Novel difluoroquercetin derivative compound and use thereof

Abstract

The present invention relates to a novel difluoroquercetin derivative compound and use thereof. In the present invention, eight novel compounds were prepared by introducing substituents at the 3-O and 7-O positions of 3',4'-difluoroquercetin, and it has been confirmed that the novel compounds exhibit inhibitory activity against carbapenemases AmpC, KPC-2, OXA-48, VIM-2, and NDM-1, and exhibit the effects of reducing antibiotic resistance to and enhancing antibacterial activity against Carbapenemase-producing Pseudomonas aeruginosa (CPPA) when used in combination with aztreonam (ATM), which is an existing antibiotic. Therefore, the difluoroquercetin derivative compound can be effectively used in related industries.

Description

Novel difluoroquercetin derivative compounds and uses thereof

[0001] The present invention relates to novel difluoroquercetin derivative compounds and their uses.

[0002] Since the discovery of penicillin and streptomycin, over 5,000 types of antibiotics have been discovered. Among them, about 50 types of antibiotics have been developed into approximately 400 formulations and are currently used in clinical practice as major treatments for infectious diseases. Antibiotics are broadly classified into beta-lactam and non-beta-lactam types based on their chemical structure, and more specifically into penicillin, cephalosporin, quinolone, and aminoglycoside classes. Additionally, antibiotics are sometimes classified according to their mechanism of action into cell wall synthesis inhibitors, nucleic acid synthesis inhibitors, and protein synthesis inhibitors.

[0003] The demand for the development of new antibiotics is increasing significantly due to the emergence of multidrug-resistant bacteria that are simultaneously resistant to multiple antibiotics, and if current trends continue, antibiotic resistance is predicted to become one of the biggest problems facing 21st-century medicine.

[0004] The six statutory types of healthcare-associated infections [vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-resistant Enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Pseudomonas aeruginosa (MRPA), multidrug-resistant Acinetobacter baumannii (MRAB), carbapenem-resistant Enterobacteriaceae (CRE), etc.] listed in the Korea Centers for Disease Control and Prevention's "Guidelines for the Management of Multidrug-Resistant Infections" are representative multidrug-resistant bacteria, and 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 coccus that causes pneumonia (especially hospital-acquired pneumonia), skin and soft tissue infections, and bacteremia; while it is one of the most common pathogens, 35–66% of S. aureus cases have been identified as MRSA since the 1990s. Meanwhile, vancomycin-resistant strains have been discovered in Enterococcus, a type of Gram-positive coccus, and the incidence of hospital-acquired VRE in the United States has increased more than 20-fold, from 0.3% in 1989 to 7.9% in 1993. Furthermore, New Delhi metallo-beta-lactamase-1 (NDM-1), identified in 2008, was found to be a superbug of hospital-acquired infections that is resistant to any existing antibiotic.

[0005] It is known that resistant strains acquire resistance to antibiotics through methods such as ① altering the structure of antibiotics via antibiotic-degrading and antibiotic-altering enzymes, ② lowering intracellular antibiotic concentrations by activating efflux through antibiotic influx inhibition / efflux pumps, or ③ changing target proteins to which antibiotics bind through mutation. Bacteria effectively counter antibiotics by mobilizing two or more of these resistance mechanisms, and it is also known that the degree of resistance increases as more mechanisms are mobilized. Therefore, antibiotics designed to combat resistant strains must be able to evade the development of resistance by either ① inhibiting novel bacterial targets previously unknown or ② inhibiting diverse target groups.

[0006] Accordingly, the inventors prepared eight novel compounds by introducing substituents at the 3-O and 7-O positions of 3',4'-difluoroquercetin derivatives, confirmed the carbapenem inhibitory activity against multidrug-resistant Pseudomonas aeruginosa, and confirmed the effect of increasing the antibacterial activity of the antibiotic Aztreonam against Pseudomonas aeruginosa, thereby completing the present invention.

[0007] The object of the present invention is to provide a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0008] [Chemical Formula 1]

[0009]

[0010] In the above chemical formula 1,

[0011] R1 is a straight-chain or branched-chain C1-10 alkyl group, a straight-chain or branched-chain C1-10 cycloalkyl group, a straight-chain or branched-chain C1-10 alkyl-cycloalkyl group, or a straight-chain or branched-chain C1-10 arylalkyl group, and

[0012] R2 is any one selected from a straight-chain or branched C1-10 alkyl group, a straight-chain or branched C1-10 alkoxy group, a straight-chain or branched C1-10 alkylamine group, a straight-chain or branched C1-10 alkylamide group, a phosphate, a sulfate, a nitrate, a C3-10 cycloalkyl group, a straight-chain or branched C1-10 cycloalkylamide group, a C3-10 aryloxy group, a C3-10 arylamine group, and a substituted or unsubstituted 3-10 atom heterocyclic group comprising one or more heteroatoms selected from N, O, S, Se and Te.

[0013] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of multidrug-resistant Pseudomonas aeruginosa infection comprising a compound represented by the above chemical formula 1 as an active ingredient.

[0014] Another objective of the present invention is to provide a food composition for preventing or improving multidrug-resistant Pseudomonas aeruginosa infection comprising a compound represented by the above chemical formula 1 as an active ingredient.

[0015] Another objective of the present invention is to provide a method for increasing the antimicrobial activity of an antibiotic, comprising the steps of: mixing a compound represented by Formula 1 and an antibiotic; and treating multidrug-resistant Pseudomonas aeruginosa.

[0016] Another objective of the present invention is to provide a method for treating multidrug-resistant Pseudomonas aeruginosa infection comprising the step of administering a pharmaceutically effective amount of the above compound to an individual.

[0017] In order to achieve the above objective,

[0018] The present invention provides a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0019] [Chemical Formula 1]

[0020]

[0021] In the above chemical formula 1,

[0022] R1 is a straight-chain or branched-chain C1-10 alkyl group, a straight-chain or branched-chain C1-10 cycloalkyl group, a straight-chain or branched-chain C1-10 alkyl-cycloalkyl group, or a straight-chain or branched-chain C1-10 arylalkyl group, and

[0023] R2 is any one selected from a straight-chain or branched C1-10 alkyl group, a straight-chain or branched C1-10 alkoxy group, a straight-chain or branched C1-10 alkylamine group, a straight-chain or branched C1-10 alkylamide group, a phosphate, a sulfate, a nitrate, a C3-10 cycloalkyl group, a straight-chain or branched C1-10 cycloalkylamide group, a C3-10 aryloxy group, a C3-10 arylamine group, and a substituted or unsubstituted 3-10 atom heterocyclic group comprising one or more heteroatoms selected from N, O, S, Se and Te.

[0024] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of multidrug-resistant Pseudomonas aeruginosa infection comprising a compound represented by the above chemical formula 1 as an active ingredient.

[0025] In addition, the present invention provides a food composition for preventing or improving multidrug-resistant Pseudomonas aeruginosa infection comprising a compound represented by the above chemical formula 1 as an active ingredient.

[0026] In addition, the present invention provides a method for increasing the antibacterial activity of an antibiotic, comprising the steps of: mixing a compound represented by the above chemical formula 1 with an antibiotic; and treating multidrug-resistant Pseudomonas aeruginosa with the mixture.

[0027] In addition, the present invention provides a method for treating multidrug-resistant Pseudomonas aeruginosa infection comprising the step of administering a pharmaceutically effective amount of the above compound to an individual.

[0028] The quercetin derivative compounds of the present invention were prepared by introducing substituents at the 3-O and 7-O positions of 3',4'-difluoroquercetin to produce eight novel compounds, and their inhibitory activity against carbapenemases AmpC, KPC-2, OXA-48, VIM-2, and NDM-1 was confirmed. Additionally, when combined with the existing antibiotic aztreonam (ATM), the effect of reducing antibiotic resistance and enhancing antimicrobial activity against carbapenemase-producing Pseudomonas aeruginosa (CPPA) was confirmed, making them useful for related industries.

[0029] Figure 1 shows the carbapenemase inhibitory activity of the compound of the present invention.

[0030] Figure 2 shows the drug efflux pump inhibitory activity of the compound of the present invention and the antibacterial activity of the compound of the present invention in combination with aztreonam against NDM-1 producing Pseudomonas aeruginosa strain PA-017.

[0031] Figure 3 shows the antibacterial activity of compound 599 of the present invention in combination with aztreonam against 26 carbapenem-resistant Pseudomonas aeruginosa.

[0032] Figure 4 shows the change in the distribution of minimum inhibitory concentrations for 26 carbapenem-resistant Pseudomonas aeruginosa strains according to the concentration of aztreonam in combination with compound 599 of the present invention and aztreonam.

[0033] Figure 5 shows the antibacterial activity of compound 596 of the present invention in combination with aztreonam against 26 carbapenem-resistant Pseudomonas aeruginosa.

[0034] Figure 6 shows the change in the distribution of minimum inhibitory concentrations for 26 carbapenem-resistant Pseudomonas aeruginosa strains according to the concentration of aztreonam in combination with compound 596 of the present invention.

[0035] Figure 7 is a table showing the cumulative antibacterial activity of aztreonam according to the dose of compound 599 of the present invention.

[0036] Figure 8 is a graph showing the cumulative antibacterial activity of aztreonam according to the dose of compound 599 of the present invention.

[0037] Figure 9 is a table showing the cumulative antibacterial activity of aztreonam according to the dose of compound 596 of the present invention.

[0038] Figure 10 is a graph showing the cumulative antibacterial activity of aztreonam according to the dose of compound 596 of the present invention.

[0039] Figure 11 shows the 50% inhibition concentration and 90% inhibition concentration of aztreonam against all strains in combination with compound 599 of the present invention.

[0040] Figure 12 shows the 50% inhibition concentration and 90% inhibition concentration of aztreonam against all strains in combination with compound 599 of the present invention.

[0041] Figure 13 shows the 50% and 90% inhibition concentrations of aztreonam against all strains in combination with compounds 599 and 596 of the present invention, compared with the 50% and 90% inhibition concentrations of a commercial antibiotic-resistance inhibitor combination.

[0042] Figure 14 shows a time-kill assay of compound 596 of the present invention in combination with an antibiotic.

[0043] Figure 15 shows the intracellular hemolysis rate by the compound of the present invention.

[0044] Figure 16 shows the cell viability rate by the compound of the present invention.

[0045] Hereinafter, the present invention will be described in detail with reference to the attached drawings and embodiments thereof. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited by this. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalent scope interpreted therefrom.

[0046] Furthermore, the terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as “comprising” a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components.

[0047] The terms used in the present invention are explained below.

[0048] The present invention provides a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0049] [Chemical Formula 1]

[0050]

[0051] In the above chemical formula 1,

[0052] R1 may be a straight-chain or branched C1-10 alkyl group, a straight-chain or branched C1-10 cycloalkyl group, a straight-chain or branched C1-10 alkyl-cycloalkyl group, or a straight-chain or branched C1-10 arylalkyl group, preferably a straight-chain or branched C1-10 alkyl group or a straight-chain or branched C1-10 alkyl-cycloalkyl group, and more preferably , or It could be.

[0053] R2 may be any one selected from a substituted or unsubstituted 3-10 atom heterocyclic group comprising a straight-chain or branched C1-10 alkyl group, a straight-chain or branched C1-10 alkoxy group, a straight-chain or branched C1-10 alkylamine group, a straight-chain or branched C1-10 alkylamide group, a phosphate, a sulfate, a nitrate, a C3-10 cycloalkyl group, a straight-chain or branched C1-10 cycloalkylamide group, a C3-10 aryloxy group, a C3-10 arylamine group, and one or more heteroatoms selected from N, O, S, Se, and Te, and preferably comprises a straight-chain or branched C1-10 alkylamine group, a straight-chain or branched C1-10 alkylamide group, a phosphate, a sulfate, a nitrate, a straight-chain or branched C1-10 cycloalkylamide group, or an N atom. It may be a substituted or unsubstituted 3-10 atom heterocyclic group, and more preferably , , , , , , or It could be.

[0054] In the present invention, “pharmaceuticalally acceptable” means not significantly stimulating the organism and not inhibiting the biological activity and properties of the administered active substance.

[0055] The term "pharmaceuticalally acceptable salt" above means that, within the scope of sound medical judgment, these salts are used in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, and similarities, and are proportional to a reasonable advantage-to-disadvantage ratio. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which are incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts of amino groups formed from inorganic acids such as hydrochloric acid, hydrobromide, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts are adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, maleate, malieate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, Includes pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valeraate salts, and similar substances.

[0056] Salts derived from suitable bases include alkali metals, alkaline earth metals, ammonium, and N+(C1-4 alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and similar ones. In addition, pharmaceutically acceptable salts include, when appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxyls, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0057] According to one embodiment of the present invention, the compound may be one selected from among compounds represented by the following chemical formulas 2 to 9:

[0058] [Chemical Formula 2]

[0059]

[0060] The compound represented by the above chemical formula 2 of the present invention can be named 3-(2-cyclohexylethoxy)-2-(3,4-difluorophenyl)-5-hydroxy-4-oxo-4H-chromene-7-yl dihydrogen phosphate (hereinafter, “Compound 591”).

[0061] [Chemical Formula 3]

[0062]

[0063] The compound represented by the above chemical formula 3 of the present invention may be named 3-(2-cyclohexylethoxy)-2-(3,4-difluorophenyl)-5-hydroxy-4-oxo-4H-chromene-7-yl hydrogen sulfate (hereinafter, “Compound 599”).

[0064] [Chemical Formula 4]

[0065]

[0066] The compound represented by the above chemical formula 4 of the present invention can be named 2-((((3-(2-cyclohexylethoxy)-2-(3,4-difluorophenyl)-5-hydroxy-4-oxo-4H-cromen-7-yl)oxy)carbonyl)amino)acetic acid (hereinafter, “Compound 602”).

[0067] [Chemical Formula 5]

[0068]

[0069] The compound represented by the above chemical formula 5 of the present invention can be named 7-((1H-tetrazole-5-yl)methoxy)-2-(3,4-difluorophenyl)-3-(3,3-dimethylbutoxy)-5-hydroxy-4H-cromen-4-one (hereinafter, “Compound 596”).

[0070] [Chemical Formula 6]

[0071]

[0072] The compound represented by the above chemical formula 6 of the present invention may be named N-cyclohexyl-2-((2-(3,4-difluorophenyl)-3-(3,3-dimethylbutoxy)-5-hydroxy-4-oxo-4H-cromen-7-yl)oxy)acetamide (hereinafter, “Compound 560”).

[0073] [Chemical Formula 7]

[0074]

[0075] The compound represented by the above chemical formula 7 of the present invention may be named 2-((3-(2-cyclopentylethoxy)-2-(3,4-difluorophenyl)-5-hydroxy-4-oxo--4H-cromen-7-yl)oxy)acetamide (hereinafter, “Compound 569”).

[0076] [Chemical Formula 8]

[0077]

[0078] The compound represented by the above chemical formula 8 of the present invention can be named 7-(2-([1,4'-bipiperidin]-1'-yl)-2-oxoethoxy)-2-(3,4-difluorophenyl)-3-(3,3-dimethylbutoxy)-5-hydroxy-4H-cromen-4-one (hereinafter, “Compound 561”).

[0079] [Chemical Formula 9]

[0080]

[0081] The compound represented by the above chemical formula 9 of the present invention can be named 2-(3,4-difluorophenyl)-3-(3,3-dimethylbutoxy)-5-hydroxy-7-(2-(4-methylpiperazine-1-yl)ethoxy)-4H-cromen-4-one (hereinafter, “Compound 566”).

[0082] In the present invention, the compounds represented by Chemical Formulas 2 to 9 can be prepared by the following Reaction Formula 1 and Reaction Formula 2.

[0083] [Reaction Equation 1]

[0084]

[0085] [Reaction Equation 2]

[0086]

[0087] According to one embodiment of the present invention, the compound may inhibit carbapenemase activity.

[0088] The “Carbapenem” of the present invention is the most commonly used antibiotic for treating severe bacterial infections and is used for multidrug-resistant (MDR) bacterial infections. Carbapenemase is an enzyme capable of degrading or neutralizing carbapenem-class antibiotics; carbapenem-class antibiotics exhibit potent effects primarily against Gram-negative bacteria such as Escherichia coli and Klebsiella pneumonia, and are effective in treating bacterial infections.

[0089] According to one embodiment of the present invention, the carbapenem degrading enzyme may be AmpC, KPC-2, OXA-48, VIM-2, or NDM-1.

[0090] “AmpC (Ampicillin C)” of the present invention is an enzyme capable of degrading ampicillin, and bacteria that produce it show resistance to cephalosporin class and other β-lactam antibiotics. AmpC is found in large quantities in intestinal bacteria (e.g., Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Citrobacter spp., etc.).

[0091] The “KPC-2 (Klebsiella pneumoniae carbapenemase-2)” of the present invention is an enzyme of Klebsiella pneumoniae that is resistant to carbapenem antibiotics, and it imparts resistance to carbapenem antibiotics and degrades the antibiotics, thereby enabling the bacteria to survive.

[0092] The “OXA-48 (oxacillin-hydrolyzing β-lactamase-48)” of the present invention is an enzyme that induces resistance to carbapenem antibiotics and is found in pathogens such as Klebsiella pneumoniae, Escherichia coli, and Enterobacter. OXA-48 degrades carbapenem antibiotics, thereby preventing them from inhibiting bacteria.

[0093] “VIM-2 (Verona Integron-encoded Metallo-β-lactamase-2)” of the present invention is an enzyme belonging to the metallo-β-lactamase family, which is mainly found in multidrug-resistant bacteria (MDR), and can degrade various beta-lactam antibiotics, including carbapenem antibiotics.

[0094] “NDM-1 (New Delhi Metallo-β-lactamase-1)” of the present invention is an enzyme belonging to the metallo-β-1-lactamase family, which can induce resistance to beta-lactam antibiotics and, in particular, confer resistance to carbapenem antibiotics such as meropenem.

[0095] According to one embodiment of the present invention, the compound may inhibit a drug ejection pump.

[0096] The “Efflux pump” of the present invention is an active transporter that moves unwanted substances out of a cell and is an important component of bacteria that removes antibiotics. It can transport heavy metals, organic contaminants, compounds produced by plants, quorum sensing signals, bacterial metabolites, and neurotransmitters. The efflux system effluxes unwanted toxic substances by pumping them out through specific efflux pumps via an energy-dependent mechanism (active transport). Some efflux systems are drug-specific, while others can accommodate multiple drugs using small multiple drug resistance (SMR) transporters.

[0097] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of multidrug-resistant Pseudomonas aeruginosa infection comprising a compound represented by the above chemical formula 1 as an active ingredient.

[0098] The term “prevention” as used in this invention refers to any act of suppressing the symptoms of a specific disease or delaying its progression through the administration of the composition of this invention.

[0099] The term “treatment” as used in this invention refers to any act of improving or beneficially altering the symptoms of a specific disease through the administration of the composition of this invention.

[0100] The pharmaceutical composition of the present invention may additionally include an adjuvant in addition to the active ingredient. Any adjuvant known in the art may be used without limitation, but, for example, Freund's complete or incomplete adjuvant may be further included to increase the effect.

[0101] The pharmaceutical composition according to the present invention may be prepared in a form in which an active ingredient is incorporated into a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention are not limited to these, but may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0102] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external formulations, suppositories, or sterile injectable solutions, each according to conventional methods.

[0103] When formulating, the product may be prepared using diluents or excipients such as commonly used fillers, volume expanders, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules, and such solid dosage forms may be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the active ingredient. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, and syrups, and may contain various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, in addition to commonly used diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Water-insoluble solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Bases for suppositories may include Witepsol, Tween 61, cocoa paste, laurin paste, glycerogelatin, etc.

[0104] The pharmaceutical composition according to the present invention may be administered to an individual by various routes. Any mode of administration may be anticipated, for example, by oral, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

[0105] The dosage of the pharmaceutical composition according to the present invention is selected by taking into consideration the age, weight, gender, physical condition, etc. of the individual. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the subject, and preferably, it is included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg / mL. If the concentration is less than 0.01 μg / mL, pharmaceutical activity may not appear, and if it exceeds 5,000 μg / mL, it may exhibit toxicity to the human body.

[0106] The “Pseudomonas aeruginosa” of the present invention is a Gram-negative aerobic bacterium and a pathogenic microorganism frequently found in various environments. It can cause various infections and is one of the pathogens that is very difficult to treat due to its resistance to several antibiotics.

[0107] According to one embodiment of the present invention, the compound may increase the antibacterial activity of the antibiotic.

[0108] According to one embodiment of the present invention, the antibiotic may be aztreonam.

[0109] The “Aztreonam” of the present invention is a beta-lactam antibiotic primarily used as a treatment for Gram-negative bacteria. It is mainly used to treat various types of infections, such as respiratory infections, urinary tract infections, abdominal infections, and sepsis; however, resistance may occur against bacteria that produce some Metallo-β-lactamase (MBL) or Extended-Spectrum Beta-Lactamase (ESBL).

[0110] According to one embodiment of the present invention, the compound may inhibit Pseudomonas aeruginosa in combination with an antibiotic.

[0111] According to one embodiment of the present invention, the Pseudomonas aeruginosa may be a GES (Guiana-Extended-Spectrum beta-lactamase) producing bacterium, a VIM (Verona integron-encoded metallo-β-lactamase) producing bacterium, an IMP (imipenemase-type metallo-β-lactamase) producing bacterium, an NDM (New Delhi metallo-β-lactamase) producing bacterium, or a co-producing bacterium of IMP and NDM.

[0112] According to one embodiment of the present invention, the compound may reduce the minimum inhibitory concentration (MIC) of an antibiotic against Pseudomonas aeruginosa by 2 to 64 times.

[0113] In addition, the present invention provides a food composition for preventing or improving multidrug-resistant Pseudomonas aeruginosa infection comprising a compound represented by the above chemical formula 1 as an active ingredient.

[0114] As used in the present invention, the term “improvement” refers to any action that at least reduces parameters related to the condition being treated, such as the degree of symptoms.

[0115] In addition to containing the active ingredient of the present invention, the food composition of the present invention may contain various flavoring agents or natural carbohydrates, etc., as additional ingredients, as in conventional food compositions.

[0116] Examples of the natural carbohydrates described above include monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; polysaccharides, e.g., dextrin, cyclodextrin, etc., and conventional sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. The flavoring agents described above may advantageously use natural flavoring agents (taumatin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention may be formulated in the same manner as the pharmaceutical composition described above and used as a functional food or added to various foods. Foods to which the composition of the present invention may be added include, for example, beverages, meat, chocolate, food products, confectionery, pizza, ramen, other noodles, chewing gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

[0117] In addition, the above food composition may contain, in addition to the extract which is an active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, the food composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages.

[0118] In addition, the present invention provides a method for increasing the antimicrobial activity of an antibiotic, comprising the steps of: mixing a compound represented by the above chemical formula 1 with a carbapenem antibiotic; and treating multidrug-resistant Pseudomonas aeruginosa with the mixture.

[0119] The recombinant AmpC, OXA-48 (residues 23-265) and VIM-2 (residues 27-266) of the present invention were expressed in Escherichia coli using a known method [Lee, S.; Lee, T.; Kim, MK; Ahn, JH; Jeong, S.; Park, K.-H.; Chong, Y. Potentiation of Antibiotic Activity of Aztreonam against Metallo-β-Lactamase-Producing Multidrug-Resistant Pseudomonas aeruginosa by 3-O-Substituted Difluoroquercetin Derivatives. Pharmaceutics 2024, 16, 185]. KPC-2 and NDM-1 were commercially purchased from LifeSpan Biosciences, Inc. (LS-G26598) and RayBiotech, Inc. (230-00554), respectively.

[0120] In addition, the present invention provides a method for treating multidrug-resistant Pseudomonas aeruginosa infection comprising the step of administering a pharmaceutically effective amount of the above compound to an individual.

[0121] The therapeutic method of the present invention comprises administering the recombinant peptide or the recombinant vector to an individual in a therapeutically effective amount. It is preferable to apply a specific therapeutically effective amount for a specific individual differently depending on various factors, including the specific composition (such as the type and degree of the response to be achieved and whether other agents are used in some cases), the individual's age, body weight, general health status, gender and diet, time of administration, route of administration and secretion rate of the composition, duration of treatment, and drugs used together or concurrently with the specific composition, as well as similar factors well known in the pharmaceutical field. The daily dosage is 0.0001 to 100 mg / kg based on the amount of the pharmaceutical composition of the present invention, preferably 0.01 to 100 mg / kg, and may be administered 1 to 6 times a day. However, it is obvious to those skilled in the art that the dosage or administration amount of each active ingredient must be such that it does not contain an excessively high content of each active ingredient to cause side effects. Therefore, it is preferable to determine the effective amount of the composition suitable for the purpose of the present invention by considering the aforementioned matters.

[0122] The above-mentioned individual is applicable to any mammal, and said mammal includes not only humans and primates, but also livestock such as cattle, pigs, sheep, horses, dogs, and cats.

[0123] The compounds of the present invention may be administered to mammals, such as rats, mice, livestock, and humans, by various routes. All modes of administration are expected, for example, orally, rectally or intravenously, intramuscularly, subcutaneously, intrathecally, or intracerebroventricularly.

[0124] All microorganisms used in the present invention were collected from 26 carbapenemase-producing P. aeruginosa clinical isolates, blaGES, blaVIM, blaIMP, blaNDM, and blaIMP / NDM, from MBL-positive patients at Hallym University Kangnam Sacred Heart Hospital.

[0125] The present invention will be explained in more detail below through examples. These examples are merely for the purpose of explaining the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited to these examples.

[0126] <Example 1> Preparation of 3-O-alkylated and 7-O-alkylated 3',4'-difluoroquercetin derivative compounds

[0127] The 3',4'-difluoroquercetin derivative compounds of the present invention are based on the previously reported technology (Pharmaceutics 2024, 16(2), 185), and as shown in Reaction Schemes 1 and 2 below, compounds 526, 549, and 545 were prepared by substituting 3,3-dimethylbutyl (hereinafter 'Compound 526'), 2-cyclopentylethyl (hereinafter 'Compound 549'), or 2-cyclohexylethyl (hereinafter 'Compound 545') at the 3rd hydroxyl position (R1) of a 3-O-alkyl 3',4'-difluoroquercetin derivative, and then regioselectively introducing a polar functional group at the 7th hydroxyl position to produce the following eight compounds (hereinafter compounds 591, 599, 602, 596, 560, 569, 561, 566) was synthesized.

[0128] [Reaction Equation 1]

[0129]

[0130] [Reaction Equation 2]

[0131]

[0132] 1-1. Preparation of Compound 591

[0133] 디Compound 545 (100 mg, 1 eq, 0.24 mmol), diethylchlorophosphite (38 μL, 1.1 eq, 0.26 mmol), and triethylamine (50 μL, 1.5 eq, 0.36 mmol) were added to 5 mL of chloromethane and stirred at room temperature for 3 hours. A saturated aqueous ammonium chloride solution was added to the reaction mixture to complete the reaction, and the organic layer obtained by extraction with dichloromethane was washed with saturated brine, dehydrated with magnesium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (Hexanes:EtOAc = 3:1) to obtain a yellow solid intermediate (110 mg, 0.20 mmol, 83% yield).

[0134] Intermediate (50 mg, 1 eq, 0.09 mmol) and bromotrimethylsilane (71.6 μL, 6 eq, 0.54 mmol) were added to 5 mL of dichloromethane and stirred at room temperature for 72 hours. After adding methanol to the reaction solution, the residue obtained by vacuum distillation was triturated with dichloromethane to obtain yellow solid compound 591 (24 mg, 0.05 mmol, 53% yield).

[0135] 1H NMR (500 MHz, methanol-d4) δ 8.04 (ddd, J = 2.2, 7.8, 12.0 Hz, 1H), 7.94-7.90 (m, 1H), 7.46 (ddd, J = 8.6, 8.6, 10.3 Hz, 1H), 6.94 (dd, J = 1.0, 2.1 Hz, 1H), 6.64 (dd, J = 0.9, 2.1 Hz, 1H), 4.09 (t, J = 6.5 Hz, 2H), 1.70-1.63 (m, 5H), 1.56 (q, J = 6.6 Hz, 2H), 1.44-1.35 (m, 1H), 1.24-1.13 (m, 3H), 0.92-0.84 (m, 2H).

[0136] 13C NMR (125 MHz, methanol-d4) δ 180.6, 163.0, 158.6 (d, J = 5.8 Hz), 157.5, 155.8, 153.1 (dd, J = 12.6, 251.9 Hz), 151.3 (dd, J = 12.9, 245.5 Hz), 140.4, 128.9 (dd, J = 3.9, 6.7 Hz), 127.1 (dd, J = 3.7, 6.9 Hz), 119.3 (d, J = 20.1 Hz), 118.8 (d, J = 17.9 Hz), 109.2, 104.2 (d, J = 5.7 Hz), 101.1 (d, J = 5.4 Hz), 72.1, 38.5, 35.4, 34.3, 27.6, 27.4.

[0137] FAB-MS calcd for C23H24F2O8P [M+H]+ 497.1, found 497.5.

[0138] 1-2. Preparation of Compound 599

[0139] 테 Compound 545 (0.26 mmol), 2,2,2-trichloroethanesulfonyl chloride (0.26 mmol), triethylamine (0.38 mmol), and DMAP (0.26 mmol) were added to 5 mL of trihydrofuran and stirred at room temperature for 2 hours. After adding a saturated aqueous ammonium chloride solution to the reaction solution, the mixture was extracted with ethyl acetate. The extracted organic layer was washed with saturated brine, dehydrated with magnesium sulfate, filtered, and subjected to vacuum distillation. The residue was purified by silica gel column chromatography (Hexanes:Et2O = 10:1) to obtain a yellow solid intermediate.

[0140] To 2 mL of tetrahydrofuran, intermediate (0.08 mmol), methanol (2 mL), ammonium formate (0.47 mmol), and 10% Pd / C (4 mg) were added, and after installing a hydrogen balloon, the mixture was stirred vigorously at room temperature for 1 hour. The reaction solution was filtered through a short Celite pad, and the residue was triturated with ether to obtain compound 599 in the form of a yellow oil (59% yield).

[0141] 1H NMR (500 MHz, methanol-d4) δ 8.06 (ddd, J = 2.2, 7.9, 12.0 Hz, 1H), 7.95-7.92 (m, 1H), 7.47 (ddd, J = 8.6, 8.6, 10.4 Hz, 1H), 7.06 (d, J = 2.1 Hz, 1H), 6.71 (d, J = 2.1 Hz, 1H), 4.09 (t, J = 6.5 Hz, 2H), 1.70-1.63 (m, 5H), 1.56 (q, J = 6.6 Hz, 2H), 1.44-1.37 (m, 1H), 1.24-1.13 (m, 3H), 0.92-0.84 (m, 2H).

[0142] 13C NMR (125 MHz, methanol-d4) δ 180.6, 162.5, 160.3, 157.4, 155.8, 153.0 (dd, J = 12.7, 251.8 Hz), 151.3 (dd, J = 13.1, 245.4 Hz), 140.3, 129.1-129.0 (m), 127.1-127.0 (m), 119.3 (d, J = 20.2 Hz), 118.8 (d, J = 17.7 Hz), 109.0, 104.3, 100.0, 72.1, 38.5, 35.4, 34.3, 27.6, 27.3.

[0143] FAB-MS calcd for C23H23F2O8S [M+H]+ 497.1, found 497.5.

[0144] 1-3. Preparation of Compound 602

[0145] 테 Compound 545 (0.24 mmol), tert-butyl 2-isocyanatoacetate (0.24 mmol), and triethylamine (0.26 mmol) were added to 5 mL of trihydrofuran and stirred at room temperature for 2 hours. Saturated aqueous ammonium chloride solution was added to the reaction solution and diluted with ethyl acetate. The organic layer was washed with saturated salt water, dehydrated with magnesium sulfate, filtered, and the residue obtained by vacuum distillation was purified by silica gel column chromatography (Hexanes:EtOAc = 4:1) to obtain a yellow solid intermediate.

[0146] 2 mL of dichloromethane was mixed with an intermediate (0.10 mmol) and trifluoroacetic acid (1 mL), stirred at room temperature for 1 hour, and then distilled under reduced pressure. The residue was triturated with ether to obtain a yellow solid compound 602 (35% yield).

[0147] 1H NMR (500 MHz, methanol-d4) δ 8.02 (ddd, J = 2.1, 7.8, 11.9 Hz, 1H), 7.92-7.89 (m, 1H), 7.44 (ddd, J = 8.6, 8.6, 9.9 Hz, 1H), 6.93 (d, J = 2.0 Hz, 1H), 6.57 (d, J = 2.0 Hz, 1H), 4.07 (t, J = 6.5 Hz, 2H), 3.93 (s, 2H), 1.68-1.66 (m, 5H), 1.55 (q, J = 6.6 Hz, 2H), 1.43-1.35 (m, 1H), 1.25-1.13 (m, 3H), 0.91-0.84 (m, 2H).

[0148] 13C NMR (125 MHz, acetone-d6) δ 180.2, 171.1, 162.1, 158.0, 156.7, 155.1, 154.4, 152.4 (dd, J = 12.7, 251.0 Hz), 150.7 (dd, J = 12.8, 244.7 Hz), 140.1, 128.8 (dd, J = 4.0, 6.9 Hz), 127.1 (dd, J = 3.6, 7.1 Hz), 119.0 (d, J = 20.1 Hz), 118.6 (d, J = 17.7 Hz), 109.3, 104.8, 101.3, 71.5, 42.9, 38.1, 35.0, 33.8, 27.2, 26.9.

[0149] FAB-MS calcd for C26H26F2NO8 [M+H]+ 518.2, found 518.5.

[0150] 1-4. Preparation of Compound 596

[0151] Compound 526 (0.51 mmol), 5-(chloromethyl)-2-(tetrahydro-2H-pyran-2-yl)-2H-tetrazole (0.46 mmol), and potassium carbonate (0.77 mmol) were added to 10 mL of dimethylformamide and stirred at room temperature for 12 hours. An aqueous ammonium chloride solution saturated with ethyl acetate was added to the reaction solution and extracted with ethyl acetate. The extracted organic layer was washed with saturated brine, dehydrated with magnesium sulfate, filtered, and subjected to vacuum distillation. The residue obtained was purified by silica gel column chromatography (Hexanes:CH2Cl2:acetone = 16:3:1) to obtain a yellow solid intermediate.

[0152] An intermediate (0.090 mmol) was added to methanol (5 mL) containing 0.5 M hydrochloric acid and stirred at 0 °C for 2 hours. The reaction solution was triturated with ether to obtain a yellow solid compound 596 (94% yield).

[0153] 1H NMR (500 MHz, acetone-d6) δ 8.11 (ddd, J = 2.1, 7.9, 12.2 Hz, 1H), 8.03-8.00 (m, 1H), 7.56 (ddd, J = 8.6, 8.6, 10.3 Hz, 1H), 6.90 (d, J = 2.2 Hz, 1H), 6.46 (d, J = 2.2 Hz, 1H), 5.69 (s, 2H), 4.21 (t, J = 7.5 Hz, 2H), 1.72 (t, J = 7.5 Hz, 2H), 0.96 (s, 9H).

[0154] 13C NMR (125 MHz, acetone-d6) δ 179.8, 164.6, 162.8, 157.6, 154.7, 154.4, 152.3 (dd, J = 12.6, 250.9 Hz), 150.8 (dd, J = 12.7, 244.7 Hz), 140.1, 128.8 (dd, J = 3.8, 7.0 Hz), 126.8 (dd, J = 3.7, 7.0 Hz), 118.7, 118.6 (d, J = 3.4 Hz), 107.4, 99.4, 94.0, 71.3, 61.3, 44.0, 29.9, 29.9.

[0155] FAB-MS calcd for C23H23F2N4O5 [M+H]+ 473.2, found 473.5.

[0156] 1-5. 화합물 560 제조

[0157] Compound 526 (500 mg, 1.26 mmol), ethyl bromoacetate (0.15 mL, 1.39 mmol), and potassium carbonate (262 mg, 1.89 mmol) were added to 10 mL of dimethylformamide and stirred at 50 °C for 6 hours. After cooling the reaction mixture to room temperature, it was diluted with ethyl acetate, saturated aqueous ammonium chloride solution was added, and washed with saturated salt water. After dehydrating the organic layer with magnesium sulfate, the residue obtained by filtration and vacuum distillation was purified by silica gel column chromatography (Hex:CH2Cl2:EtOAc = 6:1:1) to obtain a yellow solid first intermediate (400 mg, 0.83 mmol, 66% yield).

[0158] The first intermediate (200 mg, 0.42 mmol) and 2N sodium hydroxide aqueous solution (3 mL) were added to 3 mL of tetrahydrofuran and 3 mL of methanol, and stirred at room temperature for 2 hours. The reaction mixture was acidified by adding 2N hydrochloric acid, and then extracted with ethyl acetate. The extracted organic layer was washed with saturated brine, dehydrated with magnesium sulfate, filtered, and vacuum distilled. The residue was purified by tritration with ether to obtain a second intermediate (156 mg, 0.35 mmol, 83% yield) as a yellow solid.

[0159] To 10 mL of dichloromethane, the second intermediate (277 mg, 0.62 mmol), cyclohexylamine (62 mg, 0.62 mmol), DMAP (151 mg, 1.24 mmol), and EDC.HCl (237 mg, 1.24 mmol) were added and stirred at room temperature for 12 hours. The residue obtained by vacuum distillation of the reactants was purified by silica gel column chromatography (CH2Cl2:MeOH = 50:1) to obtain a third intermediate (280 mg, 0.38 mmol, 62 yield) as a yellow solid.

[0160] 3 mL of dichloromethane was mixed with the third intermediate (100 mg, 0.14 mmol) and trifluoroacetic acid (3 mL), and stirred at room temperature for 12 hours. The residue obtained by vacuum distillation of the reactants was treated with an ether containing 2N hydrochloric acid. The solid obtained by filtering the precipitate was washed with acetone to obtain the yellow solid compound 560 (36 mg, 0.06 mmol, 43% yield).

[0161] 1H NMR (500 MHz, CDCl3) δ 12.54 (s, 1H), 8.00 (ddd, J = 2.2, 7.7, 11.8 Hz, 1H), 7.92-7.89 (m, 1H), 7.30 (ddd, J = 8.7, 8.7, 9.8 Hz, 1H), 6.54 (d, J = 2.3 Hz, 1H), 6.38 (d, J = 2.3 Hz, 1H), 4.79 (d, J = 13.6 Hz, 1H), 4.75 (d, J = 13.7 Hz, 1H), 4.61 (d, J = 13.5 Hz, 1H), 4.11 (t, J = 7.7 Hz, 2H), 3.96 (d, J = 13.8 Hz, 1H), 3.11-3.05 (m, 1H), 2.66-2.61 (m, 1H), 2.50 (s, 5H), 1.96-1.88 (m, 2H), 1.69 (t, J = 7.7 Hz, 2H), 1.61-1.59 (m, 4H), 1.55-1.47 (m, 1H), 1.47-1.40 (m, 3H), 0.93 (s, 9H).

[0162] 1-6. Preparation of Compound 569

[0163] Ethyl bromoacetate (1.39 mmol) and potassium carbonate (1.89 mmol) were added to a solution in which compound 549 (1.26 mmol) was dissolved in 10 mL of dimethylformamide, and the mixture was stirred at 50 °C for 6 hours. After cooling the reaction mixture to room temperature, it was diluted with ethyl acetate, an aqueous solution of saturated ammonium chloride was added, and the mixture was washed with saturated salt water. After dehydrating the organic layer with magnesium sulfate, the residue obtained by filtration and vacuum distillation was purified by silica gel column chromatography (Hex:CH2Cl2:EtOAc = 6:1:1) to obtain the intermediate in the form of a yellow solid. The intermediate obtained above (200 mg, 0.20 mmol) was dissolved in tetrahydrofuran (3 mL) and methanol (3 mL), an ammonia solution (0.5 mL) was added, and the mixture was stirred at room temperature for 24 hours. The reaction solution was acidified by treating it with 2N hydrochloric acid and stirred at room temperature for 30 minutes. After filtering the precipitate, it was washed with ethyl acetate to obtain compound 569 (52 mg, 0.11 mmol, 55% yield) in the form of a yellow solid.

[0164] 1H NMR (500 MHz, DMSO-d6) δ 12.46 (s, 1H), 8.11 (ddd, J = 2.1, 7.9, 12.0 Hz, 1H), 7.96-7.94 (m, 1H), 7.70 (ddd, J = 8.6, 8.6, 10.5 Hz, 1H), 7.62 (s, 1H), 7.47 (s, 1H), 6.80 (d, J = 2.2 Hz, 1H), 6.45 (d, J = 2.3 Hz, 1H), 4.58 (s, 2H), 4.03 (t, J = 6.6 Hz, 2H), 1.88-1.79 (m, 1H), 1.70-1.63 (m, 4H), 1.59-1.51 (m, 2H), 1.48-1.40 (m, 2H), 1.07-1.00 (m, 2H).

[0165] 1-7. Preparation of Compound 561

[0166] Compound 526 (500 mg, 1.26 mmol), ethyl bromoacetate (0.15 mL, 1.39 mmol), and potassium carbonate (262 mg, 1.89 mmol) were added to 10 mL of dimethylformamide and stirred at 50 °C for 6 hours. After cooling the reaction mixture to room temperature, it was diluted with ethyl acetate, an aqueous solution of saturated ammonium chloride was added, and the mixture was washed with saturated salt water. After dehydrating the organic layer with magnesium sulfate, the residue obtained by filtration and vacuum distillation was purified by silica gel column chromatography (Hex:CH2Cl2:EtOAc = 6:1:1) to obtain a first intermediate of yellow solid (400 mg, 0.83 mmol, 66% yield).

[0167] The first intermediate (200 mg, 0.42 mmol) and 2N aqueous sodium hydroxide solution (3 mL) were added to tetrahydrofuran (3 mL) and methanol (3 mL), and stirred at room temperature for 2 hours. The reaction mixture was acidified by adding 2N hydrochloric acid and then extracted with ethyl acetate. The extracted organic layer was washed with saturated brine, dehydrated with magnesium sulfate, filtered, and vacuum distilled. The residue was purified by tritration with ether to obtain a second intermediate (156 mg, 0.35 mmol, 83% yield) as a yellow solid.

[0168] The second intermediate (277 mg, 0.62 mmol), 4-piperidinopiperidine (104 mg, 0.62 mmol), DMAP (151 mg, 1.24 mmol), and EDC.HCl (237 mg, 1.24 mmol) were added to 10 mL of dichloromethane and stirred at room temperature for 12 hours. The residue obtained by vacuum distillation of the reactants was purified by silica gel column chromatography (CH2Cl2:MeOH = 50:1) to obtain a third intermediate (280 mg, 0.38 mmol, 62 yield) as a yellow solid.

[0169] 3 mL of dichloromethane was mixed with the third intermediate (100 mg, 0.14 mmol) and trifluoroacetic acid (3 mL), and stirred at room temperature for 12 hours. The residue obtained by vacuum distillation of the reactants was treated with an ether containing 2N hydrochloric acid. The solid obtained by filtering the precipitate was washed with acetone to obtain the yellow solid compound 561 (36 mg, 0.06 mmol, 43% yield).

[0170] 1H NMR (500 MHz, CDCl3) δ 12.54 (s, 1H), 8.00 (ddd, J = 2.2, 7.7, 11.8 Hz, 1H), 7.92-7.89 (m, 1H), 7.30 (ddd, J = 8.7, 8.7, 9.8 Hz, 1H), 6.54 (d, J = 2.3 Hz, 1H), 6.38 (d, J = 2.3 Hz, 1H), 4.79 (d, J = 13.6 Hz, 1H), 4.75 (d, J = 13.7 Hz, 1H), 4.61 (d, J = 13.5 Hz, 1H), 4.11 (t, J = 7.7 Hz, 2H), 3.96 (d, J = 13.8 Hz, 1H), 3.11-3.05 (m, 1H), 2.66-2.61 (m, 1H), 2.50 (s, 5H), 1.96-1.88 (m, 2H), 1.69 (t, J = 7.7 Hz, 2H), 1.61-1.59 (m, 4H), 1.55-1.47 (m, 1H), 1.47-1.40 (m, 3H), 0.93 (s, 9H).

[0171] 1-8. Preparation of Compound 566

[0172] Compound 526 (0.26 mmol), 2-(4-methylpipyrazine-1-yl)-ethanol (0.28 mmol), triphenylphosphine (0.51 mmol), and DIAD (0.51 mmol) were added to 5 mL of tetrahydrofuran and stirred at room temperature for 1 hour. The residue obtained by vacuum distillation of the reaction solution was purified by silica gel column chromatography (CH2Cl2:MeOH:NH4OH(aq) = 100:10:1) to obtain compound 566 in the form of a yellow oil (29% yield).

[0173] 1H NMR (500 MHz, CDCl3) δ 12.51 (s, 1H), 7.99 (ddd, J = 2.2, 7.8, 11.8 Hz, 1H), 7.92-7.89 (m, 1H), 7.30 (ddd, J = 8.6, 8.6, 9.7 Hz, 1H), 6.45 (d, J = 2.2 Hz, 1H), 6.36 (d, J = 2.2 Hz, 1H), 4.17 (t, J = 5.8 Hz, 2H), 4.11 (t, J = 7.8 Hz, 2H), 2.85 (t, J = 5.8 Hz, 2H), 2.63 (br s, 4H), 2.49 (br s, 4H), 2.31 (s, 3H), 1.70 (t, J = 7.9 Hz, 2H), 0.93 (s, 9H).

[0174] 13C NMR (125 MHz, CDCl3) δ 178.9, 164.8, 162.1, 156.6, 153.2, 151.6 (dd, J = 12.6, 253.6 Hz), 150.1 (dd, J = 12.8, 246.8 Hz), 139.2, 127.6-127.6 (m), 125.3-125.2 (m), 117.9 (d, J = 20.5 Hz), 117.5 (d, J = 17.4 Hz), 106.2, 98.5, 92.7, 71.0, 66.7, 56.7, 55.0, 53.5, 46.0, 43.3, 29.7, 29.6.

[0175] FAB-MS calcd for C28H35F2N2O5 [M+H]+ 517.2, found 517.6.

[0176] <Example 2> Analysis of Carbapenemase Activity

[0177] 2-1. Carbapenemase Inhibitory Activity

[0178] The inhibitory activity of carbapenemase AmpC, KPC-2, OXA-48, VIM-2, and NDM-1 was confirmed using eight compounds of the present invention.

[0179] Specifically, 100 μL of each of eight compounds at concentrations ranging from 70 to 0.1 μM (in PBS) were mixed with 200 μL of β-lactamase stock (2% DMSO), and nitrocepin (final concentration 70 μM), a substrate of AmpC, KPC-2, OXA-48, and VIM-2, and imipenem (final concentration 100 μM), a substrate of NDM-1, were added to each well of a 96-well plate. After incubating each plate at 37°C for 20 minutes, the plate containing the synthesized substances and β-lactamase was transferred to another plate containing the substrate. The hydrolytic enzyme reaction was measured for 15 minutes using a Cytation 5 imaging multimode reader (Bio-Tek Instruments Inc., Winooski, VT, USA) at 486 nm for nitrocepin and 300 nm for imipenem. The hydrolysis rate in the presence of the inhibitor was determined when no further increase in reaction rate occurred. The IC50 value was calculated using non-linear sigmoidal dose-responses analysis with GraphPad Prism 5 (GraphPad Software, Boston, USA).

[0180] As a result, as shown in Figure 1, the IC50 of compounds 599, 602, 596, and 560 against NDM-1 were 8.6, 21.6, 5.8, and 7.6 μM, respectively. In particular, compound 596, which contains a tetrazole derivative, showed inhibitory activity against KPC-2, OXA-48, VIM-2, and NDM-1, and compound 599, which contains a sulfate derivative, showed inhibitory activity against AmpC, KPC-2, OXA-48, and NDM-1.

[0181] 2-2. Drug Elution Pump Inhibitory Activity

[0182] Clinical Pseudomonas aeruginosa strains (PA-017, blaNDM) producing NDM (5 mL, LB medium) were grown to the mid-log phase, then centrifuged (3,500 rpm, 15 min) to obtain a bacterial pellet, washed twice with PBS, and resuspended to an OD600 of 0.3 (PBS). Each well of a flat-bottomed black 96-well plate was treated with bacterial suspension (182 μL), glucose [4 μL, final concentration = 0.4% (w / v)], a compound or positive control CCCP (carbonyl cyanide-m-chlorophenylhydrazone) (4 μL), and EtBr (10 μL, final concentration = 1 μg / mL). While incubating the plate at 37 ℃, the fluorescence of EtBr (λex 480 nm / λem 630 nm) was recorded every 2 minutes for 60 minutes using a Cytation 5 imaging multimode reader (BioTek Instruments, Inc., Winooski, VT, US).

[0183] As a result, as shown in Figure 2, high EtBr fluorescence was observed in compounds 602 and 566 compared to the positive control CCCP, and it was confirmed that EtBr fluorescence similar to the fluorescence intensity of CCCP was observed in compounds 569 and 561.

[0184] Therefore, compounds 602 and 566 exhibit higher drug efflux pump inhibitory activity than CCCP, and compounds 569 and 561 can be confirmed to have drug efflux pump inhibitory activity similar to CCCP.

[0185] 2-3. Enhancement of Aztreonam (ATM) Antimicrobial Activity (Checkerboard Assay)

[0186] Using ATM-591, ATM-599, ATM-602, ATM-596, ATM-560, ATM-569, ATM-561, and ATM-566 groups, each formed by combining eight compounds of the present invention with aztreonam, the antibacterial activity of aztreonam against Pseudomonas aeruginosa strain PA-017 and blaNDM, which produce metallobettalactamase NDM-1, was analyzed using a Checkerboard assay.

[0187] Specifically, 100 μL of sterile CAMHB medium was added to a clear sterile 96-well microplate, and the bacterial strain (final inoculum = 5 × 10⁵ CFU / mL) was inoculated. Samples of each compound and aztreonam were prepared by doubling the concentration from 512 μg / mL to 0.06 μg / mL, and applied to the plate inoculated with bacteria along the horizontal and vertical axes, respectively, until the final broth volume was 200 μL. The plates were incubated at 37°C for 18–24 hours, and after determining the MIC values ​​for the aztreonam-only group (MICATM), compound-only group (MICtest), aztreonam / compound combination group (MICATM+test), and compound / aztreonam combination group (MICtest+ATM), the fractional inhibitory concentration index (FICI) was calculated using the following Equation 1.

[0188]

[0189] (When FICI is 0.5 or less, the relationship between aztreonam and the compound is a synergistic effect, and

[0190] If FICI is between 0.5 and 1.0, it is an additive effect, and

[0191] If FICI is 4.0 or higher, it is an antagonistic effect.

[0192] As a result, as shown in Figure 2, it was confirmed that the antimicrobial activity (FD) of ATM-599 and ATM-596 groups increased by 64 times and 16 times, respectively, the hydrophobicity index cLogD 7.4 decreased to 1.14 and 1.90, and the polar surface area (PSA) increased to 119.4 and 119.5.

[0193] Therefore, among the 8 compounds, compounds 599 and 596, which introduce polar substituents sulfate and tetrazole at the 7th position of the existing skeleton, can be confirmed to inhibit resistance to the antibiotic aztreonam (ATM) and increase the efficacy of the antibiotic.

[0194] 2-4. Inhibitory activity against carbapenem-resistant Pseudomonas aeruginosa

[0195] Using the ATM-599 and ATM-596 groups of the present invention, the enhancement of the antimicrobial activity of aztreonam by compounds 599 and 596 was analyzed in 26 clinical strains of carbapenem-resistant Pseudomonas aeruginosa (CRPA) (3 GES (Guiana-Extended-Spectrum beta-lactamase) strains; 1 VIM (Verona integron-encoded metallo-β-lactamase) strain; 7 IMP (Imipenemase-type metallo-β-lactamase) strains; 13 NDM (New Delhi metallo-β-lactamase) strains; and 2 IMP / NDM strains). As shown in Table 1 below, out of a total of 26 strains, 15 were aztreonam-resistant and 11 were aztreonam-susceptible strains. According to Examples 2-3 above, the efficacy of enhancing the antibacterial activity of aztreonam by compounds 599 and 596 in 26 clinical Pseudomonas aeruginosa strains was verified through a checkerboard assay.

[0196]

[0197] As a result, as shown in Figures 3 to 6, it was confirmed that compounds 599 and 596 enhanced the antibacterial activity of aztreonem in 24 strains, excluding PA-035 or PA-003, at a concentration of 8 mg / L, thereby reducing the minimum inhibitory concentration (MIC) of aztreonem to 8 mg / L (clinical breakpoint) or less.

[0198] In addition, as shown in Figures 7 to 10, it was confirmed that the cumulative bacterial inhibition rate was 96% in the ATM-599 and ATM-596 groups by combining 8 mg / L of compound 599 or compound 596 with 8 mg / L of aztreonem.

[0199] In addition, as shown in Figures 11 and 12, ATM-599 and ATM-596 groups showed bacterial inhibitory efficacy (MIC50) against 50% of all CPPA strains (n ​​= 26) at concentrations of 2 mg / L and 4 mg / L, which are lower than the clinical breakpoint (8 mg / L), and bacterial inhibitory ability (MIC90) against 90% of all CPPA strains at a concentration of 8 mg / L, and it was confirmed that for aztreonam-resistant CPPA (n = 15), the MIC50 and MIC90 were 4 mg / L and 16 mg / L, respectively.

[0200] 2-5. Comparison with Commercial Antibiotics

[0201] To compare the carbapenem resistance recovery effects of the ATM-599 and ATM-596 groups of the present invention with those of commercial antibiotics, commercially approved FDA-approved antibiotics (ceftazidime (CAZ), ceftolozane (C), imipenem (IPM)) and beta-lactamase inhibitors (avibactam (AVI), tazobactam (T), relebactam (REL)) were purchased commercially, and ceftazidime-avibactam (CAZ-AVI group), ceftozolane-tazobactam (C / T group), and imipenem-relebactam (IPM-REL group) in combination with antibiotics and beta-lactamase inhibitors were prepared, and the clinical breakpoint of each combination antibiotic (aztreonam: 8 mg / L, ceftazidime: 8 mg / L, The performance of susceptibility recovery against CPPA strains was measured using ceftolozane (4 mg / L, imipenem: 2 mg / L).

[0202] As a result, as shown in Figure 13, the proportion of CPPA strains recovering susceptibility to the commercial antibiotics ceftazidim, ceftolozane, and imipenem in the CAZ-AVI, C / T, and IPM-REL groups was 10 to 14%, but in the ATM-599 and ATM-596 groups, the proportion of CPPA strains recovering susceptibility to aztreonam was found to have increased significantly to 96%.

[0203] 2-6. Time-kill assay

[0204] Time-kill tests were conducted on wild-type ATCC 27853 (Pseudomonas aeruginosa) and carbapenem-resistant Pseudomonas aeruginosa strains (CRPA) that produce IMP, VIM, GES, and NDM using aztreonam (AZT) monotherapy group and aztreonam / compound 596 combination therapy group (ATM-596).

[0205] Specifically, each Pseudomonas aeruginosa strain was placed in a conical tube, and aztreonam and compound 596 (8 mg / L) at concentrations of 1 / 4 MIC, 1 / 2 MIC, 1 MIC, or 2 MIC were combined to a final volume of 2 mL, and then incubated at 37 °C. Aliquots (50 μL) of the culture were collected at intervals of 0, 3, 6, and 24 hours, serially diluted in 0.85% NaCl solution, and then incubated in BAP to measure the number of bacterial colonies.

[0206] As a result, as shown in Figure 14, all Pseudomonas aeruginosa strains were significantly reduced by ATM-596 at concentrations of 0.5 MIC and 1 MIC, and in particular, ATCC 27853, IMP, GES, and NDM Pseudomonas aeruginosa strains were reduced by more than 2 log10 CFU / mL by ATM-596 at concentrations of 1 / 4 MIC, 1 / 2 MIC, 1 MIC, or 2 MIC.

[0207] <Example 3> Cytotoxicity Analysis

[0208] To verify the safety of compounds 599 and 596 of the present invention, hemolysis caused by the compounds in Human Red Blood Cells (HRBC) and cytotoxicity in HEK293 cells were analyzed.

[0209] Specifically, HEK293 cells were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution at 37°C and 5% CO2. After treating 96-well plates at a density of 1×10⁴ cells / well and incubating for 24 hours, compound 5 was added at a concentration of 0.5-128 mg / L and incubated for 24 hours. After adding 10 μL of EZ-cytox solution (Do-GenBio, KR) to the wells and incubating for 1 hour, WST-1 analysis was performed by measuring absorbance at 450 nm using a Cytation 5 imaging multimode reader (BioTek Instruments, Inc., Winooski, VT, US). Additionally, washed human red blood cells (hRBCs, Innovative Research, Novi, MI, US) were diluted in PBS (pH 7.4) at a volume ratio of 1:100 and 250 μL aliquots were added to 1.5 mL microcentrifuge tubes. Compound 5 was serially diluted to concentrations of 4–128 mg / L, and 250 μL aliquots were taken and mixed with the hRBCs. The mixture was incubated at 37 °C for 60 minutes, centrifuged at 1700×g for 5 minutes, and 50 μL of the supernatant was transferred to a clear, flat-bottomed 96-well plate. Absorbance was measured at 405 nm using a Citation 5 imaging multimode reader (BioTek Instruments, Inc., Winooski, VT, US). TritonX-100 (10%) was used as a positive control, and PBS as a negative control. % hemolysis was calculated using the following Equation 2.

[0210]

[0211] (ODtest: Measured absorbance, OD-PBS: Absorbance of PBS, ODTritonX: Absorbance of non-ionic membrane disruptor)

[0212] As a result, as shown in Figures 15 and 16, it was confirmed that compounds 599 and 596 did not exhibit cytotoxicity or erythrocyte hemolysis up to a concentration of 32 mg / L.

[0213] Accordingly, eight novel compounds were prepared by introducing substituents at the 3-O and 7-O positions of 3',4'-difluoroquercetin derivative compounds of the present invention, and their inhibitory activity against carbapenemases AmpC, KPC-2, OXA-48, VIM-2, and NDM-1 was confirmed. Additionally, when combined with the existing antibiotic aztreonam (ATM), the effect of reducing antibiotic resistance and enhancing antimicrobial activity against carbapenemase-producing Pseudomonas aeruginosa (CPPA) was confirmed.

[0214] As described above, specific embodiments of the present invention have been described in detail; however, those skilled in the art who understand the spirit of the present invention will be able to easily propose other inventions that are inferior or other embodiments included within the scope of the spirit of the present invention by adding, changing, or deleting other components within the same spirit. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention is defined by the claims set forth below rather than by the detailed description above, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included within the scope of the present invention.

Claims

1. A compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] In the above chemical formula 1, R1 is a straight-chain or branched-chain C1-10 alkyl group, a straight-chain or branched-chain C1-10 cycloalkyl group, a straight-chain or branched-chain C1-10 alkyl-cycloalkyl group, or a straight-chain or branched-chain C1-10 arylalkyl group, and R2 is any one selected from a straight-chain or branched C1-10 alkyl group, a straight-chain or branched C1-10 alkoxy group, a straight-chain or branched C1-10 alkylamine group, a straight-chain or branched C1-10 alkylamide group, a phosphate, a sulfate, a nitrate, a C3-10 cycloalkyl group, a straight-chain or branched C1-10 cycloalkylamide group, a C3-10 aryloxy group, a C3-10 arylamine group, and a substituted or unsubstituted 3-10 atom heterocyclic group comprising one or more heteroatoms selected from N, O, S, Se and Te.

2. In Paragraph 1, The above R1 is , or A compound or a pharmaceutically acceptable salt thereof that is.

3. In Paragraph 1, The above R2 is , , , , , , or A compound or a pharmaceutically acceptable salt thereof that is.

4. In Paragraph 1, A compound or a pharmaceutically acceptable salt thereof, wherein the above compound is one selected from the compounds represented by the following chemical formulas 2 to 9: [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] [Chemical Formula 7] [Chemical Formula 8] [Chemical Formula 9] 5. In Paragraph 1, The above compound is a compound or a pharmaceutically acceptable salt thereof that inhibits carbapenemase activity.

6. In Paragraph 5, The above carbapenemase is a compound or a pharmaceutically acceptable salt thereof which is AmpC, KPC-2, OXA-48, VIM-2, or NDM-1.

7. In Paragraph 1, The above compound is a compound or a pharmaceutically acceptable salt thereof that inhibits a drug efflux pump.

8. A pharmaceutical composition for the prevention or treatment of multidrug-resistant Pseudomonas aeruginosa infection comprising a compound represented by the following chemical formula 1 as an active ingredient. [Chemical Formula 1] In the above chemical formula 1, R1 and R2 are as defined in Paragraph 1.

9. In Paragraph 8, A composition in which the above compound increases the antibacterial activity of an antibiotic.

10. In Paragraph 9, A composition wherein the above antibiotic is one or more selected from the group consisting of Aztreonam, Ceftazidime, Ceftolozane, Imipenem, and combinations thereof.

11. In Paragraph 8, A composition that inhibits Pseudomonas aeruginosa when used in combination with an antibiotic.

12. In Paragraph 11, A composition in which the above-mentioned Pseudomonas aeruginosa is one or more selected from GES (Guiana-Extended-Spectrum beta-lactamase) producing bacteria, VIM (Verona integron-encoded metallo-β-lactamase) producing bacteria, IMP (Imipenemase-type metallo-β-lactamase) producing bacteria, NDM (New Delhi metallo-β-lactamase) producing bacteria, and co-producing bacteria consisting of combinations thereof.

13. In Paragraph 8, A composition in which the above compound reduces the minimum inhibitory concentration (MIC) of an antibiotic against Pseudomonas aeruginosa by 2 to 64 times.

14. A food composition for preventing or improving multidrug-resistant Pseudomonas aeruginosa infection comprising a compound represented by the following chemical formula 1 as an active ingredient. [Chemical Formula 1] In the above chemical formula 1, R1 and R2 are as defined in Paragraph 1.

15. A step of mixing a compound represented by the following chemical formula 1 and an antibiotic; and A method for increasing the antimicrobial activity of an antibiotic, comprising the step of treating the above mixture with multidrug-resistant Pseudomonas aeruginosa. [Chemical Formula 1] In the above chemical formula 1, R1 and R2 are as defined in Paragraph 1.

16. A method for treating multidrug-resistant Pseudomonas aeruginosa infection comprising the step of administering a pharmaceutically effective amount of a compound of any one of claims 1 to 15 to an individual.

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