is, the butylamide group or 3-oxododecaneamide group corresponding to a tail part is the same, and the substituent of a head part differs from each other.

As described above, the antimicrobial amide derivative of the present invention represented by Structural Formulas 1 or 2 has a chemical structure similar to that of bacteria inducing bacteria, and thus competitively acts with a self-inducing agent on a regulatory protein that induces bacterial growth or formation of a biofilm. Thereby inhibiting the expression of the gene. Accordingly, the antimicrobial amide derivative of the present invention functions as a quorum sensing antagonist that interferes with the communication of bacteria.

As described above, the antimicrobial amide derivative of the present invention is an excellent quorum-sensing antagonist of Gram-negative bacteria, which can prevent the growth of microorganisms by interfering with communication between microorganisms and effectively block the formation of biofilms. Therefore, the antimicrobial amide derivative of the present invention can be applied to a device, a medical device or an implant in a human body in which the biofilm is easily formed, 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 Amide Derivatives

The antimicrobial amide derivative represented by Structural Formula 1 was prepared according to the following Schemes (1) to (4). In addition, the antimicrobial amide derivative represented by Structural Formula 2 was prepared according to the following Schemes (1) to (4 ').

Example  One

Tetrahydrofuran, a solvent, aminomethyl polystyrene beads (aminomethyl polystyrene bead, 1) 2.2 mmol / g, 2,4,6-trichloro-1,3,5-triazine (2,4,6-trichloro-1, 2 equivalents of 3,5-triazine) and 5 equivalents of N, N-diisopropylethylamine (DIPEA) were added and reacted according to Scheme (1) for about 10 minutes. Got it. The ninhydrine test was performed on the primary resultant (2) to confirm that the amine group disappeared. The solvent was filtered off and the remaining primary product (2) was washed with tetrahydrofuran, methylene chloride and methanol, dried and stored at about 4 ° C.

After swelling the primary resultant (2) with tetrahydrofuran, 1.5 equivalents of N-methylmorpholine (NMM) was added thereto and reacted according to Scheme (2) to obtain a secondary resultant (3). 2 equivalents of butyric acid was added to the secondary product (3), and reacted according to the reaction formula (3) under a nitrogen atmosphere for about 3 hours to obtain a tertiary product (4). The third resultant (4) was washed with tetrahydrofuran and dimethylformamide and dried. To the tetrahydrofuran solvent, add the third product (4), 1.2 equivalents of 4-chloro-2-hydroxyaniline and 1 equivalent of N-methylmorpholine, and react for about 3 hours. Reacted accordingly. The solid was filtered off and the remaining filtrate was removed with amine using a cation exchange resin to obtain a 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. In the hydrogen nuclear magnetic resonance spectrum, the chemical shift (δ) is 10.00 (s, 1H), 8.8 (s, 1H), 7.67 (d, J = 6.6, 1H), 6.74 (d, J = 2.4, 1H), 6.68 ( s, 1H), 2.33 (t, 2H), 1.73 (m, 2H) and 1.00 (t, 3H). In addition, in the mass spectrum obtained by using High Resolution Mass Spectroscopy (HRMS), a peak was found at 213.2768 corresponding to C ₁₀ H ₁₂ ClNO ₂ (M ⁺ + 1, 213.2768). Therefore, the final result (5) was confirmed to be normal- (4-chloro-2-hydroxy phenyl) butylamide (N- (4-chloro-2-hydroxyphenyl) butyramide) represented by the following formula (3).

...... (3)

...... (3)

Example 2

The final product (5) was obtained in substantially the same manner as in Example 1 except for using pyrimidin-2-amine instead of 4-chloro-2-hydroxyaniline.

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 10.30 (s, 1H), 8.9 (d, 2H), 7.69 (t, 1H), 2.28 (t, 2H), 1.71 (m, 2H), and 1.00 ( t, 3H). In addition, in the mass spectrum, Peak appeared at 165.1949 corresponding to C ₈ H ₁₁ N ₃ O (M ⁺ + 1, 165.1950). Therefore, the final result (5) was confirmed to be normal- (pyrimidin-2-yl) butylamide (N- (pyrimidin-2-yl) butyramide) represented by the following structural formula 4.

...... (4)

...... (4)

Example 3

The final product (5) was obtained in substantially the same manner as in Example 1 except for using 2-methylaniline instead of 4-chloro-2-hydroxyaniline.

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 shift (δ) is 10.01 (s, 1H), 7.67 (d, J = 6.6, 1H), 7.17 (m, 1H), 7.07 (d, J = 6.4, 1H), 6.89 (m, 1H), 2.38 (s, 3H), 2.33 (t, 2H), 1.73 (m, 2H) and 1.00 (t, 3H). In the mass spectrum, the peak was found at 177.2463 corresponding to C ₁₁ H ₁₅ NO (M ⁺ + 1, 177.2462). Therefore, the final result (5) was confirmed to be normal-ortho- toryl butyamide (No-tolylbutyramide) represented by the following structural formula 5.

...... (5)

...... (5)

Example 4

The final product (5) was obtained in substantially the same manner as in Example 1 except for using 2-methoxyaniline instead of 4-chloro-2-hydroxyaniline.

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, Chemical Shift (δ) is 10.01 (s, 1H), 7.73 (d, J = 7.6, 1H), 7.27 (m, 1H), 7.17 (d, J = 6.6, 1H), 6.99 ( m, 1H), 3.89 (s, 3H), 2.34 (t, 2H), 1.73 (m, 2H) and 1.00 (t, 3H). In the mass spectrum, the peak was found at 193.2457 corresponding to C ₁₁ H ₁₅ NO ₂ (M ⁺ + 1, 193.2456). As a result, the final product (5) was confirmed to be normal- (2-methoxyphenyl) butylamide (N- (2-methoxyphenyl) butyramide) represented by the following formula (6).

...... (6)

(6)

Example 5

The final product (5) was obtained in substantially the same manner as in Example 1 except for using 2-aminobenzenethiol instead of 4-chloro-2-hydroxyaniline.

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, Chemical Shift (δ) is 10.01 (s, 1H), 7.63 (d, J = 6.9, 1H), 7.29 (m, 1H), 7.18 (d, J = 6.7, 1H), 6.96 ( m, 1H), 3.00 (s, 1H), 2.34 (t, 2H), 1.73 (m, 2H) and 1.00 (t, 3H). C ₁₀ H ₁₃ NOS in the mass spectrum The peak was found at 195.2853, corresponding to (M ⁺ + 1, 195.2854). As a result, the final product (5) was confirmed to be normal- (2-mercapto phenyl) butylamide (N- (2-mercaptophenyl) butyramide) represented by the following formula (7).

...... (7)

(7)

Example 6

The final product (5) was obtained in substantially the same manner as in Example 1, except that 2- (methylthio) benzenamine was used instead of 4-chloro-2-hydroxyaniline. It was.

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 shift (δ) is 10.01 (s, 1H), 7.31-7.17 (m, 2H), 7.05 (m, 2H), 2.51 (s, 3H), 2.34 (t, 2H), 1.73 (m, 2H) and 1.00 (t, 3H). The mass spectrum showed a peak at 209.3122, which corresponds to C ₁₁ H ₁₅ NOS (M ⁺ + 1, 209.3122). As a result, the final product (5) was confirmed to be normal- (2- (methylthio) phenyl) butylamide (N- (2- (methylthio) phenyl) butyramide) represented by the following structural formula (8).

...... (8)

...... (8)

Example 7

The final result (5) was obtained in substantially the same manner as in Example 1 except for using 2-hydroxymethylaniline instead of 4-chloro-2-hydroxyaniline.

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, Chemical Shift (δ) is 10.01 (s, 1H), 7.78 (d, J = 7.1, 1H), 7.39 (m, 1H), 7.17 (d, J = 6.7, 1H), 6.95 ( m, 1H), 5.4 (s, 1H), 4.82 (s, 2H), 2.34 (t, 2H), 1.73 (m, 2H) and 1.00 (t, 3H). The mass spectrum showed a peak at 193.2457, which corresponds to C ₁₁ H ₁₅ NO ₂ (M ⁺ + 1, 193.2456). As a result, the final product (5) was confirmed to be normal- (2- (hydroxymethyl) phenyl) butylamide (N- (2- (hydroxymethyl) phenyl) butyramide) represented by the following structural formula (9).

...... (9)

(9)

Example 8

The final product (5) was obtained in substantially the same manner as in Example 1, except that 4-chloro-2-methylaniline was used instead of 4-chloro-2-hydroxyaniline. Obtained.

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 10.01 (s, 1H), 7.66 (d, J = 6.9, 1H), 7.17 (d, J = 6.8, 1H), 7.14 (s, 1H), 2.41 ( s, 3H), 2.33 (t, 2H), 1.73 (m, 2H) and 1.00 (t, 3H). The mass spectrum showed a peak at 211.691, corresponding to C ₁₁ H ₁₄ ClNO (M ⁺ + 1, 211.691). As a result, the final product (5) was confirmed to be normal- (4-chloro-2-methylphenyl) butylamide (N- (4-chloro-2-methylphenyl) butyramide) represented by the following structural formula (10).

...... (10)

...... (10)

Example 9

First, 3,3-ethylene dioxononanoic acid (8) used in the preparation of the antimicrobial amide derivative represented by Structural Formula 2 was synthesized according to the following scheme.

Specifically, 21.6 g (71.5 mmol) of bis-trimethylsilyl malonate was added to 100 mL of anhydrous diethyl ether, followed by cooling to a temperature of -78 ° C. To the cooled solution was slowly added 44.7 mL of diethyl ether containing n-butyl lithium (nBuLi) at a concentration of 1.6 M (71.5 mmol), and maintained at a temperature of -60 ° C or lower. 5 mL (35.75 mmol) of decanoyl chloride was added rapidly, followed by stirring at a temperature of −10 ° C. for 30 minutes. To this, 150 mL of cold 5% sodium hydrogen carbonate (NaHCO ₃ , sodium bicarbonate) solution was added and stirred vigorously for 30 minutes. The aqueous layer was separated and the pH was adjusted to 2 using cold 4N sulfuric acid. 50 mL of diethyl ether was added to the aqueous solution layer, and extracted twice. Magnesium sulfate (MgSO ₄ ) was added to remove water. It dried under reduced pressure and obtained the primary compound (6) which is a white solid. The primary compound (6) was purified by recrystallization using hexane (hexane). The yield of the primary composite 6 was about 87%.

35 mmol of the primary compound (6) was dissolved in 150 mL of a mixed solvent containing benzene and methanol in a volume ratio of 4: 1, and then (trimethylsilyl) diazomethane (TMSCHN ₂ ) was added to 2 M (42 mmol). Add 21 mL of diethyl ether in concentration over about 10 minutes. After reacting for 30 minutes, the mixture was dried under reduced pressure to obtain methyl-3-oxododecanoate, a secondary compound (7) in the form of a yellow oil, in a yield of 95%.

32.5 mmol of methyl-3-oxododecanoate) was dissolved in 125 mL of benzene, and 20.2 g (325 mmol) of ethylene glycol and 0.617 g (3.25 mmol) of para-toluenesulfonic acid (pTsOH) were added thereto. Heated for 24 hours. After drying under reduced pressure, the mixture was diluted with 100 mL of diethyl ether. The organic layer was washed twice with 25 mL of 10% aqueous sodium hydroxide (NaOH) solution, twice with 25 mL of saturated sodium chloride (NaCl) aqueous solution, and then water was removed with magnesium sulfate (MgSO ₄ ) to obtain an oil. The oil was saponified for 6 hours using 150 mL of a 1N sodium hydroxide (NaOH) aqueous solution and 75 mL of methanol. After drying under reduced pressure and cooling, the mixture was adjusted to pH 2 with hydrochloric acid. The organic component was extracted with 75 mL of diethyl ether, and water was removed with magnesium sulfate (MgSO ₄ ) to obtain 3,3-ethylene dioxononanoic acid (3,3-ethylene dioxononanoic acid) as a tertiary compound (8) in 50% yield. )

On the other hand, the secondary resultant (3) was obtained by substantially the same method as in Example 1. Instead of the butyric acid of Example 1, the 3,3-ethylene dioxonanoic acid synthesized as described above was reacted with the secondary product (3) for about 3 hours under nitrogen atmosphere according to reaction formula (3 '). ) Thereafter, the final result 5 'was obtained in substantially the same manner as in Example 1.

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, Chemical Shift (δ) is 10.00 (s, 1H), 8.8 (s, 1H), 7.67 (d, J = 6.6, 1H), 6.74 (d, J = 2.4, 1H), 6.68 ( s, 1H), 3.43 (s, 2H), 2.53 (t, 2H), 1.63 (m, 2H), 1.30-1.26 (m, 12H) and 0.90 (t, 3H). The mass spectrum showed a peak at 339.8621, corresponding to C ₁₈ H ₂₆ ClNO ₃ (M ⁺ + 1, 339.8621). As a result, the final product (5 ') was normal- (4-chloro-2-hydroxyphenyl) -3-oxododecaneamide (N- (4-chloro-2-hydroxyphenyl) -3 represented by the following structural formula 11). -oxododecanamide).

...... (11)

...... (11)

Example 10

The final product 5 'was obtained in substantially the same manner as in Example 9 except that pyrimidin-2-amine was used instead of 4-chloro-2-hydroxyaniline.

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 10.30 (s, 1H), 8.9 (d, 2H), 7.69 (t, 1H), 3.43 (s, 2H), 2.53 (t, 2H), and 1.63 (m). , 2H), 1.30-1.26 (m, 12H) and 0.90 (t, 3H). The mass spectrum showed a peak at 291.3935, corresponding to C ₁₆ H ₂₅ N ₃ O ₂ (M ⁺ + 1, 291.3935). As a result, the final result (5 ') is 3-oxo-normal- (pyrimidin-2-yl) dodecaneamide represented by the following Structural Formula 12 (3-oxo-N- (pyrimidin-2-yl) dodecanamide) It was confirmed.

...... (12)

...... (12)

Example 11

The final product 5 'was obtained in substantially the same manner as in Example 9 except for using 2-methylaniline instead of 4-chloro-2-hydroxyaniline.

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, Chemical Shift (δ) is 10.01 (s, 1H), 7.67 (d, J = 6.6, 1H), 7.17 (m, 1H), 7.07 (d, J = 6.4, 1H), 6.89 ( m, 1H), 3.43 (s, 2H), 2.52 (m, 2H), 2.38 (s, 3H), 1.63 (m, 2H), 1.30-1.26 (m, 12H) and 0.90 (t, 3H). . The mass spectrum showed a peak at 303.4448, corresponding to C ₁₉ H ₂₉ NO ₂ (M ⁺ + 1, 303.4448). As a result, the final result (5 ') was confirmed to be 3-oxo-normal-o-toryldodecaneamide represented by the following structural formula (13).

...... (13)

...... (13)

Example 12

The final resultant 5 'was obtained in substantially the same manner as in Example 9 except for using 2-methoxyaniline instead of 4-chloro-2-hydroxyaniline.

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, Chemical Shift (δ) is 10.01 (s, 1H), 7.73 (d, J = 7.6, 1H), 7.27 (m, 1H), 7.17 (d, J = 6.6, 1H), 6.99 ( m, 1H), 3.89 (s, 3H), 3.43 (s, 2H), 2.53 (t, 2H), 1.63 (m, 2H), 1.30-1.26 (m, 12H) and 0.90 (t, 3H). . The mass spectrum showed a peak at 319.4442, corresponding to C ₁₉ H ₂₉ NO ₃ (M ⁺ + 1, 319.4442). As a result, the final product (5 ') was confirmed to be normal- (2-methoxyphenyl) -3-oxododecaneamide (N- (2-methoxyphenyl) -3-oxododecanamide) represented by the following structural formula (14).

...... (14)

...... (14)

Example 13

The final product 5 'was obtained in substantially the same manner as in Example 9 except for using 2-aminobenzenethiol instead of 4-chloro-2-hydroxyaniline.

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, Chemical Shift (δ) is 10.01 (s, 1H), 7.63 (d, J = 6.9, 1H), 7,29 (m, 1H), 7.18 (d, J = 6.7, 1H), 6.96 (m, 1H), 3.43 (s, 2H), 3.00 (s, 1H), 2.53 (t, 2H), 1.63 (m, 2H), 1.30-1.26 (m, 12H), and 0.90 (t, 3H) Appeared. The mass spectrum showed a peak at 321.4839, corresponding to C ₁₈ H ₂₇ NO ₂ S (M ⁺ + 1, 321.4839). As a result, the final product (5 ') was confirmed to be normal- (2-mercaptophenyl) -3-oxododecaneamide (N- (2-mercaptophenyl) -3-oxododecanamide) represented by the following structural formula (15).

...... (15)

...... (15)

Example 14

The final product (5 ') was obtained in substantially the same manner as in Example 9, except that 2- (methylthio) benzeneamine was used instead of 4-chloro-2-hydroxyaniline. Obtained.

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 shift (δ) is 10.01 (s, 1H), 7.31-7.17 (m, 2H), 7.05 (m, 2H), 3.43 (s, 2H), 2.53-2.51 (m, 5H) , 1.63 (m, 2H), 1.30-1.26 (m, 12H) and 0.90 (t, 3H). The mass spectrum showed a peak at 335.5108 corresponding to C ₁₉ H ₂₉ NO ₂ S (M ⁺ + 1, 335.5108). As a result, the final result (5 ') was normal- (2- (methylthio) phenyl) -3-oxododecaneamide represented by the following structural formula 16 (N- (2- (methylthio) phenyl) -3-oxododecanamide) It was confirmed to be.

...... (16)

...... (16)

Example 15

The final product 5 'was obtained in substantially the same manner as in Example 9 except for using 2-hydroxymethylaniline instead of 4-chloro-2-hydroxyaniline.

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, Chemical Shift (δ) is 10.01 (s, 1H), 7.78 (d, J = 7.1, 1H), 7.39 (m, 1H), 7.17 (d, J = 6.7, 1H), 6.95 ( m, 1H), 5.4 (s, 1H), 4.82 (s, 2H), 3.43 (s, 2H), 2.53 (t, 2H), 1.63 (m, 2H), 1.30-1.26 (m, 12H) and 0.90 (t, 3H). The mass spectrum showed a peak at 319.4442, corresponding to C ₁₉ H ₂₉ NO ₃ (M ⁺ + 1, 319.4442). As a result, the final result (5 ') was normal- (2- (hydroxymethyl) phenyl) -3-oxododecaneamide represented by the following structural formula 17 (N- (2- (hydroxymethyl) phenyl) -3-oxododecanamide ).

...... (17)

...... (17)

Example 16

The final result (5 ′) was substantially the same as Example 9, except that 4-chloro-2-methylaniline was used instead of 4-chloro-2-hydroxyaniline. Obtained.

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, Chemical Shift (δ) is 10.01 (s, 1H), 7.66 (d, J = 6.9, 1H), 7.17 (d, J = 6.8, 1H), 7.14 (s, 1H), 3.43 ( s, 2H), 2.53 (t, 2H), 2.41 (s, 3H), 1.63 (m, 2H), 1.30-1.26 (m, 12H) and 0.90 (t, 3H). The mass spectrum showed a peak at 337.8895, corresponding to C ₁₉ H ₂₈ ClNO ₂ (M ⁺ + 1, 337.8896). As a result, the final product (5 ') was normal- (4-chloro-2-methylphenyl) -3-oxododecaneamide represented by the following structural formula 18 (N- (4-chloro-2-methylphenyl) -3-oxododecanamide ).

...... (18)

...... (18)

Functional Evaluation as a Quorum Sensing Antagonist

The antimicrobial amide derivatives obtained in Examples 1 to 16 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 an autoinducer, 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 KYC6 strain and 100uL of the antimicrobial amide derivatives prepared in Examples 1 to 16 were added to 5 mL of LB culture medium, and then cultured at about 30 ° C. for about 24 hours. In addition, Agrobacterium tumefaciens A136 strain was placed in an LB culture medium containing 50ug / mL spectinomycin and 4.5ug / mL tetracycline, and incubated overnight at about 30 ° C. Also, as a comparative example, Agrobacterium Agrobacterium tumefaciens KYC6 strain was cultured by adding distilled water to the culture medium of the tumefaciens KYC6 strain instead of the antimicrobial amide 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 culture medium instead of the antimicrobial amide derivative.

1 to 5 are photographs showing the color change of the culture medium using the distilled water of Comparative Example and the antimicrobial amide derivatives prepared in Examples 1, 5, 9 and 13, respectively.

As shown in Figures 1 to 5, 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 the medium cultured using the antimicrobial amide derivative according to the present invention showed a light cyan color or almost no cyan color in the region where the Agrobacterium tumefaciens A136 strain is located. Although not shown in the drawings, it was observed that even in medium cultured with the antimicrobial amide derivatives prepared in the remaining examples, light blue-green color or almost blue-green color appeared in the region where the Agrobacterium tumefaciens A136 strain is located. Therefore, From the antibacterial amide 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 antimicrobial amide derivative of the present invention has an excellent function as a quorum sensing antagonist.

Evaluation of inhibitory and antimicrobial activity on biofilm formation

Inhibition of the biofilm formation of the antimicrobial amide derivatives prepared in Examples 1 to 16 was evaluated. The specimen was placed in a nutrient medium containing P. aeruginosa , a Gram-negative indicator microorganism with strong surface adhesion, and the antimicrobial amide derivatives prepared in Examples 1 to 16 at a concentration of about 10 μmol / L. The total number of bacteria of the microorganisms attached to it was measured. In addition, in Comparative Example 1, the specimen was placed in a nutrient medium containing distilled water instead of the antimicrobial amide 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-oxododecanoyl homoserine lactone, which is known as an autoinducer, and after 4 hours, the specimen was attached to the specimen. The total bacterial count of the microorganisms 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 1.2 × 10 ⁷ 16 Example 2 4.1 × 10 ⁷ 55 Example 3 2.4 × 10 ⁷ 32 Example 4 2.3 × 10 ⁷ 31 Example 5 1.9 × 10 ⁷ 25 Example 6 2.3 × 10 ⁷ 31 Example 7 1.8 × 10 ⁷ 24 Example 8 2.4 × 10 ⁷ 32 Example 9 1.0 × 10 ⁷ 13 Example 10 3.9 × 10 ⁷ 52 Example 11 2.3 × 10 ⁷ 31 Example 12 2.1 × 10 ⁷ 28 Example 13 1.6 × 10 ⁷ 21 Example 14 2.0 × 10 ⁷ 27 Example 15 1.5 × 10 ⁷ 20 Example 16 2.0 × 10 ⁷ 27 Comparative Example 1 7.5 × 10 ⁷ 100 Comparative Example 2 1.2 × 10 ⁸ 140

Referring to Table 1, in the nutrient medium containing the antimicrobial amide derivatives prepared in Examples 1 to 16, the number of Pseudomonas aeruginosa adhered was much smaller than that of the nutrient medium containing distilled water or self-inducing substance according to Comparative Example 1 or 2. Able to know.

The antimicrobial amide derivative of the present invention described above has excellent performance as a quorum sensing antagonist that interferes with communication between individuals of Gram-negative bacteria such as Pseudomonas aeruginosa. As a result, the gene expression of the microorganisms may be disturbed, thereby effectively blocking the formation of the biofilm and preventing the growth of the microorganisms. In particular, the antimicrobial amide derivative of the present invention has a mechanism of inhibiting the growth of bacteria by interfering with communication between bacteria, and even if the amount of 1 / 1,000 to 1 / 100,000 compared to the antimicrobial agent having a mechanism for killing bacteria It is effective in inhibiting bacterial growth. Therefore, it can be effectively applied to various life tools and medical equipment that need to block the occurrence of bacteria in advance or 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)

An antimicrobial amide derivative represented by the following structural formula (1) or (2).

...... (One)

...... (2) (In the above formulas 1 and 2, R ₁ and R ₂ each independently represent an aromatic hydrocarbon group or an aromatic hydrocarbon group containing a hetero atom) The antimicrobial amide derivative according to claim 1, wherein the compound represented by Formula 1 is represented by the following Formulas 3 to 10.

...... (3)

...... (4)

...... (5)

(6)

(7)

...... (8)

(9)

...... (10) The antimicrobial amide derivative according to claim 1, wherein the compound represented by Formula 2 is represented by the following Formulas 11 to 18.

...... (11)

...... (12)

...... (13)

...... (14)

...... (15)

...... (16)

...... (17)

...... (18) A method for preventing the formation of a biofilm comprising contacting an antimicrobial amide derivative represented by the following Structural Formula 1 or 2 with a microorganism to inhibit the formation of the biofilm.

...... (One)

...... (2) (In the above formulas 1 and 2, R ₁ and R ₂ each independently represent an aromatic hydrocarbon group or an aromatic hydrocarbon group containing a hetero atom) The method of claim 4, wherein the microorganism comprises Gram-negative bacteria.

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