Novel symmetrical fluorotriazine compounds and medical or biological uses thereof

Abstract

The present invention relates to a compound selected from novel triazine derivative compound, a pharmaceutically or sitologically acceptable salt thereof, a solvate thereof, or a stereoisomer thereof, and a use thereof. With a potent antibacterial effect, the amphiphilic triazine compound according to the present invention can be safely and effectively used as an excellent antibacterial composition and a therapeutic composition for microbial infectious diseases.

Description

New symmetrical fluorotriazine compounds and their medical or biological uses

[0001] The present invention provides novel symmetrical fluorotriazine compounds and their medical or biological uses.

[0002] The present invention was studied as part of the "Development of antibiotic candidate using Norspermidine-based HSA (High Sensitive Amphipatic) synthesis technology with low resistance induction as a multidrug-resistant bacteria target" project of the Small and Medium Business Technology Information Promotion Agency of the Ministry of SMEs and Startups [S3318346 (RS-2022-TI024265)] of the Startup Growth Technology Development Project.

[0003] Enterococcus is a gram-positive coccus (coccus) that forms diplococci or short chains. It grows well even on media without blood. The most common enterococcus detected in humans is Enterococcus faecalis, followed by Enterococcus faecium and Enterococcus avium. Enterococcus faecium is more resistant to antibiotics than Enterococcus faecalis, making it a significant cause of hospital-acquired infections.

[0004] Antibiotics are secondary metabolites produced by microorganisms that kill or inhibit the growth of microorganisms at very low concentrations. Initially, synthetic substances such as sulfonamides and penicillin, produced by the penicillin mold, were primarily used. Since then, many substances exhibiting antibacterial activity have been discovered, synthesized, and used. To date, numerous antibiotics have been isolated from natural products or synthesized organically, enabling the treatment of numerous diseases and infections. However, the global overuse of antibiotics has led to an increase in the emergence and rate of antibiotic resistance in bacteria. Furthermore, the diversification of infection routes is accelerating the spread of resistant bacteria, posing a serious social problem.

[0005] In clinical practice, the need for development of new drugs to replace vancomycin and imipenem, which are the last weapons against antibiotic-resistant bacteria, is being emphasized as the number of resistant bacteria such as Enterococcus faecium and Acinetobacter baumannii increases.

[0006] The purpose of the present invention is to provide a novel triazine derivative compound.

[0007] Another object of the present invention is to provide an antibacterial composition comprising the novel compound described above.

[0008] Another object of the present invention is to provide a composition for preventing or treating a pathogenic microbial infection disease comprising the novel compound described above.

[0009] To achieve the above purpose, the present invention provides a compound selected from a triazine derivative compound represented by the following chemical formula 1, a pharmaceutically or food-wise acceptable salt thereof, a solvate thereof, or a stereoisomer thereof:

[0010] [Chemical Formula 1]

[0011]

[0012] In the above chemical formula 1, R1 and R2 may be the same or different, and are hydrogen, (C1-C5) alkyl and -(CH2) n -R 11 One of them, but at least one is -(CH2) n -R 11 , and n is one of 0 to 5, and R 11 is any one of substituted (C3-C12)cycloalkyl, substituted (C5-C12)aryl and CHF2, wherein the substituted (C3-C12)cycloalkyl and (C5-C12)aryl are substituted with at least one trifluoromethyl or fluorine, or are heterocyclic compounds in which R1 and R2 are connected to each other to form a 3- to 12-membered ring including substituted nitrogen or oxygen, and the substituted heterocyclic compound is fluorine, trifluoromethyl or -(CH2) m -R 21 is substituted with one or more, and m is one of 0 to 3, and R 21 It may be a (C5-C12)aryl substituted with trifluoromethyl.

[0013] The present invention provides an antibacterial composition comprising, as an active ingredient, a compound selected from the triazine derivative compound, a pharmaceutically or food-wise acceptable salt thereof, a solvate thereof, or a stereoisomer thereof.

[0014] The present invention provides a pharmaceutical composition, cosmetic composition, health food composition, animal feed or feed additive composition comprising the above antibacterial composition.

[0015] The present invention provides a pharmaceutical composition for preventing or treating a pathogenic microbial infection disease, comprising as an active ingredient a compound selected from the triazine derivative compound, a pharmaceutically acceptable salt thereof, a solvate thereof, or a stereoisomer thereof.

[0016] In addition, the present invention provides a health functional food composition for preventing or improving a pathogenic microbial infection disease, comprising as an active ingredient a compound selected from the triazine derivative compound, a food-acceptable salt thereof, a solvate thereof, or a stereoisomer thereof.

[0017] The present invention relates to a novel triazine derivative compound, a pharmaceutically or food-wise acceptable salt thereof, a solvate thereof or a stereoisomer thereof, and a compound selected from the group consisting of a triazine derivative compound and a medical or biological use thereof, wherein the compound can be utilized as an antibacterial composition having excellent antibacterial activity against various strains, particularly antibiotic-resistant strains.

[0018] The compound according to the present invention has the advantage of being able to continuously exhibit excellent antibacterial activity without antibiotic resistance, unlike conventional antibiotics.

[0019] In addition, the compound can be used as a composition for treating infectious diseases caused by the various strains described above, and can more effectively prevent, improve, or treat the disease.

[0020] Figures 1a and 1b show a schematic diagram of the synthesis of a novel fluorotriazine derivative compound.

[0021] Figure 2 shows data from a cytotoxicity evaluation of a novel fluorotriazine derivative compound using the MRC-5 cell line.

[0022] Figure 3 shows data for evaluating the cytotoxicity of a novel fluorotriazine derivative compound using a HepG2 cell line.

[0023] Figures 4a to 4f show data confirming the antibacterial efficacy of a novel fluorotriazine derivative compound using the A549 cell line.

[0024] Figure 5 is data confirming the bactericidal effect of a novel fluorotriazine derivative compound against Staphylococcus aureus (S. aureus).

[0025] Figure 6 shows the in vivo antibacterial activity of a novel fluorotriazine derivative compound. (a) is a survival curve of Balb / c mice infected with various concentrations of Enterococcus faecium (E. Faecium), and (b) is a survival curve of 5×10 9 Survival curves of Balb / c mice treated with various doses (5, 10, and 15 mg / kg) of PDL-16 after inoculation with 10 CFU of E. Faecium (P = 0.005).

[0026] Figure 7 shows the pathogen content and body weight changes in a mouse model of intramuscular E. Faecium infection. (a) is a CFU analysis graph, and (b) is a body weight measurement graph.

[0027] Figure 8 shows the plasma concentrations after intravenous injection of 2 mg / kg and oral administration of 20 mg / kg (n = 4, P < 0.0001).

[0028] Figure 9 schematically illustrates the experimental process for confirming the effect of inhibiting the development of antibiotic resistance against Acinetobacter baumannii.

[0029] Figure 10 is a graph confirming the effect of inhibiting the development of antibiotic resistance through an experiment according to Figure 9.

[0030] Hereinafter, the present invention will be described in more detail.

[0031]

[0032] The present invention provides a compound selected from a triazine derivative compound represented by the following chemical formula 1, a pharmaceutically or food-wise acceptable salt thereof, a solvate thereof, or a stereoisomer thereof:

[0033] [Chemical Formula 1]

[0034]

[0035] In the above chemical formula 1, R 1 and R 2may be the same or different, and are hydrogen, (C1-C5)alkyl and -(CH2) n -R 11 One of them, but at least one is -(CH2) n -R 11 , and n is one of 0 to 5, and R 11 is any one of substituted (C3-C12)cycloalkyl, substituted (C5-C12)aryl and CHF2, wherein the substituted (C3-C12)cycloalkyl and (C5-C12)aryl are substituted with one or more trifluoromethyl or fluorine, or R 1 and R 2 A heterocyclic compound that is connected to each other to form a 3- to 12-membered ring including substituted nitrogen or oxygen, and the substituted heterocyclic compound is fluorine, trifluoromethyl or -(CH2) m -R 21 is substituted with one or more, and m is one of 0 to 3, and R 21 It may be a (C5-C12)aryl substituted with trifluoromethyl.

[0036] In the above chemical formula 1, R 1 and R 2 may be the same or different, and are hydrogen, methyl and -(CH2) n -R 11 One of them, but at least one is -(CH2) n -R 11 , and the above n is 0 or 1, and the above R 11 is any one of substituted (C4-C6)cycloalkyl, substituted phenyl and CHF2, wherein the substituted (C4-C6)cycloalkyl and phenyl are substituted with 1 or 2 trifluoromethyl or fluorine, or R 1 and R 2A heterocyclic compound which is connected to each other to form a 5-membered or 6-membered ring including unsubstituted or substituted nitrogen or oxygen, and the substituted heterocyclic compound is fluorine or -CH2-R 21 This is one or more substituted, and the above R 21 may be a trifluoromethyl substituted phenyl.

[0037] In the above chemical formula 1, R 1 Silver -(CH2) n -R 11 , and the above n is 0 or 1, and the above R 11 is any one of substituted (C4-C6)cycloalkyl, substituted phenyl and CHF2, wherein the substituted (C4-C6)cycloalkyl and phenyl are substituted with 1 or 2 trifluoromethyl or fluorine, and R 2 can be hydrogen or methyl.

[0038] Preferably, the triazine derivative compound may be selected from the following structures:

[0039] , , , , , , , , , , , and

[0040]

[0041] The above compound can be used in the form of a pharmaceutically or food-wise acceptable salt within the range having the same efficacy.

[0042] As used herein, “pharmaceutically or food-wise acceptable” means a salt that is non-toxic to cells or humans exposed to the composition and has a safety and efficacy profile suitable for administration to humans.

[0043] The above salt may be used in the form of either a basic salt or an acid salt that is pharmaceutically or food-wise acceptable. For example, the basic salt may include any one of an organic base salt and an inorganic base salt, and may be selected from the group consisting of, but not limited to, sodium salt, potassium salt, calcium salt, lithium salt, magnesium salt, cesium salt, aminium salt, ammonium salt, triethylaminium salt, and pyridinium salt. The acid salt is useful as an acid addition salt formed by a free acid. Inorganic acids and organic acids can be used as free acids. Inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, sulfurous acid, phosphoric acid, etc., and organic acids include citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, benzenesulfonic acid, camphorsulfonic acid, oxalic acid, malonic acid, glutaric acid, acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, aspartic acid, etc.

[0044] The compound may include not only pharmaceutically or food-wise acceptable salts, but also all salts, hydrates, and solvates that can be prepared by conventional methods, and addition salts may be prepared by conventional methods. For example, the compound may be dissolved in a water-miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile, and an excess of an organic base or an aqueous base solution of an inorganic base may be added, followed by precipitation or crystallization. Alternatively, the compound may be prepared by evaporating the solvent or the excess base from the mixture and then drying to obtain an addition salt, or by suction filtration of the precipitated salt.

[0045]

[0046] The present invention provides an antibacterial composition comprising, as an active ingredient, a compound selected from the triazine derivative compound, a pharmaceutically or food-wise acceptable salt thereof, a solvate thereof, or a stereoisomer thereof.

[0047] As used herein, the term "antimicrobial composition" refers to a substance that kills or inhibits the growth of microorganisms, and may also be referred to as an antimicrobial agent or antibacterial agent. Antimicrobial agents can be classified as antibiotics for bacteria or antifungals for fungi, depending on the microorganism they act on. They can also be classified as bactericidal agents, which kill microorganisms, or bacteriostatic agents, which simply inhibit the growth of microorganisms, depending on their function.

[0048] The composition may have antibacterial activity against one or more bacteria selected from the group consisting of Enterococcus faecium, Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and antibiotic-resistant strains thereof.

[0049] The above antibiotic may be a beta-lactam antibiotic, and specifically, may be, but is not limited to, a penicillin, vancomycin, cephalosporin, or carbapenem antibiotic.

[0050] Preferably, the antibiotic may be selected from vancomycin, imipenem, methicillin, or ampicillin, but is not limited thereto.

[0051] Preferably, the antibiotic-resistant strain may be selected from, but is not limited to, vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus (MRSA), or multiple drug-resistant Staphylococcus aureus (MDRSA).

[0052] The composition may contain the compound at a concentration of 0.0001 to 10 μM, and may exhibit excellent antibacterial activity while being safe from cytotoxicity within the above range, so the above range is preferred.

[0053] The above composition can exhibit excellent antibacterial activity continuously without development of resistance, especially against Gram-negative bacteria such as Acinetobacter baumannii.

[0054] The above antibacterial composition can be widely used as a pharmaceutical composition, cosmetic composition, health food composition, animal feed or feed additive composition, etc. to achieve the purposes of antibacterial, sterilization, antiseptic, antifouling, etc.

[0055]

[0056] The present invention provides a pharmaceutical composition, cosmetic composition, health food composition, animal feed or feed additive composition comprising the above antibacterial composition.

[0057] The above cosmetic composition is not particularly limited in its formulation, and may be formulated as cosmetics such as, for example, a softening toner, an astringent toner, a nourishing toner, a nourishing cream, a massage cream, an essence, an eye cream, an eye essence, a cleansing cream, a cleansing foam, a cleansing water, a pack, a powder, a body lotion, a body cream, a body oil, and a body essence, and may be applied in a form that is applied to the skin or in a form that is absorbed into the skin using microneedles, etc.

[0058] In the above cosmetic composition, pharmaceutically or cosmetically acceptable carriers may vary depending on the formulation, but include hydrocarbons such as petrolatum, liquid paraffin, and gelling hydrocarbons (also known as plastibase); animal or vegetable oils such as medium-chain fatty acid triglycerides, lard, hard fat, and cacao fat; higher fatty acid alcohols and fatty acids and esters thereof such as cetanol, stearyl alcohol, stearic acid, and isopropyl palmitate; water-soluble bases such as macrogol (polyethylene glycol), 1,3-butylene glycol, glycerol, gelatin, sucrose, and sugar alcohols; emulsifiers such as glycerin fatty acid esters, polyoxyl stearate, and polyoxyethylene hydrogenated castor oil; adhesives such as acrylic acid esters and sodium alginate; propellants such as liquefied petroleum gas and carbon dioxide; Preservatives such as parahydroxybenzoic acid esters can be mentioned, and the cosmetics of the present invention can be manufactured using these according to conventional methods. In addition to these, stabilizers, fragrances, colorants, pH adjusters, diluents, surfactants, preservatives, antioxidants, etc. can also be blended as needed. The cosmetics of the present invention can be applied to local wounds using conventional methods.

[0059] The above animal feed is a substance that supplies organic or inorganic nutrients necessary to sustain the life of an individual and raise the individual, and may contain nutrients such as energy, protein, lipid, vitamins, and minerals required by the individual consuming the feed.

[0060] The above feed is a feed of a known composition generally used in animal breeding, and may include all commercially available general feeds, but is not particularly limited thereto.

[0061] Additionally, feed additives are a general term for substances added in trace amounts to feed for nutritional or specific purposes, and may include general feed additives, such as salt, bone meal, calcium phosphate, mineral mixtures, vitamins, amino acids, antibiotics, hormones, and other feed additives for special purposes. The above feed additives may be added to livestock feed, but are not limited thereto.

[0062]

[0063] The present invention provides a pharmaceutical composition for preventing or treating a pathogenic microbial infection disease, comprising as an active ingredient a compound selected from the triazine derivative compound, a pharmaceutically acceptable salt thereof, a solvate thereof, or a stereoisomer thereof.

[0064] The above pathogenic microbial infection disease may be an infection disease caused by one or more bacteria selected from the group consisting of Enterococcus faecium, Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and antibiotic-resistant strains thereof, and may be selected from, but not limited to, abscess, dermatitis, osteoarthritis, bacteremia, pneumonia, toxic shock syndrome, food poisoning, high fever, sepsis, edematous disease, mastitis, or enterocolitis.

[0065] Alternatively, the disease may be an allergic disease caused by Enterococcus faecium, and may be specifically any one selected from the group consisting of, but not limited to, hypersensitivity, allergic rhinitis, asthma, allergic conjunctivitis, allergic dermatitis, atopic dermatitis, contact dermatitis, and urticaria.

[0066]

[0067] The above pharmaceutical composition may further comprise one or more additives selected from the group consisting of suitable carriers, excipients, disintegrants, sweeteners, coating agents, bulking agents, lubricants, glidants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders and lubricants commonly used in pharmaceutical manufacturing.

[0068] Specifically, carriers, excipients, and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like. These solid preparations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like, into the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Base materials for suppositories include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

[0069] The above pharmaceutical composition can be administered to a subject in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes.

[0070] Preferably, the pharmaceutical composition may be a topical skin preparation formulated as a lotion, ointment, gel, cream, patch or spray, but is not limited thereto.

[0071] The dosage of the active ingredient according to the present invention may vary depending on the condition and weight of the subject, the type and degree of the disease, the drug form, the route and period of administration, and may be appropriately selected by a person skilled in the art, and the daily dosage may be 0.01 mg / kg to 200 mg / kg, preferably 0.1 mg / kg to 200 mg / kg, and more preferably 0.1 mg / kg to 100 mg / kg. Administration may be once a day or divided into several times, and the scope of the present invention is not limited thereby.

[0072]

[0073] In addition, the present invention provides a health functional food composition for preventing or improving a pathogenic microbial infection disease, comprising a compound selected from the triazine derivative compound, a pharmaceutically acceptable salt thereof, a solvate thereof, or a stereoisomer thereof as an active ingredient.

[0074] Corresponding features can be substituted in the above-described part.

[0075]

[0076] The above health functional food may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents 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 colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0077] In addition, it may contain fruit pulp for the production of natural fruit juice, synthetic fruit juice, and vegetable drinks. These ingredients may be used independently or in combination. Furthermore, the health functional food composition may be in the form of any one of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, drink, alcohol, and vitamin complex.

[0078] In addition, the above health functional food may additionally contain food additives, and its suitability as a "food additive" is determined by the specifications and standards for the relevant item in accordance with the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise specified. Examples of items listed in the above "Food Additives Codex" include chemically synthesized products such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kohlrabi pigment, and guar gum; mixed preparations such as sodium L-glutamate preparations, alkaline agents added to noodles, preservative preparations, and tar color preparations.

[0079] At this time, the content of the effective ingredient added to the food during the process of manufacturing the health functional food can be appropriately increased or decreased as needed, and preferably, it can be added so that it is included in an amount of 1 to 90 parts by weight per 100 parts by weight of the food.

[0080] Alternatively, the health functional food composition of the present invention can have a more excellent antibacterial effect against the pathogenic microorganisms by consuming it in the form of inner beauty food. The inner beauty is referred to as an edible cosmetic or beauty food, and refers to a food that changes the skin constitution to a healthy one by absorbing various skin-friendly ingredients into the body. Just as one selects cosmetics that suit one's skin type, one can select and consume inner beauty food that suits one's skin condition and lifestyle.

[0081]

[0082] In addition, the present invention provides an antibiotic composition comprising a compound selected from the triazine derivative compound, a pharmaceutically acceptable salt thereof, a solvate thereof, or a stereoisomer thereof as an active ingredient.

[0083] In addition, the present invention provides an antibacterial adjuvant comprising a compound selected from the triazine derivative compound, a pharmaceutically acceptable salt thereof, a solvate thereof, or a stereoisomer thereof as an active ingredient.

[0084] Hereinafter, to aid understanding of the present invention, examples and other embodiments will be described in detail. However, the following examples and other embodiments merely illustrate the content of the present invention and are not intended to limit the scope of the present invention. The examples and other embodiments of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0085]

[0086] [Synthesis Example] Synthesis of Triazine Derivatives

[0087] 1. General Procedure (a)

[0088] To a stirred solution of cyanuric chloride (1 eq) in dichloromethane (DCM; 5 volumes / weight of starting material) was added amine (2.1 eq) very slowly and stirred at room temperature for 3 h. After the reaction was completed by TLC, the reaction mixture was quenched with water (5 eq) and extracted with DCM (3 times) to obtain the crude product. Di-tert-butyl(azanediylbis(propane-3,1-diyl))dicarbamate (1.2 eq) was added to the crude mixture in 1,4-dioxane (5 eq) and refluxed for 16 h. After the reaction was completed, the crude reaction mass was evaporated and extracted with ethyl acetate and water (3 times, 5 eq). The crude mixture was purified by silica gel column chromatography using 20 to 30% ethyl acetate in a hexane mixture to obtain compound 5.

[0089]

[0090] 2. General Procedure (b)

[0091] 2-1) Synthesis of compound 7: di-tert-butyl (((4,6-dichloro-1,3,5-triazin-2-yl)azanediyl)bis(propane-3,1-diyl))dicarbamate

[0092] To a stirred solution of cyanuric chloride (compound 1, 1 eq.) and DIEA (3 eq.) in DCM (5 vol.) at 0 °C was added compound 4 (1 mol) dissolved in CH2Cl2 (2 volumes / weight) dropwise over 30 min while maintaining the temperature. The mixture was stirred for an additional 3 h at 0 °C. The reaction mixture was quenched with water (5 vol.) and extracted with DCM (3×). The combined organic extracts were washed with brine (1 vol.), dried over Na2SO4, and evaporated. The resulting residue was purified by silica gel column chromatography using methanol (1-3%) in DCM to give compound 7 as a white solid (91%).

[0093] 1 H NMR (400 MHz, CDCl3) δ 5.08 (t,J= 6.0 Hz, 2H), 3.61 (t,J= 6.4 Hz, 4H), 3.11 (q,J= 6.4 Hz, 4H), 1.80 (p,J= 6.4 Hz, 4H), 1.47 (s, 18H). 13 C NMR (100 MHz, CDCl3) δ 170.1, 164.7, 156.0, 79.3, 45.0, 37.4, 28.4, 27.7. Maldi-tofm / zcalcd for C 19 H 32 Cl2N6O4: 478.1, found 501.2(M+Na) + .

[0094]

[0095] 2-2) Synthesis of compound 5

[0096] To a stirred solution of the compound 7 (1 equiv) and DIEA (3 equiv) dissolved in 1,4-dioxane (5 vol) were slowly added various amines (2.1 equiv) at room temperature and stirred for 1 h. The mixture was then heated at reflux for 16 h. After completion of the reaction, the reaction mixture was evaporated under vacuum. The resulting mixture was dissolved in water (5 vol) and EtOAc (5 vol) and extracted with EtOAc (2×). The combined organic layers were washed with brine (2 vol), dried over anhydrous Na2SO4, filtered, and evaporated. The residue was purified by flash chromatography on silica gel using 20-30% ethyl acetate in hexane mixture to give compound 5.

[0097]

[0098] Compound 5a: Di-tert-butyl (((4,6-bis((4-(trifluoromethyl)cyclohexyl)amino)-1,3,5-triazin-2-yl)azanediyl)bis(propane-3,1-diyl))dicarbamate

[0099]

[0100] Compound 5a was synthesized according to the general procedure (a). Yield 67%; pale yellow solid; 1 H NMR (400 MHz, CDCl3) δ 5.12 - 4.42 (m, 2H), 4.27 - 4.03 (m, 1H), 3.85 - 3.37 (m, 4H), 2.86-3.34 (m, 4H), 2.44 - 1.89 (m, 6H), 1.87 - 1.52 (m, 11H), 1.44 (s, 18H), 1.38 - 1.05 (m, 5H).

[0101]

[0102] Compound 5b: Di-tert-butyl (((4,6-bis(((4-(trifluoromethyl)cyclohexyl)methyl)amino)-1,3,5-triazin-2-yl)azanediyl)bis(propane-3,1-diyl))dicarbamate

[0103]

[0104] Compound 5b was synthesized according to the general procedure (a). Yield 74%; pale yellow solid; 1 H NMR (400 MHz, CDCl3) δ 5.58 (s, 1H), 5.44 - 4.20 (m, 2H), 3.68 - 3.47 (m, 4H), 3.46 - 2.94 (m, 8H), 2.21 - 1.85 (m, 5H), 1.83 - 1.65 (m, 16H), 1.44 (s, 18H), 1.37 - 1.15 (m, 1H), 1.11 - 0.74 (m, 2H).

[0105]

[0106] Compound 5c: Di-tert-butyl (((4,6-bis((4-(trifluoromethyl)phenyl)amino)-1,3,5-triazin-2-yl)azanediyl)bis(propane-3,1-diyl))dicarbamate

[0107]

[0108] Compound 5c was synthesized according to the general procedure (a). Yield 66%; white solid. 1H NMR (400 MHz, CDCl3) δ 7.88 - 7.65 (m, 2H), 7.63 - 7.5 (m, 2H), 7.16-7.10 (s, 2H), 3.76 - 3.46 (m, 4H), 3.29 - 2.98 (m, 4H), 1.89 - 1.75 (m, 4H), 1.48 -1.43 (m, 18H).

[0109]

[0110] Compound 5d: Di-tert-butyl (((4,6-bis((4-(trifluoromethyl)phenethyl)amino)-1,3,5-triazin-2-yl)azanediyl)bis(propane-3,1-diyl))dicarbamate

[0111]

[0112] Compound 5d was synthesized according to the general procedure (b). Yield 72%; white solid. 1 H NMR (400 MHz, CDCl3) δ 7.65 - 7.44 (m, 4H), 7.43 - 7.27 (m, 4H), 5.35 - 4.65 (m, 2H), 3.80 - 3.35 (m, 8H), 3.23 - 2.80 (m, 8H), 1.85 - 1.66 (m, 4H), 1.55 - 1.31 (m, 18 H).

[0113]

[0114] Compound 5e: Di-tert-butyl (((4,6-bis((2,2-difluoroethyl)amino)-1,3,5-triazin-2-yl)azanediyl)bis(propane-3,1-diyl))dicarbamate

[0115]

[0116] Compound 5e was synthesized according to the general procedure (a). Yield 56%; white solid. 1 H NMR (400 MHz, CDCl3) δ 5.91 (tt,J= 56.4, 4.1 Hz, 2H), 3.86 - 3.64 (m, 4H), 3.63 - 3.38 (m, 4H), 3.23 - 2.89 (m, 4H), 1.85 - 1.61 (m, 4H), 1.45 (s, 18H).

[0117]

[0118] Compound 5f: Di-tert-butyl (((4,6-bis((3,3-difluorocyclobutyl)amino)-1,3,5-triazin-2-yl)azanediyl)bis(propane-3,1-diyl))dicarbamate

[0119]

[0120] Compound 5f was synthesized according to the general procedure (a). Yield 51%; white solid. 1 H NMR (400 MHz, CDCl3) δ 5.00 (s, 2H), 4.38 - 4.16 (m, 2H), 3.72 - 3.29 (m, 4H), 3.26 - 2.82 (m, 8H), 2.79 - 2.34 (m, 4H), 1.80 - 1.67 (m, 4H), 1.46 (s, 18H).

[0121]

[0122] Compound 5g: Di-tert-butyl (((4,6-bis((4-fluorobenzyl)amino)-1,3,5-triazin-2-yl)azanediyl)bis(propane-3,1-diyl))dicarbamate

[0123]

[0124] Compound 5g was synthesized according to the general procedure (b). Yield 64%; white solid. 1 H NMR (400 MHz, CDCl3) δ 7.38 - 7.11 (m, 4H), 7.09 - 6.74 (m, 4H), 5.56 - 4.85 (m, 2H), 4.54 (d,J= 5.9 Hz, 4H), 3.52 (t,J= 6.2 Hz, 4H), 3.16 - 2.87 (m, 4H), 1.82 - 1.59 (m, 4H), 1.54 - 1.28 (m, 18H).

[0125]

[0126] Compound 5h: Di-tert-butyl (((4,6-bis(4-(trifluoromethyl)piperidin-1-yl)-1,3,5-triazin-2-yl)azanediyl)bis(propane-3,1-diyl))dicarbamate

[0127]

[0128] Compound 5h was synthesized according to the general procedure (b). Yield: 62%; white solid: 1H NMR (400 MHz, CDCl3) δ 5.23 - 5.04 (m, 2H), 4.90 - 4.79 (m, 4H), 3.82 - 3.39 (m, 4H), 3.18 - 2.94 (m, 4H), 2.78 (t,J= 12.3 Hz, 4H), 2.25 (tdd,J= 12.0, 7.8, 3.8 Hz, 2H), 2.00 - 1.83 (m, 4H), 1.72 (p,J= 6.0 Hz, 4H), 1.52 (ddd,J= 25.5, 12.8, 4.3 Hz, 4H), 1.43 (s, 18H).

[0129]

[0130] Compound 5i: Di-tert-butyl (((4,6-bis(4-(4-(trifluoromethyl)benzyl)piperazin-1-yl)-1,3,5-triazin-2-yl)azanediyl)bis(propane-3,1-diyl))dicarbamate

[0131]

[0132] Compound 5i was synthesized according to the general procedure (b). Yield 80%; white solid; 1 H NMR (400 MHz, CDCl3) δ 7.57 (d,J= 8.0 Hz, 4H), 7.46 (d,J= 8.0 Hz, 4H), 5.22 (s, 4H), 3.85 - 3.69 (m, 8H), 3.60 - 3.51 (m, 8H), 3.03 (dd,J= 11.5, 5.9 Hz, 4H), 2.34-2.56 (m, 4H), 1.68 (p,J= 6.5 Hz, 4H), 1.39 (s, 18H).

[0133]

[0134] Compound 5j: Di-tert-butyl (((4,6-bis(3,3-difluoropiperidin-1-yl)-1,3,5-triazin-2-yl)azanediyl)bis(propane-3,1-diyl))dicarbamate

[0135]

[0136] Compound 5j was synthesized according to the general procedure (a). Yield 61%; white solid; 1 H NMR (400 MHz, CDCl3) δ 4.02 (t,J= 11.9 Hz, 4H), 3.84 - 3.72 (m, 4H), 3.58 (t,J= 6.6 Hz, 4H), 3.08 (dd,J= 11.8, 5.8 Hz, 4H), 2.13 - 1.97 (m, 4H), 1.84 - 1.67 (m, 8H), 1.43 (s, 18H).

[0137]

[0138] Compound 5k: Di-tert-butyl (((4,6-bis(3,3-difluoropyrrolidin-1-yl)-1,3,5-triazin-2-yl)azanediyl)bis(propane-3,1-diyl))dicarbamate

[0139]

[0140] Compound 5k was synthesized according to the general procedure (b). Yield 52%; white solid; 1H NMR (400 MHz, CDCl3) δ 5.44 (s, 2H), 4.01 - 3.71 (m, 4H), 3.60 (t,J= 6.2 Hz, 4H), 3.07 (ddd,J= 12.0, 8.1, 5.2 Hz, 4H), 2.46 - 2.28 (m, 4H), 1.71 (p,J= 8.0 Hz, 4H), 1.56 - 1.51 (m, 4H), 1.43 (s, 18H).

[0141]

[0142] Compound 5l: Di-tert-butyl (((4,6-bis((3-fluorobenzyl)(methyl)amino)-1,3,5-triazin-2-yl)azanediyl)bis(propane-3,1-diyl))dicarbamate

[0143]

[0144] Compound 5l was synthesized according to the general procedure (b). Yield 64%; white solid; 1 H NMR (400 MHz, CDCl3) δ 7.51 - 7.13 (m, 2H), 7.10 - 6.69 (m, 6H), 4.96 - 4.51 (m, 4H), 3.79 - 3.32 (m, 4H), 3.26 - 2.82 (m, 10H), 1.84 - 1.63 (m, 4H), 1.41 (s, 18H).

[0145]

[0146] 3. General Procedure (c)

[0147] To a stirred solution of the above compound 5 dissolved in DCM (1 mol) (5 volumes), diethyl ether or methanol or dioxane (3 equivalents) was added dropwise 2 m HCl at 0°C for 30 minutes, and the resulting residue was stirred for 2 to 3 hours. The resulting solid was filtered, washed with acetonitrile (1 X 3 ml) and DCM (2 X 2 ml), and dried to obtain the desired final compound 6.

[0148]

[0149] Compound 6a: N2,N2-bis(3-aminopropyl)-N4,N6-bis(4-(trifluoromethyl)cyclohexyl)-1,3,5-triazine-2,4,6-triamine n-hydrochloride (PDL-13)

[0150]

[0151] Compound 6a was synthesized according to the general procedure (c). Yield 48%; white solid. 1 H NMR (400 MHz, MeOD) δ 4.26 (s, 1H), 3.95 - 3.57 (m, 5H), 3.19 - 3.07 (m,1H), 3.05 - 2.88 (m, 4H), 2.34 - 2.14 (m, 3H), 2.11 - 1.93 (m, 8H), 1.91 - 1.56 (m, 8H), 1.55 - 1.26 (m, 3H).

[0152]

[0153] Compound 6b: N2,N2-bis(3-aminopropyl)-N4,N6-bis((4-(trifluoromethyl)cyclohexyl)methyl)-1,3,5-triazine-2,4,6-triamine n-hydrochloride (PDL-14)

[0154]

[0155] Compound 6b was synthesized according to the general procedure (c). Yield 62%; white solid. 1 H NMR (400 MHz, MeOD) δ 3.86 - 3.60 (m, 5H), 3.44 - 3.35 (m, 1H), 3.20 - 3.11 (m, 1H), 3.10 - 2.91 (m, 5H), 2.33 - 2.17 (m, 1H), 2.05 (ddd,J= 12.9, 12.4, 7.0 Hz, 10H), 1.80 - 1.51 (m, 10H), 1.41 - 1.25 (m, 2H), 1.14 - 0.97 (m, 1H).

[0156]

[0157] Compound 6c: N2,N2-bis(3-aminopropyl)-N4,N6-bis(4-(trifluoromethyl)phenyl)-1,3,5-triazine-2,4,6-triamine n-hydrochloride (PDL-15)

[0158]

[0159] Compound 6c was synthesized according to the general procedure (c). Yield 56%; white solid. 1H NMR (400 MHz, MeOD) δ 7.87 (d,J= 8.2 Hz, 4H), 7.70 (d,J= 8.5 Hz, 4H), 3.81 (t,J= 7.2 Hz, 4H), 3.02 (t,J= 7.2 Hz, 4H), 2.14 - 2.05 (m, 4H).

[0160]

[0161] Compound 6d: N2,N2-bis(3-aminopropyl)-N4,N6-bis(4-(trifluoromethyl)phenethyl)-1,3,5-triazine-2,4,6-triamine n-hydrochloride (PDL-16)

[0162]

[0163] Compound 6d was synthesized according to the general procedure (c). Yield 51%; white solid. 1 H NMR (400 MHz, MeOD) δ 7.62 (d,J=8.0 Hz, 4H), 7.48 (d,J=7.9 Hz, 4H), 3.80 - 3.61 (m, 8H), 3.05 - 2.95 (m, 8H), 2.14 - 1.94 (m, 4H).

[0164]

[0165] Compound 6e: N2,N2-bis(3-aminopropyl)-N4,N6-bis(2,2-difluoroethyl)-1,3,5-triazine-2,4,6-triamine n-hydrochloride (PDL-18)

[0166]

[0167] Compound 6e was synthesized according to the general procedure (c). Yield 82%; white solid. 1 H NMR (400 MHz, MeOD) δ 6.32 - 5.81 (m, 2H), 3.90 (t, J= 15.3 Hz, 4H), 3.81 - 3.66 (m, 4H), 3.07 - 2.93 (m, 4H), 2.14 - 2.00 (m, 4H).

[0168]

[0169] Compound 6f: N2,N2-bis(3-aminopropyl)-N4,N6-bis(3,3-difluorocyclobutyl)-1,3,5-triazine-2,4,6-triamine (PDL-19)

[0170]

[0171] Compound 6f was synthesized according to the general procedure (c). Yield 56%; white solid. 1 H NMR (400 MHz, MeOD) δ 4.41 - 4.13 (m, 2H), 3.81 - 3.62 (m, 4H), 3.14 - 2.96 (m, 8H), 2.81 - 2.62 (m, 4H), 2.11 - 1.99 (m, 4H).

[0172]

[0173] Compound 6g: N2,N2-bis(3-aminopropyl)-N4,N6-bis(4-fluorobenzyl)-1,3,5-triazine-2,4,6-triamine n-hydrochloride (PDL-22)

[0174]

[0175] Compound 6g was synthesized according to the general procedure (c). Yield 78%; white solid. 1 H NMR (400 MHz, MeOD) δ 7.48 - 7.21 (m, 4H), 7.15 - 6.91 (m, 4H), 4.62 (s, 4H), 3.69 (t,J= 7.2 Hz, 4H), 2.89 (t,J= 8.0 Hz, 4H), 1.98 (dt,J= 14.6, 7.2 Hz, 4H).

[0176]

[0177] Compound 6h: N1-(3-aminopropyl)-N1-(4,6-bis(4-(trifluoromethyl)piperidin-1-yl)-1,3,5-triazin-2-yl)propane-1,3-diamine n-hydrochloride)(PDL-12)

[0178]

[0179] Compound 6h was synthesized according to the general procedure (c). Yield 82%; white solid. 1 H NMR (400 MHz, MeOD) δ 4.61 (d,J= 12.8 Hz, 4H), 3.75 (t,J= 7.1 Hz, 4H), 3.11 (t,J= 12.2 Hz, 4H), 3.05 - 2.96 (m, 4H), 2.69 - 2.49 (m, 2H), 2.04 (dt,J= 11.0, 8.7 Hz, 8H), 1.60 (p,J= 8 Hz, 4H).

[0180]

[0181] Compound 6i: N1-(3-aminopropyl)-N1-(4,6-bis(4-(4-trifluoromethyl)benzyl)piperazin-1-yl)-1,3,5-triazin-2-yl)propane-1,3-diamine n-hydrochloride (PDL-17)

[0182]

[0183] Compound 6i was synthesized according to the general procedure (c). Yield 83%; white solid. 1 H NMR (400 MHz, MeOD) δ 7.90 - 7.77 (m, 8H), 4.53 (s, 4H), 3.45-3.51 (m, 4H), 3.58 - 3.42 (m, 4H), 3.41 - 3.27 (m, 8H), 3.10-3.25 (m, 4H), 3.02 - 2.89 (m, 4H), 2.06 - 1.95 (m, 4H).

[0184]

[0185] Compound 6j: N1-(3-aminopropyl)-N1-(4,6-bis(3,3-difluoropiperidin-1-yl)-1,3,5-triazin-2-yl)propane-1,3-diamine n-hydrochloride (PDL-21)

[0186]

[0187] Compound 6j was synthesized according to the general procedure (c). Yield 78%; white solid. 1H NMR (400 MHz, MeOD) δ 4.05 (t,J= 11.7 Hz, 4H), 3.87 - 3.77 (m, 4H), 3.73 (t,J= 6.8 Hz, 4H), 2.98 (t,J= 4.0 Hz, 4H), 2.18 - 1.98 (m, 8H), 1.87 - 1.73 (m, 4H).

[0188]

[0189] Compound 6k: N1-(3-aminopropyl)-N1-(4,6-bis(3,3-difluoropyrrolidin-1-yl)-1,3,5-triazin-2-yl)propane-1,3-diamine n-hydrochloride)(PDL-20)

[0190]

[0191] Compound 6k was synthesized according to the general procedure (c). Yield 74%; white solid. 1 H NMR (400 MHz, CDCl3) δ 4.04 (t,J= 12.6 Hz, 4H), 3.91 (t,J= 7.4 Hz, 4H), 3.73 (t,J= 7.0 Hz, 4H), 2.99 (d,J= 8.0 Hz, 4H), 2.53 (d,J= 8.0 Hz, 4H), 2.10 - 1.99 (m, 4H).

[0192]

[0193] Compound 6l: N2,N2-bis(3-aminopropyl)-N4,N6-bis(3-fluorobenzyl)-N4,N6-dimethyl-1,3,5-triazine-2,4,6-triamine n-hydrochloride (PDL-24)

[0194]

[0195] Compound 6l was synthesized according to the general procedure (c). Yield 74%; white solid. 1 H NMR (400 MHz, MeOD) δ 7.36 (s, 2H), 7.21 - 6.80 (m, 6H), 3.83 - 3.58 (m, 4H), 3.35 - 3.27 (m, 4H), 3.22 (s, 6H), 3.07 - 2.73 (m, 4H), 2.15 - 1.87 (m, 4H).

[0196]

[0197] [Preparation Example] Control Group Information

[0198] As a control, vancomycin hydrochloride was purchased from Sigma (V2002), and PCN-1801 (N2,N2-bis(3-aminopropyl)-N4,N6-bis(naphthalen-1-ylmethyl)-1,3,5-triazine2,4,6-triamine), the parent of the derivatives, was manufactured and used directly by the inventor.

[0199]

[0200] [Example 1] Confirmation of cytotoxicity

[0201] 1. Cell line information

[0202] 1) MRC-5

[0203] Origin: Human

[0204] Organ: Lung

[0205] Morphology: Fibroblast

[0206] Medium: Minimum essential medium, 25 mM HEPES, 25 mM NaHCO3, 10% Fetal bovine serum (FBS)

[0207] KCLB number: 10171 (KCLB: Korean Cell Line Bank)

[0208] 2) HepG2

[0209] Origin: Human

[0210] Organ: liver

[0211] Morphology: epithelial-like

[0212] Medium: Minimum essential medium, 25mM HEPES, 25mM NaHCO3, Fetal bovine Serum (FBS) 10%

[0213] KCLB number: 88065 (KCLB: Korean Cell Line Bank)

[0214]

[0215] 2. Test Procedure

[0216] Sufficient MRC-5 and HepG2 cell numbers were secured through subculture. The medium in the T-75 cell culture flask was then removed. 5 mL of phosphate-buffered saline (PBS) was added to the T-75 cell culture flask, gently shaken, and then removed. Then, 3 mL of 0.25% Trypsin-EDTA was added and incubated for 5 minutes at 36°C in a 0.5% CO2 environment. Attached cells were suspended by pipetting, and the cell suspension was then collected. Medium was added to the cell suspension to a volume of 10 mL and centrifuged at 1800 rpm for 3 minutes. The supernatant was removed, 1 mL of medium was added, and the cells were resuspended. 10 μL of the cell suspension was collected and mixed with 10 μL of Trypan Blue. 10 μL of a mixture of cell suspension and Trypan Blue was added to a hematocytometer, and the number of cells per volume was determined under an optical microscope. 1 × 10 4Cells / 100 μL were dispensed into 1 well of a 96-well cell culture plate. The mixture was cultured for more than 12 hours at 36°C in a 0.5% CO2 environment. The medium was then removed and replaced with 99 μL / well of fresh medium. The compound synthesized in the above synthesis example was serially diluted from 10 mM → 5 mM → 2.5 mM → 1.25 mM → 0.625 mM → 0.3125 mM → 0.15625 mM. Then, 1 μL of each compound was added to the well to obtain a final concentration of 100 μM → 50 μM → 25 μM → 12.5 μM → 6.25 μM → 3.125 μM → 1.5625 μM, and the mixture was cultured for 24 hours at 36°C in a 0.5% CO2 environment. Afterwards, 10 μL of CCK-8 (cell counting kit 8) was added to each well and incubated for 1 hour and 30 minutes in a 0.5% CO2 environment at 36°C. The absorbance was then measured at a wavelength of 450 nm.

[0217]

[0218] IC using Nonlinear regression using Graph Pad Prism5 50 The value was calculated.

[0219]

[0220] 3. Results

[0221] Cytotoxicity was confirmed using the MRC-5 cell line, a normal fibroblast cell line, and the HepG2 cell line, a liver cancer cell line. As a result, according to Figures 2 and 3, vancomycin, which was used as a control, did not show cell death even at the maximum concentration used in the test, PCN-1801 also did not show cell death, and fluorotriazine derivatives showed cell death, but the half lethal concentration (LD 50: Lethal dose 50) was calculated, and it was confirmed that all substances showed a half-lethal concentration above 10 μM, and thus it was confirmed that there was no cytotoxicity below 10 μM.

[0222]

[0223] [Example 2] Confirmation of antibacterial activity

[0224] 1. Cell line information

[0225] A549

[0226] Origin: Human

[0227] Organ: Lung

[0228] Morphology: epithelial

[0229] Medium: RPMI-1640 with L-glutamine (300 mg / L) 25mM HEPES and 25mM NAHCO3, 10% Fetal Bovine Serum (FBS)

[0230] KCLB number: 10185 (KCLB: Korean Cell Line Bank)

[0231]

[0232] 2. Test Procedure

[0233] The above A549 cell line was cultured 1 to 2 times to proliferate and stabilize the cells, and 3 x 10 5 Cells / well were dispensed into 6 cell culture plates (2 mL) and cultured for 12 h in a 0.5% CO2 environment at 37°C. After confirming the stabilization of cells and their attachment as single cells, Enterococcus Faecium was added at a density of 1 × 10 7 CFU / mL, or 1×10 8CFU / mL. After 30 minutes, the compound synthesized in the above synthetic example was treated at various concentrations (stock 10 mM). Cultured for 24 hours at 36°C in a 0.5% CO2 environment. Calcein AM (stock concentration: 2 mM) was treated to 2 μM and cultured for 40 minutes at 37°C in a 0.5% CO2 environment. 0.5 μM DAPI was treated. Photographs were taken using an inverted fluorescence microscope. The captured images were sectioned into 500 μm × 500 μm sections to confirm the number of dead cells, DAPI statin (blue), and viable cells, Calcein (green), and diagrammed.

[0234]

[0235] 3. Results

[0236] As a result, in order to first confirm the cell killing ability of Enterococcus faecium (E. Faecium), 1×10 7 CFU / mL and 1×10 8 Cell killing ability was confirmed by treating CFU / mL alone. 1×10 8 In the group treated with CFU / mL, it was confirmed that all cells died within 24 hours, and when evaluating the antibacterial efficacy of the substance, all experimental groups were treated with 1×10 8 CFU / mL was treated (Fig. 4a).

[0237] To determine the effects of the test substances on the cells, vancomycin, PCN-1801, and PDL-16 were treated alone and changes were observed after 24 hours. As a result, it was confirmed that none of the test substances had any effect on the cells (Fig. 4b).

[0238] To compare and evaluate the antibacterial activity of vancomycin, PCN-1801 and PDL-16, the same number (1×10 8E. Faecium was treated with 10 μM of all three substances. The ratio of surviving cells after 24 hours (number of green-stained cells / green-stained cells + blue-stained cells) was measured, and vancomycin was 60.65 ± 5.30, PCN-1801 was 55.44 ± 5.31, and PDL-16 was 73.37 ± 4.92 (Fig. 4c and 4d).

[0239] In order to evaluate the antibacterial efficacy of PDL-16, the infected cell line was treated in a concentration-dependent manner and the antibacterial efficacy was evaluated. As a result, it was confirmed that the 1 μM PDL-16 treatment group showed an antibacterial efficacy similar to that of 10 μM vancomycin (Fig. 4e and Fig. 4f).

[0240] PDL-16 was found to selectively kill E. Faecium.

[0241]

[0242] [Example 3] Confirmation of bactericidal effect

[0243] To verify the bactericidal effect of PDL-16, bacterial killing against Staphylococcus aureus (S. aureus) was investigated.

[0244] As a result, as shown in Fig. 5, PDL-16 was shown to be able to completely kill pathogens within 24 hours, proving its potential as a disinfectant.

[0245]

[0246] [Example 4] Confirmation of in vivo antibacterial effect

[0247] As in the above-described examples, after confirming the in vitro antibacterial efficacy of PDL-16, an in vivo survival experiment was performed using a Balb / c mouse model infected with Enterococcus faecium (E. Faecium).

[0248] To determine the minimum bacterial concentration that causes mortality, mice were inoculated with various concentrations of E. Faecium (1 × 10 8 , 1×10 9 , 2×10 9 , and 5×10 9 CFU). Referring to Figure 6(a), 5×10 9 The CFU dose was determined to be the minimum concentration at which mice died from infection.

[0249] Subsequently, this concentration was used for co-treatment with PDL-16 at doses of 5, 10, and 15 mg / kg in a mouse model. Referring to Fig. 6(b), mice not treated with PDL-16 died after 60 hours, whereas mice administered PDL-16 did not die even after 80 hours, demonstrating the in vivo protective antimicrobial effect of PDL-16.

[0250]

[0251] [Example 5] Confirmation of changes in weight and pathogen content in an intramuscular infection model

[0252] E. Faecium5×10 4 Changes in weight and pathogen content were investigated using an animal Balb / C model in which CFU / mL was slowly injected into the left thigh.

[0253] Referring to Fig. 7(a), the PDL-16 10 mg / kg treatment group showed a statistically significantly lower pathogen content than the vehicle group after 24 hours of administration.

[0254] Referring to Figure 7(b), it can be confirmed that the body weights of all test groups were measured as normal, and that there were no diseases or behavioral abnormalities caused by the drug.

[0255]

[0256] [Example 6] ADME (Absorption, Distribution, Metabolism, Excretion) Analysis of PDL-16

[0257] 1. CYP inhibition

[0258] Table 1 below shows the inhibitory effects of compound PDL-16 and reference compound Ketoconazole on various cytochrome P450 isoenzymes.

[0259] Compound IC 50 (μM)CYP1A2CYP2C9CYP2C19CYP2D6CYP3A4PDL-16>10>10>10>10>10Ketoconazole (reference)92.296.191.195.228.6

[0260]

[0261] Referring to Table 1 above, PDL-16 had an IC of 10 μM or more for all cytochrome P450 isoenzymes tested (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). 50 , indicating that PDL-16 had minimal inhibitory effects on this enzyme at the concentrations tested. This suggests that PDL-16 may have a low propensity to induce cytochrome P450-related drug-drug interactions, whereas ketoconazole may pose a significant risk for such interactions due to its potent inhibitory ability, particularly toward CYP3A4. These results are important for understanding the metabolic profile and stability of PDL-16 in pharmacological applications.

[0262]

[0263] 2. Metabolic stability analysis

[0264] Metabolic stability analysis of PDL-16 was evaluated using human liver microsomes, a predictive measure of hepatic clearance of a drug in vivo (Table 2).

[0265] Compound Human liver microsomal stability (% remaining after 30 min) PDL-16 95.7 Verapamil (reference) 9.7

[0266]

[0267] Referring to Table 2 above, PDL-16 exhibited high stability, with 95.7% of the compound remaining after 30 minutes of incubation, indicating slow hepatic metabolism. In contrast, verapamil, a known substrate for CYP enzymes, showed significant metabolism, with only 9.7% remaining, indicating rapid hepatic clearance. This marked difference in microsomal stability suggests that PDL-16 may exhibit reduced metabolic degradation compared to verapamil, which may extend its duration of action in vivo.

[0268]

[0269] 3. Plasma protein binding analysis

[0270] Table 3 below summarizes the results of the plasma protein binding analysis.

[0271]

[0272]

[0273] Referring to Table 3 above, PDL-16 exhibited a very high plasma protein binding rate of 99.8%. This is similar to the protein-bound anticoagulant warfarin, which exhibits a binding rate of 99.4%. In contrast, dexamethasone, a corticosteroid used as a reference, exhibited a significantly lower binding rate of 64.1%. The high protein binding of PDL-16 may imply a limited volume of distribution and prolonged half-life, potentially affecting pharmacokinetics and dosage requirements.

[0274]

[0275] 4. Plasma stability analysis

[0276] Table 4 below summarizes the results of the plasma stability analysis.

[0277]

[0278]

[0279] Referring to Table 4 above, PDL-16 maintained high plasma stability, with 98.2% remaining at 30 minutes and 94.9% remaining at 120 minutes. This stability is similar to that of another reference compound, enalapril, which exhibited higher stability (98.9%) at 30 minutes but maintained a similar proportion (92.5%) at 120 minutes. In contrast, procaine exhibited rapid plasma degradation, with only 1.3% remaining at 5 minutes and less than 1% remaining at 10 minutes. The high plasma stability of PDL-16 suggests that it has a slower degradation rate in human plasma, which may allow for less frequent dosing intervals in clinical settings.

[0280]

[0281] 5. Pharmacokinetic profile of PDL-16

[0282] Table 5 below shows the PK (pharmacokinetic) parameters after intravenous injection of 2 mg / kg dose (n = 4).

[0283]

[0284]

[0285] Table 6 below shows the PK parameters after oral administration of 20 mg / kg dose (n = 4).

[0286]

[0287]

[0288] Referring to Tables 5 and 6 above, the pharmacokinetic profiles for PDL-16 showed different behaviors depending on the various administration routes in rats.

[0289] When administered intravenously, PDL-16 exhibited a prolonged mean terminal half-life of 21.8 hours with a significant area under the curve, suggesting extensive systemic circulation and slow elimination. In contrast, oral administration demonstrated a similar terminal half-life but a significantly lower AUC, potentially reflecting the oral route's influence due to first-pass metabolism. Nevertheless, oral bioavailability was moderately good at 41.8%, demonstrating a good absorption rate. Comparing intravenous and oral administration allows for the assessment of the route of administration's impact on drug bioavailability and systemic exposure. These findings contribute to understanding the pharmacokinetics of PDL-16, and these data encourage further exploration of its pharmacodynamics and long-term effects, as well as dose optimization for maximum efficacy and safety in future clinical applications. This may include controlled-release formulations to mitigate the rapid metabolism observed with oral administration.

[0290]

[0291] Also, referring to Figure 8, the comparative plasma concentration profiles of PDL-16 after intravenous (IV) and oral (PO) administration showed that intravenous administration of 2 mg / kg of PDL-16 achieved high plasma concentrations immediately, but oral dose of 20 mg / kg showed a delayed and attenuated peak indicating absorption and first-pass metabolism effects. These data demonstrate the influence of the route of administration on the pharmacokinetics of PDL-16, as oral administration resulted in lower plasma concentrations compared to intravenous administration, which can be used to determine an appropriate dosing strategy for therapeutic purposes.

[0292]

[0293] [Example 7] Confirmation of inhibition of antibiotic resistance in Gram-negative bacteria

[0294] To confirm the inhibitory effect of PDL-16 on the development of antibiotic resistance against Acinetobacter baumannii, a Gram-negative bacterium, a serial passage assay was performed.

[0295] In the same order as Fig. 9, the parent culture was first established through a preliminary experiment, including the inoculation amount, incubation time, culture volume, and initial concentration. This was inoculated into a medium supplemented with antibiotics (imipenem, PDL-16). The maximum concentration cultured on day 1 was set as the sub-MIC, and the sub-MIC set on day 1 was used as the inoculation strain on day 2 to perform passage 2. This process was continued for more than 20 days (passage 20) (Table 7).

[0296] Table 7 below shows the amounts of imipenem and PDL-16 added to the medium up to passage 20.

[0297]

[0298]

[0299] As a result of the analysis, referring to Fig. 10, the sub-MIC value increased rapidly after day 15 (passage 15) in the medium treated with imipenem, whereas the medium treated with PDL-16 maintained a low sub-MIC value until day 20.

[0300] Through this, it was confirmed that, unlike existing antibiotics, PDL-16 does not induce resistance to PDL-16 in Acinetobacter baumannii (A. baumannii).

[0301]

[0302] Overall, PDL-16 was not cytotoxic in mammalian cells and did not induce resistance in Gram-negative bacteria. Furthermore, it possesses a promising pharmacokinetic profile, with a long half-life and moderate oral bioavailability. Minimal inhibition of cytochrome P450 enzymes, high metabolic stability, and significant plasma protein binding are particularly encouraging, suggesting a low potential for drug-drug interactions and a prolonged half-life that could potentially reduce dosing frequency. These characteristics suggest that PDL-16 is a promising agent for the treatment of resistant bacterial infections.

[0303]

[0304] The foregoing description of the present invention is for illustrative purposes only. Those skilled in the art will readily appreciate that modifications to other specific embodiments can be made without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0305] The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. A compound selected from a triazine derivative compound represented by the following chemical formula 1, a pharmaceutically or food-wise acceptable salt thereof, a solvate thereof, or a stereoisomer thereof: [Chemical Formula 1] In the above chemical formula 1, R 1 and R 2 may be the same or different, and are hydrogen, (C1-C5) alkyl and -(CH 2 ) n -R 11 One of them, but at least one is -(CH 2 ) n -R 11 , and n is one of 0 to 5, and R 11 Silver substituted (C3-C12)cycloalkyl, substituted (C5-C12)aryl and CHF 2 and the substituted (C3-C12)cycloalkyl and (C5-C12)aryl are substituted with one or more trifluoromethyl or fluorine, R 1 and R 2 A heterocyclic compound which is connected to each other to form a 3- to 12-membered ring including substituted nitrogen or oxygen, and the substituted heterocyclic compound is fluorine, trifluoromethyl or -(CH 2 ) m -R 21 is substituted with one or more of m, wherein m is one of 0 to 3, and R 21 is a (C5-C12)aryl substituted with trifluoromethyl.

2. In claim 1, In the above chemical formula 1, R 1 and R 2 may be the same or different, and are hydrogen, methyl and -(CH 2 ) n -R 11 One of them, but at least one is -(CH 2 ) n -R 11 , and the above n is 0 or 1, and the above R 11 Silver substituted (C4-C6)cycloalkyl, substituted phenyl and CHF 2 and the substituted (C4-C6)cycloalkyl and phenyl are substituted with 1 or 2 trifluoromethyl or fluorine, R 1 and R 2 is a heterocyclic compound which is connected to each other to form a 5-membered ring or a 6-membered ring including unsubstituted or substituted nitrogen or oxygen, and the substituted heterocyclic compound is fluorine or -CH 2 -R 21 This one or more is substituted, and the above R 21 A compound selected from a triazine derivative compound, a pharmaceutically or food additively acceptable salt thereof, a solvate thereof or a stereoisomer thereof, characterized in that silver trifluoromethyl is substituted phenyl.

3. In claim 1, In the above chemical formula 1, R 1 Silver -(CH 2 ) n -R 11 , and the above n is 0 or 1, and the above R 11 Silver substituted (C4-C6)cycloalkyl, substituted phenyl and CHF 2 and the substituted (C4-C6)cycloalkyl and phenyl are substituted with 1 or 2 trifluoromethyl or fluorine, and R 2 A compound selected from a triazine derivative compound, a pharmaceutically or food scienceally acceptable salt thereof, a solvate thereof or a stereoisomer thereof, characterized in that it is hydrogen or methyl.

4. In claim 1, The above triazine derivative compound is, A compound selected from a triazine derivative compound, a pharmaceutically or food-wise acceptable salt thereof, a solvate thereof or a stereoisomer thereof, characterized by being selected from the following structures: , , , , , , , , , , , and .

5. An antibacterial composition comprising, as an active ingredient, a compound selected from the triazine derivative compound of claim 1, a pharmaceutically or food science-wise acceptable salt thereof, a solvate thereof, or a stereoisomer thereof.

6. In claim 5, The above composition, An antibacterial composition characterized in that it exhibits antibacterial activity against one or more bacteria selected from the group consisting of Enterococcus Faecium, Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and antibiotic-resistant strains thereof.

7. In claim 5, The above composition, An antibacterial composition characterized in that the compound is contained in a concentration of 0.0001 to 10 μM.

8. A pharmaceutical product comprising an antibacterial composition according to any one of claims 5 to 7.

9. A cosmetic composition comprising an antibacterial composition according to any one of claims 5 to 7.

10. A health food composition comprising an antibacterial composition according to any one of claims 5 to 7.

11. An animal feed or feed additive composition comprising an antimicrobial composition according to any one of claims 5 to 7.

12. A pharmaceutical composition for preventing or treating a pathogenic microbial infection disease, comprising as an active ingredient a compound selected from the triazine derivative compound of claim 1, a pharmaceutically acceptable salt thereof, a solvate thereof, or a stereoisomer thereof.

13. In claim 12, The above pathogenic microbial infectious diseases are, A pharmaceutical composition characterized in that the disease is an infectious disease caused by one or more bacteria selected from the group consisting of Enterococcus Faecium, Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and antibiotic-resistant strains thereof.

14. A health functional food composition for preventing or improving a pathogenic microbial infection disease, comprising as an effective ingredient a compound selected from the triazine derivative compound of claim 1, a food-chemically acceptable salt thereof, a solvate thereof, or a stereoisomer thereof.

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