KR102361131B1 - Composition for antibacterial activity of multi-drug resistant bacteria - Google Patents

Abstract

The present invention relates to an antibacterial composition having antibacterial activity against antibiotic-resistant strains, and more particularly, to a composition comprising an antimicrobial compound exhibiting antibacterial activity targeting the cell membrane of multidrug-resistant bacteria of gram-positive bacteria. The composition contains triphenylphosphonium (TPP ⁺ ), which is a positively charged lipid-affinity cationic compound and targets mitochondria, and thus targets the cell membrane anionic lipids of bacteria, Gram-positive bacteria, particularly Staphylococcus aureus and It shows a low MIC value for metacillin-resistant Staphylococcus aureus and shows antibacterial activity by suppressing the outflow of antibiotics introduced into cells by reducing the expression of drug outflow genes. applicable.

Description

Antibacterial composition having antibacterial activity against antibiotic-resistant strains {Composition for antibacterial activity of multi-drug resistant bacteria}

The present invention relates to an antimicrobial composition having antimicrobial activity against antibiotic-resistant strains, and more particularly, to a composition comprising an antimicrobial compound exhibiting antimicrobial activity by targeting the cell membrane of Gram-positive multidrug-resistant bacteria.

Bacterial infection is one of the most common and fatal causes of human diseases. In diseases caused by bacterial infection, the use of antibiotics is inevitable. After the discovery of the first antibiotic penicillin, numerous types of antibiotics have been developed and used for the treatment of bacterial infections. However, in recent years, such misuse and abuse of antibiotics is regarded as a global problem due to the emergence of antibiotic-resistant strains and further, multi-drug resistant bacteria (MDR) that are resistant to various antibiotics. The emergence of such multidrug-resistant bacteria limits the types of antibiotics that can be used in bacterial infectious diseases, and thus causes great difficulties in disease treatment and at the same time causes burdens such as extension of treatment period and increase in medical costs. There is this.

To overcome these resistant bacteria, many new antibiotics have been developed so far, and about 160 antibacterial agents are being used clinically. Currently, there are 100 registered antibiotics in Korea, but antibiotics that are effective against resistant bacteria are sulphonamides and penicillin, followed by aminoglycosides, tetracyclines, cephalosporins, macrolides, glycopeptides, quinolones, and carbapenems. The need to develop antibiotics to combat resistant bacterial infections is increasing.

In 2002, vancomycin-resistant S. aureus, which showed severe resistance to vancomycin, which the U.S. Centers for Disease Control said in the last treatment for Staphylococcus aureus, the most common cause of infection in humans, VRSA) has been reported for the first time in the world, significantly increasing the possibility of the spread of multidrug-resistant bacteria. In particular, after vancomycin resistant enterococcus (VRE) was first discovered in Europe in 1988, vancomycin intermediate-resistant S. aureus (VISA) occurred in France and Korea. As it is emerging as a global problem, the development of a new concept of antibiotics is urgently required.

In order to overcome these major multidrug-resistant bacteria, many studies such as various anti-toxic, nanomaterials and drug delivery systems are being conducted (Korean Patent Registration No. 10-1599587). Recently, research on antibacterial substances using antibacterial peptides and bacteriophages has been actively conducted. In particular, in the development of antibiotics using antimicrobial peptides, the positively charged portion of the peptide binds to the negatively charged bacterial cell membrane to change the potential of the cell membrane, or to form a pore in the cell membrane itself to improve membrane permeability. It is made due to the antibacterial action mechanism that destroys cells by changing the membrane and fluidity. As such, various efforts have been made to develop antibiotics, but these methods still have many difficulties until they become drugs for clinical use.

In order to solve the above problems, the present inventors have previously used triphenylphosphonium (TPP ⁺ ) as a strategy for targeting anionic lipids in the cell membrane of bacteria to ciproproxacin, which is used to treat gram-negative and gram-positive bacteria. to synthesize two types of ciproproxacin derivatives chemically combined with the carboxylic acid of ciproproxacin through -ester and -amide bonds, By confirming the antibacterial activity, the present invention was completed.

Accordingly, an object of the present invention is to provide an antibacterial composition comprising a compound having antibacterial properties against antibiotic-resistant strains.

In order to achieve the above object,

The present invention provides an antibacterial composition for antibiotic-resistant strains comprising a compound represented by the following [Formula 1].

[Formula 1]

A description of the structure and substituents of [Formula 1] will be described later.

In one embodiment of the present invention, D is flumequine, oxolinic acid, rosoxacin, cinoxacin, nalidixic acid, piromidic acid ), pipemitic acid, ciprofloxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin (ofloxacin) Pefloxacin, rufloxacin, enoxacin, balofloxacin, grepafloxacin, levofloxacin, pazufloxacin, sparfloxacin sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, gatifloxacin, moxifloxacin, sitafloxacin, sitafloxacin Roxacin (prulifloxacin), besifloxacin (besifloxacin), gemifloxacin (gemifloxacin), trovafloxacin (trovafloxacin), delafloxacin (delafloxacin), danofloxacin (danofloxacin), difloxacin (difloxacin) It may be selected from the group consisting of rofloxacin, ibafloxacin, marbofloxacin, orbifloxacin and sarafloxacin.

In another embodiment of the present invention, the strain may be a gram-positive strain.

In another embodiment of the present invention, the Gram-positive bacteria may be Entrococcus faecium or Staphylococcus aureus.

In another embodiment of the present invention, the Entrococcus pecium may be vancomycin-resistant Entrococcus pecium (VRE).

In another embodiment of the present invention, the Staphylococcus aureus may be methicillin-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Staphylococcus aureus (VISA).

In another embodiment of the present invention, the compound of [Formula 1] may be a compound represented by the following [Formula 2] or [Formula 3].

[Formula 2]

[Formula 3]

In another embodiment of the present invention, the composition can directly target the cell membrane of Gram-positive bacteria.

In another embodiment of the present invention, the composition may inhibit biofilm formation of Gram-positive bacteria.

In another embodiment of the present invention, the composition may suppress the outflow of drugs introduced into cells by reducing the expression of norA , sepA or medA genes.

The composition comprising the antimicrobial compound according to the present invention is a positively charged lipid-affinity cationic compound and contains triphenylphosphonium (TPP ⁺ ) that targets mitochondria, so that it targets anionic lipids in the cell membrane of bacteria. , Gram-positive bacteria, particularly Staphylococcus aureus and metacillin-resistant Staphylococcus aureus, show a low MIC value, and inhibit the outflow of antibiotics introduced into cells by reducing the expression of drug outflow genes, thereby showing antibacterial activity. Various biological applications are possible, such as in the medical industry or food additives.

1 is a graph showing the MIC results for viewing the antibacterial activity of ciproproxacin derivatives CFX-ester-PPh ₃ and CFX-amide-PPh ₃ against multi-drug-resistant Gram-negative and positive bacteria.
Figure 2 shows a biofilm formation inhibition screening graph of ciproproxacin derivatives CFX-ester-PPh ₃ and CFX-amide-PPh ₃ for multidrug-resistant Gram-negative bacteria.
Figure 3 shows a biofilm formation inhibition screening graph of ciproproxacin derivatives CFX-ester-PPh ₃ and CFX-amide-PPh ₃ for multi-drug-resistant Gram-positive bacteria.
Figure 4 is treated with ciproproxacin derivatives CFX-ester-PPh ₃ and CFX-amide-PPh ₃ in MSSA and MRSA, and shows a graph showing the antibacterial effect over time.
5 is a graph showing the growth curve in the MIC of Gram-positive Staphylococcus aureus and methicillin Staphylococcus aureus.
6 shows TEM images for morphological observation of ciproproxacin derivatives CFX-ester-PPh ₃ and CFX-amide-PPh ₃ treated with MSSA and MRSA.
7 is a graph showing the results of confirming the mRNA expression patterns of multiple efflux pump-related genes for the ciproproxacin derivatives CFX-ester-PPh ₃ and CFX-amide-PPh ₃ treated with MSSA and MRSA through qRT-PCR. .
8 is a summary of the results of the antimicrobial effect on the compound according to the present invention and its multidrug-resistant bacteria.

Hereinafter, the present invention will be described in more detail.

The present inventors have previously used triphenylphosphonium (TPP ⁺ ) as a strategy for targeting anionic lipids in the cell membrane of bacteria to ciproproxacin, which is used to treat gram-negative and positive bacteria, -ester and -amide ( The present invention was completed by synthesizing two types of ciproproxacin derivatives chemically combined with the carboxylic acid of ciproproxacin through amide) binding and confirming antibacterial activity against multidrug-resistant bacteria.

Accordingly, the present invention provides an antibacterial composition for antibiotic-resistant strains comprising a compound represented by the following [Formula 1].

[Formula 1]

In the above [Formula 1],

Where D is a fluoroquinolone-based antibiotic skeleton with high stability, flumequine, oxolinic acid, rosoxacin, cinoxacin, nalidixic acid, pyrimidic acid ( piromidic acid, pipemdic acid, ciprofloxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin (ofloxacin) ), pefloxacin, rufloxacin, enoxacin, balofloxacin, grepafloxacin, levofloxacin, pazufloxacin (pazufloxacin) Lefloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, gatifloxacin, moxifloxacin, sitafloxacin prulifloxacin, besifloxacin, gemifloxacin, trovafloxacin, delafloxacin, danofloxacin, difloxacin , may be selected from enrofloxacin, ibafloxacin, marbofloxacin, orbifloxacin and sarafloxacin, and the fluoroquinolone antibiotic skeleton terminal A hydroxyl group of may be connected to X.

Wherein X is any one selected from O, S and NR (wherein R is any one selected from hydrogen, an alkyl group having 1 to 10 carbon atoms, an aryl group having 6 to 10 carbon atoms, and a heteroaryl group having 2 to 10 carbon atoms) and preferably O or NR.

In this case, when X is an amide group (NR), the stability in the body of the compound of [Formula 1] according to the present invention is more excellent.

In addition, L is a linking group with the target group, and may be any one selected from an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, and a polyalkylene glycol group.

In addition, Q may be any one selected from Group 15 elements such as N. P, As and Sb, R1 to R3 are the same as or different from each other, and each independently an alkyl group having 1 to 10 carbon atoms, 2 to 10 carbon atoms It may be any one selected from an alkenyl group, an aryl group having 6 to 10 carbon atoms, and a heteroaryl group having 2 to 10 carbon atoms.

In addition, A- may be any one anion selected from halogen, hydroxyl, carboxylate, sulfate, sulfurmate, sulfonate, phosphate, phosphonate, boronate and (poly)ethyleneoxy.

In addition, the halogen may be any one selected from fluorine (F), chlorine (Cl), bromine (Br), and iodine (I).

According to an embodiment of the present invention, if a specific compound of [Formula 1] is exemplified, it may be a compound of the following [Formula 2] or [Formula 3], whereby the scope of [Formula 1] according to the present invention is limited it is not

[Formula 2] - CFX-ester-PPh ₃

[Formula 3] - CFX-amide-PPh ₃

In one embodiment, the CFX-ester-PPh ₃ and CFX-amide-PPh ₃ are characterized in that they include a triphenylphosphonium structure based on ciproproxacin, and the triphenylphosphoni The structure has the properties of a positively charged lipophilic cationic compound and can target the mitochondria.

The CFX-amide-PPh ₃ is a modification of the binding site of CFX-ester-PPh ₃ to an amide group rather than an ester, and can further improve stability in the body.

In another embodiment of the present invention, the antibacterial composition according to the present invention may be a composition having antibacterial use against Gram-positive bacteria, and the Gram-positive bacteria may be Entrococcus faecium or Staphylococcus aureus.

The Entrococcus specium or Staphylococcus aureus may be a multidrug-resistant strain, but is not limited thereto, but Vancomycin-resistant E. faecalis (VRE), Methicillin-resistant Staphylococcus aureus (Methicillin) -resistance S. aureus (MRSA)), heterologous resistance VISA (Heteroresistant VISA (hVISA)) and vancomycin-resistant Staphylococcus aureus (Vancomycin-intermediate resistance S. aureus (VISA)) Any one or more selected from the group consisting of uniformity, , preferably vancomycin-resistant Entrococcus faecium (VRE), methicillin-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Staphylococcus aureus (VISA).

In another embodiment of the present invention, the composition may inhibit the biofilm formation of Gram-positive bacteria or inhibit the expression of norA , sepA or medA gene to prevent the outflow of a drug introduced into the cell, and the cell membrane of Gram-positive bacteria Antibacterial activity can be exhibited by direct targeting.

In the present invention, as described above, it was specifically confirmed that the antimicrobial compound according to the present invention exhibits an antibacterial effect on multidrug-resistant bacteria.

According to the present invention, through the examples, the compound represented by CFX-ester-PPh ₃ or CFX-amide-PPh ₃ is expected to exhibit antibacterial activity against bacteria having a mitochondrial-like cell membrane due to the mitochondrial targeting property, and multidrug-resistant bacteria As a result of performing antimicrobial activity screening for 14 species, it was confirmed that CFX-ester-PPh ₃ and CFX-amide-PPh ₃ had a low MIC against Gram-positive multidrug-resistant bacteria (see Example 3), and CFX-ester The effect of treatment with -PPh ₃ and CFX-amide-PPh ₃ on the inhibition of biofilm formation formed by multidrug-resistant bacteria was investigated, and as a result, it was confirmed that the biofilm was significantly inhibited in Gram-positive multidrug-resistant strains (conduct see example 4).

In another embodiment of the present invention, it was confirmed that most of the bacteria were killed within 6 hours when the compounds represented by CFX-ester-PPh ₃ and CFX-amide-PPh ₃ were treated with Gram-positive multidrug-resistant bacteria, especially CFX-amide -PPh ₃ It was confirmed that the compound represented by ciprofloxacin and CFX-ester-PPh ₃ showed a killing effect more than twice as quickly, and the antibacterial effect of the compound represented by CFX-ester-PPh ₃ and CFX-amide-PPh ₃ As a result of investigating the mechanism of activity in terms of morphology and genetics, it was found that the compound of CFX-ester-PPh ₃ or CFX-amide-PPh ₃ exhibits antibacterial effect by directly targeting the cell membrane of Gram-positive multidrug-resistant bacteria in terms of morphology. In terms of genetics, it was confirmed that antibiotic leakage was inhibited by reducing the expression of norA , sepA or medA genes, which are multi-drug efflux pumps (see Example 5).

From the results of the above Examples, it was confirmed that the compound according to the present invention effectively targets the cell membrane of Gram-positive multidrug-resistant bacteria by cationic lipid affinity properties, thereby exhibiting excellent antibacterial activity, and based on this, the antimicrobial compound of the present invention or the compound It was confirmed that the pharmaceutical composition comprising the composition can have an antibacterial effect for effectively controlling multi-drug-resistant bacteria compared to conventional antibiotics.

When the antimicrobial composition according to the present invention is a pharmaceutical composition, the antimicrobial compound represented by the above [Formula 1] or a salt thereof may be included as an active ingredient, and may also include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in formulation, and includes, but is not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and the like. It does not, and may further include other conventional additives such as antioxidants and buffers, if necessary. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules, or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, formulations can be preferably made according to each component using the method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in the formulation, but may be formulated as an injection, inhalant, external preparation for skin, or oral ingestion.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, dermally, nasally, or applied to the respiratory tract) according to a desired method, and the dosage may vary depending on the condition and weight of the patient, and the disease. Although it varies depending on the degree, drug form, administration route and time, it may be appropriately selected by those skilled in the art.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , can be determined according to factors including sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical field. The composition according to the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

When the antimicrobial composition according to the present invention is a food composition, it can be used for antibacterial use against Gram-positive bacteria, preferably methicillin-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Staphylococcus aureus (VISA). The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food, food additives and feed, and includes animals including humans or livestock. target for eating. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

Food compositions of this type can be prepared in various forms according to conventional methods known in the art. Common foods include, but are not limited to, beverages (including alcoholic beverages), fruits and their processed foods (eg, canned fruit, bottled, jam, marmalade, etc.), fish, meat and their processed foods (eg, ham, sausages) Corned beef, etc.), breads and noodles (eg udon noodles, soba noodles, ramen, spagate, macaroni, etc.), fruit juice, various drinks, cookies, syrup, dairy products (eg butter, cheese, etc.), edible vegetable oils and fats, margarine , vegetable protein, retort food, frozen food, various seasonings (eg, soybean paste, soy sauce, sauce, etc.) may include a composition containing the antimicrobial compound represented by the above [Formula 1] or a salt thereof as an active ingredient.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, these Examples and the like are intended to explain the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereby.

Example 1. CFX-ester-PPh ₃ synthesis of

CFX-ester-PPh ₃ was synthesized through the synthesis route of the following [Scheme 1]. Compounds 1 and 2 of Scheme 1 were synthesized according to a conventionally known method.

[Scheme 1]

(1) Synthesis of compound 3

Compound 1 (1 g, 1.975 mmol) and Compound 2 (852 mg, 1.975 mmol) were dissolved in a dimethylformamide solvent, and potassium carbonate (819 mg, 5.925 mmol) was slowly added thereto. After stirring at 50 °C for 12 hours, the solvent is evaporated under reduced pressure. After separation and purification by column chromatography, it was dissolved in 50 mL of a mixed solution (methanol/distilled water = 1/9), NaBF ₄ was added, stirred for 1 hour, extracted with dichloromethane and distilled water, and then dissolved in methanol and Dowex® 1X8 chloride After addition, the mixture was stirred for 6 hours, and the solvent was evaporated after filtering.

(2) Synthesis of CFX-ester-PPh ₃

After dissolving compound 3 in 18 mL of the mixed solution (1,4-dioxane/dichloromethane = 4/1), add 6 mL of 4 N HCl in Dioxane drop by drop at 0 °C. After stirring at room temperature for 12 hours, the solvent was evaporated under reduced pressure to synthesize CFX-ester-PPh ₃ .

¹ H NMR (CDCl ₃ , 500 MHz): δ8.50 (s, 1H), 7.87-7.70 (m, 15H), 7.66 (d, J = 13.1 Hz, 1H), 7.36 (d, J = 7.1 Hz, 1H), 4.29 - 4.23 (m, 2H), 3.69- 3.60 (m,2H), 3.59-3.53 (m, 1H), 3.50-3.42 (m, 4H), 3.30-3.20 (m, 4H), 1.78- 1.65 (m, 6H), 1.61-1.53 (m, 2H), 1.39 (d, J = 5.9 Hz, 2H), 1.14 - 1.07 (m, 2H) ppm.

Example 2. CFX-amide-PPh ₃ synthesis of

CFX-amide-PPh ₃ was synthesized through the synthesis route of the following [Scheme 2]. Compounds 1 and 2 of Scheme 2 were synthesized according to a conventionally known method.

[Scheme 2]

(1) Synthesis of compound 4

Compound 1 (1 g, mmol) was dissolved in 35 mL of 7 N NH ₃ methanol solution and stirred at room temperature for 3 days. Then, the solvent is evaporated under reduced pressure, and separated and purified by column chromatography.

(2) Synthesis of compound 5

Compound 2 (1 g, 2.318 mmol), EDC hydrochloride (667 mg, 3.477 mmol), and 1-Hydroxybenzotriazole hydrate (470 mg, 3.477 mmol) were dissolved in a dimethylformamide solvent and stirred at room temperature for 30 minutes. After adding DMAP (425 mg, 3.477 mmol) and compound 4 (1.025 g, 2.318 mmol), the mixture was stirred for 12 hours and then the solvent was evaporated under reduced pressure. After separation and purification by column chromatography, dissolve in 50 mL of a mixed solution (methanol/distilled water = 1/9), add NaBF4, stir for 1 hour, extract with dichloromethane and distilled water, dissolve in methanol, add Dowex® 1X8 chloride, and then 6 hours After stirring, the solvent is evaporated after filtering.

(3) Synthesis of CFX-amide-PPh ₃

에서 4N HCl in Dioxane 6mL 을 한 방울씩 넣어준다. 상온에서 12시간 동안 교반 한 뒤 감압하여 용매를 증발시켜 CFX-amide-PPh₃를 합성하였다. After dissolving compound 5 in 18 mL of mixed solution (1,4-dioxane/dichloromethane = 4/1), 0

Add 6mL of 4N HCl in Dioxane drop by drop. After stirring at room temperature for 12 hours, the solvent was evaporated under reduced pressure to synthesize CFX-amide-PPh ₃ .

1H NMR (MeOD, 500 MHz): δ 8.88 (s, 1H), 7.95 - 7.74 (m, 17H),7.69 (d, J = 7.2 Hz, 1H) 3.85 - 3.79 (m, 1H), 3.69 - 3.64 ( m, 4H), 3.54 - 3.49 (m, 4H), 3.49 - 3.41 (m, 4H), 1.77 - 1.70 (m, 2H), 1.70 - 1.58 (m, 4H), 1.53 - 1.42 (m, 4H) ppm .

Example 3. Comparison of minimum inhibitory concentrations of ciproxacin derivatives against Gram-negative and positive multidrug-resistant strains

The present inventors performed screening for antibacterial activity against 14 multidrug-resistant bacteria targeting the two ciproproxacin derivatives synthesized in Examples 1 and 2 above.

Specifically, for the screening of antimicrobial activity against multidrug-resistant bacteria, a sensitivity test method according to the CLSI (clinical and laboratory standard institute, 2016) guidelines was performed, and accordingly, the minimum antibiotic concentration and minimum inhibitory concentration that can inhibit bacterial growth (Minimum Inhibitory Concentration, MIC) was investigated. In order to investigate the MIC according to the sensitivity test method, broth microdilution was used. The medium was CAMHB (Cation-Adjust Muller Hilton broth), which was prepared by dissolving 22 g of CAMHB powder in 1 L of sterile distilled water, followed by autoclaving at 121 ° C. at a pressure of 15 lb psi for 15 minutes at high pressure. The 6 standard strains used in this experiment were purchased from the american type culture collection (ATCC) and the culture collection of antibiotics resistant microbes (CCARM), and the 8 resistant strains were purchased from Asan Medical Center (AMC, Asan Medical Center). A list of strains is shown in [Table 1] below.

Species strains Antibiotic resistance Gram negative bacteria Escherichia coli ATCC ^® 25922 Carbapenem-susceptible E. coli AMCEC 22365 Carbapenem-resistant E. coli (NDM-1 type) Klebsiella pneumoniae ATCC ^® 13883 Carbapenem-susceptible Klebsiella pneumoniae AMCKP 24272 Carbapenem-resistant K. pneumoniae (KPC type) Pseudomonas aeruginosa ATCC ^® 27853 Carbapenem-susceptible Pseudomonas aeruginosa (CSPA) CCARM 2321 Carbapenem-resistant P. aeruginosa (CRPA) Acinetobacter baumannii ATCC ^® 19606 Carbapenem-susceptible Acinetobacter baumannii (CSAB) AMCAB 643 Carbapenem-resistant A. baumannii (CRAB) Gram positive bacteria Enterococcus faecium ATCC ^® 29212 Vancomycin-susceptible E. faecium (VSE) CCARM 5024 Vancomycin-resistant E. faecium (VRE) Staphylococcus aureus ATCC ^® 29213 Methicillin-susceptible S. aureus (MSSA) AMCSA 5016 MRSA (ST5) ATCC ^® 700698 (Mu3) Heteroresistant VISA (hVISA) ATCC ^® 700698 (Mu50) Vancomycin-intermediate resistance S. aureus (VISA)

To prepare the culture solution shown in [Table 1], 14 multidrug-resistant bacteria were cultured on a blood agar plate for 24 hours at 37° C., and 16 test strains cultured overnight were colonies of each bacteria. A suspension of 0.5 McFarland standard turbidity was prepared. Next, each compound was diluted using CAMHB medium. Ciproproxacin had a maximum concentration of 512 μg/mL and ciproproxacin derivatives CFX-ester-PPh ₃ and CFX-amide-PPh ₃ had a maximum concentration of 179.05 μg It was gradually diluted 2-fold from /mL and dispensed into 96-well microplates (round bottom). Then, in a 96-well microplate containing the compound, diluted 1/10 of the prepared bacterial solution with 0.5 McFarland standard turbidity was inoculated at 10 μL (approximately 5 x 10 ⁵ CFU/mL based on E. coli ). After inoculation, incubated for 20 hours at 37°C using an incubator, the minimum concentration of the compound that inhibited proliferation was visually observed and the MIC was read, and the results are shown in [Table 2] and FIG. 1 .

type or strain CLSI breakpoints to CFX
susceptibility (μg/mL) QC range
for CFX MIC test MIC (μg/mL) CFX CFX-ester-PPh ₃ CFX-amide-PPh ₃ Gram-negative Carbapenem-susceptible E. coli ≤1 0.004-0.015 0.004 5.56 5.56 Carbapenem-resistant E. coli (NDM-1 type) ≤1 - 16 >178.05 89.02 Carbapenem-susceptible K. pneumoniae ≤1 - 0.031 22.25 44.51 Carbapenem-resistant K. pneumoniae (KPC type) ≤1 - 32 >178.05 178.05 Carbapenem-susceptible P. aeruginosa (CSPA) ≤1 - 0.25-0.125 89.02 89.02 Carbapenem-resistant P. aeruginosa (CRPA) ≤1 0.25-2.0 32 >178.05 178.05 Carbapenem-susceptible A. baumannii (CSAB) ≤1 0.5 89.02 89.02 Carbapenem-resistant A. baumannii (CRAB) ≤1 64 89.02 89.02 Gram-positive Vancomycin-susceptible E. faecium (VSE) ≤1 0.25-2.0 1.0-0.5 178.05 44.51 Vancomycin-resistant E. faecalis (VRE) ≤1 - 256 89.02 22.25 Methicillin-susceptible S. aureus (MSSA) ≤1 0.12-0.5 0.5 22.25 2.78 MRSA (ST5 5016) ≤1 128 11.12 2.78 Heteroresistant VISA (hVISA) ≤1 64 11.12 11.12 Vancomycin-intermediate resistance S. aureus (VISA) ≤1 64 44.51 11.12

As shown in [Table 2] and FIG. 1, the two ciproproxacin derivatives had no antibacterial activity in gram-negative multidrug-resistant bacteria, but showed significantly superior effects in gram-positive multidrug-resistant bacteria.

In other words, it showed a relatively low MIC value against Gram-negative susceptible strains (standard strains; E. coli , K. pneumoniae, P. aeruginosa , A. baumanii ), but showed a high MIC value for multidrug-resistant Gram-negative strains. On the other hand, VRE, MRSA, and VISA of Gram-positive multidrug-resistant bacteria showed a very low MIC value compared to ciproproxacin, confirming significant antibacterial activity.

Example 4. Comparative test of biofilm inhibition against gram-negative and positive multidrug-resistant strains of ciproxacin derivatives

The present inventors performed a crystal violet staining method to see the biofilm formation inhibitory effect on 14 types of the two ciproproxacin derivatives synthesized in Examples 1 and 2 above.

The 6 standard strains used in the experiment were purchased from the american type culture collection (ATCC) and the culture collection of antibiotics resistant microbes (CCARM), and the 8 resistant strains were purchased from the Asan Medical Center (AMC). The main list is shown in [Table 1] above.

Specifically, 14 strains were cultured in TSB (tryptic soy broth) at 37° C. for 24 hours, and the overnight culture medium was prepared as a suspension of 0.5 McFarland standard turbidity in fresh TSB. Next, each compound was diluted using TSB medium containing 0.1% glucose, and ciproproxacin had a maximum concentration of 256 µg/mL and two ciproproxacin derivatives had a maximum concentration of 512 µg/mL, gradually 2-fold. It was diluted stepwise and dispensed into 96-well microplates. Then, in a 96-well microplate containing the compound, diluted 1/10 of the prepared bacterial solution with 0.5 McFarland standard turbidity was inoculated at 10 μL (about 5 x 10 ⁵ CFU/mL based on E. coli). After inoculation, after incubation for 20 hours at 37 °C using an incubator, the culture medium and planktonic bacteria were removed and the 96-well microplates were carefully washed 3 times with buffered saline (PBS, phophate-buffered saline, pH7.4) and , and dried completely in a dryer at 50° C. for about 2 hours. The dried 96-well microplate was stained with 200 µL of 1.0% crystal violet at room temperature for 10 minutes, and then carefully washed with sterile distilled water. To quantify the stained biofilm mass, 200 μL of 33% dilute acetic acid (AcOH) was added to each well and incubated with shaking for 20 min. The biofilm mass was measured at absorbance at 600 nm using a Tecan Microplate Reader provided by Spark. The biofilm formation inhibition efficiency was calculated by the following [Calculation Formula], and the results are shown in Table 2, FIGS. 2 and 3 .

[formula]

Inhibition(%) = OD _{Positive-control} - OD _Experimental /OD _{Positive-control}

type or strain Biofilm formation of Inhibition
(Inhibition rate %) CFX CFX-ester
-PPh ₃ CFX-amide
-PPh ₃ Gram negative bacteria E. coli <0.5 (96.49%) 32 (76.0%) 8 (85.4%) Carbapenem-resistant E. coli (CRE NDM-1 type) - - - K. pneumoniae - - - Carbapenem-resistant K. pneumoniae (CRE KPC type) 128 (74.8%) >512 (28.5%) 256 (51.2%) Carbapenem-susceptible P. aeruginosa (CSPA) 2 (73.2%) 64 (50.1%) 256 (54.0%) Carbapenem-resistant P. aeruginosa (CRPA) <0.5 (80.3%) 64 (74.4%) 32 (56.5%) Carbapenem-susceptible A. baumannii (CSAB) 256 (66.0%) >512 (48.2%) 256 (53.4%) Carbapenem-resistant A. baumannii (CRAB, A643) - - - Gram positive bacteria Vancomycin-susceptible E. faecium (VSE) <0.5 (65.0%) 64 (60.0%) 64 (91.5%) Vancomycin-resistant E. faecalis (VRE) - - - Methicillin-susceptible S. aureus (MSSA) <0.5 (89.49%) 16 (50.5%) 4 (73.5%) MRSA (ST5 5016) 64 (60.8%) 8 (52.5%) 4 (76.4%) Heterogeneous VISA (hVISA, Mu3) - - - Vancomycin-intermediate resistance S. aureus (VISA) - - -

- means that no biofilm is formed. As shown in Table 3 and FIG. 2, in the case of Gram-negative multidrug-resistant bacteria, ciproproxacin derivatives (CFX-ester-PPh ₃ and CFX-amide-PPh ₃ ) formed a biofilm compared to ciproproxacin. The inhibitory effect was not excellent.

On the other hand, in the case of Gram-positive multidrug-resistant bacteria, particularly in the case of MRSA, as shown in [Table 3] and FIG. 3, ciproproxacin confirmed the biofilm formation inhibitory activity of about 60.8% at a high drug concentration of 64 μg/mL However, it was confirmed that the derivatives of ciproproxacin (CFX-ester-PPh ₃ and CFX-amide-PPh ₃ ) exhibited an equally or significantly superior biofilm formation inhibitory effect even at a much lower concentration than this.

Based on the above results, through the MIC and biofilm formation inhibition test, the ciproproxoxin derivatives (CFX-ester-PPh ₃ and CFX-amide-PPh ₃ ) were confirmed to have excellent antibacterial effects in gram-positive antibiotic-resistant bacteria, and these results Based on the ciproproxacin derivatives CFX-ester-PPh ₃ and CFX-amide-PPh ₃ , the following experiment was additionally performed using S. aureus and MRSA to elucidate the antibiotic mode of action.

Example 5. Confirmation of mechanism of ciproxacin derivatives against Gram-positive bacteria

Based _on the MIC results and the biofilm formation inhibition efficiency results, the present inventors added _{ciproproxacin} to Methicillin sensitive S. aureus and resistant strain MRSA ST5 5016, which exhibited high antibacterial effect against ciproproxacin derivatives CFX-ester-PPh ₃ and CFX-amide-PPh ₃ while having resistance to As a result, the mechanism of the derivative was confirmed through the following examples.

5-1. Time-kill curve experiment

For the selected Methicillin sensitive S. aureus (MSSA) and MRSA ST5 5016, time-kill curve assay was performed to confirm the effect of the ciproproxacin derivatives CFX-ester-PPh ₃ and CFX-amide-PPh ₃ over time. carried out.

Specifically, the selected strains MSSA and MRSA ST5 5016 were cultured in MHB medium at 37 ° C for 24 hours, inoculated with the culture medium in fresh _MHB medium at a ratio of 1/10, and 0.5 to 0.7 ( about 10 ⁶ CFU/mL) were cultured with shaking. In a strain culture medium of about 10 ⁶ CFU/mL, ciproproxacin and ciproproxacin derivatives CFX-ester-PPh ₃ and CFX-amide-PPh ₃ were added to 0.5x MIC, 1.0x MIC as shown in Table 4 below. , after treatment with 2.0x MIC, incubated with shaking at 37 °C.

MSSA MRSA ST5 5016 CFX CFX-ester-PPh ₃ CFX-amide-PPh ₃ CFX CFX-ester-PPh ₃ CFX-amide-PPh ₃ 0.5x MIC 0.25 11.12 1.39 64 5.56 1.39 1.0x MIC 0.5 22.25 2.78 128 11.12 2.78 2.0x MIC 1.0 44.51 5.56 256 22.25 5.56

After incubation, 10 μL of the culture medium was taken at 0, 3, and 6 hours, spread on LB plate medium, and cultured at 37 ° C for 24 hours, and the number of generated colonies was counted. times, the average value of the number of colonies was obtained. The counted colonies were converted to CFU/mL and displayed as a graph.

As a result, as shown in FIG. 4 , it was confirmed that the number of bacteria gradually increased as time passed in the condition in which the compound, which was a negative control, was not treated in MSSA (a) and MRSA (b). CFX-amide-PPh ₃ showed the highest antibacterial activity in both MSSA (a) and MRSA (b). In the case of CFX-amide-PPh ₃ , it was confirmed that MSSA (a) and MRSA (b) at all concentrations inhibited growth within 3 hours, and almost died within 6 hours. In the case of CFX-ester-PPh ₃ , similar to CFX-amide-PPh ₃ in MRSA(b), almost apoptosis was confirmed within 6 hours, but in MSSA(a), 1.0x MIC and 2.0x MIC within 6 hours It was confirmed that 50% of growth was inhibited. On the other hand, when treated with the conventional antibiotic ciproproxacin, MSSA (a) was observed to inhibit growth within 6 hours at all concentrations, and in the case of MRSA (b), 0.5x MIC and 1.0x MIC for 3 hours The number of bacteria was kept constant within, and it was confirmed that most of the MRSA was killed within 6 hours at 2.0x MIC.

That is, based on these results, it was confirmed that the ciproproxacin derivative CFX-amide-PPh ₃ had a high antibacterial effect showing a growth inhibitory effect within 6 hours at 0.5x MIC.

In addition, by treating the MIC values of ciproproxacin CFX-ester-PPh ₃ and CFX-amide-PPh ₃ in MSSA and MRSA, and measuring the OD over 0, 3, 6, 9, 12, 24 hours, Growth curves for the strain were obtained, and as a result, as shown in FIG. 5 , MSSA and MRSA of the control group not treated with CFX-ester-PPh ₃ and CFX-amide-PPh ₃ entered the logarithmic growth phase 3 hours after incubation. that was observed. On the other hand, in the group treated with CFX-ester-PPh ₃ and CFX-amide-PPh ₃ , growth inhibition was observed within 3 hours at each MIC value.

5-2. Morphological observation through transmission electron microscopy (TEM)

To confirm the antimicrobial action mechanism of the ciproproxacin CFX-ester-PPh ₃ and CFX-amide-PPh ₃ , the morphological change of MRSA ST5 5016 after treatment with CFX-ester-PPh ₃ and CFX-amide-PPh ₃ Transmission electron microscopy (TEM, transmission electron microscopy) was used for imaging, and the results are shown in FIG. 6 .

Specifically, to observe TEM, MRSA ST5 5016 was cultured in MHB medium until the logarithmic growth phase, and then diluted and inoculated in fresh MHB medium at a ratio of 1:10. Then, ciproproxacin and its CFX-ester-PPh ₃ and CFX-amide-PPh ₃ were treated with 0.5x MIC concentration and cultured for 6 hours, and then the bacteria were obtained by centrifugation. The bacterial pellet from which the supernatant was removed was washed 3 times with PBS and then fixed at 4°C using Karnovsky's fixative for 24 hours. After that, it was washed again with PBS 3 times, fixed again with 2% osmium tetraoxide (OsO ₄ , osmium tetroxide) for 1 hour, and then dehydrated with ethanol (EtOH) and replaced with 100% propylene oxide , Propylene oxide: Polymerized for one day at 60 ℃ in a 1:1 ratio of Spurr's resin. The solidified tissue was made into 1 μm ultra-thin sections, stained with 3% uranyl acetate, and observed by TEM.

As a result of observing MRSA treated with ciproproxacin CFX-ester-PPh ₃ and CFX-amide-PPh ₃ through TEM, CFX-ester-PPh ₃ and Significant morphological changes were observed when CFX-amide-PPh ₃ was treated. In the MRSA of the control group that was not treated with the drug, a coccus form with an intact septum and smooth surface features of bacteria was confirmed. However, significant membrane deformation of irregular and rough surfaces was observed by treatment with CFX-ester-PPh ₃ and CFX-amide-PPh ₃ . In particular, MRSA treated with CFX-amide-PPh ₃ confirmed the breakdown of the cell membrane due to severe membrane damage, and this observed a phenomenon in which the contents in the cytoplasm were partially leaked out.

5-3. qRT-PCR for Mechanism Analysis in Genetics

To analyze the antibacterial action mechanism of ciproproxacin CFX-ester-PPh ₃ and CFX-amide-PPh ₃ from a genetic point of view, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed. did In general, in antibiotics, antibiotics that have entered the cell through the multi-drug efflux pump flow back out of the cell, and the multi-drug efflux pump exhibits a characteristic dependent on proton motive force (PMF). Based on this fact, the present inventors Ciproproxacin derivatives CFX-ester-PPh ₃ and CFX-amide-PPh ₃ were expected to inhibit drug efflux by reducing PMF. Expression patterns of norA, sepA and medA genes, which are PMF-related multidrug efflux pumps was confirmed by qRT-PCR.

In order to perform qRT-PCR, the strain was cultured by treating the drug as in [Example 5-2] described above, and after culturing, all strains were obtained and RNA was extracted for qRT-PCR. Total RNA extraction was performed using the easy-Blue RNA extraction kit, and was performed according to the method provided in the kit. After total RNA extraction, cDNA was synthesized from RNA extracted from MSSA and MRSA using a high-capacity cDNA reverse transcription kit. Using cDNA as a template, qRT-PCR was performed to view the expression patterns of norA , sepA , and medA genes. As a result, as shown in FIG. 7, the expression of norA gene was significantly increased when ciproproxacin was treated, but CFX-ester-PPh ₃ and CFX-amide-PPh ₃ which are ciproproxacin derivatives were treated. In the group, it was confirmed that the mRNA expression of norA significantly decreased in both MSSA and MRSA. In MRSA, the expression patterns of sepA and medA genes were similar to those of the norA gene, indicating that when CFX-ester-PPh ₃ and CFX-amide-PPh ₃ were treated, mRNA expression was decreased compared to the group treated with ciproproxacin. seemed On the other hand, it was confirmed that the sepA and medA genes were not expressed in MSSA.

The description of the present invention described is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above are only for describing the present invention in more detail, and the scope of the present invention is not limited by these embodiments.

Claims (10)

Antibacterial composition for antibiotic-resistant strain comprising a compound represented by the following [Formula 1]:
[Formula 1]

In the above [Formula 1],
D is ciprofloxacin and is linked to X,
X is any one selected from O and NR, wherein R is hydrogen,
L is an alkylene group having 1 to 10 carbon atoms,
Q is P,
R ₁ to R ₃ are a phenyl group,
A ^- is any one anion selected from halogen, hydroxyl, carboxylate, sulfate, sulfurmate, sulfonate, phosphate, phosphonate, boronate and (poly)ethyleneoxy. delete According to claim 1,
The strain is characterized in that the gram-positive bacteria, antibacterial composition. 4. The method of claim 3,
The Gram-positive bacteria are Entrococcus faecium ( Enterococcus faecium ) or Staphylococcus aureus ( Staphylococcus aureus ), characterized in that the antibacterial composition. 5. The method of claim 4,
The Entrococcus pecium bacteria is an antibacterial composition, characterized in that the vancomycin-resistant Entrococcus pecium bacteria. 5. The method of claim 4,
The Staphylococcus aureus is an antibacterial composition, characterized in that it is methicillin-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Staphylococcus aureus (VISA). According to claim 1,
The compound of [Formula 1] is an antibacterial composition, characterized in that it is a compound represented by the following [Formula 2] or [Formula 3]:
[Formula 2]

[Formula 3]

According to claim 1,
The composition is characterized in that it directly targets the cell membrane of Gram-positive bacteria, antibacterial composition. According to claim 1,
The composition is characterized in that it inhibits the biofilm formation of Gram-positive bacteria, antibacterial composition. According to claim 1,
The composition is norA , by reducing the expression of sepA or medA gene, characterized in that to inhibit the outflow of the drug introduced into the cell, antibacterial composition.

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