Pharmaceutical composition for preventing or treating multidrug-resistant gram-negative bacterial infections comprising 3',4'-difluorokercetin derivative compounds

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of multidrug-resistant Gram-negative bacterial infections comprising a 3',4'-difluoroquercetin derivative compound as an active ingredient. The composition comprises 28 novel compounds prepared by introducing various substituents to the 3-O position of 3',4'-difluoroquercetin. The effects of improving drug efflux pump inhibitory activity and carbapenemase inhibitory activity of the compounds were confirmed, and the effect of restoring the antibacterial activity of the antibiotic against carbapenem-resistant Enterobacteriaceae (CRE) when used in combination with the existing carbapenem antibiotic meropenem (MEM) was confirmed, which can be usefully utilized in related industries.

Description

Technology Field

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of multidrug-resistant Gram-negative bacterial infections comprising a 3',4'-difluoroquercetin derivative compound as an active ingredient. Background Technology

[0002] Since the discovery of penicillin and streptomycin, over 5,000 types of antibiotics have been discovered, and among them, about 50 types of antibiotics have been developed into approximately 400 types of preparations and are actually used in clinical practice as a major treatment for infectious diseases. Antibiotics are broadly classified according to their chemical structure into beta-lactams ( Antibiotics are classified into -lactam and non-beta-lactam types, and more specifically into penicillin, cephalosporin, quinolone, and aminoglycoside types. 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 It is a Gram-positive coccus that causes pneumonia (especially hospital-acquired pneumonia), skin and soft tissue infections, and bacteremia; although it is one of the most common pathogens, since the 1990s S. aureus It is being revealed that 35-66% of cases are MRSA. Meanwhile, vancomycin-resistant strains of Enterococcus, a type of Gram-positive coccus, have been discovered, 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. Additionally, New Delhi metallo-beta-lactamase-1 (NDM-1), identified in 2008, has been found to be a superbug of hospital-acquired infections that is resistant to any existing antibiotics.

[0005] It is known that resistant strains acquire resistance to antibiotics through methods such as 1) altering the structure of antibiotics using antibiotic-degrading enzymes 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 intended to combat resistant strains must be able to avoid the development of resistance by 1) inhibiting novel bacterial targets previously unknown, or 2) inhibiting various target groups.

[0006] Accordingly, the inventors prepared a novel compound by introducing a substituent at the 3-O position of a 3',4'-difluoroquercetin derivative, and confirmed the effect of inhibiting drug efflux pumps and carbapenem degrading enzymes and restoring the antibacterial activity of antibiotics against carbapenem-resistant Enterobacteriaceae (CRE), thereby completing the present invention. The problem to be solved

[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] R is COCH2C(CH3)3, CH2OCOC(CH3)3, CH2OCOOCH(CH3)2, CH2(4- t BuPh), CH2(4- iPrPh), (CH2)2OH, (CH2)2O(CH2)2OH, (CH2)2O(CH2)2O(CH2)2OH, (CH2)2O(CH2)2NH2, (CH2)2(thiophen-2-yl), (CH2)2N(CH3)2, (CH2)2N(CH2)2, (CH2)2N + (CH3)2CH2CO2, (CH2)2(4-methylpiperazin-1-yl), (CH2)2N(CH2)4NH, (CH2)2N(CH2)4, (CH2)2N(CH2)5, (CH2)2N(CH2)40, ( S It is one selected from )-(CH2)2(1-methylpyrrolidin-2-yl), (CH2)2CF3, (CH2)2C(CH3)3, (CH2)2CH(CH3)2, (CH2)2CH(CH2)2, (CH2)2CH(CH2)4, (CH2)2CH(CH2)5, CH2CH(CH2)4, (CH2)3CH(CH2)4, (CH2)2CH(CH2)40 and (CH2)2CH(CH2)4NH.

[0012] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of multidrug-resistant Gram-negative bacterial infections comprising a compound represented by the above chemical formula 1 as an active ingredient.

[0013] Another objective of the present invention is to provide a food composition for preventing or improving multidrug-resistant Gram-negative bacterial infections 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 method for increasing the antibacterial activity of an antibiotic, comprising the steps of: mixing a compound represented by Formula 1 and a beta-lactam antibiotic; and treating multidrug-resistant Gram-negative bacteria with the mixture. means of solving the problem

[0015] In order to achieve the above objective,

[0016] The present invention provides a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0017] [Chemical Formula 1]

[0018]

[0019] In the above chemical formula 1,

[0020] R is COCH2C(CH3)3, CH2OCOC(CH3)3, CH2OCOOCH(CH3)2, CH2(4- t BuPh), CH2(4- i PrPh), (CH2)2OH, (CH2)2O(CH2)2OH, (CH2)2O(CH2)2O(CH2)2OH, (CH2)2O(CH2)2NH2, (CH2)2(thiophen-2-yl), (CH2)2N(CH3)2, (CH2)2N(CH2)2, (CH2)2N + (CH3)2CH2CO2, (CH2)2(4-methylpiperazin-1-yl), (CH2)2N(CH2)4NH, (CH2)2N(CH2)4, (CH2)2N(CH2)5, (CH2)2N(CH2)40, ( S It is one selected from )-(CH2)2(1-methylpyrrolidin-2-yl), (CH2)2CF3, (CH2)2C(CH3)3, (CH2)2CH(CH3)2, (CH2)2CH(CH2)2, (CH2)2CH(CH2)4, (CH2)2CH(CH2)5, CH2CH(CH2)4, (CH2)3CH(CH2)4, (CH2)2CH(CH2)40 and (CH2)2CH(CH2)4NH.

[0021] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of multidrug-resistant Gram-negative bacterial infections comprising a compound represented by the above chemical formula 1 as an active ingredient.

[0022] In addition, the present invention provides a food composition for preventing or improving multidrug-resistant Gram-negative bacterial infections comprising a compound represented by the above chemical formula 1 as an active ingredient.

[0023] 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 a beta-lactam antibiotic; and treating multidrug-resistant Gram-negative bacteria with the mixture. Effects of the invention

[0024] The novel quercetin derivative compounds of the present invention were prepared by introducing various substituents to the 3-O position of 3',4'-difluoroquercetin to produce 28 novel compounds. The drug efflux pump and carbapenem degrading enzyme inhibitory effects of the compounds were confirmed, and the effect of restoring the antibacterial activity of the antibiotic against carbapenem-resistant Enterobacteriaceae (CRE) when used in combination with the existing carbapenem antibiotic meropenem (MEM) was confirmed, so they can be usefully utilized in related industries. Brief explanation of the drawing

[0025] Figure 1 shows the EtBr fluorescence intensity in EC-001blaNDM according to the compound of the present invention. Figure 2 shows the drug efflux pump inhibitory activity of the compound of the present invention. Figure 3 shows the NDM-1 inhibitory activity of the compound of the present invention. Figure 4 shows the molecular binding modes of the compounds of the present invention for AcrB, NDM-1, and KPC-2. Figure 5 shows the time-kill curves for E. coli-012 and blaNDM-1 in combination with the compound of the present invention and the antibiotic meropenem. Figure 6 shows the NPN fluorescence intensity according to the compound of the present invention. Figure 7 shows the Kaplan-Meier survival curves of E. coli-012 and blaNDM-1 infected mouse models in combination with the compound of the present invention and the antibiotic meropenem. Specific details for implementing the invention

[0026] 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.

[0027] 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 may include additional components rather than excluding other components.

[0028] The terms used in the present invention are explained below.

[0030] The present invention provides a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0031] [Chemical Formula 1]

[0032]

[0033] In the above chemical formula 1,

[0034] R is COCH2C(CH3)3, CH2OCOC(CH3)3, CH2OCOOCH(CH3)2, CH2(4- t BuPh), CH2(4- iPrPh), (CH2)2OH, (CH2)2O(CH2)2OH, (CH2)2O(CH2)2O(CH2)2OH, (CH2)2O(CH2)2NH2, (CH2)2(thiophen-2-yl), (CH2)2N(CH3)2, (CH2)2N(CH2)2, (CH2)2N + (CH3)2CH2CO2, (CH2)2(4-methylpiperazin-1-yl), (CH2)2N(CH2)4NH, (CH2)2N(CH2)4, (CH2)2N(CH2)5, (CH2)2N(CH2)40, ( S It is one selected from )-(CH2)2(1-methylpyrrolidin-2-yl), (CH2)2CF3, (CH2)2C(CH3)3, (CH2)2CH(CH3)2, (CH2)2CH(CH2)2, (CH2)2CH(CH2)4, (CH2)2CH(CH2)5, CH2CH(CH2)4, (CH2)3CH(CH2)4, (CH2)2CH(CH2)40 and (CH2)2CH(CH2)4NH.

[0035] 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.

[0036] 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.

[0037] Salts derived from suitable bases are alkali metals, alkaline earth metals, ammonium, and N + (C 1-4 It includes alkyl)tetraphosphates. 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, hydroxylates, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0038] According to one embodiment of the present invention, the compound may inhibit a drug ejection pump.

[0039] 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.

[0040] According to one embodiment of the present invention, the compound may inhibit carbapenem degrading enzyme activity.

[0041] The "Carbapenemase" of the present invention is a β-Lactamase capable of hydrolyzing penicillin, cephalosporin, monobactam, and carbapenem, and said β-Lactamase is an enzyme produced by bacteria that provide multiple resistance to β-lactam antibiotics. said carbapenem is an effective class of antibiotics most commonly used for the treatment of severe bacterial infections and is used for known or suspected multiple drug-resistant (MDR) bacterial infections.

[0042] According to one embodiment of the present invention, the carbapenem degrading enzyme may include KPC-2, OXA-48, VIM-2, or NDM-1.

[0043] "KPC-2 ( of the present invention Klebsiella pneumoniae carbapenemase-2” is Klebsiella pneumoniae resistant to carbapenem antibiotics ( Klebsiella pneumoniae It is an enzyme that confers resistance to carbapenem antibiotics and breaks down antibiotics, allowing bacteria to survive.

[0044] 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.

[0045] "VIM-2 (Verona Integron-encoded Metallo-β-lactamase-2)" of the present invention is an enzyme belonging to the metallo-β-lactamase family, found mainly in multidrug-resistant bacteria (MDR), and can degrade various beta-lactam antibiotics, including carbapenem antibiotics.

[0046] "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.

[0048] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of multidrug-resistant Gram-negative bacterial infections comprising a compound represented by the above chemical formula 1 as an active ingredient.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] According to one embodiment of the present invention, the multidrug-resistant Gram-negative bacteria may include carbapenem-resistant Gram-negative bacteria or carbapenem-resistant enterobacteria.

[0058] The "Carbapenemase" of the present invention is an enzyme that confers resistance to carbapenem antibiotics, and carbapenem antibiotics may include imipenem, meropenem, ertapenem, doripenem, etc.

[0059] According to one embodiment of the present invention, the composition may further include an antibiotic.

[0060] According to one embodiment of the present invention, the compound may increase the antibacterial activity of the antibiotic.

[0061] According to one embodiment of the present invention, the antibiotic may include meropenem.

[0062] The "Meropenem" of the present invention is a drug belonging to the carbapenem class of antibiotics and is primarily used to treat severe infections. Carbapenem antibiotics possess potent efficacy in treating bacteria resistant to other antibiotics, playing a significant role, particularly in hospital-acquired infections or multidrug-resistant (MDR) infections.

[0063] According to one embodiment of the present invention, the compound may be used in combination with an antibiotic to inhibit multidrug-resistant bacteria.

[0064] According to one embodiment of the present invention, the compound may reduce the minimum inhibitory concentration of an antibiotic by 4 to 64 times.

[0065] According to one embodiment of the present invention, the compound may reduce the number of bacteria.

[0067] In addition, the present invention provides a food composition for preventing or improving multidrug-resistant Gram-negative bacterial infections comprising a compound represented by the above chemical formula 1 as an active ingredient.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0073] 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 a beta-lactam antibiotic; and treating multidrug-resistant Gram-negative bacteria with the mixture.

[0075] New Delhi metallo-β-lactamase (NDM)-producing (blaNDM) carbapenem-resistant Escherichia coli (E. coli) strains (EC-001, EC-012, EC-013, EC-014, EC-033, EC-040), NDM-producing Klebsiella pneumoniae (KPC-001, KP-018, KP-020), Klebsiella pneumoniae carbapenemase (KPC)-producing (blaKPC) Escherichia coli (E. coli) strains (EC-010, EC-022, EC-023), and KPC-producing (blaKPC) Klebsiella pneumoniae (KP-009, KP-022) strains were provided by Asan Medical Center (Seoul, Korea).

[0076] All animal experiments were approved by the KRIBB’s Institutional Animal Care and Use Committee (approval code: KRIBB-AEC23204).

[0078] The present invention will be explained in more detail below through the following examples. However, these examples are merely illustrative of the invention, and the scope of the invention is not limited by these examples.

[0080] <Example 1> General synthesis method of 3-O-alkylated 3',4'-difluoroquercetin

[0081] The 3',4'-difluoroquercetin (compound 2) of the present invention was prepared from quercetin (compound 1) as shown in the following reaction scheme A, with reference to previously known literature (Cho et al., 2012).

[0082] [Reaction Equation A]

[0083]

[0084] In addition, compounds 3 to 31 of the following chemical formula 1 were synthesized using method A or method B in order to introduce the following 28 types of alkylating agents (R) into the 3-O position of compound 2 as shown in reaction scheme 1 below.

[0085] [Chemical Formula 1]

[0086]

[0087] 상기 화학식 1에서,

[0088] R은 COCH2C(CH3)3, CH2OCOC(CH3)3, CH2OCOOCH(CH3)2, CH2(4-) t BuPh), CH2(4- i PrPh), (CH2)2OH, (CH2)2O(CH2)2OH, (CH2)2O(CH2)2O(CH2)2OH, (CH2)2O(CH2)2NH2, (CH2)2(thiophen-2-yl), (CH2)2N(CH3)2, (CH2)2N(CH2)2, (CH2)2N + (CH3)2CH2CO2, (CH2)2(4-methylpiperazin-1-yl), (CH2)2N(CH2)4NH, (CH2)2N(CH2)4, (CH2)2N(CH2)5, (CH2)2N(CH2)40, S )-(CH2)2(1-methylpyrrolidin-2-yl), (CH2)2CF3, (CH2)2C(CH3)3, (CH2)2CH(CH3)2, (CH2)2CH(CH2)2, (CH2)2CH(CH2)4, (CH2)2CH(CH2)5, CH2CH(CH2)4, (CH2)3CH(CH2)4, (CH2)2CH(CH2)40 및 (CH2)2CH(CH2)4NH 중에서 선택되는 하나이다.

[0089] [반응식 1]

[0090]

[0091] 1-1. Method A

[0092] N , N100 mg (0.33 mmol, 1.0 equivalent) of cooled 3',4'-difluoroquercetin, alkyl halide (RX, 1.1 equivalent), and K2CO3 (1.3 equivalent) were added to 3 mL of dimethylformamide (DMF) and stirred at 100 °C for 2 hours. After natural cooling to room temperature, the mixture was treated with a saturated aqueous solution of NH4Cl and diluted with ethyl acetate (EtOAc). The organic layer was washed with brine, dried with MgSO4, filtered, and concentrated under reduced pressure to obtain an oil-phase concentrate. The concentrate was washed with an organic solvent, ground, or purified by silica gel column chromatography to obtain compounds substituted with alkyl substituents at the 3-O position. Some compounds underwent a protecting group removal step to obtain the final product.

[0094] 1-2. Method B

[0095] 100 mg (0.33 mmol) of cooled 3',4'-difluoroquercetin, alcohol (1.5 equivalents), PPh3 (1.5 equivalents), and diisopropyl azodicarboxylate (DIAD, 1.5 equivalents) were sequentially added to 5 mL of tetrahydrofuran (THF), stirred at room temperature for 1 hour, and then concentrated under reduced pressure to obtain an oil-phase concentrate. The concentrate was washed with an organic solvent, ground, or purified by silica gel column chromatography to obtain a compound substituted with an alkyl substituent at the 3-O position.

[0097] <Example 2> Synthesis of 2-(3,4-difluorophenyl)-5,7-dihydroxy-4-oxo-4H-chromene-3-yl 3,3-dimethylbutanoate (Compound 3)

[0098] 200 mg (0.65 mmol) of 3',4'-difluoroquercetin (compound 2), 0.12 mL (0.85 mmol) of tert-butylacetyl chloride, and 135 mg (9.8 mmol) of K2CO3 were added to 5 mL of DMF and stirred for 1 hour. The reaction mixture was then treated with a saturated solution of NH4Cl and diluted with ethyl acetate (EtOAc). The organic layer was washed with brine, dried with MgSO4, filtered, and concentrated under reduced pressure to obtain the oily residue. The residue was purified by silica gel column chromatography (Hexanes:EtOAc=6:1) to obtain 90 mg (0.23 mmol, 34% yield) of a yellow solid.

[0099] 1 H NMR (500 MHz, CDCl3): δ 12.08 (s, 1H), 7.73 (ddd, J = 2.2, 7.5, 11.0 Hz, 1H), 7.67-7.64 (m, 1H), 7.32 (ddd, J = 8.5, 8.5, 9.7 Hz, 1H), 6.27 (d, J = 2.1 Hz, 1H), 6.23 (d, J = 2.1 Hz, 1H), 2.53 (s, 2H), 1.12 (s, 9H).

[0100] 13 C NMR (125 MHz, CDCl3): δ 175.6, 170.6, 163.6, 162.1, 156.9, 154.6, 152.3 (dd, J = 12.0, 254.3 Hz), 150.4 (dd, J = 12.0, 249.6 Hz), 131.7, 126.4 (dd, J = 3.5, 6.0 Hz), 125.5 (dd, J = 3.5, 6.0 Hz), 118.2 (d, J = 19.3 Hz), 118.0 (d, J = 16.8 Hz), 105.4, 99.7, 94.5, 47.4, 31.1, 29.6.

[0101] FAB-MS (m / z): found 405.4 [M+H] + , calcd for C21 H 18 F2O6404.1.

[0103] <Example 3> Synthesis of ((2-(3,4-difluorophenyl)-5,7-dihydroxy-4-oxo-4H-chromene-3-yl)oxy)methyl pivalate (Compound 4)

[0104] In the above method A, the reaction was carried out using iodomethyl pivalate as an alkylating agent. After purification by column chromatography (Hexanes:EtOAc=6:1), a yellow solid was obtained.

[0105] Yield: 35%

[0106] 1 H NMR (500 MHz, acetone-d6): δ 12.39 (s, 1H), 8.14 (ddd, J = 2.3, 7.9, 12.2 Hz, 1H), 8.05-8.02 (m, 1H), 7.55 (ddd, J = 8.6, 8.6, 10.4 Hz, 1H), 6.58 (d, J = 2.1 Hz, 1H), 6.32 (d, J = 2.1 Hz, 1H), 5.87 (s, 2H), 0.90 (s, 9H).

[0107] 13 C NMR (125 MHz, acetone-d6): δ 178.6, 177.6, 165.6, 163.1, 157.9, 154.1, 152.4 (dd, J = 13.1, 250.8 Hz), 150.7 (dd, J = 13.3, 244.9 Hz), 137.2, 128.5 (dd, J = 3.6, 7.1 Hz), 127.2 (dd, J = 3.6, 7.3 Hz), 119.3 (d, J = 19.1 Hz), 118.5 (d, J = 16.9 Hz), 105.8, 99.9, 94.9, 88.0, 39.1, 26.8.

[0108] FAB-MS (m / z): found 421.4 [M+H] + , calcd for C 21 H 18 F2O7420.1.

[0110] <Example 4> Synthesis of ((2-(3,4-difluorophenyl)-5,7-dihydroxy-4-oxo-4H-chromene-3-yl)oxy)methyl isopropyl carbonate (Compound 5)

[0111] In the above method A, the reaction was carried out using chloromethyl isopropyl carbonate as an alkylating agent. After purification by column chromatography (Hexanes:EtOAc=6:1), a yellow solid was obtained.

[0112] Yield: 37%

[0113] 1 H NMR (500 MHz, acetone-d6): δ 12.37 (s, 1H), 8.08 (ddd, J = 2.3, 7.9, 12.1 Hz, 1H), 7.99-7.96 (m, 1H), 7.55 (ddd, J = 8.6, 8.6, 10.4 Hz, 1H), 6.58 (d, J = 2.1 Hz, 1H), 6.32 (d, J = 2.1 Hz, 1H), 5.79 (s, 2H), 4.59 (sept, J = 6.3 Hz, 1H), 1.08 (d, J = 6.3 Hz, 6H).

[0114] 13 C NMR (125 MHz, acetone-d6): δ 178.4, 165.6, 163.1, 158.0, 154.8, 154.5, 152.4 (dd, J = 12.0, 250.8 Hz), 150.6 (dd, J = 12.0, 244.8 Hz), 136.8, 128.5-128.4 (m), 127.2 (dd, J = 3.6, 7.1 Hz), 119.2 (d, J = 19.3 Hz), 118.5 (d, J = 18.0 Hz), 105.9, 99.9, 94.9, 90.6, 73.1, 21.5.

[0115] FAB-MS (m / z): found 423.3 [M+H] + , calcd for C 20 H 16 F2O8422.1.

[0117] <Example 5> Synthesis of 3-(4-tert-butyl-benzyloxy)-2-(3,4-difluorophenyl)-5,7-dihydroxy-4H-chromen-4-one (Compound 6)

[0118] In the above method A, the reaction was carried out using 4-tert-butylbenzyl bromide as an alkylating agent. After purification by column chromatography (Hexanes:acetone = 6:1), a pale yellow solid was obtained.

[0119] Yield: 67%

[0120] 1 H NMR (500 MHz, acetone-d6): δ 12.66 (s, 1H), 7.90 (ddd, J = 2.2, 7.9, 12.1 Hz, 1H), 7.87-7.84 (m, 1H), 7.44 (ddd, J = 8.6, 8.6, 10.5 Hz, 1H), 7.32 (d, J = 8.3 Hz, 2H), 7.27 (d, J = 8.3 Hz, 2H), 6.53 (d, J = 2.0 Hz, 1H), 6.31 (d, J = 2.0 Hz, 1H), 5.13 (s, 2H), 1.28 (s, 9H).

[0121] 13 C NMR (125 MHz, acetone-d6): δ 179.6, 165.3, 163.3, 157.9, 154.8, 152.1, 152.1 (dd, J = 13.3, 250.8 Hz), 150.5 (dd, J = 13.3, 244.9 Hz), 138.6, 134.3, 129.8, 128.9 (dd, J = 3.6, 7.3 Hz), 126.9-126.8 (m), 125.9, 118.8 (d, J = 20.4 Hz), 118.2 (d, J = 18.0 Hz), 106.1, 99.7, 94.7, 74.9, 35.1, 31.5.

[0122] FAB-MS (m / z): found 453.4 [M+H] + , calcd for C 26 H 22 F2O5452.1.

[0124] <Example 6> Synthesis of 2-(3,4-difluorophenyl)-5,7-dihydroxy-3-(4-isopropyl-benzyloxy)-4H-chromen-4-one (Compound 7)

[0125] In the above method A, the reaction was carried out using 4-isopropylbenzyl bromide as an alkylating agent. After purification by column chromatography (Hexanes:acetone = 6:1), a pale yellow solid was obtained.

[0126] Yield: 60%

[0127] 1 H NMR (500 MHz, acetone-d6): δ 12.65 (s, 1H), 7.91 (ddd, J = 2.2, 7.9, 12.1 Hz, 1H), 7.87-7.84 (m, 1H), 7.43 (ddd, J = 8.6, 8.6, 10.5 Hz, 1H), 7.26 (d, J = 8.1 Hz, 2H), 7.15 (d, J = 8.1 Hz, 2H), 6.52 (d, J = 2.1 Hz, 1H), 6.30 (d, J = 2.1 Hz, 1H), 5.13 (s, 2H), 2.87 (sept, J = 7.0 Hz, 1H), 1.20 (d, J = 7.0 Hz, 6H).

[0128] 13 C NMR (125 MHz, acetone-d6): δ 179.6, 165.2, 163.2, 157.9, 154.7, 152.1 (dd, J = 13.1, 250.8 Hz), 150.5 (dd, J = 13.3, 244.8 Hz), 149.9, 138.6, 134.7, 130.1, 128.8 (dd, J = 3.6, 7.3 Hz), 127.0, 126.8 (dd, J = 3.6, 7.3 Hz), 118.7 (d, J = 20.4), 118.2 (d, J = 18.0 Hz), 106.0, 99.6, 94.7, 74.9, 34.5, 24.2.

[0129] FAB-MS (m / z): found 439.4 [M+H] + , calcd for C 25 H20F2O5438.1.

[0131] <Example 7> Synthesis of 2-(3,4-difluorophenyl)-5,7-dihydroxy-3-(2-hydroxyethoxy)-4H-chromen-4-one (Compound 8)

[0132] In the above method A, the reaction was carried out using 2-bromoethanol as an alkylating agent. After purification by column chromatography (Hexanes:acetone = 3:1), a yellow solid was obtained.

[0133] Yield: 43%

[0134] 1 H NMR (500 MHz, acetone-d6): δ 12.49 (s, 1H), 8.29 (ddd, J = 2.2, 7.9, 12.4 Hz, 1H), 8.15-8.12 (m, 1H), 7.52 (ddd, J = 8.6, 8.6, 10.4 Hz, 1H), 6.57 (d, J = 2.1 Hz, 1H), 6.29 (d, J = 2.1 Hz, 1H), 4.23 (t, J = 4.6 Hz, 2H), 3.81 (t, J = 4.6 Hz, 2H).

[0135] 13 C NMR (125 MHz, acetone-d6): δ 179.8, 165.5, 163.1, 157.9, 154.0, 152.3 (dd, J = 13.3, 250.8 Hz), 150.8 (dd, J = 13.3, 244.9 Hz), 139.6, 128.9-128.8 (m), 126.8 (dd, J = 3.6, 7.3 Hz), 119.0 (d, J = 20.4 Hz), 118.5 (d, J = 16.8 Hz), 105.9, 99.7, 94.8, 75.5, 61.8.

[0136] FAB-MS (m / z): found 351.3 [M+H] + , calcd for C 17 H 12 F2O6350.1.

[0138] <Example 8> Synthesis of 2-(3,4-difluorophenyl)-5,7-dihydroxy-3-[2-(2-hydroxyethoxy)ethoxy]-4H-chromen-4-one (Compound 9)

[0139] In the above method B, the reaction was carried out using diethylene glycol as an alkylating agent. After purification by column chromatography (Hexanes:acetone = 3:1), a yellow solid was obtained.

[0140] Yield: 35%

[0141] 1 H NMR (500 MHz, DMSO-d6): δ 12.45 (s, 1H), 10.98 (s, 1H), 8.25 (ddd, J = 2.2, 8.0, 12.4 Hz, 1H), 8.08-8.05 (m, 1H), 7.63 (ddd, J = 8.7, 8.7, 10.5 Hz, 1H), 6.51 (d, J = 2.1 Hz, 1H), 6.22 (d, J = 2.1 Hz, 1H), 4.59 (t, J = 5.4 Hz, 1H), 4.26-4.25 (m, 2H), 3.63-3.61 (m, 2H), 3.44 (dt, J = 5.2, 5.3 Hz, 2H), 3.34 (t, J = 5.4 Hz, 2H).

[0142] 13 C NMR (125 MHz, DMSO-d6): δ 178.1, 164.6, 161.2, 156.4, 152.6, 150.8 (dd, J = 14.4, 253.1 Hz), 149.2 (dd, J = 13.3, 243.6 Hz), 137.9, 127.5 (dd, J = 3.6, 7.3 Hz), 126.2-126.1 (m), 118.0 (d, J = 21.6 Hz), 117.8 (d, J = 17.9 Hz), 104.4, 98.8, 94.0, 72.2, 71.6, 69.4, 60.1.

[0143] FAB-MS (m / z): found 395.3 [M+H] + , calcd for C 19 H 16 F2O7394.1.

[0145] <Example 9> Synthesis of 2-(3,4-difluorophenyl)-5,7-dihydroxy-3-{2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}-4H-chromen-4-one (Compound 10)

[0146] In the above method B, the reaction was carried out using triethylene glycol as an alkylating agent. After purification by column chromatography (Hexanes:acetone = 1:1), a yellow solid was obtained.

[0147] Yield: 38%

[0148] 1 H NMR (500 MHz, DMSO-d6): δ 12.45 (s, 1H), 8.25 (ddd, J = 2.3, 8.0, 12.4 Hz, 1H), 8.06-8.03 (m, 1H), 7.63 (ddd, J = 8.7, 8.7, 10.5 Hz, 1H), 6.50 (d, J = 2.1 Hz, 1H), 6.22 (d, J = 2.1 Hz, 1H), 4.58 (s, 1H), 4.26-4.25 (m, 2H), 3.63-3.61 (m, 2H), 3.46-3.42 (m, 6H), 3.37 (t, J = 5.3 Hz, 2H).

[0149] 13 C NMR (125 MHz, DMSO-d6): δ 178.1, 164.6, 161.2, 156.4, 152.5, 150.7 (dd, J = 12.0, 250.8 Hz), 149.2 (dd, J = 13.1, 244.8 Hz), 137.9, 127.6-127.5 (m), 126.1 (dd, J = 3.6, 7.3 Hz), 118.0 (d, J = 20.4 Hz), 117.8 (d, J = 18.0 Hz), 104.4, 98.8, 94.0, 72.4, 71.5, 69.7, 69.7, 69.4, 60.2.

[0150] FAB-MS (m / z): found 439.4 [M+H] + , calcd for C 21 H20F2O8438.1.

[0152] <Example 10> Synthesis of 3-[2-(2-aminoethoxy)ethoxy]-2-(3,4-difluorophenyl)-5,7-dihydroxy-4H-chromen-4-one (Compound 11)

[0153] 10-1. Synthesis of Benzyl (2-(2-iodoethoxy)ethyl)carbamate

[0154] 1 mL (8.97 mmol) of cooled diglycolamine, 1.25 mL (8.97 mmol) of triethylamine (TEA), and 1.4 mL (9.87 mmol) of benzyl chloroformate were added to 25 mL of THF and stirred at room temperature for 1 hour, after which the reaction was terminated by treatment with a saturated aqueous solution of NH4Cl. The organic layer was washed with brine and dried with MgSO4, filtered, and concentrated under reduced pressure to obtain a concentrate, which was then purified by silica gel column chromatography (CH2Cl2:EtOAc=1:1) to obtain 1.53 g of colorless oil-phase Cbz-protected diglycolamine.

[0155] 1.53 g of Cbz-protected diglycolamine was dissolved in 20 mL of CH2Cl2, 0.98 mL (1.1 equivalents) of TEA and 0.55 mL of methanesulfonyl chloride (MsCl) were added, and after stirring for 30 minutes, the reaction was terminated by treatment with a saturated aqueous solution of NH4Cl. The organic layer was washed with brine and dried with MgSO4, and a concentrate was obtained by filtration and vacuum concentration. The concentrate was dissolved in 20 mL of acetone, 1.11 g of NaI was added, and the mixture was heated for 2 hours and 30 minutes. After cooling to room temperature, it was diluted with EtOAc, washed with a saturated aqueous solution of Na2S2O3 and brine, dried with MgSO4, filtered, and vacuum concentrated. The residue was purified by silica gel column chromatography (Hexanes:EtOAc=4:1) to obtain 1.18 g of benzyl (2-(2-iodoethoxy)ethyl)carbamate, an alkyl halide.

[0157] 10-2. Compound 11 Synthesis

[0158] In Method A above, the reaction was carried out using benzyl(2-(2-iodoethoxy)ethyl)carbamate as an alkylating agent. To remove the Cbz protecting group, 43 mg (0.09 mmol) of benzyl(2-(2-iodoethoxy)ethyl)carbamate and 4 mg of 10% Pd / C precipitate were added to 3 mL of CH2Cl2 and 3 mL of methanol in a 1:1 volume ratio mixed solvent, and stirred for 24 hours under H2 (balloon). The reaction mixture was filtered through a short Celite pad and concentrated under reduced pressure. It was purified by column chromatography (CH2Cl2:MeOH:NH4OH(aq.)=90:10:1) to obtain a yellow solid.

[0159] Yield: 47% (16 mg, 0.04 mmol).

[0160] 1 H NMR (500 MHz, methanol-d4): δ 8.14 (ddd, J = 2.3, 7.9, 12.3 Hz, 1H), 7.97-7.94 (m, 1H), 7.44 (ddd, J = 8.6, 8.6, 10.3 Hz, 1H), 6.27 (d, J = 2.1 Hz, 1H), 6.09 (d, J = 2.1 Hz, 1H), 4.17-4.15 (m, 2H), 3.78-3.76 (m, 2H), 3.67-3.65 (m, 2H), 3.05-3.03 (m, 2H).

[0161] 13 C NMR (125 MHz, methanol-d4): δ 179.6, 168.5, 163.0, 158.6, 154.6, 153.0 (dd, J = 13.3, 250.8 Hz), 151.3 (dd, J = 13.1, 244.8 Hz), 139.5, 129.0 (dd, J = 3.6, 6.0 Hz), 126.8 (dd, J = 3.6, 7.1 Hz), 119.2 (d, J = 20.4 Hz), 118.8 (d, J = 18.0 Hz), 105.5, 100.8, 95.4, 73.1, 71.2, 68.3, 40.7.

[0162] FAB-MS (m / z): found 394.3 [M+H] + , calcd for C 19 H 17 F2NO6393.1.

[0164] <Example 11> Synthesis of 2-(3,4-difluorophenyl)-5,7-dihydroxy-3-[2-(thiophene-2-yl)ethoxy]-4H-chromen-4-one (Compound 12)

[0165] 2-(thiophene-2-yl)ethanol, TEA (1.1 equivalents), and MsCl (1.1 equivalents) were added to CH2Cl2 and stirred for 1 hour, after which the reaction was terminated by treatment with a saturated aqueous solution of NH4Cl. The organic layer was washed with brine and dried with MgSO4, filtered, and concentrated under reduced pressure to obtain 2-(thiophene-2-yl)ethyl methanesulfonate.

[0166] In the above method A, the reaction was carried out using 2-(thiophene-2-yl)ethyl methanesulfonate as an alkylating agent. After purification by column chromatography (Hexanes:EtOAc=6:1), a yellow solid was obtained.

[0167] Yield: 47%

[0168] 1 H NMR (500 MHz, acetone-d6): δ 12.58 (s, 1H), 7.97 (ddd, J = 2.2, 7.9, 12.1 Hz, 1H), 7.90-7.86 (m, 1H), 7.39 (ddd, J = 8.6, 8.6, 10.5 Hz, 1H), 7.26 (dd, J = 1.5, 4.8 Hz, 1H), 6.94-6.91 (m, 2H), 6.53 (d, J = 2.1 Hz, 1H), 6.28 (d, J = 2.1 Hz, 1H), 4.40 (t, J = 6.6 Hz, 2H), 3.27 (t, J = 6.6 Hz, 2H).

[0169] 13C NMR (125 MHz, acetone-d6): δ 179.5, 165.2, 163.2, 157.8, 154.0, 152.1 (dd, J = 13.3, 250.8 Hz), 150.6 (dd, J = 12.0, 244.8 Hz), 141.3, 139.3, 128.8-128.7 (m), 127.7, 126.8 (dd, J = 3.6, 7.3 Hz), 126.5, 124.7, 118.6 (d, J = 19.1 Hz), 118.4 (d, J = 18.0 Hz), 106.0, 99.7, 94.7, 73.8, 31.2.

[0170] FAB-MS (m / z): found 417.3 [M+H] + , calcd for C 21 H 14 F2O5S 416.1.

[0172] <Example 12> Synthesis of 2-(3,4-difluorophenyl)-3-[2-(dimethylamino)ethoxy]-5,7-dihydroxy-4H-chromen-4-one (Compound 13)

[0173] As an alkylating agent in the above method B N , N The reaction was carried out using dimethyl ethanolamine. After washing the concentrate with acetone, a yellow solid was obtained.

[0174] Yield: 26%

[0175] 1 H NMR (500 MHz, DMSO-d6): δ 12.29 (s, 1H), 8.11 (ddd, J = 2.1, 7.8, 11.7 Hz, 1H), 7.98-7.95 (m, 1H), 7.66 (ddd, J = 8.6, 8.6, 10.2 Hz, 1H), 6.57 (d, J = 2.0 Hz, 1H), 6.30 (d, J = 2.1 Hz, 1H), 4.28 (t, J = 4.7 Hz, 2H), 3.46 (t, J = 4.9 Hz, 2H), 2.89 (s, 6H).

[0176] 13C NMR (125 MHz, DMSO-d6): δ 177.7, 165.0, 161.1, 156.6, 153.6, 151.0 (dd, J = 12.0, 250.8 Hz), 149.4 (dd, J = 13.1, 244.8 Hz), 137.3, 127.1 (dd, J = 3.6, 7.1 Hz), 126.3 (dd, J = 3.6, 7.1 Hz), 118.3 (d, J = 16.8 Hz), 117.9 (d, J = 19.1 Hz), 104.4, 99.2, 94.3, 66.4, 55.9, 42.6.

[0177] FAB-MS (m / z): found 378.3 [M+H] + , calcd for C 19 H 17 F2NO5377.1.

[0179] <Example 13> Synthesis of 3-[2-(aziridin-1-yl)ethoxy]-2-(3,4-difluorophenyl)-5,7-dihydroxy-4H-chromen-4-one (Compound 14)

[0180] In the above method B, the reaction was carried out using 1-(2-hydroxyethyl)ethyleneimide as an alkylating agent. After washing the concentrate with acetone, a yellow solid was obtained.

[0181] Yield: 28%

[0182] 1 H NMR (500 MHz, DMSO-d6): δ 12.31 (s, 1H), 8.13 (ddd, J = 2.2, 7.9, 11.9 Hz, 1H), 7.99-7.96 (m, 1H), 7.65 (ddd, J = 8.7, 8.7, 10.4 Hz, 1H), 6.56 (d, J = 2.1 Hz, 1H), 6.29 (d, J = 2.1 Hz, 1H), 4.23 (t, J = 5.2 Hz, 2H), 3.93 (t, J = 6.2 Hz, 2H), 3.36 (t, J = 6.2 Hz, 2H), 3.28 (t, J = 5.2 Hz, 2H).

[0183] 13C NMR (125 MHz, DMSO-d6): δ 177.8, 165.0, 161.1, 156.6, 153.4, 151.0 (dd, J = 12.0, 250.8 Hz), 149.4 (dd, J = 13.3, 244.8 Hz), 137.5, 127.2 (dd, J = 3.6, 7.1 Hz), 126.3-126.3 (m), 118.2 (d, J = 16.8 Hz), 117.9 (d, J = 19.1 Hz), 104.3, 99.1, 94.2, 68.3, 48.3, 46.8, 40.3.

[0184] FAB-MS (m / z): found 376.3 [M+H] + , calcd for C 19 H 15 F2NO5375.1.

[0186] <Example 14> Synthesis of 2-((2-((2-(3,4-difluorophenyl)-5,7-dihydroxy-4-oxo-4H-chromene-3-yl)oxy)ethyl)dimethylammonium)acetate (Compound 15)

[0187] 14-1. Step 1

[0188] 310 mg (1.00 mmol) of compound 2, 2-chloro-N,N-dimethylethylamine hydrochloride (1.1 equivalents), and K2CO3 (2.5 equivalents) were added to 10 mL of DMF and stirred at 90 °C for 2 hours. After cooling to room temperature, the mixture was treated with a saturated aqueous solution of NH4Cl and extracted with EtOAc. The organic layer was washed with brine, dried with MgSO4, filtered, and concentrated under reduced pressure. The mixture was purified by column chromatography (CH2Cl2:MeOH = 30 to 10:1) to obtain a yellow solid.

[0189] Yield: 65%

[0190] 1H NMR (500 MHz, CDCl3): δ 12.46 (s, 1H), 8.19 (ddd, J = 2.3, 7.8, 12.0 Hz, 1H), 7.98-7.95 (m, 1H), 7.31-7.26 (m, 1H), 6.64 (d, J = 2.2 Hz, 1H), 6.47 (d, J = 2.2 Hz, 1H), 5.25 (s, 2H), 4.18 (t, J = 5.5 Hz, 2H), 3.50 (s, 3H), 2.67 (t, J = 5.6 Hz, 2H), 2.30 (s, 6H).

[0192] 14-2. Stage 2

[0193] 250 mg (0.59 mmol) of the yellow solid and 0.26 mL (1.78 mmol) of tert-butylbromoacetate were added to 5 mL of DMF and stirred at room temperature for 12 hours, and the product was filtered and washed with EtOAc to obtain a light brown solid ammonium bromide salt.

[0194] Yield: 77%

[0195] 1 H NMR (500 MHz, DMSO-d6): δ 12.18 (s, 1H), 8.14 (ddd, J = 2.2, 7.9, 11.9 Hz, 1H), 7.98-7.95 (m, 1H), 7.70 (ddd, J = 8.6, 8.6, 10.4 Hz, 1H), 6.94 (d, J = 2.1 Hz, 1H), 6.54 (d, J = 2.2 Hz, 1H), 5.35 (s, 2H), 4.59 (s, 2H), 4.43-4.41 (m, 2H), 4.04-4.02 (m, 2H), 3.41 (s, 3H), 3.36 (s, 6H), 1.38 (s, 9H).

[0197] 14-3. Step 3

[0198] 100 mg (0.16 mmol) of the above light brown solid was treated with 5 mL of 0.5 M hydrochloric acid MeOH solution and stirred at 70 °C for 24 hours. After cooling to room temperature and concentrating under reduced pressure, the solution was neutralized with a saturated aqueous solution of NaHCO3 and purified by reverse-phase chromatography in a C18 reaction bed to obtain a yellow solid.

[0199] Yield: 30%

[0200] 1 H NMR (500 MHz, methanol-d4): δ 7.93 (ddd, J = 2.2, 7.8, 11.8 Hz, 1H), 7.91-7.87 (m, 1H), 7.44 (ddd, J = 8.6, 8.6, 10.3 Hz, 1H), 6.08 (d, J = 2.0 Hz, 1H), 5.95 (d, J = 2.0 Hz, 1H), 4.33-4.32 (m, 2H), 4.24 (s, 2H), 4.12-4.11 (m, 2H), 3.49 (s, 6H).

[0201] 13 C NMR (125 MHz, methanol-d4): δ 178.9, 177.5, 169.3, 162.6, 159.3, 153.4, 152.7 (dd, J = 12.0, 250.8 Hz), 151.4 (dd, J = 13.3, 246.0 Hz), 138.0, 129.3 (dd, J = 3.5, 6.0 Hz), 126.7-126.6 (m), 118.9 (d, J = 18.0 Hz), 118.4 (d, J = 19.3 Hz), 104.2, 102.6, 97.8, 67.0, 66.0, 64.0, 53.1.

[0202] FAB-MS (m / z): found 436.3 [M+H] + , calcd for C 21 H 19 F2NO7435.1.

[0204] <Example 15> Synthesis of 2-(3,4-difluorophenyl)-5,7-dihydroxy-3-[2-(4-methylpiperazine-1-yl)ethoxy]-4H-chromen-4-one (Compound 16)

[0205] In the above method B, the reaction was carried out using 4-methylpiperazine-1-ethanol as an alkylating agent. After grinding the concentrate with ethyl acetate, a white solid was obtained.

[0206] Yield: 54%

[0207] 1 H NMR (500 MHz, DO): δ 7.66-7.60 (m, 2H), 7.29 (ddd, J = 8.6, 8.6, 10.0 Hz, 1H), 6.03 (d, J = 2.1 Hz, 1H), 6.00 (d, J = 2.1 Hz, 1H), 4.13 (t, J = 4.6 Hz, 2H), 3.79 (br s, 8H), 3.64 (t, J = 4.5 Hz, 2H), 3.10 (s, 3H).

[0208] 13 C NMR (125 MHz, DO): δ 177.7, 163.5, 159.8, 156.1, 154.1, 151.8 (dd, J = 12.0, 253.1 Hz), 149.8 (dd, J = 13.1, 244.8 Hz), 136.8, 125.8 (dd, J = 3.6, 7.3 Hz), 125.7-125.6 (m), 118.1 (d, J = 18.0 Hz), 117.2 (d, J = 20.5 Hz), 104.3, 99.1, 94.5, 66.2, 56.7, 50.5, 49.5, 42.9.

[0209] FAB-MS (m / z): found 433.4 [M+H] + , calcd for C 22 H 22 F2N2O5432.1.

[0211] <Example 16> Synthesis of 2-(3,4-difluorophenyl)-5,7-dihydroxy-3-[2-(piperazine-1-yl)ethoxy]-4H-chromen-4-one (Compound 17)

[0212] In the above method B, the reaction was carried out using 1-piperazine ethanol as an alkylating agent. After grinding the concentrate with ethyl acetate, a yellow solid was obtained.

[0213] Yield: 46%

[0214] 1 H NMR (500 MHz, DO): δ 7.77 (ddd, J = 2.0, 7.7, 11.5 Hz, 1H), 7.73-7.71 (m, 1H), 7.37-7.32 (m, 1H), 6.23 (d, J = 2.0 Hz, 1H), 6.12 (d, J = 2.0 Hz, 1H), 4.14 (t, J = 4.8 Hz, 2H), 3.80-3.74 (m, 4H), 3.73-3.69 (m, 4H), 3.61 (t, J = 4.6 Hz, 2H).

[0215] 13 C NMR (125 MHz, DMSO-d6): δ 177.7, 165.0, 161.1, 156.6, 153.5, 151.0 (dd, J = 13.3, 250.8 Hz), 149.5 (dd, J = 13.3, 244.8 Hz), 137.4, 127.1-127.1 (m), 126.4-126.4 (m), 118.3 (d, J = 16.8 Hz), 117.9 (d, J = 19.1 Hz), 104.3, 99.2, 94.3, 66.3, 55.3, 48.3, 48.3.

[0216] FAB-MS (m / z): found 419.4 [M+H] + , calcd for C 21 H20F2N2O5418.1.

[0218] <Example 17> Synthesis of 2-(3,4-difluorophenyl)-5,7-dihydroxy-3-[2-(pyrrolidin-1-yl)ethoxy]-4H-chromen-4-one (Compound 18)

[0219] In the above method A, the reaction was carried out using 1-(2-chloroethyl)pyrrolidine hydrochloride as an alkylating agent. After grinding the concentrate with ethanol, a pale yellow solid was obtained.

[0220] Yield: 32%

[0221] 1H NMR (500 MHz, DMSO-d6): δ 12.32 (s, 1H), 11.18 (s, 1H), 10.31 (s, 1H), 8.13 (ddd, J = 2.2, 7.9, 11.8 Hz, 1H), 7.98-7.96 (m, 1H), 7.67 (ddd, J = 8.7, 8.7, 10.4 Hz, 1H), 6.56 (d, J = 2.1 Hz, 1H), 6.29 (d, J = 2.1 Hz, 1H), 4.27 (t, J = 4.8 Hz, 2H), 3.67 (br s, 2H), 3.56-3.50 (m, 2H), 3.11 (br s, 2H), 2.01 (br s, 2H), 1.93 (br s, 2H).

[0222] 13 C NMR (125 MHz, DMSO-d6): δ 177.7, 164.9, 161.1, 156.6, 153.6, 151.0 (dd, J = 12.0, 249.5 Hz), 149.4 (dd, J = 12.0, 244.8 Hz), 137.3, 127.7-127.7 (m), 126.3-126.3 (m), 118.3 (d, J = 18.0 Hz), 117.9 (d, J = 19.3 Hz), 104.4, 99.1, 94.3, 67.4, 53.5, 53.3, 22.6.

[0223] FAB-MS (m / z): found 404.4 [M+H] + , calcd for C 21 H 19 F2NO5403.1.

[0225] <Example 18> Synthesis of 2-(3,4-difluorophenyl)-5,7-dihydroxy-3-[2-(piperidin-1-yl)ethoxy]-4H-chromen-4-one (Compound 19)

[0226] In the above method A, the reaction was carried out using 1-(2-chloroethyl)piperidine hydrochloride as an alkylating agent. After grinding the concentrate with ethanol, a yellow solid was obtained.

[0227] Yield: 34%

[0228] 1H NMR (500 MHz, DMSO-d6): δ 12.29 (s, 1H), 11.25 (s, 1H), 10.25 (s, 1H), 8.11 (ddd, J = 2.2, 7.8, 11.8 Hz, 1H), 7.97-7.94 (m, 1H), 7.67 (ddd, J = 8.6, 8.6, 10.5 Hz, 1H), 6.57 (d, J = 2.0 Hz, 1H), 6.31 (d, J = 2.1 Hz, 1H), 4.33 (t, J = 4.7 Hz, 2H), 3.57-3.51 (m, 2H), 3.45-3.41 (m, 2H), 3.02-2.95 (m, 2H), 1.83-1.75 (m, 4H), 1.73-1.69 (m, 1H), 1.42-1.32 (m, 1H).

[0229] 13 C NMR (125 MHz, DMSO-d6): δ 178.1, 165.0, 161.2, 156.4, 152.3, 150.0 (dd, J = 12.0, 250.8 Hz), 149.2 (dd, J = 13.3, 244.8 Hz), 138.0, 127.7-127.7 (m), 126.0-126.0 (m), 118.1 (d, J = 19.3 Hz), 117.9 (d, J = 16.9 Hz), 104.3, 99.0, 94.0, 68.6, 57.7, 53.8, 34.0, 24.9.

[0230] FAB-MS (m / z): found 418.4 [M+H] + , calcd for C 22 H 21 F2NO5417.1.

[0232] <Example 19> Synthesis of 2-(3,4-difluorophenyl)-5,7-dihydroxy-3-(2-morpholinoethoxy)-4H-chromen-4-one (Compound 20)

[0233] In the above method B, the reaction was carried out using 4-(2-hydroxyethyl)morpholine as an alkylating agent. After grinding the concentrate with ethyl acetate, a white solid was obtained.

[0234] Yield: 60%

[0235] 1 H NMR (500 MHz, DMSO-d6): δ 12.26 (s, 1H), 11.25 (s, 1H), 11.15 (s, 1H), 8.11 (ddd, J = 2.2, 7.8, 11.8 Hz, 1H), 7.98-7.95 (m, 1H), 7.65 (ddd, J = 8.6, 8.6, 10.4 Hz, 1H), 6.57 (d, J = 2.1 Hz, 1H), 6.30 (d, J = 2.1 Hz, 1H), 4.35 (t, J = 4.6 Hz, 2H), 4.00-3.98 (m, 2H), 3.87-3.83 (m, 2H), 3.58-3.52 (m, 4H), 3.24-3.19 (m, 2H).

[0236] 13 C NMR (125 MHz, DMSO-d6): δ 177.7, 165.0, 161.1, 156.5, 153.6, 151.0 (dd, J = 13.1, 250.8 Hz), 149.4 (dd, J = 13.3, 246.0 Hz), 137.3, 127.1-127.1 (m), 126.4-126.3 (m), 118.2 (d, J = 16.8 Hz), 117.9 (d, J = 19.1 Hz), 104.3, 99.1, 94.3, 66.0, 63.2, 55.4, 51.3.

[0237] FAB-MS (m / z): found 420.3 [M+H] + , calcd for C 21 H 19 F2NO6419.1.

[0239] <Example 20> Synthesis of 2-(3,4-difluorophenyl)-5,7-dihydroxy-3-[2-(1-methylpyrrolidin-2-yl) ethoxy]-4H-chromen-4-one (Compound 21)

[0240] In the above method B, the reaction was carried out using 2-(2-hydroxyethyl)-1-methylpyrrolidine as an alkylating agent. After grinding the concentrate with ethyl acetate, a yellow solid was obtained.

[0241] Yield: 35%

[0242] 1H NMR (500 MHz, methanol-d4): δ 8.04 (ddd, J = 2.2, 7.7, 11.8 Hz, 1H), 7.99-7.95 (m, 1H), 7.51 (ddd, J = 8.6, 8.6, 10.2 Hz, 1H), 6.48 (d, J = 2.1 Hz, 1H), 6.26 (d, J = 2.2 Hz, 1H), 4.07 (t, J = 5.2 Hz, 2H), 3.87 (ddd, J = 5.5, 7.7, 11.7 Hz, 1H), 3.74-3.68 (m, 1H), 3.28-3.22 (m, 1H), 3.06 (s, 3H), 2.45-2.38 (m, 1H), 2.26 (td, J = 5.2, 5.5 Hz, 2H), 2.23-2.16 (m, 2H), 2.03-1.95 (m, 1H).

[0243] 13 C NMR (125 MHz, methanol-d4): δ 180.1, 167.0, 163.0, 158.7, 155.8, 153.3 (dd, J = 13.1, 252.0 Hz), 151.6 (dd, J = 12.0, 242.4 Hz), 139.4, 128.6-128.6 (m), 127.1-127.0 (m), 119.2 (d, J = 18.0 Hz), 118.9 (d, J = 20.4 Hz), 105.8, 100.4, 95.3, 70.6, 69.0, 57.5, 40.2, 30.4, 29.7, 22.3.

[0244] FAB-MS (m / z): found 418.4 [M+H] + , calcd for C 22 H 21 F2NO5417.1.

[0246] <Example 21> Synthesis of 2-(3,4-difluorophenyl)-5,7-dihydroxy-3-(3,3,3-trifluoropropoxy)-4H-chromen-4-one (Compound 22)

[0247] In the above method A, the reaction was carried out using 1,1,1-trifluoro-3-iodopropane as the alkylating agent. After purifying the concentrate by silica gel column chromatography (Hexanes:EtOAc=4:1), a pale yellow solid was obtained.

[0248] Yield: 50%

[0249] 1 H NMR (500 MHz, acetone-d6): δ 12.48 (s, 1H), 8.07 (ddd, J = 2.2, 7.9, 12.1 Hz, 1H), 8.01-7.98 (m, 1H), 7.54 (ddd, J = 8.6, 8.6, 10.4 Hz, 1H), 6.54 (d, J = 2.1 Hz, 1H), 6.29 (d, J = 2.1 Hz, 1H), 4.39 (t, J = 6.3 Hz, 2H), 2.78 (qt, J = 4.9, 6.3 Hz, 2H).

[0250] 13 C NMR (125 MHz, acetone-d6): δ 179.3, 165.4, 161.2, 157.9, 154.3, 152.3 (dd, J = 13.3, 250.9 Hz), 150.7 (dd, J = 13.1, 245.9 Hz), 139.0, 128.7-128.6 (m), 127.5 (q, J = 273.6 Hz), 126.9 (dd, J = 3.6, 7.3 Hz), 118.8 (d, J = 19.1 Hz), 118.6 (d, J = 18.0 Hz), 106.0, 99.8, 94.8, 66.2 (q, J = 3.8 Hz), 35.0 (q, J = 22.8 Hz).

[0251] FAB-MS (m / z): found 403.3 [M+H] + , calcd for C 18 H 11 F5O5402.1.

[0253] <Example 22> Synthesis of 2-(3,4-difluorophenyl)-3-(3,3-dimethylbutoxy)-5,7-dihydroxy-4H-chromen-4-one (Compound 23)

[0254] In the above method A, the reaction was carried out using 1,1,1-trifluoro-3-iodopropane as the alkylating agent. After purifying the concentrate by silica gel column chromatography (Hexanes:EtOAc=6:1), a yellow solid was obtained.

[0255] Yield: 67%

[0256] 1 H NMR (500 MHz, acetone-d6): δ 12.48 (s, 1H), 8.07 (ddd, J = 2.2, 7.9, 12.1 Hz, 1H), 8.01-7.98 (m, 1H), 7.54 (ddd, J = 8.6, 8.6, 10.4 Hz, 1H), 6.54 (d, J = 2.1 Hz, 1H), 6.29 (d, J = 2.1 Hz, 1H), 4.39 (t, J = 6.3 Hz, 2H), 2.78 (qt, J = 4.9, 6.3 Hz, 2H).

[0257] 13 C NMR (125 MHz, acetone-d6): δ 179.3, 165.4, 161.2, 157.9, 154.3, 152.3 (dd, J = 13.3, 250.9 Hz), 150.7 (dd, J = 13.1, 245.9 Hz), 139.0, 128.7-128.6 (m), 127.5 (q, J = 273.6 Hz), 126.9 (dd, J = 3.6, 7.3 Hz), 118.8 (d, J = 19.1 Hz), 118.6 (d, J = 18.0 Hz), 106.0, 99.8, 94.8, 66.2 (q, J = 3.8 Hz), 35.0 (q, J = 22.8 Hz).

[0258] FAB-MS (m / z): found 403.3 [M+H] + , calcd for C 18 H 11 F5O5402.1.

[0260] <Example 23> Synthesis of 2-(3,4-difluorophenyl)-5,7-dihydroxy-3-(3-methylbutoxy)-4H-chromen-4-one (Compound 24)

[0261] In the above method A, the reaction was carried out using 1-bromo-3-methylbutane as an alkylating agent. After purifying the concentrate by silica gel column chromatography (Hexanes:EtOAc=6:1), a yellow solid was obtained.

[0262] Yield: 70%

[0263] 1 H NMR (500 MHz, acetone-d6): δ 12.63 (s, 1H), 8.11 (ddd, J = 2.2, 7.9, 12.2 Hz, 1H), 8.04-8.01 (m, 1H), 7.56 (ddd, J = 8.6, 8.6, 10.5 Hz, 1H), 6.54 (d, J = 2.1 Hz, 1H), 6.29 (d, J = 2.0 Hz, 1H), 4.15 (t, J = 6.6 Hz, 2H), 1.79 (t sept, J = 6.6, 6.7 Hz, 1H), 1.61 (dt, J = 6.7, 6.8 Hz, 2H), 0.91 (d, J = 6.7 Hz, 6H).

[0264] 13 C NMR (125 MHz, acetone-d6): δ 179.6, 165.2, 163.2, 157.8, 154.0, 152.2 (dd, J = 13.1, 250.8 Hz), 150.7 (dd, J = 13.3, 244.8 Hz), 139.7, 129.0-128.9 (m), 126.7-126.7 (m), 118.6 (d, J = 19.1 Hz), 118.5 (d, J = 15.6 Hz), 106.1, 99.6, 94.7, 71.9, 39.6, 25.5, 22.7.

[0265] FAB-MS (m / z): found 377.4 [M+H] + , calcd for C 20 H 18 F2O5376.1.

[0267] <Example 24> Synthesis of 3-(2-cyclopropylethoxy)-2-(3,4-difluorophenyl)-5,7-dihydroxy-4H-chromen-4-one (Compound 25)

[0268] 2-cyclopropylethanol cooling solution, TEA (1.1 equivalents), and MsCl (1.1 equivalents) were added to CH2Cl2 and stirred for 1 hour, after which the reaction was terminated by treatment with a saturated aqueous solution of NH4Cl. The organic layer was washed with brine and dried with MgSO4, and 2-cyclopropylethyl methanesulfonate was obtained by filtration and concentration under reduced pressure.

[0269] In the above method A, the reaction was carried out using 2-cyclopropylethyl methanesulfonate as an alkylating agent. After purifying the concentrate by silica gel column chromatography (Hexanes:acetone = 6:1), a yellow solid was obtained.

[0270] Yield: 51%

[0271] 1 H NMR (500 MHz, acetone-d6): δ 12.61 (s, 1H), 8.13 (ddd, J = 2.2, 7.9, 12.2 Hz, 1H), 8.06-8.03 (m, 1H), 7.54 (ddd, J = 8.6, 8.6, 10.4 Hz, 1H), 6.53 (d, J = 2.1 Hz, 1H), 6.27 (d, J = 2.1 Hz, 1H), 4.18 (t, J = 6.7 Hz, 2H), 1.63 (td, J = 6.7, 6.8 Hz, 2H), 0.85-0.77 (m, 1H), 0.46-0.42 (m, 2H), 0.10-0.07 (m, 2H).

[0272] 13 C NMR (125 MHz, acetone-d6): δ 179.6, 165.2, 163.2, 157.8, 153.9, 152.2 (dd, J = 12.0, 250.8 Hz), 150.7 (dd, J = 13.1, 244.8 Hz), 139.7, 129.0-128.9 (m), 126.7 (dd, J = 3.6, 7.3 Hz), 118.7 (d, J = 20.5 Hz), 118.5 (d, J = 16.8 Hz), 106.1, 99.6, 94.7, 73.5, 35.6, 8.4, 4.7.

[0273] FAB-MS (m / z): found 375.3 [M+H] + , calcd for C 20 H 16 F2O5374.1.

[0275] <Example 25> Synthesis of 3-(2-cyclopentylethoxy)-2-(3,4-difluorophenyl)-5,7-dihydroxy-4H-chromen-4-one (Compound 26)

[0276] In the above method B, the reaction was carried out using 2-cyclopentylethanol as an alkylating agent. After purifying the concentrate by silica gel column chromatography (Hexanes:EtOAc=6:1), a yellow solid was obtained.

[0277] Yield: 70%

[0278] 1 H NMR (500 MHz, CDCl3): δ 12.52 (s, 1H), 7.97 (ddd, J = 2.2, 7.7, 11.7 Hz, 1H), 7.90-7.86 (m, 1H), 7.29 (ddd, J = 8.6, 8.6, 9.6 Hz, 1H), 6.45 (d, J = 2.2 Hz, 1H), 6.34 (d, J = 2.1 Hz, 1H), 4.00 (t, J = 7.0 Hz, 2H), 1.84 (tqu, J = 7.5, 8.3 Hz, 1H), 1.73 (td, J = 7.0, 7.1 Hz, 2H), 1.73-1.67 (m, 2H), 1.61-1.53 ​​(m, 2H), 1.52-1.43 (m, 2H), 1.08-1.01 (m, 2H).

[0279] 13 C NMR (125 MHz, CDCl3): δ 179.1, 163.2, 162.2, 157.0, 154.0, 151.9 (dd, J = 13.1, 254.4 Hz), 150.2 (dd, J = 13.3, 248.4 Hz), 139.1, 127.5-127.4 (m), 125.5 (dd, J = 3.6, 6.0 Hz), 118.1 (d, J = 20.5 Hz), 117.7 (d, J = 16.9 Hz), 105.9, 99.6, 94.3, 73.2, 36.6, 36.2, 32.7, 25.1.

[0280] FAB-MS (m / z) found 403.4 [M+H] + , calcd for C 22 H 20 F2O5402.1.

[0282] <Example 26> Synthesis of 3-(2-cyclohexylethoxy)-2-(3,4-difluorophenyl)-5,7-dihydroxy-4H-chromen-4-one (Compound 27)

[0283] In the above method B, the reaction was carried out using 2-cyclohexylethanol as an alkylating agent. After purifying the concentrate by silica gel column chromatography (Hexanes:EtOAc=10:1), a yellow solid was obtained.

[0284] Yield: 66%

[0285] 1 H NMR (500 MHz, CDCl3): δ 12.56 (s, 1H), 7.97 (ddd, J = 2.2, 7.7, 11.6 Hz, 1H), 7.89-7.85 (m, 1H), 7.30 (ddd, J = 8.6, 8.6, 9.7 Hz, 1H), 6.42 (d, J = 2.1 Hz, 1H), 6.31 (d, J = 2.1 Hz, 1H), 4.06 (t, J = 6.8 Hz, 2H), 1.68-1.61 (m, 5H), 1.59 (dt, J = 6.8, 6.8 Hz, 2H), 1.42-1.34 (m, 1H), 1.21-1.07 (m, 3H), 0.90-0.83 (m, 2H).

[0286] 13 C NMR (125 MHz, CDCl3): δ 179.1, 162.8, 162.4, 156.9, 153.8, 151.8 (dd, J = 13.1, 254.4 Hz), 150.2 (dd, J = 13.3, 247.3 Hz), 139.1, 127.6-127.5 (m), 125.5 (dd, J = 3.6, 6.0 Hz), 118.2 (d, J = 19.1 Hz), 117.6 (d, J = 18.0 Hz), 106.1, 99.5, 94.2, 71.6, 37.5, 34.4, 33.3, 26.6, 26.3.

[0287] FAB-MS (m / z) found 417.4 [M+H] + , calcd for C 23 H 22 F2O5416.1.

[0289] <Example 27> Synthesis of 3-(cyclopentylmethoxy)-2-(3,4-difluorophenyl)-5,7-dihydroxy-4H-chromen-4-one (Compound 28)

[0290] In the above method B, the reaction was carried out using cyclopentylmethanol as an alkylating agent. After purifying the concentrate by silica gel column chromatography (Hexanes:acetone = 6:1), a yellow solid was obtained.

[0291] Yield: 34%

[0292] 1 H NMR (500 MHz, acetone-d6): δ 12.61 (s, 1H), 8.09 (ddd, J = 2.2, 7.9, 12.2 Hz, 1H), 8.02-7.99 (m, 1H), 7.54 (ddd, J = 8.6, 8.6, 10.5 Hz, 1H), 6.52 (d, J = 2.1 Hz, 1H), 6.27 (d, J = 2.1 Hz, 1H), 3.98 (d, J = 7.0 Hz, 2H), 2.30 (tqu, J = 7.5, 7.6 Hz, 1H), 1.77-1.70 (m, 2H), 1.61-1.51 (m, 4H), 1.36-1.29 (m, 2H).

[0293] 13 C NMR (125 MHz, acetone-d6): δ 179.6, 165.2, 163.2, 157.8, 153.9, 152.2 (dd, J = 12.0, 250.8 Hz), 150.7 (dd, J = 12.0, 244.8 Hz), 139.6, 129.0-128.9 (m), 126.8 (dd, J = 3.6, 7.3 Hz), 118.8 (d, J = 20.4 Hz), 118.5 (d, J = 18.0 Hz), 106.1, 99.6, 94.7, 77.5, 40.6, 30.0, 26.1.

[0294] FAB-MS (m / z) found 389.3 [M + H] + , calcd for C 21 H 18 F2O5388.1.

[0296] <Example 28> Synthesis of 3-(3-cyclopentylpropoxy)-2-(3,4-difluorophenyl)-5,7-dihydroxy-4H-chromen-4-one (Compound 29)

[0297] In the above method B, the reaction was carried out using 3-cyclopentyl-1-propanol as an alkylating agent. After purifying the concentrate by silica gel column chromatography (Hexanes:EtOAc=4:1), a yellow solid was obtained.

[0298] Yield: 55%

[0299] 1 H NMR (500 MHz, acetone-d6): δ 12.61 (s, 1H), 8.10 (ddd, J = 2.2, 7.9, 12.2 Hz, 1H), 8.02-7.99 (m, 1H), 7.54 (ddd, J = 8.6, 8.6, 10.4 Hz, 1H), 6.52 (d, J = 2.1 Hz, 1H), 6.27 (d, J = 2.1 Hz, 1H), 4.12 (t, J = 6.5 Hz, 2H), 1.78-1.68 (m, 5H), 1.62-1.55 (m, 2H), 1.53-1.45 (m, 2H), 1.40-1.36 (m, 2H), 1.09-1.02 (m, 2H).

[0300] 13 C NMR (125 MHz, acetone-d6): δ 179.6, 165.2, 163.2, 157.8, 154.0, 152.2 (dd, J = 12.0, 250.8 Hz), 150.7 (dd, J = 13.3, 244.9 Hz), 139.6, 129.0 (dd, J = 3.5, 6.0 Hz), 126.8 (dd, J = 3.6, 6.0 Hz), 118.8 (d, J = 20.4 Hz), 118.5 (d, J = 16.9 Hz), 106.1, 99.6, 94.7, 73.8, 40.6, 33.2, 33.1, 29.9, 25.7.

[0301] FAB-MS (m / z): found 417.4 [M+H] + , calcd for C 23 H 22 F2O5416.1.

[0303] <Example 29> Synthesis of 2-(3,4-difluorophenyl)-5,7-dihydroxy-3-[2-(tetrahydro-2H-pyran-4-yl)ethoxy]-4H-chromen-4-one (Compound 30)

[0304] In the above method B, the reaction was carried out using 2-(tetrahydro-2H-pyran-4-yl)ethanol as an alkylating agent. After purifying the concentrate by silica gel column chromatography (Hexanes:acetone = 3:1), a pale yellow solid was obtained.

[0305] Yield: 56%

[0306] 1 H NMR (500 MHz, DMSO-d6): δ 12.46 (s, 1H), 8.04 (ddd, J = 2.2, 7.9, 12.0 Hz, 1H), 7.89-7.86 (m, 1H), 7.66 (ddd, J = 8.6, 8.6, 10.6 Hz, 1H), 6.45 (d, J = 2.1 Hz, 1H), 6.21 (d, J = 2.1 Hz, 1H), 4.03 (t, J = 6.0 Hz, 2H), 3.80-3.77 (m, 2H), 3.16 (dt, J = 2.0, 11.9 Hz, 2H), 1.61-1.50 (m, 1H), 1.53 (t, J = 5.9 Hz, 2H), 1.49-1.46 (m, 2H), 1.13-1.05 (m, 2H).

[0307] 13 C NMR (125 MHz, DMSO-d6): δ 178.1, 164.6, 161.3, 156.5, 153.1, 150.7 (dd, J = 12.0, 249.5 Hz), 149.2 (dd, J = 13.1, 244.8 Hz), 138.0, 127.6 (dd, J = 3.6, 7.3 Hz), 126.1-126.0 (m), 118.0 (d, J = 18.0 Hz), 117.8 (d, J = 19.3 Hz), 104.5, 98.8, 94.0, 69.6, 67.0, 36.3, 32.4, 31.1.

[0308] FAB-MS (m / z): found 419.3 [M+H] + , calcd for C 22 H20 F2O6418.1.

[0310] <Example 30> Synthesis of 2-(3,4-difluorophenyl)-5,7-dihydroxy-3-[2-(piperidin-4-yl)ethoxy]-4H-chromen-4-one (Compound 31)

[0311] In the above method B, the reaction was carried out using 1-Boc-4-(2-hydroxyethyl)piperidine as an alkylating agent. After washing the concentrate with diethylether (Et2O), a yellow solid was obtained.

[0312] Yield: 51%

[0313] 1 H NMR (500 MHz, DMSO-d6): δ 12.46 (s, 1H), 11.13 (s, 1H), 8.90-8.88 (m, 1H), 8.68-8.62 (m, 1H), 8.07 (ddd, J = 2.2, 7.9, 12.0 Hz, 1H), 7.92-7.89 (m, 1H), 7.68 (ddd, J = 8.6, 8.6, 10.5 Hz, 1H), 6.52 (d, J = 2.1 Hz, 1H), 6.26 (d, J = 2.1 Hz, 1H), 4.03 (t, J = 6.2 Hz, 2H), 3.23-3.21 (m, 2H), 2.79-2.72 (m, 2H), 1.79-1.76 (m, 2H), 1.73-1.66 (m, 1H), 1.58 (td, J = 6.3, 6.5 Hz, 2H), 1.34-1.26 (m, 2H).

[0314] 13 C NMR (125 MHz, DMSO-d6): δ 178.0, 164.7, 161.2, 156.5, 153.0, 150.7 (dd, J = 12.0, 250.8 Hz), 149.2 (dd, J = 13.3, 244.8 Hz), 138.0, 127.6-127.5 (m), 126.1-126.0 (m), 118.1 (d, J = 18.0 Hz), 117.7 (d, J = 19.1 Hz), 104.4, 98.9, 94.1, 69.7, 43.0, 35.5, 30.0, 28.1.

[0315] FAB-MS (m / z): found 418.4 [M+H] + , calcd for C 22 H 21 F2NO5417.1.

[0317] <Example 31> Ethidium bromide (EtBr) elimination activity

[0318] 31-1. Activity of increased EtBr accumulation due to drug efflux pump inhibition

[0319] Ethidium bromide (EtBr) is a fluorescent probe susceptible to efflux, which accumulates within bacterial cells and binds to cytoplasmic components to emit fluorescence. For 14 clinical strains of carbapenem-resistant Enterobacteriaceae (CRE), the degree of EtBr accumulation effluxed by drug efflux pumps was measured using compounds 3 to 31, a control (compound 2), and a positive control, carbonyl cyanide 3-chlorophenylhydrazone (CCCP).

[0320] As a result, as shown in Figure 1, no fluorescence was observed in the negative control group without compound treatment due to the rapid efflux of EtBr by the drug efflux pump; however, fluorescence due to EtBr accumulation increased in compounds 4, 6, 23, 24, 26, and 27, and in particular, fluorescence due to EtBr accumulation increased as the concentration of compound 23 increased (EC 50 It was confirmed that =1.2 mg / L).

[0322] 31-2. Inhibitory activity of EtBr accumulation

[0323] Since the accumulation of EtBr reflects the balance between inflow and outflow into the cell, the degree of inhibition of outflow pump activity was measured by monitoring the fluorescence intensity of compound 23 at concentrations of 0.25–32 mg / L after loading EtBr into bacterial cells.

[0324] As a result, as shown in Figure 2, EtBr fluorescence decreased over time in the control EC-001blaNDM strain treated with DMSO and in CRE strains treated with Compound 23 at concentrations of 0.25, 0.5, 1, and 2 mg / L, whereas in CRE strains treated with Compound 23 at concentrations of 4 mg / L or higher, fluorescence remained constant (EC 50 It was confirmed that it appeared as =2.7 mg / L.

[0326] <Example 32> Carbapenemase Inhibitory Activity

[0327] 32-1. Metallo-β-lactamase (MBL) Multi-Target Fluorescence Detection

[0328] Carbapenem degrading enzymes KPC-2 (class A; LifeSpan Biosciences, Inc. (LSBio; product code LS-G26598)), OXA-48 (class C, residues 23265; Bioneer, Inc. (Seoul, Korea)), VIM-2 (residues 27266; ​​Bioneer, Inc. (Seoul, Korea)) and NDM-1 (class B; RayBiotech, Inc. (product code 230-00554)) were commercially purchased or obtained by applying previously known expression and purification methods, and the activities of carbapenem degrading enzymes KPC-2, OXA-48, VIM-2, and NDM-1 were monitored using nitrocepin or imipenem as fluorescent substrates.

[0329] Specifically, 100 μL of PBS solution containing compounds 3 to 31 at concentrations of 0.1 to 70 μM and 100 μL of diluted enzyme stock solution were mixed, and the mixture containing a final concentration of 2% DMSO was added to a 96-well plate and incubated at 37 °C for 20 minutes. Each well plate was treated with 70 μM nitrocepin for KPC-2, OXA-48, and VIM-2, and 100 μM imipenem for NDM-1 as fluorescent substrates, and substrate degradation was monitored for 15 minutes at a wavelength of 486 nm (nitrocepin) or 300 nm (imipenem) using a Cytation 5 imaging multimode reader (BioTek Instruments, Inc., Winooski, VT, USA). The enzyme inhibition rate at each concentration was calculated by comparing it to the degradation rate (100%) in the absence of inhibitors, and the IC50 of compounds 3 to 31 was determined from the sigmoid dose-response curves. 50 The values ​​were derived using GraphPad Prism 5 (GraphPad Software, Boston, USA).

[0330]

[0331] As a result, as shown in Table 1 above, compounds 3, 4, 7, 23, and 25 to 29 exhibited inhibitory activity against carbapenemases for KPC, OXA-48, VIM-2, and NDM-1, and it was confirmed that compounds 5, 6, and 24 also exhibited some inhibitory activity. Meanwhile, compounds 8-22, 30, and 31, which have substituents containing nitrogen or oxygen, did not exhibit carbapenemase inhibitory activity, confirming that there are highly hydrophilic binding sites on the target enzymes.

[0333] 32-2. NDM-1 Inhibitory Activity

[0334] Zn of NDM-1 2+The binding site is surrounded by hydrophobic residues, including tryptophan (Trp93), which emits intrinsic fluorescence. Since tryptophan fluorescence depends on Trp93 interacting with hydrophobic active site residues, structural changes occurring at the active site due to the binding of the inhibitor reduce tryptophan fluorescence.

[0335] Specifically, 100 μL of 2 μM NDM-1 HEPES solution (50 mM, pH 7.5) was treated with 0, 1, 5, 10, 20, and 50 μM concentrations of compound 23 or compound 27, respectively, and incubated at 37 °C for 20 minutes. Fluorescence emission spectra were measured by measuring the fluorescence change of tryptophan at wavelengths of 300 to 400 nm (Em: 280 nm) using a Cytation 5 imaging multimode reader (BioTek Instruments, Inc., Winooski, VT, USA).

[0336] As a result, as shown in Figure 3, it was confirmed that the fluorescence intensity decreased when compound 23 or 27 was treated at a concentration of 10 μM or higher, and that the fluorescence intensity decreased as the concentration increased. Therefore, it can be confirmed that compounds 23 and 27 bind to the active site of carbapenemase NDM-1.

[0338] <Example 33> Molecular Docking Study

[0339] Using compound 23 of the present invention, molecular docking studies were conducted to analyze the characteristic binding mode of binding to carbapenem degrading enzymes.

[0340] Specifically, the three-dimensional structures of AcrB, NDM-1, and KPC-2 were obtained from the Protein Data Bank (PDB ID: 3W9H, 6V1M, 6V1J), respectively (Nakashima et al. 2013; Hecker et al. 2013) and modeled using Vega ZZ (Pedretti et al. 2021). Using the AutoDock Vina module of UCSF Chimera (Pettersen et al. 2004), compound 23 was docked to the AcrB protein and β-lactamase (NDM-1 and KPC-2), which are three-part efflux pumps of E. coli. The grid spacing was set to 1.0 A for the target protein, and the grid map size was set to AcrB (X 37.15, Y 38.96, Z 37.70), NDM-1 (X 32.91, Y 15.25, Z 14.44), and KPC-2 (X 18.97, Y 36.13, Z 19.33). The center of the grid box was set to AcrB (X 53.94, Y 27.06, Z 45.70), NDM-1 (X 16.76, Y 12.66, Z 3.87), and KPC-2 (X 22.25, Y 15.37, Z 8.33). Docking was performed using the default parameters of the Lamarckian genetic algorithm, and the binding modes between the ligand and target protein were analyzed using ChimeraX's interactive ViewDockX tool.

[0341] As a result, as shown in Figure 4, in the case of the AcrB structure, compound 23 was recognized by a phenylalanine hydrophobic trap, and the aromatic ring was in close contact with Phe178, Phe610, and Phe628, which corresponded to the binding mode of the pyridopyrimidine derivative, and it was confirmed that the terminal tert-butyl group of compound 23 was docked into a channel-like structure surrounded by Phe178, Val612, Phe615, and Gln176.

[0342] In addition, for NDM-1 and KPC-2, the 4-CO and 5-OH groups of compound 23 bonded to the binding site of NDM-1 in the same manner as QPX7728, and it was confirmed that the B-ring (3,4-difluorophenyl) and 3-O substituents of compound 23 formed additional hydrophobic bonds with His250 and Lys211 for NDM-1, and with Trp105 and Thr216 for KPC-2.

[0344] <Example 34> Antimicrobial Activity Analysis

[0345] 34-1. Enhancement of the Antimicrobial Activity of the Carbapenem Antibiotic Meropenem (MEM)

[0346] The antibiotic activity of compounds 23 and 27 of the present invention and compounds 2 and 25 as controls was evaluated using the broth microdilution checkerboard synergy assay, and each of the above compounds was used in combination with the carbapenem antibiotic MEM against NDM-1 or KPC-producing carbapenem-resistant Escherichia coli ( E. coli ) and Klebsiella pneumoniae ( K. pneumoniae The minimum inhibitory concentration (MIC) of the strain was measured.

[0347] Specifically, a bacterial inoculum (5×10⁻⁶) prepared with 100 μL of CAMHB in a 96-well microplate 5 Compounds 3 to 31 and MEM were treated at concentrations of 0.06 to 512 μg / mL, respectively, in CFU / mL, and incubated at 37°C for 18 to 24 hours, after which the minimum inhibitory concentration (MIC) was visually determined. The analysis of MICMEM (when combined with compounds), MICMEM (MEM alone) + MICtitle compounds (when combined with MEM), or MIC (title compound alone) was performed using the fractional inhibitory concentration index (FICI), and the combinations were classified as synergistic (FICIF ≤ 0.5), additive (0.5 < FICI ≤ 1.0), non-additive (1.0 < FICI ≤ 4.0), or antagonistic (FICI > 4.0) depending on the FICI value.

[0348]

[0349] As a result, as shown in Table 2 above, all 14 clinical strains exhibited strong resistance to MEM, and while single-target inhibitor compound 2 or compound 25 failed to restore the antibiotic activity of MEM, multi-target inhibitor compound 23 or compound 27 restored the antimicrobial resistance of MEM and were found to reduce the minimum inhibitory concentration of MEM by 4 to 64 times in 8 to 9 of the 14 strains, respectively.

[0351] 34-2. Bactericidal activity

[0352] Time-kill curve experiments were conducted on E. coli-012 and blaNDM-1 for a 24-hour culture period using MEM 128 mg / L and compound 23 32 mg / L, either alone or in combination.

[0353] Specifically, a bacterial suspension of EC-012blaNDM (106 CFU / mL) was prepared from cells undergoing exponential growth. The bacterial suspension was placed in a conical tube and treated with meropenem (MEM), compound 23, or a mixture of MEM and compound 23, and incubated at 37 °C. After 0, 1, 2, 4, 8, and 24 hours, 50 μL was taken, serially diluted in 0.85% NaCl solution, and incubated in BAP to count the bacteria.

[0354] As a result, as shown in Figure 5, when 1 / 8 MIC or 1 / 4 MIC was used alone, the initial bacterial count decreased and then increased again after 8 hours; however, when 1 / 8 MIC or 1 / 4 MIC was used in combination with Compound 23, E. coli It was confirmed that -012 and blaNDM-1 decreased by more than 3 log10 CFU over 24 hours.

[0356] 34-3. Analysis of the Mechanism of Antibiotic Synergy Effects

[0357] The synergistic effect between antibiotics and antibiotic adjuvants is attributed to off-target effects resulting from increased outer membrane permeability (Opperman and Nguyen, 2015). To confirm the effect of the multi-target inhibitor Compound 23 on increasing outer membrane permeability, the fluorescence intensity of N-phenyl-1-naphthylamine (NPN) was measured in NDM-1-producing CRE strains EC-012 and blaNDM after treatment with Compound 23 or the positive control polymyxin B at concentrations of 2, 4, 8, 16, 32, or 64 μg / L. An increase in NPN fluorescence due to NPN absorption into the surrounding cytoplasm indicates outer membrane permeability.

[0358] Specifically, the EC-012blaNDM strain was cultured in CAMHB, washed, and resuspended in a buffer solution (5 mM HEPES, 5 mM glucose, pH 7.4). Then, 107 CFU / mL of the bacterial suspension was dispensed into 152 μL of HEPES in a 96-well microplate, 40 μL of NPN (in 50 μM HEPES), and compound 23 at concentrations of 2, 4, 8, 16, 32, or 64 μg / mL. Fluorescence was measured at a wavelength of 420 nm (Em: 350 nm) using a Cytation 5 imaging multimode reader (BioTek Instruments, Inc., Winooski, VT, USA).

[0359] As a result, as shown in Figure 6, compound 23 showed no change in NPN fluorescence intensity compared to the control group, and fluorescence was significantly increased in bacterial cells treated with polymyxin B using an outer membrane permeable agent.

[0360] Therefore, it can be confirmed that the synergistic effect of the multi-target inhibitor compound 23 and the antibiotic is not an off-target effect related to increased outer membrane permeability.

[0362] <Example 35> Analysis of in vivo antimicrobial activity

[0363] NDM is produced by using compound 23 of the present invention and MEM in combination. E. coli The antimicrobial activity of mice infected with -033 and blaNDM was evaluated. The animal experiments were approved by the KRIBB Institutional Animal Care and Use Committee (approval code: KRIBB-AEC23204).

[0364] Specifically, 6-week-old female C57BL / 6J mice (body weight 20-22 g) 4.0×10 7 CFU / mouse E. coli Infection was performed by mixing the -033, blaNDM strain and 5% porcine mucin, and after 1 hour, treatment was performed for 5 days with MEM (25 mg / kg / day) and compound 23 (10 mg / kg / day, 20 mg / kg / day, 40 mg / kg / day), with 4 animals placed in each treatment group.

[0365] As a result, as shown in Figure 7, all mice in the control group that were not treated with antibiotics or treated with MEM alone died within 2 days, but it was confirmed that 25% of mice treated with compound 23 (10 mg / kg and 40 mg / kg) and MEM survived after 5 days.

[0367] Accordingly, 28 novel compounds were prepared by introducing various substituents to the 3-O position of 3',4'-difluoroquercetin, and the drug efflux pump inhibitory activity and carbapenem degrading enzyme inhibitory activity of the compounds were confirmed. Additionally, the effect of restoring the antibacterial activity of the antibiotic against carbapenem-resistant Enterobacteriaceae (CRE) was confirmed when used in combination with the existing carbapenem antibiotic meropenem (MEM).

[0369] 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

Claim 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, R is COCH2C(CH3)3, CH2OCOC(CH3)3, CH2OCOOCH(CH3)2, CH2(4- t BuPh), CH2(4- i PrPh), (CH2)2OH, (CH2)2O(CH2)2OH, (CH2)2O(CH2)2O(CH2)2OH, (CH2)2O(CH2)2NH2, (CH2)2(thiophen-2-yl), (CH2)2N(CH3)2, (CH2)2N(CH2)2, (CH2)2N + (CH3)2CH2CO2, (CH2)2(4-methylpiperazin-1-yl), (CH2)2N(CH2)4NH, (CH2)2N(CH2)4, (CH2)2N(CH2)5, (CH2)2N(CH2)40, ( S It is one selected from )-(CH2)2(1-methylpyrrolidin-2-yl), (CH2)2CF3, (CH2)2C(CH3)3, (CH2)2CH(CH3)2, (CH2)2CH(CH2)2, (CH2)2CH(CH2)4, (CH2)2CH(CH2)5, CH2CH(CH2)4, (CH2)3CH(CH2)4, (CH2)2CH(CH2)40 and (CH2)2CH(CH2)4NH. Claim 2 In claim 1, the compound is a compound or a pharmaceutically acceptable salt thereof that inhibits a drug ejection pump. Claim 3 In claim 1, the compound is a compound or a pharmaceutically acceptable salt thereof that inhibits carbapenemase activity. Claim 4 In paragraph 3, the carbapenem degrading enzyme is a compound or a pharmaceutically acceptable salt thereof comprising KPC-2, OXA-48, VIM-2, or NDM-1. Claim 5 A pharmaceutical composition for the prevention or treatment of multidrug-resistant Gram-negative bacterial infections comprising a compound represented by the following chemical formula 1 as an active ingredient. [Chemical Formula 1] In the above chemical formula 1, R is COCH2C(CH3)3, CH2OCOC(CH3)3, CH2OCOOCH(CH3)2, CH2(4- t BuPh), CH2(4- i PrPh), (CH2)2OH, (CH2)2O(CH2)2OH, (CH2)2O(CH2)2O(CH2)2OH, (CH2)2O(CH2)2NH2, (CH2)2(thiophen-2-yl), (CH2)2N(CH3)2, (CH2)2N(CH2)2, (CH2)2N + (CH3)2CH2CO2, (CH2)2(4-methylpiperazin-1-yl), (CH2)2N(CH2)4NH, (CH2)2N(CH2)4, (CH2)2N(CH2)5, (CH2)2N(CH2)40, ( S It is one selected from )-(CH2)2(1-methylpyrrolidin-2-yl), (CH2)2CF3, (CH2)2C(CH3)3, (CH2)2CH(CH3)2, (CH2)2CH(CH2)2, (CH2)2CH(CH2)4, (CH2)2CH(CH2)5, CH2CH(CH2)4, (CH2)3CH(CH2)4, (CH2)2CH(CH2)40 and (CH2)2CH(CH2)4NH. Claim 6 A pharmaceutical composition according to claim 5, wherein the multidrug-resistant Gram-negative bacteria include carbapenem-resistant Gram-negative bacteria or carbapenem-resistant enterobacteria. Claim 7 A pharmaceutical composition according to claim 5, wherein the composition further comprises an antibiotic. Claim 8 A pharmaceutical composition according to claim 5, wherein the compound increases the antibacterial activity of an antibiotic. Claim 9 A pharmaceutical composition according to claim 8, wherein the antibiotic comprises meropenem. Claim 10 A pharmaceutical composition according to claim 5, wherein the compound inhibits multidrug-resistant bacteria when used in combination with an antibiotic. Claim 11 A pharmaceutical composition according to claim 5, wherein the compound reduces the minimum inhibitory concentration of an antibiotic by 4 to 64 times. Claim 12 In paragraph 5, the above compound is a pharmaceutical composition that reduces the number of bacteria. Claim 13 A food composition for preventing or improving multidrug-resistant Gram-negative bacterial infections comprising a compound represented by the following chemical formula 1 as an active ingredient. [Chemical Formula 1] In the above chemical formula 1, R is COCH2C(CH3)3, CH2OCOC(CH3)3, CH2OCOOCH(CH3)2, CH2(4- t BuPh), CH2(4- i PrPh), (CH2)2OH, (CH2)2O(CH2)2OH, (CH2)2O(CH2)2O(CH2)2OH, (CH2)2O(CH2)2NH2, (CH2)2(thiophen-2-yl), (CH2)2N(CH3)2, (CH2)2N(CH2)2, (CH2)2N + (CH3)2CH2CO2, (CH2)2(4-methylpiperazin-1-yl), (CH2)2N(CH2)4NH, (CH2)2N(CH2)4, (CH2)2N(CH2)5, (CH2)2N(CH2)40, ( S It is one selected from )-(CH2)2(1-methylpyrrolidin-2-yl), (CH2)2CF3, (CH2)2C(CH3)3, (CH2)2CH(CH3)2, (CH2)2CH(CH2)2, (CH2)2CH(CH2)4, (CH2)2CH(CH2)5, CH2CH(CH2)4, (CH2)3CH(CH2)4, (CH2)2CH(CH2)40 and (CH2)2CH(CH2)4NH. Claim 14 A method for increasing the antimicrobial activity of an antibiotic, comprising the steps of: mixing a compound represented by the following chemical formula 1 with a beta-lactam antibiotic; and treating multidrug-resistant Gram-negative bacteria with the mixture. [Chemical Formula 1] In the above chemical formula 1, R is COCH2C(CH3)3, CH2OCOC(CH3)3, CH2OCOOCH(CH3)2, CH2(4- t BuPh), CH2(4- i PrPh), (CH2)2OH, (CH2)2O(CH2)2OH, (CH2)2O(CH2)2O(CH2)2OH, (CH2)2O(CH2)2NH2, (CH2)2(thiophen-2-yl), (CH2)2N(CH3)2, (CH2)2N(CH2)2, (CH2)2N + (CH3)2CH2CO2, (CH2)2(4-methylpiperazin-1-yl), (CH2)2N(CH2)4NH, (CH2)2N(CH2)4, (CH2)2N(CH2)5, (CH2)2N(CH2)40, ( S It is one selected from )-(CH2)2(1-methylpyrrolidin-2-yl), (CH2)2CF3, (CH2)2C(CH3)3, (CH2)2CH(CH3)2, (CH2)2CH(CH2)2, (CH2)2CH(CH2)4, (CH2)2CH(CH2)5, CH2CH(CH2)4, (CH2)3CH(CH2)4, (CH2)2CH(CH2)40 and (CH2)2CH(CH2)4NH.

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