Novel inflammasome inhibitor, and composition comprising same for preventing or treating hyperuricemia

Abstract

The present invention relates to: a novel derivative compound of febuxostat; and a composition comprising same as an active ingredient for preventing or treating hyperuricemia, specifically gout. The compound of the present invention significantly inhibits NLRP3 inflammasome activity of uric acid-induced macrophages even at a low dose, and significantly reduces inflammatory cytokines and neutrophils in lesion sites of a gout animal model, thereby effectively inhibiting inflammatory responses caused by uric acid accumulation, and thus can be effectively used for long-term administration for the treatment of gout, which is a chronic systemic disease.

Description

Novel inflammasome inhibitor and composition for the prevention or treatment of hyperuricemia containing the same

[0001] The present invention relates to novel febuxostat derivative compounds and a method for preventing or treating inflammatory damage caused by hyperuricemia, specifically gout, using the same. The present invention was made possible through research projects (2710001164 and 2710018234) of the National Research Foundation of Korea funded by the Ministry of Science and ICT.

[0002]

[0003] An inflammatory response refers to a reaction of cells or blood vessels triggered by damage or stimulation to the body, signifying a complex immune response of the human body generated by pathogenic substances such as pathogens, autoantigens, and virus-infected cells. Furthermore, the inflammatory response encompasses the entire process of reconstructing damaged biological tissues, including the removal of various causative substances—such as microorganisms and toxins—that caused the damage, as well as the elimination of necrotic cells and tissues resulting from the injury.

[0004] Meanwhile, gout, known to induce an inflammatory response, is a representative metabolic disease caused by hyperuricemia, in which uric acid, the end product of purine metabolism, accumulates in the blood and its concentration rises. Uric acid accumulated between the blood and joints forms urate crystals that are deposited in the tissues surrounding the joints and subcutaneous tissues, thereby causing excessive inflammatory responses and pain.

[0005] Febuxostat is an inhibitor of xanthine oxidase, which is involved in purine metabolism, and has been verified to have significant therapeutic effects on hyperuricemia and gout by reducing uric acid production. However, side effects such as cardiovascular death, liver dysfunction, chest pain, and dyspnea have been reported due to long-term administration of Febuxostat; consequently, the Ministry of Food and Drug Safety recently added a 'warning' section to the product leaflet for Febuxostat as a gout treatment and restricted its use. In particular, while Febuxostat is currently widely used as a gout treatment, research on inflammatory responses mediated by various inflammatory cells, such as macrophages and neutrophils, remains insufficient. Currently, Febuxostat is administered at a high dose of 80 mg for adults, and there is a growing demand for the development of more efficient alternative drugs that offer high therapeutic efficacy while reducing cardiovascular side effects at lower concentrations.

[0006]

[0007] Throughout this specification, numerous papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

[0008]

[0009] The inventors have made diligent research efforts to develop an excellent small molecule therapeutic agent capable of efficiently controlling hyperuricemia caused by the accumulation of uric acid in the blood due to abnormal purine metabolism, and the excessive inflammatory response resulting therefrom. As a result, the present invention was completed by discovering that a Febuxostat derivative compound represented by Chemical Formula 1 described below significantly inhibits the activity of the NLRP3 inflammasome involved in the inflammatory mechanism of hyperuricemia and significantly reduces inflammatory cytokines and neutrophils in a gout animal model.

[0010] Therefore, the objective of the present invention is to provide a novel derivative compound of febuxostat and a composition for the prevention or treatment of hyperuricemia containing the same as an active ingredient.

[0011]

[0012] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings.

[0013]

[0014] According to one aspect of the present invention, the present invention provides a compound represented by the following chemical formula 1:

[0015] Chemical formula 1

[0016]

[0017] In the above formula, R1 is a C1-C7 alkyl; L1 is a direct bond or -NH-, A is a heteroaryl of a 5-9 ring that is unsubstituted or substituted with one or more substituents selected from the group consisting of C1-C5 alkyl and -NR3R4 (R3 and R4 are each independently hydrogen or C1-C7 alkyl); and R2 is a C1-C5 hydroxyalkyl or -COOR5 (R5 is hydrogen or C1-C3 alkyl).

[0018] The inventors have made diligent research efforts to develop an excellent small molecule therapeutic agent capable of efficiently controlling the excessive inflammatory response caused by hyperuricemia, specifically gout, which is caused by the accumulation of uric acid in the blood due to abnormal purine metabolism. As a result, it was discovered that a Febuxostat derivative compound represented by Chemical Formula 1 can efficiently suppress the uric acid-induced inflammatory response by significantly inhibiting the NLRP3 inflammasome activity of macrophages induced by uric acid and significantly reducing inflammatory cytokines and neutrophils within the lesion site of a gout animal model.

[0019] In this specification, the term “alkyl” means a straight-chain or branched saturated hydrocarbon group, including, for example, methyl, ethyl, propyl, isopropyl, etc. C1-C7alkyl means an alkyl group having alkyl units having 1 to 7 carbon atoms, and when C1-C7alkyl is substituted, the number of carbon atoms of the substituent is not included.

[0020] In this specification, the term “heteroaryl” refers to a heterocyclic aromatic group containing oxygen, sulfur, or nitrogen as a heteroatom within the ring. The number of heteroatoms included within the ring is 1-3, specifically 1-2. The term “heteroaryl of a pentagonal-nine-membered ring” refers to a heteroaryl in which the number of atoms forming the ring, including both carbon and heteroatoms, is 5 to 9, and the ring may be a monocycle or a two-membered bicycle.

[0021] In this specification, the term “hydroxyalkyl” means a straight-chain or branched saturated hydrocarbon group substituted with a hydroxyl group. C1-C5 hydroxyalkyl means an alkyl group in which any one hydrogen of an alkyl unit having 1 to 5 carbon atoms is substituted with a hydroxyl group.

[0022] According to a specific embodiment of the present invention, A is a heteroaryl of a 5-9 ring that is unsubstituted or substituted with -NR3R4 (R3 is hydrogen and R4 is C1-C7 alkyl).

[0023] More specifically, the heteroaryl of the 5-9-membered ring is selected from the group consisting of benzothiazole, thiophene, furan, pyrrole, and imidazopyridine.

[0024] According to a more specific embodiment of the present invention, when the heteroaryl of the 5-9-membered ring is a benzothiazole, the L1 is -NH-.

[0025] According to a more specific embodiment of the present invention, when the heteroaryl of the 5-9-membered ring is furan, the R2 is a C1-C5 hydroxyalkyl.

[0026]

[0027] According to a more specific embodiment of the present invention, when the heteroaryl of the 5-9-membered ring is imidazopyridine, the heteroaryl of the 5-9-membered ring is substituted with -NR3R4 (R3 is hydrogen and R4 is C1-C7 alkyl).

[0028] According to a specific embodiment of the present invention, the compound represented by Formula 1 is selected from the group consisting of compounds represented by Formulas 2 to 6 below:

[0029] Chemical formula 2 Chemical formula 3

[0030]

[0031] Chemical formula 4 Chemical formula 5

[0032]

[0033] Chemical formula 6

[0034] .

[0035] According to another aspect of the present invention, the present invention provides a composition for the prevention or treatment of hyperuricemia comprising the compound of the present invention described above or a pharmaceutically acceptable salt thereof as an active ingredient.

[0036] According to another aspect of the present invention, the present invention provides a method for preventing or treating hyperuricemia comprising the step of administering the compound of the present invention or a pharmaceutically acceptable salt thereof to a subject.

[0037] In this specification, the term “hyperuricemia” refers to a metabolic syndrome in which blood uric acid levels are abnormally increased due to a purine metabolism disorder or impaired uric acid (UA) excretion disorder. Specifically, it refers to a pathological condition in which, under normal purine diet conditions, the fasting serum uric acid level exceeds 420 μM for men and 360 μM for women for at least 2 days. When blood uric acid levels rise, uric acid crystals form and accumulate, causing severe inflammation which can lead to gout.

[0038] According to a specific embodiment of the present invention, the hyperuricemia is gout.

[0039] In this specification, the term “gout” refers to a metabolic joint disease caused by the deposition of urate crystals in the joints due to an increase in blood uric acid concentration. When urate crystals are deposited in the cartilage, tendons, and surrounding tissues of the joints, they cause inflammation of the joints, which is accompanied by severe pain.

[0040] In this specification, the term “prevention” means suppressing the occurrence of a disease or illness in subjects who have not been diagnosed with having such a disease or illness but are at risk of developing such a disease or illness.

[0041] In this specification, the term “treatment” means (a) inhibition of the progression of a disease, illness, or symptom; (b) alleviation of a disease, illness, or symptom; or (c) elimination of a disease, illness, or symptom. The compounds of the present invention play a role in inhibiting, eliminating, or alleviating the progression of symptoms caused by hyperuricemia by significantly inhibiting the activity of the NLRP3 inflammasome, a key factor in the inflammatory mechanism caused by hyperuricemia, in macrophages stimulated by the gout-inducing factors MSU (monosodium urate) and LPS (lipopolysaccharide), and by significantly reducing IL-1β and neutrophil levels in gout animal models. Accordingly, the compositions of the present invention may serve as compositions for treating hyperuricemia on their own, or they may be applied as therapeutic adjuvants for hyperuricemia by being administered together with other pharmacological components having anti-inflammatory or uric acid-eliminating activity. Accordingly, in this specification, the terms “treatment” or “therapeutic agent” include the meaning of “therapeutic adjuvant” or “therapeutic adjuvant.”

[0042] In this specification, the terms “administration” or “to administer” refer to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that an equal amount is formed within the subject’s body.

[0043] In the present invention, the term “therapeutic effective amount” refers to the content of a composition in which the pharmacological component within the composition is contained in an amount sufficient to provide a therapeutic or preventive effect to an individual to whom the pharmaceutical composition of the present invention is to be administered, and includes the meaning of “preventive effective amount.”

[0044] In this specification, the term “object” includes, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons, or rhesus monkeys. Specifically, the object of the present invention is a human.

[0045] In this specification, the term “pharmaceutically acceptable salt” includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, trifluoroacetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Salts derived from suitable bases may include alkali metals such as sodium, alkaline earth metals such as magnesium, and ammonium, etc.

[0046] When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier.

[0047] Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may additionally include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0048] The pharmaceutical composition of the present invention may be administered orally or parenterally, specifically parenterally, and more specifically, intravenously, intramuscularly, or into the joint cavity.

[0049] Suitable dosages of the pharmaceutical composition of the present invention can be prescribed in various ways depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity. Preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.001-100 mg / kg for adults.

[0050] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-volume container by formulation using a pharmaceutically acceptable carrier and / or excipient, according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0051] According to another aspect of the present invention, the present invention provides a functional food composition for improving or preventing hyperuricemia comprising the compound of the present invention described above or a food-grade salt thereof as an active ingredient.

[0052] Since the compound of Formula 1 used in the present invention and hyperuricemia that can be improved or prevented by using it have already been described above, the description thereof is omitted to avoid excessive duplication.

[0053] In this specification, the term “food-grade acceptable salt” refers to a salt in which a cation and an anion are combined by electrostatic attraction and is of a form that can be used in a food composition, and specific examples thereof include the examples of “pharmaceutical-grade acceptable salt” described above.

[0054] When the composition of the present invention is prepared as a food composition, it may include not only the compound of the present invention as an active ingredient, but also carbohydrates, seasonings, and flavorings that are typically added during food manufacturing. Examples of carbohydrates include, but are not limited to, monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavorings, natural flavorings [taumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)] and synthetic flavorings (saccharin, aspartame, etc.) may be used. For example, when the food composition of the present invention is prepared as a drink, in addition to the pine bark extract which is the active ingredient of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, Eucommia ulmoides extract, jujube extract, licorice extract, etc. may be additionally included.

[0055]

[0056] The features and advantages of the present invention are summarized as follows:

[0057] (a) The present invention provides a novel derivative compound of Febuxostat and a composition for the prevention or treatment of hyperuricemia, specifically gout, comprising the same as an active ingredient.

[0058] (b) The compound of the present invention can be usefully utilized for long-term administration for the treatment of gout, a chronic disease, by significantly inhibiting uric acid-induced NLRP3 inflammasome activity in macrophages even at low doses and significantly reducing inflammatory cytokines and neutrophils in the lesion site of a gout animal model, thereby effectively suppressing the inflammatory response caused by uric acid accumulation.

[0059]

[0060] Figure 1 is a diagram illustrating the process of applying inflammatory stimulation to macrophages using uric acid.

[0061] Figure 2 shows the results of measuring the relative survival rate of macrophages according to each concentration of the compound of the present invention (Figure 2a) and the change in IL-1β expression levels by febuxostat and the compound of the present invention under inflammatory response induction conditions (LPS + uric acid) (Figure 2b), respectively.

[0062] Figure 3 shows the results of measuring cytotoxicity (Figure 3a), changes in IL-1β expression (Figure 3b), changes in TNF-α expression (Figure 3c), changes in IL-6 expression (Figure 3d), and changes in IL-10 expression (Figure 3e), respectively, in macrophages stimulated with LPS 100 ng / ml and MSU 0.5 mg / ml after treatment with the optimal concentration of the compound of the present invention.

[0063] Figure 4 shows the results of measuring cytotoxicity (Figure 4a), changes in IL-1β expression (Figure 4b), changes in TNF-α expression (Figure 4c), changes in IL-6 expression (Figure 4d), and changes in IL-10 expression (Figure 4e), respectively, in macrophages stimulated with LPS 100 ng / ml and MSU 1 mg / ml after treatment with the optimal concentration of the compound of the present invention.

[0064] Figure 5 shows the results of measuring the changes in the expression of inflammatory factors in macrophages by the compounds FT033, FT037, and FT046 of the present invention using Western blot (Figure 5a) and the results of quantifying the changes in the expression of IL-1β, the final inflammatory response product (Figure 5b), respectively.

[0065] Figure 6 is a schematic diagram of an experiment for further exploration of the anti-inflammatory mechanism in macrophages by the compound of the present invention (Figure 6a) and the results of quantifying changes in the expression of TNF-α and IL-6 (Figure 6b), respectively.

[0066] Figure 7 shows the experimental schematic (Figure 7a), the quantitative results of the number of neutrophils (Figure 7b), and the results of the cytospin experiment for measuring neutrophils (Figure 7c), respectively, to investigate the inhibitory effect of the insufficiency of the pneumatocystic gout mouse model.

[0067] Figure 8 is a schematic diagram illustrating the process of measuring changes in the expression of inflammation-related cytokines following treatment with the compounds of the present invention in macrophages stimulated by LPS and ATP.

[0068] Figure 9 shows the results of measuring changes in IL-1β expression according to each concentration of the compound of the present invention in macrophages stimulated by LPS and ATP.

[0069] Figure 10 is a figure showing the changes in IL-1β expression (Fig. 10a), TNF-α expression (Fig. 10b), IL-6 expression (Fig. 10c), and IL-10 expression (Fig. 10d), respectively, following treatment with the optimal concentration of the compound of the present invention in macrophages stimulated by LPS and ATP.

[0070] Figure 11 shows an experimental schematic diagram for evaluating changes in the expression of target factors related to the NLRP3 inflammasome mechanism of action in macrophages stimulated by LPS and ATP (Figure 11a), results of measuring changes in the expression of each inflammatory factor by Western blot (Figure 11b), and results of quantifying changes in the expression of IL-1β, the final inflammatory response product (Figure 11c), respectively.

[0071] Figure 12 is a diagram illustrating the process of measuring changes in the expression of NLRP3 inflammasome-related factors following treatment with the compounds of the present invention in macrophages stimulated by LPS and nigerisin (Figure 12a), the results of measuring changes in the expression of each factor by Western blot (Figure 12b), and the results of quantifying changes in the expression of IL-1β, the final inflammatory response product (Figure 12c), respectively.

[0072]

[0073] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0074] Examples

[0075] Structure number (chemical formula) of synthetic FT series small molecule compound Structure IUPAC name FT023(2) 2-((3-cyano-4-isobutoxyphenyl)amino)benzo[d]thiazole-6-carboxylic acid FT033(3) 5-(3-cyano-4-isobutoxyphenyl)thiophene-2-carboxylic acid FT037(4) 5-(5-(hydroxymethyl)furan-2-yl)-2-isobutoxybenzonitrileFT039(5) 5-(3-cyano-4-isobutoxyphenyl)-1H-pyrrole-2-carboxylic acid FT046(6) 3-(tert-butylamino)-2-(3-cyano-4-isobutoxyphenyl)imidazo[1,2-a]pyridine-6-carboxylic acid

[0076]

[0077] Synthetic example

[0078] Synthesis of Compound FT023

[0079] [FT02]

[0080]

[0081] (i) Synthesis of Compound H

[0082] 40 mg (0.17 mmol) of Compound F and 57 mg (0.21 mmol) of Compound G were dissolved in 2 mL of DMSO solvent, after which 0.2 mg (0.002 mmol) of CuBr and 16 mg (0.17 mmol) of tetrabutylammonium bromide were added. The reaction mixture was heated and stirred at 40°C for 6 hours. After cooling the reaction mixture to room temperature, 10 mL of water was added dropwise, and the mixture was extracted twice with 10 mL of EtOAc. The obtained EtOAc layer was dried with MgSO4, the solid was removed by filtering, and the filtrate was distilled under reduced pressure. The resulting mixture was separated and purified by silica gel column chromatography (Hex:EtOAc = 3:1 volume ratio) to obtain 35 mg (0.092 mmol, yield 53%) of Compound H.

[0083]

[0084] (ii) Synthesis of Compound FT023

[0085] Compound H40 mg (0.10 mmol) was dissolved in MeOH:THF (1 mL, 1.5 mL) solvent, then 0.63 mL (1.26 mmol) of 2 M NaOH aqueous solution was added and stirred at room temperature for 16 hours. Acidic ion exchange resin was added to the reaction mixture to adjust the pH to 3–4, and after filtration, the ion exchange resin was washed with EtOAc. All filtrates were collected and subjected to vacuum distillation to obtain FT02316 mg (0.05 mmol, yield 43%).

[0086] 1 H NMR (600 MHz, DMSO-d6)δ10.83(s,1H),8.42(d,J=1.8 Hz, 1H), 8.21 (d,J=2.8 Hz, 1H), 7.92-7.87 (m, 2H), 7.66 (d,J=8.4 Hz, 1H), 7.30 (d,J=9.2 Hz, 1H), 3.91 (d,J=6.5 Hz, 2H), 2.09-2.04 (m, 1H), 1.01 (d,J=6.7 Hz, 6H).

[0087]

[0088] Synthesis of Compound FT033

[0089] [FT033]

[0090]

[0091] (i) Synthesis of Compound M

[0092] Compound K 245 mg (0.81 mmol), Compound L 120 mg (0.54 mmol), Pd(PPh3) 463 mg (0.05 mmol), and Na2CO3 115 mg (1.09 mmol, 2 M aqueous solution) were dissolved in 5 mL of 1,4-dioxane solvent and heated and stirred at 90°C for 6 hours. After cooling the reaction mixture to room temperature, 10 mL of sat. NH4Cl(aq) was added dropwise, and the mixture was extracted twice with 10 mL of EtOAc. The obtained EtOAc layer was dried with MgSO4, the solid was removed by filtering, and the filtrate was distilled under reduced pressure. The resulting mixture was separated and purified by silica gel column chromatography (Hex:EtOAc = 8:1 volume ratio) to obtain Compound M 120 mg (0.38 mmol, yield 70%).

[0093]

[0094] (ii) Synthesis of Compound FT033

[0095] 50 mg (0.16 mmol) of compound M was dissolved in a THF:MeOH:H2O solvent (1 mL, 2 mL, 1 mL), followed by the addition of 0.95 mL (1.90 mmol) of 2 M NaOH aqueous solution, and the mixture was heated and stirred at 60°C for 10 hours. After cooling the reaction mixture to room temperature, an acidic ion exchange resin was added to adjust the pH to 3–4. The mixture was then filtered, and the ion exchange resin was washed with EtOAc. All filtrates were collected and subjected to vacuum distillation to obtain FT03340 mg (0.13 mmol, yield 84%).

[0096] 1H NMR (600 MHz, Methanol-d4)δ7.98-7.85(m,2H),7.72(d,J= 3.5 Hz, 1H), 7.41 - 7.35 (m, 1H), 7.20 (d,J= 8.3 Hz, 1H), 3.94 (d,J= 6.2 Hz, 2H), 2.21 - 2.12 (m, 1H), 1.09 (d,J= 6.6 Hz, 6H).

[0097]

[0098] Synthesis of Compound FT037

[0099] [FT037]

[0100]

[0101] (i) Synthesis of Compound FT037

[0102] 0.92 mg (0.50 mmol) of LiBH4 was added to a solution of 1.5 mL of THF solvent in which 0 mg (0.10 mmol) of compound O3 was dissolved, and the mixture was heated and stirred at 55°C for 6 hours. After cooling the reaction mixture to room temperature, 10 mL of sat. NH4Cl(aq) was added dropwise, and the mixture was extracted twice with 10 mL of EtOAc. The obtained EtOAc layer was dried with MgSO4, the solid was removed by filtering, and the filtrate was distilled under reduced pressure. The resulting mixture was separated and purified by silica gel column chromatography (Hex:EtOAc = 3:1 volume ratio) to obtain FT03716 mg (0.06 mmol, yield 58%).

[0103] 1 H NMR (600 MHz, Chloroform-d / Methanol-d4(4:1)) δ 7.84 (d,J= 2.2 Hz, 1H), 7.79 - 7.77 (m, 1H), 6.96 (d,J= 8.9 Hz, 1H), 6.52 (d,J= 3.3 Hz, 1H), 6.38 (d,J= 3.3 Hz, 1H), 4.66 (s, 2H), 3.86 (d,J= 6.5 Hz, 2H), 2.22 - 2.14 (m, 1H), 1.08 (d,J= 6.7 Hz, 6H).

[0104]

[0105] Synthesis of Compound FT039

[0106] [FT039]

[0107]

[0108] (i) Synthesis of Compound Q

[0109] Compound K 415 mg (1.38 mmol), Compound P 300 mg (0.99 mmol), Pd(PPh3) 4114 mg (0.10 mmol), and Na2CO3 209 mg (1.97 mmol, 2 M aqueous solution) were dissolved in 10 mL of 1,4-dioxane solvent and heated and stirred at 95°C for 15 hours. After cooling the reaction mixture to room temperature, 10 mL of sat. NH4Cl(aq) was added dropwise, and the mixture was extracted twice with 10 mL of EtOAc. The obtained EtOAc layer was dried with MgSO4, the solid was removed by filtering, and the filtrate was distilled under reduced pressure. The resulting mixture was separated and purified by silica gel column chromatography (Hex:EtOAc = 5:1 volume ratio) to obtain Compound Q 200 mg (0.50 mmol, yield 51%).

[0110]

[0111] (ii) Synthesis of Compound R

[0112] 200 mg (0.50 mmol) of compound Q was dissolved in 1 mL of CH2Cl2 solvent, the temperature was lowered to 0 °C, and 0.5 mL of trifluoroacetic acid was added dropwise. After stirring the reaction mixture at room temperature for 2 hours, 10 mL of sat. Na2CO3(aq) was added dropwise to the reaction mixture, and the mixture was extracted twice with 10 mL of EtOAc. The obtained EtOAc layer was dried with MgSO4, the solid was removed by filtering, and compound R120 mg (0.40 mmol, yield 80%) was obtained by vacuum distillation.

[0113]

[0114] (iii) Synthesis of Compound FT039

[0115] Dissolve compound R40 mg (0.13 mmol) in THF:H2O (1 mL, 1 mL) solvent, then LiOH . 22.5 mg (0.54 mmol) of H2O was added, and the mixture was heated and stirred at 50°C for 5 hours. After cooling the reaction mixture to room temperature, an acidic ion exchange resin was added to adjust the pH to 3–4. After filtering, the ion exchange resin was washed with EtOAc. All filtrates were collected and subjected to vacuum distillation to obtain FT03920 mg (0.07 mmol, yield 52%).

[0116] 1 H NMR (600 MHz, Chloroform-d / Methanol-d4(4:1)) δ 7.71 (d,J= 36.8 Hz, 2H), 6.94 - 6.85 (m, 2H), 6.36 (s, 1H), 3.78 (d,J= 5.2 Hz, 2H), 2.13 - 2.06 (m, 1H), 1.00 (d,J= 6.5 Hz, 6H).

[0117]

[0118] Synthesis of Compound FT046

[0119] [FT046]

[0120]

[0121] (i) Synthesis of Compound X

[0122] Compound I241 mg (1.18 mmol) and Compound AA (0.99 mmol) were dissolved in 5 mL of MeOH solvent, after which 90 mg (1.08 mmol) of pivalonitrile and 38 mg (0.20 mmol) of p-toluenesulfonic acid monohydrate were added. The reaction mixture was stirred at room temperature for 16 hours, after which 10 mL of sat. NaHCO3(aq) was added dropwise and extracted twice with 10 mL of EtOAc. The obtained EtOAc layer was dried with MgSO4, the solid was removed by filter, and the filtrate was distilled under reduced pressure. The resulting mixture was separated and purified by silica gel column chromatography (Hex:EtOAc = 2:1 volume ratio) to obtain Compound U227 mg (0.54 mmol, yield 55%).

[0123]

[0124] (ii) Synthesis of Compound FT046

[0125] 30 mg (0.07 mmol) of Compound X was dissolved in THF:MeOH:H2O (0.5 mL, 1 mL, 0.5 mL) solvent, followed by the addition of 34 mg (0.86 mmol, 2 M aqueous solution) of NaOH, and the mixture was heated and stirred at 60°C for 10 hours. After cooling the reaction mixture to room temperature, an acidic ion exchange resin was added to adjust the pH to 3–4. The mixture was then filtered, and the ion exchange resin was washed with EtOAc. All filtrates were collected and subjected to vacuum distillation to obtain FT04622 mg (0.05 mmol, yield 76%).

[0126] 1H NMR (600 MHz, Chloroform-d / Methanol-d4(4:1)) δ 8.91 (s, 1H), 8.17 (d,J= 2.2 Hz, 1H), 8.08 (dd,J= 8.8, 2.2 Hz, 1H), 7.69 (d,J= 9.3 Hz, 1H), 7.43 (d,J= 9.1 Hz, 1H), 6.97 (d,J= 8.9 Hz, 1H), 3.79 (d,J= 6.4 Hz, 2H), 2.12 - 2.03 (m, 1H), 0.98 (d,J= 6.7 Hz, 6H), 0.97 (s, 9H).

[0127]

[0128] Experimental method

[0129] Differentiation of bone marrow-derived macrophages

[0130] After isolating bone marrow cells from mouse bone marrow, differentiation medium was prepared by adding 10% L929 cell line culture medium to high-glucose DMEM (Biowest, France) containing 10% fetal bovine serum (Biowest, France) and 1% penicillin / streptomycin (Biowest, France). Subsequently, 10 ml of the above differentiation medium was placed in a 90 x 15 mm Petri dish (SPL life science, Korea) and cultured for 3 days in an incubator under 5% CO2 and 37°C conditions. Afterward, macrophages were obtained by adding an additional 10 ml of the differentiation medium to the Petri dish and culturing for an additional 6 or 7 days. For use in experiments, the macrophages were isolated using trypsin-EDTA (Biowest, France) and then placed in each well of a 96-well cell culture plate at a rate of 1 x 10⁶ 6 200 µl were dispensed at a cell / ml cell count and cultured for 24 hours to allow the macrophages to sufficiently attach to the plate.

[0131]

[0132] Evaluation of cytotoxicity by compounds

[0133] After isolating macrophages, place 1 x 10⁶ in each well of a 96-well plate. 6 Cells were dispensed at a rate of 200 μl per cell / ml and cultured for 24 hours to allow for sufficient attachment, after which the concentration of each compound was administered according to each experimental condition. After culturing for each experimental time, the medium was replaced with a medium containing 0.5 mg / ml MTT reagent (for measuring mitochondrial activity, Abcam, UK) and cultured for 4 hours. The resulting purple formazan crystals were dissolved in 200 μl DMSO, and the absorbance of the resulting solution was measured at 570 nm. The optical density (OD) values ​​of the solvent-treated cells (non; solvent control) were compared to the 100% viability to calculate the relative viability of the cells cultured with each compound (Fig. 2).

[0134]

[0135] Cytokine measurement

[0136] To evaluate the inflammatory response of macrophages induced by LPS (Sigma, USA) and MSU crystals (Sigma, USA; 0.5 or 1 mg / ml) and the anti-inflammatory effects of each compound, macrophages were isolated and placed in each well of a 96-well cell culture plate at a dose of 1 x 10⁶ 6 200 μl was dispensed at a cell / ml cell count, cultured for 24 hours to allow sufficient attachment, and the concentration of each compound was administered according to each experimental condition.

[0137] In addition, to comprehensively verify the anti-inflammatory effects on macrophages induced by various inflammatory stimuli, experiments were conducted using macrophages induced by LPS and ATP 1 mM (Sigma, USA) and macrophages induced by LPS and Nigericin 10 μM (Sigma, USA), respectively, following the same procedure.

[0138] The expression levels of inflammatory cytokines IL-1β, TNF-α, IL-6 (Invitrogen, USA) or anti-inflammatory cytokine IL-10 (BioLegend, USA) were analyzed using the cell culture supernatant from all experimental conditions in accordance with the manufacturer's protocol guidelines.

[0139]

[0140] Western blot analysis

[0141] Cell lysates were prepared using RIPA cell lysis buffer at 4°C. The total cell protein concentration of each treatment group was measured by the BCA method, and 10 μg of protein samples were separated by SDS-PAGE. Proteins separated according to molecular weight were transferred to a PVDF membrane, after which protein expression was verified as shown in Figure 5. The expression level of each protein was confirmed by visualizing protein bands using an ECL detection reagent (Thermo Scientific, Rockford, IL). The density of each protein band was quantified using an internal loading control (Actin) (Santa Cruz Biotechnology, USA).

[0142]

[0143] Experimental animals

[0144] 12-week-old female C57BL / 6J mice (Damul Science, Daejeon) were reared in an environment maintained at 50±5% humidity and 23±1℃ temperature. Feed and water were provided free of charge, and the mice were used for experiments after acclimatizing to the laboratory environment for one week. The bedding used was non-allergenic, dust-free, non-toxic, absorbent, and pathogen-free, and the cages were replaced once a week to keep the animals clean.

[0145]

[0146] Establishment of an animal model of air pouch gout induced by MSU crystals

[0147] MSU crystals were prepared based on Devi et al. (STAR ​​Protocol to create a murine subcutaneous air pouch for the study of monosodium urate crystal-induced gout (5) 102888(2024)). A mouse animal model of air pouch gout was constructed using the prepared MSU crystals. On Day 1, the dorsal area was shaved and disinfected, and 5 mL of sterile air was injected subcutaneously to form an air pouch. On Day 3, an additional 3 mL of sterile air was injected into the partially deflated air pouch to inflate it again. On Day 5, a suspension of MSU crystals at a concentration of 10 mg / mL was prepared in sterile PBS. After anesthetizing the mice, 1 mL of the suspension was injected into the air pouch to induce a local inflammatory response. Sterile PBS was injected into the control group instead of MSU. Four hours after the injection of the MSU suspension, the mice were sacrificed, and a small incision was made in the skin at the site of the air pouch. 2 mL of sterile PBS was injected into the air bag, and after massaging the air bag, 1 mL of the air bag rinsing solution was aspirated with a pipette and stored for use in the experiment.

[0148]

[0149] Establishment of experimental animal groups and drug administration

[0150] Group Composition of Experimental Animals | Air Sac Administration | Substance | Route of Administration | Concentration | Amount Air Sac Alone | PBSP | BS / DMSO | Intraperitoneal | DMSO 1.7% | 5 Air Sac + MSUMS | BS / DMSO | Intraperitoneal | DMSO 1.7% | 6 FBXMSU | Febuxostat | Intraperitoneal | 25 mpk | 6 FT-0 | 33 | MSU | FT-0 | 33 | Intraperitoneal | 25 mpk | 6 FT-0 | 37 | MSU | FT-0 | 37 | Intraperitoneal | 25 mpk | 6 Total | 29

[0151] On Day 5, PBS / DMSO was administered to the control group, Febuxostat (a commercially available gout treatment) to the positive control group, and FT-033 and FT-037 to the experimental group (drug group for efficacy evaluation) via intraperitoneal injection. To induce a local inflammatory response, 1 mL of a 10 mg / mL MSU crystal suspension prepared in sterile PBS was injected into the air sac 30 minutes after drug administration.

[0152]

[0153] Analysis indicators for experimental animals

[0154] After the autopsy, the wash fluid collected from the air sac was centrifuged to separate the supernatant, and the IL-1β concentration was measured in the supernatant. To compare and analyze the proportion of blood cells, the wash fluid collected from the air sac was centrifuged to obtain the precipitate, which was then Cytospin-treated, stained with Diff-Quick, and the blood cells were observed under a microscope.

[0155]

[0156] Experimental results

[0157] Inhibitory effect on inflammation in macrophages induced by LPS and MSU crystals

[0158] Inflammatory responses caused by hyperuricemia particularly induce the activation of macrophages present throughout the body and the subsequent secretion of inflammatory cytokines. Due to the secreted inflammatory cytokines, neutrophils accumulate around the inflammatory cells, causing the inflammatory response to become excessive and leading to chronic inflammation; a representative cytokine involved in this process is IL-1β. IL-1β is synthesized in an inactive state to form a protein complex called an inflammasome, through which it becomes activated. When macrophages are exposed to inflammatory factors, NLRP3 (NLR Family Pyrin Domain Containing 3), a type of inflammasome, activates the caspase-1 enzyme to produce active IL-1β and IL-18, thereby inducing inflammation. Therefore, the inventors sought to determine whether the febuxostat derivative compound of the present invention inhibits the production of IL-1β by inhibiting the activation of the NLRP3 inflammasome.

[0159] According to the method described above, the DMEM culture medium of macrophages attached to a 96-well plate was removed, and the cells were washed once with 1 x PBS (phosphate buffered saline). Subsequently, Opti-MEM (Thermo Fisher Scientific, USA) containing 100 ng / ml LPS was applied to each well, and the cells were incubated in an incubator at 37°C for 4 hours. Then, febuxostat and each derivative were pretreated to each well at non-cytotoxic concentrations according to the treatment process diagrammed in Figure 1, and incubated for 30 minutes. The treatment concentrations were 1.25, 2.5, 5, 10, 20, and 40 μM (febuxostat, FT023, FT037, FT039, FT046); 1.25, 2.5, 5, and 10 μM; The cell was set to FT033, and additionally treated with 1 mg / ml MSU crystals (Sigma, USA), cultured in an incubator for 6 hours, and the cell culture medium was collected. The concentration of IL-1β cytokine (Invitrogen, USA) in the collected cell culture medium was measured using ELISA (enzyme-linked immunosorbent assay).

[0160] As a result of measurement, febuxostat and each derivative (FT023, FT033, FT037, FT039, FT046) reduced the expression of IL-1β cytokine in macrophages treated under inflammation-inducing conditions (LPS + MSU crystal treatment) in a concentration-dependent manner. Finally, the five derivatives of the present invention showed IL-1β expression inhibition efficiency equivalent to or better than that of febuxostat at the same concentration (Fig. 2b).

[0161]

[0162] Changes in the expression of inflammation-related cytokines in macrophages with inflammation induced by LPS and MSU crystals

[0163] In order to comprehensively evaluate the regulatory effects of febuxostat-based derivatives on the expression of various inflammatory / anti-inflammatory cytokines, the expression of representative inflammatory cytokines IL-1β, TNF-α, IL-6, and anti-inflammatory cytokine IL-10 was examined at optimal concentrations of five derivatives (FT023, FT033, FT037, FT039, FT046) in an inflammatory response induced by stimulation of macrophages with LPS and MSU crystals (0.5 or 1 mg / ml). As a result, it was confirmed that IL-1β was significantly reduced by all five derivatives (Figs. 3b and 4b), and FT039 significantly reduced TNF-α expression (Figs. 3c and 4c). Furthermore, five derivatives significantly reduced IL-6 expression, and among them, four derivatives—FT023, FT033, FT037, and FT046—reduced IL-6 expression even more than the positive control, febuxostat (Figs. 3d and 4d). These results confirmed that the derivatives of the present invention are efficient anti-inflammatory agents capable of specifically reducing inflammasome activity and inflammatory mechanisms related to IL-1β.

[0164]

[0165] Confirmation of the mechanism of inflammation inhibition by each compound in macrophages induced by LPS and MSU crystals

[0166] To evaluate the mechanism of inhibition of inflammatory responses induced by LPS and MSU crystals using FT033, FT037, and FT046, which showed the most superior anti-inflammatory effects in macrophages, the expression of target factors related to the NLRP3 inflammasome mechanism of action was measured by Western blot. As a result, treatment with FT037 derivatives significantly reduced the expression of Caspase-1 (Cell Signaling, USA) and Caspase-11 [activated forms; Caspase-1(p20) (Cell Signaling, USA), Caspase-11(p30) (Cell Signaling, USA)], which are representative factors induced by inflammatory responses, and it was confirmed that the expression of IL-1β (Cell Signaling, USA), the final inflammatory response product regulated by these factors, was further reduced (Figs. 5a and 5b).

[0167]

[0168] Verification of additional mechanisms of action

[0169] To determine whether the anti-inflammatory effect of the compounds of the present invention in LPS-induced macrophages acts specifically only on the representative NLRP3 inflammasome inflammatory mechanism or affects other signaling pathways, macrophages were stimulated with LPS for 4 hours, treated with each derivative compound, and confirmed by measuring the expression of TNF-α and IL-6 cytokines related to other inflammatory mechanisms after 6 hours (Fig. 6a). As a result, it was confirmed that FT033 and FT037 did not affect other signaling mechanisms (Fig. 6b). This is because in specific diseases where the NLRP3 inflammasome is involved in the pathological mechanism (CAPS, catastrophic antiphospholipid syndrome), gout, type 2 diabetes, etc.), inhibiting other signaling pathways can lead to unnecessary immunosuppression or a reduction in inflammatory response, which may cause safety issues in the development of gout treatments; however, it is expected that there will be no problems when developing NLRP3 inflammasome inhibitors using these derivatives.

[0170]

[0171] Evaluation of the efficacy of derivative compounds in a gout animal model

[0172] A mouse pneumatocystic gout model was constructed using 12-week-old female C57BL / 6J mice, and the inhibitory effect on inflammatory response was further confirmed using two compounds (FT033 and FT037) among the derivative compounds of the present invention that exhibited the best anti-inflammatory activity (Fig. 7a). Through mouse necropsy, the lavage fluid collected from the pneumatocysts was centrifuged to separate the supernatant. As a result of measuring neutrophils in this supernatant, it was confirmed that the FT033 administration group (G4), which is known to specifically increase at the site of inflammation, showed a neutrophil-decreasing tendency compared to G2, and in particular, a significant decrease was confirmed in the FT037 administration group (G5) (Fig. 7b). Furthermore, the decrease in neutrophils was verified through a cytospin experiment (Fig. 7d). This suggests a high possibility that FT033 and FT037 can inhibit the inflammatory response induced by MSU crystals.

[0173]

[0174] Changes in the expression of inflammation-related cytokines in macrophages with inflammation induced by LPS and ATP

[0175] Inflammatory responses induced by LPS and ATP particularly induce the activation of macrophages present throughout the body and the subsequent secretion of inflammatory cytokines. Following the method described above, the DMEM culture medium of macrophages attached to a 96-well plate was removed, and the cells were washed once with 1 x PBS (phosphate buffered saline). Subsequently, Opti-MEM (Thermo Fisher Scientific, USA) containing 100 ng / ml LPS was applied to each well, and the cells were incubated in an incubator at 37°C for 4 hours. Then, febuxostat and each derivative were pretreated to each well at non-cytotoxic concentrations according to the treatment process diagrammed in Fig. 8, and incubated for 30 minutes. The treatment concentrations were 1.25, 2.5, 5, 10, 20, and 40 μM (febuxostat, FT023, FT037, FT039, FT046); The concentrations were set to 1.25, 2.5, 5, and 10 μM (FT033), and additionally treated with ATP (Sigma, USA). The cells were incubated in an incubator for 1 hour, and the cell culture medium was collected. The concentration of IL-1β cytokine (Invitrogen, USA) in the collected cell culture medium was measured using ELISA (enzyme-linked immunosorbent assay).

[0176] As a result of the measurements, febuxostat failed to reduce IL-1β expression under inflammation-inducing conditions (LPS + ATP treatment), whereas each derivative (FT023, FT033, FT037, FT039, FT046) reduced IL-1β cytokine expression levels at concentrations of 10, 20, or 40 μM in macrophages treated with each derivative. Finally, the five derivatives of the present invention demonstrated IL-1β expression inhibition efficiency equivalent to or better than that of febuxostat at the same concentration (Fig. 9). Furthermore, by setting the concentration effective for inhibiting the inflammatory response (LPS + ATP) of each derivative and evaluating the effects of regulating the expression of various inflammatory / anti-inflammatory cytokines, it was found that while the expression of inflammatory cytokines TNF-α and IL-6 was not affected (Figs. 10b and 10c), all five derivatives significantly reduced the expression of the representative inflammatory cytokine IL-1β compared to febuxostat (Fig. 10a). These results reaffirmed that the derivatives of the present invention are anti-inflammatory agents capable of specifically reducing inflammasome activity and the IL-1β inflammatory mechanism.

[0177]

[0178] Confirmation of the mechanism of inflammation inhibition by each compound in LPS and ATP-induced macrophages

[0179] To evaluate the mechanism of inhibition of LPS and ATP-induced inflammatory responses using FT033, FT037, and FT046, which showed the most superior anti-inflammatory effects in macrophages, the expression of target factors related to the NLRP3 inflammasome mechanism of action was measured by Western blot (Fig. 11a). As a result, FT033 and FT046 reduced the expression of Caspase-1 (p20), and in particular, treatment with the FT037 derivative significantly reduced the expression of Caspase-1 [activated form; Caspase-1 (p20)], a representative factor induced by inflammatory responses. It was also confirmed through cytokine measurements that the expression of IL-1β, the final inflammatory response product regulated by these factors, was further reduced (Figs. 11b and 11c).

[0180]

[0181] Changes in the expression of inflammation-related cytokines in macrophages with inflammation induced by LPS and nigericin

[0182] To evaluate the mechanism of inhibition of inflammatory responses induced by LPS and nigerisin using FT033, FT037, and FT046, which showed the most superior anti-inflammatory effects in macrophages, the expression of target factors related to the NLRP3 inflammasome mechanism of action was measured by Western blot. As a result, compared to other inflammatory response conditions (LPS + MSU or LPS + ATP), FT037 showed a small decrease in Caspase-1 (p20) expression, whereas FT033 and FT046 significantly reduced Caspase-1 (p20) expression. In particular, treatment with the three derivatives significantly reduced the expression of IL-1β, the final inflammatory response product regulated by Caspase-1 [activated form; Caspase-1 (p20)], a representative factor induced by inflammation. Furthermore, a significant decrease in IL-1β expression was also confirmed through cytokine measurements (Figs. 12b and 12c). Finally, the derivatives of the present invention demonstrated IL-1β expression inhibition efficiency equivalent to or better than febuxostat at the same concentration.

[0183]

[0184] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. Compound represented by the following chemical formula 1: Chemical formula 1 In the above formula, R1 is a C1-C7 alkyl; L1 is a direct bond or -NH-, A is a heteroaryl of a 5-9 ring that is unsubstituted or substituted with one or more substituents selected from the group consisting of C1-C5 alkyl and -NR3R4 (R3 and R4 are each independently hydrogen or C1-C7 alkyl); and R2 is a C1-C5 hydroxyalkyl or -COOR5 (R5 is hydrogen or C1-C3 alkyl).

2. A compound according to claim 1, wherein A is an unsubstituted or substituted heteroaryl of a 5- to 9-membered ring (R3 is hydrogen and R4 is a C1-C7 alkyl).

3. A compound according to claim 1, characterized in that the heteroaryl of the pentagonal-nine-membered ring is selected from the group consisting of benzothiazole, thiophene, furan, pyrrole, and imidazopyridine.

4. A compound according to claim 3, wherein, when the heteroaryl of the pentagon-nine-membered ring is a benzothiazole, the L1 is -NH-.

5. A compound according to claim 3, wherein, when the heteroaryl of the 5-9-membered ring is furan, the R2 is C1-C5 hydroxyalkyl.

6. A compound according to claim 3, wherein, when the heteroaryl of the pentagon-nine-membered ring is imidazopyridine, the heteroaryl of the pentagon-nine-membered ring is substituted with -NR3R4 (R3 is hydrogen and R4 is C1-C7 alkyl).

7. A compound according to claim 1, characterized in that the compound represented by Chemical Formula 1 is selected from the group consisting of compounds represented by Chemical Formulas 2 to 6 below: Chemical formula 2 Chemical formula 3 Chemical formula 4 Chemical formula 5 Chemical formula 6 .

8. A composition for the prevention or treatment of hyperuricemia comprising, as an active ingredient, a compound of any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

9. A composition according to claim 8, characterized in that the hyperuricemia is gout.

10. A functional food composition for the improvement or prevention of hyperuricemia comprising, as an active ingredient, a compound of any one of claims 1 to 7 or a food-grade salt thereof.

11. A composition according to claim 10, characterized in that the hyperuricemia is gout.

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