KR20200046287A - Idnhibition of antibacterial resistance by 3',4'-difluoroquercetin and its derivative - Google Patents

Abstract

The present invention relates to 3′,4′-difluoroquercetin having antibacterial activity on multiple drug resistant bacteria and a novel derivative thereof. A quercetin derivative compound of the present invention exhibits a significant antibacterial activity on Gram-positive multiple drug resistant bacteria, exhibits strong antibacterial activity only on Gram-negative multiple drug resistant bacteria, and a significant synergistic effect occurs when an antibiotic which does not have antibacterial activity or has low antibacterial activity and the compound of the present invention are mixed and treated in Gram-negative multiple drug resistant bacteria, thereby being able to exhibit an excellent antibacterial effect on Gram-positive multiple drug resistant bacteria, Gram-negative multiple drug resistant bacteria, and antibiotic-resistant bacteria thereof.

Description

Idhibition of antibacterial resistance by 3 ', 4'-difluoroquercetin and its derivatives

The present invention relates to the use of 3 ', 4'-difluoroquercetin (3', 4'-difluoroquercetin) and novel derivatives thereof to inhibit antibiotic resistance.

Since Penicillin and Streptomycin have been discovered, more than 5,000 antibiotics have been discovered, of which 50 antibiotics have been developed with over 400 antimicrobial agents and used in clinical practice as a major treatment for infectious diseases. have. Antibiotics are largely classified into beta-lactam and vibetalactam, and smallly penicillin, cepha, quinolone, and aminoglycoside based on their chemical structure. In addition, antibiotics are classified into cell wall synthesis inhibitors, nucleic acid synthesis inhibitors, and protein synthesis inhibitors depending on the mechanism of action.

Due to the emergence of multidrug resistant bacteria, which are simultaneously resistant to several antibiotics, the demand for new antibiotics is increasing significantly. In the current trend, antibiotic resistance is one of the biggest problems facing medicine in the 21st century. It is expected to be one.

6 types of medical-related infection statutory standards under the "Management Management Guidelines for Multi-drug-resistant Bacteria" (Vancomycin-resistant-Staphylococcus aureus), vancomycin-resistant Enterococci (VRE), methicillin Resistant Staphylococcus aureus (MRSA), Multidrug-resistant Pseudomonas aeruginosa (MRPA), Multidrug-Resistant Acinetobacter baumannii (CAAB) Carbapenem Resistant Enterobacteriaceae (CRE), etc. are representative multi-drug resistant bacteria, in particular, MRSA (methicillin-resistant Staphylococcus aureus) and VRE (vancomycin-resistant Enterococci) are the most serious representative resistant strains. S. aureus is a Gram-positive cocci that causes pneumonia (especially in-hospital pneumonia), skin and soft tissue infections, and bacteremia, but is one of the most common pathogens, but since 1990s, 35-66% of S. aureus has been identified as MRSA. On the other hand, vancomycin-resistant strains were found in Enterococcus, one of Gram-positive cocci, and the incidence of VRE in the US has increased more than 20-fold from 0.3% in 1989 to 7.9% in 1993. In addition, New Delhi Metallo Beta-lactamase-1 (NDM-1), which was confirmed in 2008, has been found to be a hospital infectious super-strong super bacteria that is resistant to any existing antibiotics.

Resistant strains are ① by changing the structure of antibiotics by antibiotic-degrading enzyme and antibiotic-altering enzyme, ② by reducing the concentration of antibiotics in the cell by inhibiting the influx of antibiotics / activating the discharge through the discharge pump, or ③ antibiotics bind It is known to obtain resistance to antibiotics, such as by changing the target protein through mutation. Bacteria use two or more of the resistance mechanisms to effectively fight antibiotics, and it is also known that the degree of resistance increases as several mechanisms are mobilized. Therefore, antibiotics to fight resistant strains should be able to avoid the occurrence of resistance, ① by inhibiting new bacterial targets unknown to date, or ② by inhibiting various target groups.

Although new concept targets are expected to be able to evade the mechanism of resistance based on the bacterial genome sequence, studies of new targets have failed to derive hits-level compounds compared to the high cost and effort, and more A serious problem is that even if you find a new target that can evade the mechanism of resistance known to date, you will eventually develop a resistant strain, for example, the linezolid, the only antibiotic approved as a new target inhibitor in the 2000s. It has been confirmed that resistance to the disease appears very quickly.

Therefore, a smarter and more effective method in antibiotic development research is a study that increases the utility of existing antibiotics with excellent efficacy and safety by developing substances that can inhibit the mechanism of resistance rather than the development of new antibiotics and combining them with existing antibiotics. have. In particular, since the mechanism of resistance is different for each resistant strain and sometimes two or more mechanisms are compounded, it is expected that the resistance inhibitory substance can more effectively cope with resistance if it can simultaneously inhibit various resistance mechanisms.

Polyphenols are secondary metabolites produced by plants to fight herbivorous insects, fungi, or bacteria, and show various antibacterial effects. Since antibiotics are substances originally created by microorganisms to prevent the survival of other microorganisms, when combined with conventional antibiotics for secondary metabolites such as polyphenols, antibiotics can increase the drug's efficacy and reduce antibiotic dosage. Due to the expectation of reducing side effects, many studies have been conducted on the combination prescription of antibiotics and polyphenols, and a substantial synergistic effect has been observed. In particular, quercetin has been reported to have activity to inhibit beta-lactamase, topoisomerase, and efflux pump related to antibiotic resistance mechanisms. It is expected to be a new way to effectively maintain the efficacy of antibiotics against various resistant strains by combined prescription with antibiotics (Planta Med. 2008, 74: 840-846; Antimicrob.Agents Chemother. 1997, 41: 992- 998; Planta Med. 2002, 68: 1140-1141).

However, despite the significant synergistic effect of quercetin on resistant strains by co-administration with antibiotics, its clinical value is not high because of the low bioavailability of quercetin, appropriate dosage that can be developed as a real drug. This is because the formulation and method of administration are not secured. In practice, it is generally reported that the blood concentration (<1 μM) that can be obtained by consuming quercetin from a diet is usually lower than the concentration of polyphenols (10-100 μM) that exhibit physiological activity in cell lines ( BioFactors, 2000, 12: 175-180).

KR 10-1325058 B1

 Cho, S. Y .; Kim, M. K .; Mok, H .; Choo, H .; Chong, Y. Separation of Quercetin's Biological Activity from Its Oxidative Property through Bioisosteric Replacement of the Catecholic Hydroxyl Groups with Fluorine Atoms. J. Agric. Food Chem. 2012, 60, 6499-6506.

The present inventors showed that the newly synthesized quercetin derivative and 3 ', 4'-difluoroquercetin exhibited antibacterial activity against multidrug resistant bacteria, and further significantly treated the multidrug resistant bacteria when mixed with antibiotics. Since it exhibits antibacterial activity with an increased level of synergistic effect, the quercetin derivative of the present invention was completed by confirming that it can be used as an active ingredient of an antimicrobial composition as a combination agent with a conventional antibiotic.

Accordingly, an object of the present invention is to provide a novel quercetin derivative having improved bioavailability by improving instability of quercetin, and showing excellent antibacterial activity against multidrug resistant bacteria.

Another object of the present invention is to provide a composition for antibacterial use of a quercetin derivative as an active ingredient.

Another object of the present invention is to provide an antimicrobial composition that exhibits excellent synergistic antimicrobial activity against multidrug resistant bacteria by administering in complex with a quercetin derivative.

In one aspect for achieving the above object, the present invention is a compound of Formula 1 or Formula 2; Or it provides a composition for antibacterial, comprising a salt thereof as an active ingredient.

[Formula 1]

[Formula 2]

In another aspect, the present invention provides a compound of Formula 1 or Formula 2; And (b) provides an antimicrobial composition comprising an antibiotic as an active ingredient.

The compounds of the present invention may be used alone for antibacterial purposes or may be used in combination with antibiotics to inhibit antibiotic resistance.

In a preferred embodiment of the present invention, the antibiotic is ampicillin (Ampicillin), vancomycin (Vancomycin), linezolid (Linezolid), erythromycin (Erythromycin), methicillin (Methicillin), oxacillin (Oxacillin), cell tamsim ( Cefotaxime, Rifampicin, Amikacin, Gentamicin, Amikacin, Kanamycin, Tobramycin, Neomycin, Ertapenem (Ertapenem) Ertapenem, Doripenem, Imipenem / Cilastatin, Meropenem, Ceftazidime, Cefepime, Ceftaroline, Ceftaroline (Ceftaroline) Ceftobiprole, Aztreonam, Piperacillin, Polymyxin B, Colistin, Ciprofloxacin, Levofloxacin, Moxifloxacin, Moxifloxacin, Moxifloxacin Gatifloxacin, Tigecycline, and combinations thereof It may be any one or more selected from the group consisting of a sieve and derivatives thereof.

In one preferred embodiment of the present invention, the antimicrobial composition may have antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria and multidrug-resistant strains thereof.

The Gram-positive bacteria may be any one or more selected from the group consisting of Staphylococcus aureus and Enterococcus sp.

The gram-negative bacteria may be any one or more selected from the group consisting of Psedomonas aeruginosa , Acinetobacter baumannii and Escherichia coli .

The multi-drug resistant strains are methicillin-sensitive yellow staphylococcus (MSSA), methicillin-resistant yellow staphylococcus (MRSA), vancomycin-resistant yellow staphylococcus (VRSA), vancomycin-moderately resistant yellow staphylococcus (VISA), vancomycin intra-growth bacterium , Vancomycin-sensitized growth bacteria (VSE), multidrug-resistant Pseudomonas aeruginosa (MRTA), carpepenem-resistant Pseudomonas aeruginosa (CRPA), cappapenem-resistant acetobacter baumani bacteria (CRAB), and selected from the group consisting of carbapenem-ingrowing bacteria It can be any one or more.

In another aspect, the present invention provides a pharmaceutical composition for antibacterial, comprising the compound of Formula 1 or Formula 2 as an active ingredient.

In addition, the present invention provides a food additive comprising the compound of Formula 1 or Formula 2 as an active ingredient.

In addition, the present invention provides a feed additive comprising the compound of Formula 1 or Formula 2 as an active ingredient.

In addition, the present invention provides a preservative composition comprising the compound of Formula 1 or Formula 2 as an active ingredient.

In addition, the present invention provides a quasi-drug composition comprising the compound of Formula 1 or Formula 2 as an active ingredient.

In another aspect, the present invention provides a compound represented by Formula 2 below.

[Formula 2]

1 is a diagram showing the synthesis process of 3 ', 4'-difluoroquercetin YC-399 of the present invention.
2 is a diagram showing the synthesis process of the newly synthesized 3 ', 4'-difluoroquercetin derivative YC-400 in the present invention.
Figure 3 shows the biofilm inhibitory effect on the multi-drug resistant bacteria of Staphylococcus aureus ( S. aureus ) of 3 ', 4'-difluoroquercetin YC-399 of the present invention,
A shows the relative biofilm production ability by Staphylococcus aureus strains,
B shows the effect of inhibiting biofilm by concentration of YC-399 on MRSA (ST-72),
C shows the effect of inhibiting biofilm by concentration of YC-399 on MRSA (ST-5),
D shows the effect of inhibiting biofilm by concentration of YC-399 on MSSA,
E shows the effect of inhibiting biofilm by concentration of YC-399 on MRSA (ST-239),
F shows the effect of inhibiting biofilm by concentration of YC-399 on hVISA,
G shows the effect of inhibiting biofilm by concentration of YC-399 on VISA,
H shows the effect of inhibiting biofilm by concentration of YC-399 on the negative control (ATCC 25923 strain MSSA).

Hereinafter, the present invention will be described in detail.

As described above, the development of antibiotics showing antimicrobial activity against multidrug-resistant bacteria resistant to antibiotics is continuously required.

In order to achieve the above object, the present invention provides a method for producing a quercetin derivative represented by Chemical Formula 2.

[Formula 2]

Specifically, the present invention is a quercetin derivative, 3 ', 4'-difluoroquercetin (3', 4'-difluoroquercetin, YC-399) known through registration patent [10-1325058] using quercetin as a starting material. Synthesis (FIG. 1), and a new quercetin derivative YC- through a substitution reaction with 1-chloro-4,4-dimethylpentan-2-one as shown in [FIG. 2] using the YC-399 compound as a starting material. 400 compounds (represented by Formula 2) were synthesized.

Next, in the present invention, as a result of confirming the antibacterial activity of the quercetin derivatives YC-399 and YC-400 by measuring the minimum growth inhibitory concentration (MIC) in Gram-positive and Gram-negative multidrug-resistant bacteria, the quercetin derivative YC-399 And YC-400 was confirmed to have high antibacterial activity against all Gram-positive multidrug-resistant bacteria, and also antibacterial activity against Gram-negative multidrug-resistant bacteria (Table 2, Table 3 and FIG. 3).

Thus, the present invention is a compound of Formula 1 or Formula 2; Or it provides a composition for antibacterial, comprising a salt thereof as an active ingredient.

[Formula 1]

[Formula 2]

The term “pharmaceutically acceptable salt” of the present invention is a compound having a relatively non-toxic and harmless effect on the patient, and the side effects caused by this salt do not degrade the beneficial efficacy of the compound represented by Formula 1 or 2 Means any and all organic or inorganic addition salts.

Acid addition salts are prepared by dissolving the compound in a conventional method, for example, an aqueous solution of excess acid, and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The molar amount of the compound and the acid or alcohol in water (eg, glycol monomethyl ether) may be heated, and then the mixture may be evaporated to dryness, or the precipitated salt may be suction filtered, but is not limited thereto.

At this time, organic and inorganic acids can be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, and tartaric acid can be used as the inorganic acid, and methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, and maleic acid can be used as the organic acid. (maleic acid), succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid (gluconic acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanic acid, hydroiodic acid, etc. can be used. , Not limited to these.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal salt or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the inexpensive compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable to manufacture sodium, potassium, or calcium salts, but is not limited thereto. Further, the corresponding silver salt may be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate), but is not limited thereto.

Pharmaceutically acceptable salts of the compounds of Formula 1 or 2 include salts of acidic or basic groups that may be present in the compounds of Formula 1 or 2, unless otherwise indicated. For example, pharmaceutically acceptable salts may include sodium, calcium, and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, and hydrogen phosphate. , Dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, etc., and preparation of salts known in the art It can be prepared through a method.

The pharmaceutically acceptable salt of the compound of Formula 1 or 2 of the present invention is a pharmaceutically acceptable salt, as long as it is a salt of the compound of Formula 1 or 2 which exhibits an antimicrobial activity equivalent to that of the compound of Formula 1 or 2 All can be used without limitation.

In another aspect, the present invention provides a compound of formula (a) Formula 1 or Formula 2; And (b) provides an antimicrobial composition comprising an antibiotic as an active ingredient.

In one embodiment of the present invention, the efficacy of inhibiting drug resistance by a quercetin derivative was evaluated by confirming the extent to which the activity of an existing antibiotic in a drug resistant strain is recovered by a quercetin derivative. As a result, the YC-399 (Chemical Formula 1) or YC-400 (Chemical Formula 2) compound of the present invention has a significant synergistic antibacterial effect against Gram-positive bacteria, Gram-negative bacteria and antibiotic-resistant strains thereof when mixed with an existing antibiotic. It confirmed that it showed (Tables 4-7). Thus, the YC-399 or YC-400 compound of the present invention can be usefully used as an antibiotic adjuvant to inhibit antibiotic resistance.

The "antibiotic" of the present invention may be used as long as it has low antibacterial activity against multidrug-resistant bacteria or is known to have no antibacterial activity in the art. Specifically, the antibiotics of the present invention include ampicillin, vancomycin, linezolid, erythromycin, methicillin, oxacillin, cetaxoxime, rifampicin (Rifampicin), Amikacin, Gentamicin, Amikacin, Kanamycin, Tobramycin, Neomycin, Ertapenem, Dory Doripenem, Imipenem / Cilastatin, Meropenem, Ceftazidime, Cefepime, Ceftaroline, Ceftobiprole, Aziz Aztreonam, Piperacillin, Polymyxin B, Colistin, Ciprofloxacin, Levofloxacin, Moxifloxacin, Gatifloxacin, Gatifloxacin , Tigecycline, combinations thereof and oils thereof One body is more preferred than one selected from the group consisting of, and the like.

The compounds of the present invention are preferably, but not limited to, antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and multidrug-resistant strains thereof.

Specifically, the Gram-positive bacteria are Staphylococcus species, Listeria species, Streptococcus species, Corynebacterium species, Lactobacillus species, Gram-positive bacteria, including Clostridium species, Enterococcus species, Erysipelothrix species and Bacillus species, are all Gram-positive bacteria known in the art Preferred are Staphylococcus aureus and Enterococcus species, but are not limited thereto.

The gram-negative bacteria are all gram-negative bacteria known in the art as gram-negative bacteria including Escherichia, Pseudomonas, Salmonella, Leptospira, and Rickettsia. And, Pseudomonas aeruginosa ( Psedomonas aeruginosa ), acetobacter baumannii ( Acinetobacter baumannii ) or E. coli ( Escherichia coli ) is more preferable, but not limited to.

The multi-drug resistant strains are methicillin-sensitive yellow staphylococcus (MSSA), methicillin-resistant yellow staphylococcus (MRSA), vancomycin-resistant yellow staphylococcus (VRSA), vancomycin-moderately resistant yellow staphylococcus (VISA), vancomycin intra-growth bacterium , Vancomycin-sensitized growth bacteria (VSE), multidrug-resistant Pseudomonas aeruginosa (MRTA), carpepenem-resistant Pseudomonas aeruginosa (CRPA), kappapenem-resistant acetobacter baumani bacteria (CRAB), and a bacterium-in-growth bacterium genus (CRE) Preferably, but not limited to.

In another aspect, the YC-399 (Chemical Formula 1) or YC-400 (Chemical Formula 2) compound of the present invention exhibits excellent antibacterial activity against multidrug-resistant bacteria by itself, and is a Gram-positive bacteria when mixed with an existing antibiotic, Since it exhibits a significant synergistic antimicrobial effect against Gram-negative bacteria and its antibiotic-resistant strains, the compound of the present invention or a combination of the compound of the present invention and an antibiotic may be usefully used as an active ingredient of an antibacterial pharmaceutical composition.

The pharmaceutical composition for antibacterial use of the present invention can be administered parenterally during clinical administration and can be used in the form of a general pharmaceutical preparation. Parenteral administration may mean administration through a non-oral route of administration such as rectal, intravenous, peritoneal, muscular, arterial, transdermal, nasal, inhalation, ocular and subcutaneous. When the pharmaceutical composition for antibacterial use of the present invention is used as a pharmaceutical, it may further contain one or more active ingredients exhibiting the same or similar functions.

That is, the pharmaceutical composition for antimicrobial use of the present invention can be actually administered in various parenteral formulations. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc., which are usually used are used. Is prepared. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As a non-aqueous solvent and a suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, Witepsol, Macrogol, Tween 61, cacao butter, Liurinji, and glycerogelatin may be used.

In addition, the pharmaceutical composition for antimicrobial use of the present invention can be used by mixing with various carriers allowed as medicaments such as physiological saline or organic solvents, such as glucose, sucrose, or dextran to increase stability or absorbency. Antioxidants such as carbohydrates, ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers can be used as medicaments.

The effective dose of the pharmaceutical composition for antibacterial use of the present invention is 0.01 to 100 mg / kg, preferably 0.1 to 10 mg / kg, and can be administered once to three times a day.

In the pharmaceutical composition of the present invention, the total effective amount of the compound of the present invention, or a combination of a compound and an antibiotic, is administered to a patient in a single dose by bolus form or infusion for a relatively short period of time. It can be administered, and multiple doses can be administered by the Fractionated treatment protocol, which is administered for a long time. Since the effective dose of the patient is determined in consideration of various factors such as the patient's age and health status, as well as the administration route and number of treatments of the drug, the present invention is considered by those skilled in the art. It may be possible to determine an appropriate effective dosage for a particular use as a pharmaceutical composition.

The antimicrobial adjuvant of the present invention may be administered with antibiotics, together with antibiotics, prior to antibiotic treatment, and after antibiotic treatment, and may be used as an adjuvant to enhance the antibacterial activity of antibiotics. It is preferable that the adjuvant has a synergistic effect on antibiotics and antibacterial activity, and it is preferable to increase the antibacterial activity of antibiotics against Gram positive bacteria, Gram negative bacteria and antibiotic resistant strains.

In another aspect, the present invention provides a food additive comprising a compound represented by Formula 1 or Formula 2.

In addition, the present invention (a) a compound represented by Formula 1 or Formula 2; And (b) antibiotics as an active ingredient.

When the compound of the present invention, or the compound and antibiotic combination agent is used as a food additive, the compound, or the compound and antimicrobial agent combination agent may be added as it is or used with other food ingredients, and may be suitably used according to conventional methods. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use. In general, the compound of the present invention, or the compound and the antimicrobial agent combination agent is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less based on the raw material. However, in the case of long-term intake, the amount may be below the above range, and since there is no problem in terms of stability, the active ingredient may be used in an amount above the above range.

There are no particular restrictions on the type of food. Examples of foods to which the above substances can be added are meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other dairy products including noodles, gums, ice cream, various soups, beverages, teas, drinks, Alcoholic beverages, vitamin complexes, and the like, and include all foods in the ordinary sense.

In another aspect, the present invention provides a feed additive comprising the compound of Formula 1 or Formula 2 as an active ingredient.

In addition, the present invention (a) a compound represented by Formula 1 or Formula 2; And (b) antibiotics as an active ingredient.

The feed composition comprising the compound of the present invention, or a compound and an antimicrobial agent complex, replaces the existing antibiotics and suppresses the growth of harmful food pathogenic bacteria to improve the health of the animal body, improve the weight gain and meat quality of the livestock, and the amount of milk produced. And increases immunity. The feed composition of the present invention may be prepared in the form of fermented feed, blended feed, pellet form and silage.

The fermented feed can be prepared by fermenting an organic substance by adding various microbial groups or enzymes other than the compound of the present invention, or a compound and an antibacterial agent complex, and the blended feed is a combination of various types of general feed and the compound and antibacterial agent of the present invention. It can be prepared by mixing. Feed in the form of pellets can be prepared by applying heat and pressure to the blended feed or the like in a pellet machine, and silage can be prepared by fermenting the greens feed into microorganisms. The wet fermented feed collects and transports organic substances such as food waste, mixes the sterilization process and excipients for moisture control at a certain rate, and then ferments it for more than 24 hours at a temperature suitable for fermentation, so that the moisture content is included in about 70%. It can be prepared by adjustment. The fermented dry feed can be prepared by adjusting the wet fermented feed to contain about 30% to 40% of moisture content through an additional drying process.

In another aspect, the present invention provides a preservative composition comprising the compound of Formula 1 or Formula 2 as an active ingredient.

In addition, the present invention provides a compound represented by the formula (a) Formula 1 or Formula 2; And (b) provides an antiseptic composition comprising an antibiotic as an active ingredient

The preservative composition includes food preservatives, cosmetic preservatives or pharmaceutical preservatives. Preservatives, cosmetic preservatives and pharmaceutical preservatives of the foods are additives used to prevent deterioration, decay, discoloration and chemical changes of pharmaceuticals, and include antiseptics and antioxidants to inhibit the growth of microorganisms such as bacteria, fungi, and yeast. And functional antibiotics such as deterring or sterilizing decaying microorganisms in pharmaceuticals. The ideal condition of the antiseptic composition should be non-toxic and should be effective even in trace amounts.

In addition, the present invention provides a quasi-drug composition for antibacterial, comprising the compound of Formula 1 or Formula 2 as an active ingredient.

In addition, the present invention (a) a compound represented by Formula 1 or Formula 2; And (b) provides an antimicrobial quasi-drug composition comprising an antibiotic as an active ingredient

When the composition of the present invention is used as a quasi-drug additive, the compound of the present invention, or a compound and an antibiotic complex agent can be added as it is or used with other quasi-drugs or quasi-drug components, and can be suitably used according to conventional methods. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use.

The quasi-drug composition of the present invention is not limited thereto, but may preferably be a disinfecting cleaner, shower foam, gagreen, wipes, detergent soap, hand wash, humidifier filler, mask, ointment, patch, or filter filler.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

[Production Example 1]

Preparation of 3 ', 4'-difluoroquercetin YC-399

[Fig. 1] is a diagram showing a method of manufacturing 3 ', 4'-difluoroquercetin (YC-399) used in the present invention, from quercetin (1) by the method described in Korean Patent Registration No. 10-1325058. 3 ', 4'-diflorocercetin (6) was prepared.

[Example 1]

Synthesis of 3 ', 4'-difluoroquercetin derivative YC-400

Figure 2 is a 3 ', 3'-difluoroorcercetin derivative YC-400 of the present invention shows a method of production from 3', 4'-difluoroquercetin (YC-399) prepared above. .

Specifically, potassium carbonate (338 mg, 2.4 mmol) was added to a solution of 500 mg (1.6 mmol) of 3 ', 4'-difluoroquercetin (YC-399) prepared in Preparation Example 1 in acetone (30 mL). And 1-chloro-4,4-dimethylpentan-2-one (363 mg, 2.4 mmol) was added, and the reaction solution was stirred at room temperature for 12 hours. Filter the solid from the reactant, distill the remaining solution under reduced pressure, and then purify the residue obtained by silica gel column chromatography (hexane: ethyl acetate = 3: 1) to obtain the desired compound 3 ', 4'-difluoroquercetin derivative YC-400 was obtained in 65% yield: ¹ H NMR (400 MHz) δ (Acetone-d6) 8.21-8.12 (m, 2H), 7.53 (dd, J = 18.9, 8.8 Hz, 1H), 6.70 (d , J = 1.8 Hz, 1H), 6.35 (d, J = 1.8 Hz, 1H), 4.90 (s, 2H), 2.54 (s, 2H), 1.07 (s, 9H).

[Example 2]

Confirmation of antibacterial activity of quercetin derivatives

<2-1> Confirmation of antibacterial activity of quercetin derivatives YC-399 and YC-400 against multidrug-resistant bacteria

In order to confirm the antibacterial activity of the quercetin derivative of the present invention, the antibacterial activity of the quercetin derivatives YC-399 and YC-400 of the present invention was measured with respect to the multi-drug resistant bacteria of Gram-negative bacteria and Gram-positive bacteria. The antimicrobial activity was measured by a minimal inhibitory concentration (MIC) in which the cells were not divided in a medium with sufficient nutrients.

The minimum growth inhibitory concentration was performed by the broth microdilution method according to the US Clinical Laboratory Standards Institute (CLSI) M7-A10. The strains listed in [Table 1] were used for the minimum growth inhibitory concentration experiment, and the strains purchased or purchased from the Korea Microbial Conservation Center (KCCM) and the antibiotic resistant strain bank (CCARM) were used as clinical strains separated from patients. . Remedy, after diluting with Cationic Adjusted Muller Hinton Broth (CAMHB) medium so that the number of bacteria becomes 5Х10 ⁵ colony-forming units (CFUs) per ml, and 200 μl of each is dispensed into a 96-well microtiter plate, 200 μl of the quercetin derivative solution diluted with CAMHB medium was added to each well. The plate was incubated for 16-24 hours according to the species at a temperature of 35 ° C ± 2 ° C under ambient air (ambient air), and then determined visually by MIC of the quercetin derivative. When the MIC value exceeded the concentration, the experiment was carried out after assuming the MIC as the concentration next to the concentration (if MIC> 256, it was considered as MIC 512).

As a result, as shown in Table 1 below, it was confirmed that the quercetin derivatives YC-399 and YC-400 have high antibacterial activity against all Gram-positive multidrug-resistant bacteria. In particular, YC-399 is a Gram-positive multi-drug resistant S.aureus , Enterococcus species, And it was found that it exhibits excellent antibacterial activity against the resistant bacteria. These compounds were confirmed to have a very high antibacterial activity selectively to all Gram-positive multidrug-resistant bacteria, it was confirmed to have a relatively low antibacterial activity to Gram-negative multidrug-resistant bacteria (Table 1).

[Table 1]

Antibacterial activity of quercetin derivatives against multidrug resistant bacteria

<2-2> Confirmation of antibacterial activity of existing antibiotics against multidrug resistant bacteria

In order to compare the antibacterial activity of the compounds of the present invention with existing antibiotics, the MIC concentration indicated by the antibiotics against the same multidrug resistant bacteria was confirmed.

Specifically, a target strain was prepared in the same manner as in Example <2-1>, and antibiotics Ampicillin (AMP), Ceftazidime (CAZ), Sepepime (FEP), and Vancomycin (Vancomycin) , VAN) and meropenem (MERO), respectively, to confirm the MIC of the antibiotic.

As a result, it was confirmed that the existing antibiotics showed low antibacterial activity against multidrug resistant bacteria (Table 2 and Table 3).

[Table 2]

Antibacterial activity of existing antibiotics in Gram-positive resistant bacteria

[Table 3]

Antibacterial activity of existing antibiotics in Gram-negative resistant bacteria

[Example 3]

Confirmation of synergy when quercetin derivatives and antibiotics are mixed with Gram-positive multidrug-resistant bacteria

By confirming that the compound of the present invention shows high antibacterial activity against Gram-positive multidrug-resistant bacteria, an antibiotic having strong antibacterial activity against Gram-negative multidrug-resistant bacteria and not having significant antibacterial activity against Gram-positive multidrug-resistant bacteria is mixed with the compound of the present invention It was confirmed that it showed significant synergistic activity when treated. A checkerboard synergy test was performed on a 96 well plate, and the minimum inhibitory concentration (MIC) of YC-399, YC-400, and antimicrobial was used by the broth microdilution method. By combining various concentrations of antibacterial agents and YC-399 and YC-400, changes in the minimum inhibitory concentrations of existing antibacterial agents by combining YC-399 and YC-400 were evaluated.

Specifically, each strain described in [Table 1] was prepared, diluted with CAMBH medium so that the number of bacteria was 5 × 10 ⁵ colony-forming units (CFUs) per ml, and 200 μl of each 96-well microtiter plate (Microtiter plate) After dispensing in), 100 µl of each compound-containing solution (step-diluted solution) diluted from the MIC value with CAMBH medium was added to each well. Thereafter, the antibiotic solution was diluted in CAMBH medium, and 100 µl was added to each well, and the same was performed under the opposite conditions to determine the MIC value of each mixed solution.

As a result, as shown in the following [Table 4] and [Table 5], the conventional antibiotics, ampicillin (AMP), ceftazidim (CAZ), cefapime (FEP) or vancomycin (Vancomycin, VAN) of the present invention The synergistic effect of the antibacterial activity administered with the compounds YC-399 or YC-400 was confirmed. When the compound of the present invention was treated at 1/2 MIC value, it was confirmed that the MIC of ceftazidim for MRSA, VISA, VISA, VSE, and VRE was 4-64 times lower. In addition, when the compound of the present invention was treated at 1/2 MIC value, the MIC of sepapim was lowered by 4-128 times compared to MRSA, VISA, and VSE, and when the compound of the present invention was treated at 1/2 MIC value, VSE, VRE , MRSA, it was confirmed that the MIC of vancomycin is 4 times lower for VISA.

For example, when the compound is mixed with antibiotics and treated with VSE at 8 mg / L at a concentration of 1/2 of the compound YC-399 MIC, it is ceftazidim when YC-399 is mixed with ceftazidim or sepipim. And it was confirmed that the MIC value of sepapim is 8 and 1 mg / L (1/32 and 1/32 of the antibiotic M IC, respectively) (Table 4).

[Table 4]

Antibacterial activity of antibiotics when treating YC-399 compound of the present invention with Gram-positive multidrug-resistant bacteria

[Table 5]

Antibacterial activity of antibiotics when treating YC-400 compounds of the present invention with Gram-positive multidrug-resistant bacteria

[Example 4]

Confirmation of synergy when quercetin derivatives and antibiotics are mixed with Gram-negative multidrug-resistant bacteria

It was confirmed that the antibiotics showing relatively strong antibacterial activity against Gram-negative multidrug-resistant bacteria showed significant synergistic activity when mixed with the compounds of the present invention. In the same manner as in Example 3, a checkerboard synergy test was performed on a 96 well plate, and the minimum inhibitory concentration (MIC) of YC-399, YC-400, and antibiotic was used for the broth microdilution method. By combining various concentrations of antibiotics and YC-399 and YC-400, changes in minimum inhibitory concentrations of existing antibiotics by combining YC-399 and YC-400 were evaluated.

As a result, as shown in [Table 6] and [Table 7], the conventional antibiotics ceftazidim (CAZ), or cepapime (FEP) are administered together with the compounds of the present invention YC-399 or YC-400. As a result of confirming the synergistic effect of one antibacterial activity, when the compound of the present invention was treated with 1/2 MIC value, it was confirmed that the MIC of the existing antibiotic against CRPA and CRAB is 4-8 times lower.

[Table 6]

Antibacterial activity of antibiotics when treating YC-399 compound of the present invention with Gram-negative multidrug resistant bacteria

[Table 7]

Antibacterial activity of antibiotics when treating YC-400 compounds of the present invention with Gram-negative multidrug resistant bacteria

[Example 5]

Confirmation of the static biofilm inhibitory effect of YC-399 on Staphylococcus aureus

Most bacteria generally exist in the form of planktonic water, but when the survival conditions are deteriorated due to nutrient loss or antibiotic attack, the phenotype is a biofilm that adheres to the solid surface and forms a colony. Will change. Bacteria in biofilms show enhanced resistance to antibiotics and host defense systems, often leading to persistent and difficult to treat infections (Kania et al . , 2008; Vlastarakos et al . , 2007). Various studies have reported that pneumococcal biofilms have remarkably high resistance to penicillin, tetracycline, lympycin, amoxicillin, erythromycin, clindamycin and levofloxacin (del Prado et al . , 2010; Garcia-Castillo et al . , 2007). Marks, etc. 　 (2012), reported that pneumococcal biofilms formed on nasopharyngeal tissues of mice are more resistant to gentamicin and penicillin G than planktonic biofilms (Marks et al . , 2012). Thus, a static biofilm formation experiment was conducted in a 95-well plate (Corning, no. 3595) to confirm the effect of inhibiting biofilm on YC-399's Staphylococcus aureus ( S. aureus ) resistant bacteria. Bacteria were inoculated in 0.5 X, tryptic soy broth (Becton Dickinson and Co.), and 1% glucose was added. Each well was inoculated with 200 uL of inoculum with YC-399 (0, 1, 5, 10, 25, and 50 mg / L), and the well corresponds to 5 X 10 ⁶ bacteria. After incubating the plate for 20 hours at 37 ° C, unsolicited bacteria are washed out using PBS buffer. The attached bacteria were fixed with 100% ethanol for 15 minutes, and then stained with 0.1% crystal violet solution for 10 minutes. After washing the plate with deionized water (dH ₂ O), each well was measured at OD ₆₀₀ using a spectrophotometer.

Figure 7 shows the effect on the biofilm growth of the Streptococcus aureus resistant bacteria of the YC-399 compound of the present invention, showing the results of measuring the optical density at 600 nm (OD ₆₀₀₎ .

Through this, it can be confirmed that the growth of the biofilm decreases from the group treated with 5 mg / L of YC-399. YC-399 has a biofilm inhibitory effect at a concentration below the minimum inhibitory concentration against MRSA (methicillin-resistant S. aureus ), so it is expected to be used as an effective antimicrobial or antimicrobial agent for overcoming multi-drug resistant infection.

Claims (11)

A compound represented by the following Chemical Formula 1 or Chemical Formula 2; Or comprising a salt thereof as an active ingredient, an antimicrobial composition.
[Formula 1]

[Formula 2]

(a) a compound of Formula 1 or Formula 2; And
(b) Antimicrobial composition comprising an antibiotic as an active ingredient.
[Formula 1]

[Formula 2]

According to claim 2,
The compound is characterized in that it is used as an antibiotic supplement to inhibit antibiotic resistance, antimicrobial composition.
According to claim 2,
The antibiotics include ampicillin, vancomycin, linezolid, erythromycin, methicillin, oxacillin, cefaxime, rifampicin, rifampicin Amikacin, Gentamicin, Amikacin, Kanamycin, Tobramycin, Neomycin, Ertapenem, Dorippenem, Imipenem / Cilastatin, Meropenem, Ceftazidime, Cefepime, Ceftaroline, Ceftobiprole, Aztreonam , Piperacillin, Polymyxin B, Colistin, Ciprofloxacin, Levofloxacin, Moxifloxacin, Gatifloxacin, Tigecycline Tigecycline ), Combinations thereof and derivatives thereof Characterized in that any one or more selected from, composition for antibacterial.
The method according to claim 1 or 2,
The antimicrobial composition is characterized in that it has antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria and multi-drug-resistant strains thereof.
The method of claim 5,
The Gram-positive bacteria are Staphylococcus aureus ( Staphylococcus aureus ) and Enterococcus sp., Characterized in that at least one selected from the group consisting of, the composition for antibacterial.
The method of claim 5,
The gram-negative bacteria, Psedomonas aeruginosa ( Psedomonas aeruginosa ), acetobacter baumannii ( Acinetobacter baumannii ), and Escherichia coli , characterized in that at least one selected from the group consisting of, antibacterial composition.
The method of claim 5,
The multi-drug resistant strains are methicillin-sensitive yellow grape algal bacteria (MSSA), methicillin-resistant yellow grape algal bacteria (MRSA), vancomycin-resistant yellow grape staphylococcus (VRSA), vancomycin-moderately resistant yellow grape staphylococcus (VISA), vancomycin intra-growth bacteria (VRE) , Vancomycin-sensitized growth bacteria (VSE), multidrug-resistant Pseudomonas aeruginosa (MRTA), carpepenem-resistant Pseudomonas aeruginosa (CRPA), kappapenem-resistant acetobacter baumani bacteria (CRAB) carbapenem-in-growth mycobacterium bacterium (CRE) It characterized in that any one or more, antibacterial composition.
The method according to claim 1 or 2,
The antimicrobial composition is characterized in that the pharmaceutical composition, antimicrobial composition.
The method according to claim 1 or 2,
The antibacterial composition is an antimicrobial composition, characterized in that any one selected from the group consisting of food additives, feed additives, antiseptic composition and quasi-drug composition.
Compound represented by the following formula (2):
[Formula 2]

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