Novel febuxostat derivative and composition for host-directed treatment comprising same

Abstract

The present invention relates to a novel febuxostat derivative compound and to a composition for preventing or treating a nontuberculous mycobacterial infection, comprising same as an active ingredient. The compound of the present invention efficiently kills nontuberculous mycobacteria, including Mycobacterium shinjukuense and Mycobacterium kansasii, which exhibit low responsiveness to conventional standard therapies and a high frequency of resistance mutation occurrence, via host-directed therapy mediated by host macrophages, and thus can be effectively used as a fundamental therapeutic composition for various complications caused by nontuberculous mycobacteria infections.

Description

Novel febuxostat derivatives and host-directed therapeutic compositions comprising the same

[0001] The present invention relates to a novel Febuxostat derivative compound and a composition for host-directed therapy comprising the same as an active ingredient.

[0002]

[0003] Mycobacterium species are divided into two groups based on their degree of pathogenicity and infectivity to the host: the tuberculosis group, which includes Mycobacterium tuberculosis and Mycobacterium leprae, which are obligate pathogens, and the nontuberculous mycobacteria (NTM), which are opportunistic pathogens.

[0004] Nontuberculous mycobacteria are widely distributed in nature, and the virulence of causing diseases varies depending on the species. For example, M. kansasii, M. avium complex, and M. abscessus are relatively virulence-prone, whereas M. fortuitum is relatively less virulence-prone. Diseases caused by nontuberculous mycobacteria present with four characteristic clinical signs: pulmonary disease, lymphadenitis, skin, soft tissue, and bone infections, and disseminated disease.

[0005] Mycobacterium shinjukuense is a novel nontuberculous mycobacterium first reported in Japan in 2011. Like previously known nontuberculous mycobacteria causing lung disease, it is slow-growing and can cause chronic lung infections. Although cases of infection with Mycobacterium shinjukuense are still few, one patient has been reported in Korea. Unlike well-known nontuberculous mycobacteria, there is no established standard treatment for Mycobacterium shinjukuense infection. However, chemotherapy for approximately one year using drugs such as isoniazid, rifampicin, ethambutol, or clarithromycin has been reported to be effective.

[0006] Patients with lung disease caused by nontuberculous mycobacteria (NTM) are treated with the antituberculosis drug rifampicin, a macrolide such as clarithromycin or azithromycin (AZM), and ethambutol, according to the ATS / IDSA and BTS guidelines. However, the efficacy of these antibiotics remains unclear, and mutant NTMM strains resistant to macrolides are being detected. Therefore, active research is underway to discover more effective therapeutic agents.

[0007] Meanwhile, in the field of tuberculosis research, research is actively underway on the possibility of treating tuberculosis through a synergistic effect with existing anti-tuberculosis drugs through a treatment method (host-directed therapy; HDT) that increases the immune response of a host infected with tuberculosis bacteria through metabolites that enable the regulation of the immune / metabolic response of host cells.

[0008]

[0009] Numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the present invention.

[0010]

[0011] The present inventors have devoted extensive research efforts to develop a small-molecule therapeutic agent that can effectively inhibit nontuberculous mycobacteria (NTM), which exhibit high infection rates and low antibiotic treatment responsiveness, and lead to lung disease, lymphadenitis, skin, soft tissue, and bone infections, resulting in high mortality worldwide. As a result, we discovered that a febuxostat derivative compound represented by the following chemical formula 1 can significantly inhibit the growth of infected nontuberculous mycobacteria by activating the host's macrophage-mediated immune response.

[0012] Accordingly, the purpose of the present invention is to provide a novel derivative compound of Febuxostat and a composition for preventing or treating tuberculosis containing the same as an active ingredient.

[0013]

[0014] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0015]

[0016] According to one aspect of the present invention, the present invention provides a compound represented by the following chemical formula 1:

[0017] Chemical Formula 1

[0018]

[0019] In the above chemical formula, R1 is C1-C7 alkyl; R2 is hydrogen or C1-C5 alkyl, and L1 is a 5-membered-9-membered heteroaryl ring which is unsubstituted or substituted with one or more substituents selected from the group consisting of C1-C7 alkyl and NR3R4 (R3 and R4 are each independently hydrogen or C1-C7 alkyl).

[0020] The present inventors have diligently researched and developed a small-molecule therapeutic agent that can effectively suppress nontuberculous mycobacteria, which exhibit high infection rates and low antibiotic treatment responsiveness, and lead to lung disease, lymphadenitis, skin, soft tissue, and bone infections, resulting in a high mortality rate worldwide. As a result, the present invention was completed by discovering that the febuxostat derivative compound of Chemical Formula 1 can significantly suppress the growth of infected nontuberculous mycobacteria by activating the host's macrophage-mediated immune response.

[0021] The term “alkyl” as used herein means a straight-chain or branched saturated hydrocarbon group, and includes, for example, methyl, ethyl, propyl, isopropyl, etc. C1-C5 alkyl means an alkyl group having an alkyl unit having 1 to 5 carbon atoms, and when C1-C5 alkyl is substituted, the carbon number of the substituent is not included.

[0022] As used herein, the term “heteroaryl” refers to a heterocyclic aromatic group containing oxygen, sulfur, or nitrogen as a heteroatom within the ring. The number of heteroatoms contained within the ring is 1-3, and specifically 1-2. The term “penta- to non-penta-membered ring heteroaryl” refers to a heteroaryl having 5 to 9 ring atoms including both carbon and heteroatoms, and the ring may be a monocycle or a bicycle.

[0023] According to a specific embodiment of the present invention, R1 is C2-C5 alkyl; R2 is hydrogen or C1-C3 alkyl. More specifically, R1 is C3-C5 alkyl; R2 is hydrogen or C1-C2 alkyl, and most specifically, R1 is C4 alkyl; R2 is hydrogen or C1 alkyl.

[0024] According to a specific embodiment of the present invention, when L1 is a 5-membered-9-membered heteroaryl substituted with C1-C7 alkyl, the C1-C7 alkyl is C2-C5 alkyl, more specifically C2-C4 alkyl, and most specifically C2 alkyl or C4 alkyl.

[0025] According to a specific embodiment of the present invention, R3 and R4 are each independently hydrogen or C2-C5 alkyl, and R3 and R4 are not both hydrogen. More specifically, R3 and R4 are each hydrogen and C3-C5 alkyl, and most specifically, R3 and R4 are each hydrogen and C4 alkyl.

[0026] According to a specific embodiment of the present invention, the heteroaryl of the 5-membered-9-membered ring is selected from the group consisting of thiophene, indole, benzimidazole, and imidazopyridine.

[0027]

[0028] More specifically, when the heteroaryl of the pentagonal-nine-membered ring is benzimidazole, R2 is C1-C3 alkyl, and most specifically C1 alkyl.

[0029]

[0030] According to a specific embodiment of the present invention, the compound represented by the chemical formula 1 is selected from the group consisting of compounds represented by the following chemical formulas 2 to 8:

[0031] Chemical Formula 2 Chemical Formula 3

[0032]

[0033] Chemical Formula 4 Chemical Formula 5

[0034]

[0035] Chemical Formula 6 Chemical Formula 7

[0036]

[0037] Chemical Formula 8

[0038] .

[0039]

[0040] According to another aspect of the present invention, the present invention provides a composition for preventing or treating a nontuberculous mycobacterial infection disease, comprising the compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient.

[0041] According to another aspect of the present invention, the present invention provides a method for preventing or treating a nontuberculous mycobacterial infection disease, comprising a step of administering to a subject the compound of the present invention or a pharmaceutically acceptable salt thereof.

[0042] In this specification, the term “nontuberculous mycobacteria (NTM) infection disease” is a comprehensive term that includes all clinical symptoms caused by infection with pathogens of the genus Mycobacterium belonging to nontuberculous mycobacteria, including, but not limited to, pulmonary disease, lymphadenitis, skin, soft tissue, and bone infections, and disseminated diseases, and includes all pathological conditions that are directly or indirectly caused by infection with nontuberculous mycobacteria.

[0043] According to a specific embodiment of the present invention, nontuberculous mycobacteria that can be prevented or treated with the composition of the present invention include Mycobacterium shinjukuense, Mycobacterium kansasii, Mycobacterium avium, Mycobacterium abscessus, Mycobacterium abscessus subsp. massiliense, Mycobacterium intracellurare, Mycobacterium chimaera, Mycobacterium Scrofulaceum, Mycobacterium chelonae, Mycobacterium fortuitum, Selected from the group consisting of Mycobacterium peregrinum, Mycobacterium ulcerans, Mycobacterium marinum, Mycobacterium Genevans, Mycobacterium simiae, Mycobacterium terrae, Mycobacterium nonchromogenicum, Mycobacterium celatum, Mycobacterium gordonae, Mycobacterium szulgai, Mycobacterium mucogenicum, and Mycobacterium aubagnens.

[0044] More specifically, the nontuberculous mycobacterium is Mycobacterium shinjukuense or Mycobacterium kansasii.

[0045] The term “prevention” as used herein means inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed as having the disease or condition but is susceptible to such disease or condition.

[0046] As used herein, the term “treatment” means (a) inhibiting the development of a disease, condition, or symptom; (b) alleviating the disease, condition, or symptom; or (c) eliminating the disease, condition, or symptom. When the composition of the present invention is administered to a subject, it acts to inhibit the development of symptoms caused by nontuberculous mycobacterial infection, eliminate them, or alleviate them by activating a macrophage-mediated immune response in the infected subject. Therefore, the composition of the present invention can be a composition for treating these diseases on its own, or can be used as an adjuvant treatment for tuberculosis when administered together with other antibiotics. Accordingly, the terms “treatment” or “therapeutic agent” as used herein include the meaning of “adjuvant treatment” or “adjuvant treatment agent.” Antibiotics that can be administered together with the composition of the present invention include, for example, rifamficin, rifapentine, isoniazid, pyrazinamide, ethambutol, streptomycin, fluoroquinolone, kanamycin, cycloserine, prothionamide, levofloxacin, moxifloxacin, ofloxacin, rifabutin, capeomycin, amikacin, ciprofloxacin, protionamide, ethionamide, cycloserine, These include, but are not limited to, thioacetazone, clofazimine, amoxicillin / clavulanate, clarithromycin, azithromycin, and linezolid.

[0047] As used herein, the term “administration” or “administer” refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the body of the subject.

[0048] In the present invention, the term “therapeutically effective amount” means the content of a composition containing a pharmacological ingredient in the composition sufficient to provide a therapeutic or preventive effect to a subject to whom the pharmaceutical composition of the present invention is to be administered, and includes a “prophylactically effective amount”.

[0049]

[0050] The term “subject” as used herein includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus macaque. Specifically, the subject of the present invention is a human.

[0051] As used herein, the term “pharmaceutically acceptable salt” includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, hydrobromic 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, and the like. Salts derived from suitable bases may include alkali metals such as sodium, alkaline earth metals such as magnesium, and ammonium.

[0052] When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.

[0053] 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 further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, preservatives, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0054] The pharmaceutical composition of the present invention can be administered orally or parenterally, and specifically can be administered orally, intravenously, or intramuscularly.

[0055] The appropriate dosage of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. The preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.001-100 mg / kg for adults.

[0056] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and the like. In this case, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0057] According to another aspect of the present invention, the present invention provides a functional food composition for improving or preventing nontuberculous mycobacterial infections, comprising the compound of the present invention or a food-related acceptable salt thereof as an active ingredient.

[0058] According to another aspect of the present invention, the present invention provides a method for improving or preventing a nontuberculous mycobacterial infection disease, comprising a step of administering to a subject the compound of the present invention or a food-related acceptable salt thereof.

[0059] The chemical formula 1 compound used in the present invention and the nontuberculous mycobacterial infection disease that can be improved or prevented by using the same have already been described above, so description thereof is omitted to avoid excessive duplication.

[0060] In this specification, the term “food-acceptable salt” means a salt in a form that can be used in a food composition among salts in which cations and anions are bonded by electrostatic attraction, and specific examples thereof include the examples of “pharmaceutically acceptable salts” described above.

[0061] When the composition of the present invention is manufactured as a food composition, it may contain not only the compound of the present invention as an active ingredient, but also carbohydrates, seasonings, and flavoring agents that are commonly 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 flavoring agents, natural flavoring agents [thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is manufactured as a drink, in addition to the pine bark extract, which is an 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. can be additionally included.

[0062]

[0063] The features and advantages of the present invention are summarized as follows:

[0064] (a) The present invention provides a novel derivative compound of Febuxostat and a composition for preventing or treating nontuberculous mycobacterial infections, comprising the same as an active ingredient.

[0065] (b) The compound of the present invention can be effectively used as a fundamental treatment composition for various complications caused by nontuberculous mycobacterial infections by efficiently killing nontuberculous mycobacteria, including Mycobacterium shinjukuense and Mycobacterium kansasii, which have low responsiveness to conventional standard treatments and a high frequency of occurrence of resistant mutations, through host-directed therapy mediated by host macrophages.

[0066]

[0067] Figure 1 is a drawing showing the results of analyzing the drug susceptibility of nontuberculous mycobacteria to FBX derivatives discovered in the present invention using a liquid dilution method, and the experimental results for Mycobacterium kansashii (Figure 1a) and Mycobacterium shinjukuense (Figure 1b) are shown, respectively. PC: positive control; NC: negative control.

[0068] Figure 2 is a drawing showing the results of measuring the growth inhibitory effect of the FBX derivative of the present invention on non-tuberculous mycobacteria in macrophages, and shows the experimental results for Mycobacterium kansashi (Figure 2a) and Mycobacterium shinjukuense (Figure 2b), respectively.

[0069]

[0070] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0071] Example

[0072] Synthetic example

[0073] Synthesis of compound FT017

[0074]

[0075] Compound E185 mg (0.91 mmol) and methyl 3,4-diaminobenzoate 182 mg (1.10 mmol) were dissolved in 10 mL of DMF, and then NaHSO3 133 mg (1.27 mmol) was added. The reaction mixture was heated and stirred at 100 °C for 16 hours. After cooling the reaction mixture to room temperature, 10 mL of water was added dropwise, and extracted twice with 10 mL of EtOAc. The obtained EtOAc layer was dried over MgSO4, and the solid was removed by filtering, and the filtrate was distilled under reduced pressure. The obtained mixture was separated and purified by silica gel column chromatography (Hex:EtOAc = 5:1 volume ratio) to obtain FT017 230 mg (0.66 mmol, yield 72%).

[0076] 1 H NMR (400MHz, DMSO-d6) δ13.27(brs, 1H), 8.49-8.43(m, 2H), 8.18(s, 1H), 7.85(dd, J=8.4, 1.6Hz, 1H), 7.68(d, J=8.5Hz, 1H), 7.48(d, J=8.9Hz, 1H), 4.03 (d, J=6.5Hz, 2H), 3.88(s, 3H), 2.16-2.06(m, 1H), 1.05(s, 3H), 1.03(s, 3H).

[0077]

[0078] Synthesis of compound FT033

[0079]

[0080] (i) Synthesis of compound M

[0081] Compound B 245 mg (0.81 mmol), Compound L 120 mg (0.54 mmol), Pd(PPh3) 463 mg (0.054 mmol), and Na2CO3 115 mg (1.09 mmol, 2 M aqueous solution) were dissolved in 5 mL of 1,4-dioxane solvent, and stirred and heated at 90°C for 6 hours. After cooling the reaction mixture to room temperature, 10 mL of sat. NH4Cl(aq) was added dropwise, and extracted twice with 10 mL of EtOAc. The obtained EtOAc layer was dried over MgSO4, and the solid was removed by filtering, and the filtrate was distilled under reduced pressure. The obtained 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%).

[0082] (ⅱ) Synthesis of compound FT033

[0083] Compound M50 mg (0.16 mmol) was dissolved in THF:MeOH:H2O (1 mL, 2 mL, 1 mL), NaOH 76 mg (1.90 mmol) was added, and the mixture was heated and stirred at 60°C for 10 hours. After cooling the reaction mixture to room temperature, acidic ion exchange resin was added to adjust the pH to 3-4, filtered, and the ion exchange resin was washed with EtOAc. The entire filtrate was collected and distilled under reduced pressure to obtain FT03340 mg (0.13 mmol, yield 84%).

[0084] 1 H NMR (600 MHz, Methanol-d4) δ7.91(d, J=5.3Hz, 1H), 7.86(tt, J=7.7, 2.9 Hz, 1H), 7.69(q, J=4.0, 3.5Hz, 1H), 7.36(q, J=4.6, 3.9Hz, 1H), 7.18(t, J=8.2Hz, 1H), 3.91(d, J=6.3Hz, 2H), 2.18-2.08(m,1H), 1.07(s, 3H), 1.06(s, 3H).

[0085]

[0086] Synthesis of compound FT041

[0087]

[0088] (i) Synthesis of compound O

[0089] Compound B 358 mg (1.19 mmol), Compound N 300 mg (0.849 mmol), Pd(PPh3) 498 mg (0.085 mmol), and Na2CO3 180 mg (1.70 mmol, 2 M aqueous solution) were dissolved in 8 mL of DMF solvent and stirred at 95°C for 16 hours. After cooling the reaction mixture to room temperature, 10 mL of sat. NH4Cl(aq) was added dropwise and extracted twice with 10 mL of EtOAc. The obtained EtOAc layer was dried over MgSO4, the solid was removed by filtering, and the filtrate was distilled under reduced pressure.

[0090] The obtained mixture was dissolved in 5 mL of CH2Cl2 solvent, and 1 mL of trifluoroacetic acid was added dropwise at 0°C. The reaction mixture was stirred at room temperature for 2 hours, then 10 mL of sat. NaHCO3(aq) was added dropwise, and extracted twice with 10 mL of EtOAc. The obtained EtOAc layer was dried over MgSO4, and the solid was removed by filtering, and the filtrate was distilled under reduced pressure. The obtained mixture was separated and purified by silica gel column chromatography (Hex: EtOAc = 1:1 volume ratio) to obtain 31 mg (0.38 mmol, yield 44%) of compound O1.

[0091] (ⅱ) Synthesis of compound FT041

[0092] Dissolve 30 mg (0.086 mmol) of compound O in THF:H2O (1 mL, 1 mL) solvent and add LiOH . 15 mg (0.34 mmol) of H2O was added 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, filtered, and the ion exchange resin was washed with EtOAc. The entire filtrate was collected and distilled under reduced pressure to obtain FT04122 mg (0.066 mmol, yield 76%).

[0093] 1 H NMR (600 MHz, Chloroform-d / Methanol-d4(4:1))δ8.49(d, J=1.5Hz, 1H), 7.85(dd, J=8.6, 1.7Hz, 1H), 7.76(dd, J=8.6, 2.4Hz, 1H), 7.72(d, J=2.4Hz, 1H), 7.39(d, J=8.4Hz, 1H),7.33(s, 1H), 6.99(d, J=8.7Hz, 1H), 3.82(d, J=6.4Hz, 2H), 2.18-2.07(m, 1H), 1.02(s, 3H), 1.01(s, 3H).

[0094]

[0095] Synthesis of compound FT046

[0096]

[0097] (i) Synthesis of compound U

[0098] Compound I241 mg (1.18 mmol) and compound T150 mg (0.985 mmol) were dissolved in 5 mL of MeOH solvent, and then 90 mg (1.08 mmol) of pivalonitrile and 38 mg (0.197 mmol) of p-toluenesulfonic acid monohydrate were added. The reaction mixture was stirred at room temperature for 16 hours, 10 mL of sat. NaHCO3(aq) was added dropwise, and extracted twice with 10 mL of EtOAc. The obtained EtOAc layer was dried over MgSO4, and the solid was removed by filtering, and the filtrate was distilled under reduced pressure. The obtained 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%).

[0099] (ⅱ) Synthesis of compound FT046

[0100] Compound U30 mg (0.071 mmol) was dissolved in THF: MeOH: H2O (0.5 mL, 1 mL, 0.5 mL) solvent, NaOH 34 mg (0.86 mmol) was added, and the mixture was heated and stirred at 60°C for 10 hours. After cooling the reaction mixture to room temperature, acidic ion exchange resin was added to adjust the pH to 3–4, filtered, and the ion exchange resin was washed with EtOAc. The entire filtrate was collected and distilled under reduced pressure to obtain FT04622 mg (0.054 mmol, yield 76%).

[0101] 1 H NMR (600 MHz, Chloroform-d / Methanol-d4(4:1))δ8.97-8.88(m, 1H), 8.21(d, J=2.3Hz, 1H), 8.12(dd, J=8.9, 2.2Hz, 1H), 7.73(d, J=9.3Hz, 1H), 7.47 (d, J=9.1Hz, 1H), 7.01(d, J=8.9Hz, 1H), 3.83(d, J=6.5Hz, 2H), 2.18-2.03(m, 1H), 1.03(s, 3H), 1.02(s, 3H), 1.01(s, 9H).

[0102]

[0103] Synthesis of compound FT060

[0104]

[0105] FT017450 mg (1.32 mmol) was dissolved in 10 mL of DMF solvent, then the temperature was lowered to 0℃, and Cs2CO3629 mg (1.93 mmol) and iodoethane155 μL (0.193 mmol) were added dropwise. The reaction mixture was stirred at room temperature for 16 hours, and the solvent was removed by distillation under reduced pressure. 10 mL of water was added dropwise to the obtained mixture, and it was extracted twice with 10 mL of CH2Cl2. The obtained CH2Cl2 layer was dried over MgSO4, and the solid was removed by filtering, and the filtrate was distilled under reduced pressure. The obtained mixture was separated and purified by silica gel column chromatography (CHCl3: MeOH = 100:1 volume ratio) to obtain 140 mg (0.39 mmol, yield 30%) of compound X and 150 mg (0.40 mmol, yield 31%) of FT060.

[0106] 1 H NMR (600 MHz, Chloroform-d) δ 8.46(s, 1H), 8.01(dd, J=8.5, 1.4 Hz, 1H), 7.89(s, 1H), 7.88(d, J=2.3 Hz, 1H), 7.40(d, J=8.5 Hz, 1H), 7.08(d, J=9.4 Hz, 1H), 4.26(q, J=7.3 Hz, 2H), 3.92(s, 3H), 3.89(d, J=6.5 Hz, 2H), 2.23-2.14 (m, 1H), 1.45(t, J=7.2 Hz, 3H), 1.08(s, 3H), 1.07 (s, 3H).

[0107]

[0108] Synthesis of compounds FT064 and FT065

[0109]

[0110] FT01740 mg (0.114 mmol) was dissolved in 2 mL of DMF solvent, the temperature was lowered to 0 °C, and 112 mg (0.34 mmol) of Cs2CO3, 19 mg (0.11 mmol) of potassium iodide, and 37 μL (0.34 mmol) of isobutyl bromide were added dropwise. The reaction mixture was heated and stirred at 80 °C for 16 hours, and the solvent was removed by distillation under reduced pressure. 10 mL of water was added dropwise to the obtained mixture, and it was extracted twice with 10 mL of CH2Cl2. The obtained CH2Cl2 layer was dried over MgSO4, the solid was removed by filtering, and the filtrate was distilled under reduced pressure. The obtained mixture was separated and purified by silica gel column chromatography (CHCl3: MeOH = 100:1 volume ratio) to obtain FT0648 mg (0.020 mmol, yield 17%) and FT06512 mg (0.030 mmol, yield 26%).

[0111] FT064: 1 H NMR (600 MHz, Chloroform-d) δ 8.17(d, J = 1.5 Hz, 1H), 8.04 (dd, J = 8.5, 1.5 Hz, 1H), 8.00-7.95(m, 1H), 7.92(d, J = 2.2 Hz, 1H), 7.84 (d, J = 8.5 Hz, 1H), 7.13(d, J = 8.8 Hz, 1H), 4.14(d, J = 7.7 Hz, 2H), 3.97 (s, 3H), 3.93(d, J = 6.5 Hz, 2H), 2.27-2.19(m, 1H), 2.19-2.12(m, 1H), 1.11 (s, 3H), 1.10 (s, 3H), 0.78 (s, 3H), 0.76 (s, 3H).

[0112] FT065: 1H NMR (600 MHz, Chloroform-d) δ 8.50 (d, J = 1.2 Hz, 1H), 8.05 (dd, J = 8.6, 1.5 Hz, 1H), 7.97-7.88 (m, 2H), 7.43 (d, J = 8.6 Hz, 1H), 7.11 (d, J = 8.7 Hz, 1H), 4.09 (d, J = 7.6 Hz, 2H), 3.95(s, 3H), 3.93 (d, J = 6.5 Hz, 2H), 2.26-2.17(m, 1H), 2.15-2.06(m, 1H), 1.11(s, 3H), 1.10(s, 3H), 0.77 (s, 3H), 0.76 (s, 3H).

[0113] Synthetic Febuxostat Derivatives Derivative Structure Molecular Weight IUPAC Name FT017 (Formula 2) 349.39 Methyl 2-(3-cyano-4-isobutoxyphenyl)-1H-benzo[d]imidazole-6-carboxylate FT033 (Formula 3) 301.365-(3-cyano-4-isobutoxyphenyl)thiophene-2-carboxylic acid FT041 (formula 4) 334.383-(3-cyano-4-isobutoxyphenyl)-1H-indole-5-carboxylic acid FT046 (formula 5) 406.493-(tert-butylamino)-2-(3-cyano-4-isobutoxyphenyl)imidazo[1,2-a]pyridine-6-carboxylic acid FT060 (Formula 6) 377.44 Methyl 2-(3-cyano-4-isobutoxyphenyl)-1-ethyl-1H-benzo[d]imidazole-5-carboxylate FT064 (Formula 7) 405.5 Methyl 2-(3-cyano-4-isobutoxyphenyl)-1-isobutyl-1H-benzo[d]imidazole-6-carboxylate FT065 (Formula 8) 405.5 Methyl 2-(3-cyano-4-isobutoxyphenyl)-1-isobutyl-1H-benzo[d]imidazole-5-carboxylate

[0114]

[0115] Preparation Example 1: Cultivation of nontuberculous mycobacteria

[0116] To cultivate nontuberculous mycobacteria, M. kansasii standard strain (ATCC_12478) and M. shinjukuense CI #2 (Clinical isolate #2;), the liquid medium used was Middlebrook 7H9 medium containing 10% OADC. After inoculating the strains into the medium, they were cultured at 37°C with stirring. The cultured strains were cultured until the absorbance at 600 nm reached 0.3-0.5, and when the absorbance reached 0.005 (approximately 5 × 10 6 A MIC (Minimal Inhibitory Concentration) test was performed to confirm the inhibition of microbial growth by diluting the solution to 10 CFU / ml.

[0117]

[0118] Preparation Example 2: Measurement of minimal inhibitory concentrations (MICs)

[0119] Nontuberculous mycobacteria prepared according to Preparation Example 1 were treated with Febuxostat (FBX) and FBX-based derivatives (Table 1) based on the Clinical and Laboratory Standards Institute (CLSI) guidelines and the Resazurin microtiter assay (REMA) to analyze the susceptibility of nontuberculous mycobacteria to each drug using the liquid dilution method (Fig. 1). Specifically, FBX (600 to 1.18 μM; 0.39 to 190 μg / ml) and FBX-based derivatives were tested in the concentration range of about 600 to 1.18 μM. 100 μl was dispensed per well in a 96-well plate, and the absorbance at 600 nm was 0.005 (about 5 × 10 6The strains diluted to 100 CFU / ml were additionally inoculated into each well at 100 μl and cultured at 37°C for 2 weeks. At this time, a negative control group (Negative Control group; NC) in which neither nontuberculous mycobacteria nor drugs were inoculated was cultured as a negative control group, and a positive control group (Positive Control group; PC) in which only each nontuberculous mycobacteria was cultured was cultured as a positive control group. After 1 week of culture, 22 μl of a color-developing solution (0.02% resazurin solution) was added to each well and cultured again at 37°C for 24 hours. After 24 hours, the antibacterial activity of the drug treated was compared with the negative control group, positive control group, and FBX control group.

[0120]

[0121] Preparation Example 3: Induction of differentiation of bone marrow-derived macrophages

[0122] After isolating bone marrow cells from the mouse bone marrow, differentiation medium was prepared by adding 10% L929 cell line culture medium to high-glucose DMEM (Biowest, France) culture medium containing 10% fetal bovine serum (Biowest, France) and 1% penicillin / streptomycin (Biowest, France). Then, 10 ml of the 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. Afterwards, macrophages were obtained by adding an additional 10 ml of the differentiation medium to the petri dish and further culturing for 6 to 7 days. In subsequent experiments, the macrophages were separated using trypsin-EDTA (Biowest, France) and then 2 x 10 cells were seeded into each well of a 48-well cell culture plate. 5 Cells were seeded and cultured for 24 hours to allow sufficient attachment of macrophages to the plate.

[0123]

[0124] Preparation Example 4: Preparation of 7H10 solid medium for culturing nontuberculous mycobacteria

[0125] In a 1 L Erlenmeyer flask, 9.5 g of DifcoTM Middlebrook 7H10 agar powder (BD bioscience, USA) was added, 450 mL of distilled water was added, and mixed well. After sterilization in an autoclave at 121°C for 15 minutes, the mixture was cooled sufficiently to 60°C, 50 mL of OADC (oleic acid + albumin + dextrose + catalase; BD bioscience, USA) was added, and 23 mL was added to a 90 x 15 mm petri dish (SPL life science, Korea), and left at room temperature for one day to solidify sufficiently, and then refrigerated until used in future experiments.

[0126]

[0127] Experimental Example 1: Drug susceptibility test (MIC) of Febuxostat-based derivatives

[0128] The susceptibility to tuberculosis was evaluated using the derivatives based on febuxostat (FT017, FT033, FT041, FT046, FT060, FT064, FT065) of Table 1 discovered in the present invention according to the method described above in Preparation Example 2. As a result, as shown in Fig. 1, when compared to the positive control group (PC) cultured only with nontuberculous mycobacteria, all seven tested derivatives failed to inhibit or kill the growth of nontuberculous mycobacteria (Mycobacterium kansasii ATCC 12478, Mycobacterium shinjukuense CI#2) at all concentrations (600-1.2 μM) or at concentrations similar to febuxostat.

[0129]

[0130] Example 2: Inhibitory effect of seven derivatives on the growth of nontuberculous mycobacteria in macrophages

[0131] Nontuberculous mycobacteria (Mycobacterium kansasii ATCC 12478, Mycobacterium shinjukuense CI#2) were added to each well of the macrophages attached to a 48-well plate at an MOI (Multiplicity of infection) of 1:3 and cultured for 4 hours, and the culture medium containing the nontuberculous mycobacteria was removed. The differentiation medium of Preparation Example 3 was treated to the plate containing each of the 7 types of derivatives and cultured for 72 hours. Here, only the differentiation medium was used as the control group (pi_72h; CTL). After the culture was completed, each well of the plate was washed with 1 x PBS (phosphate buffered saline), and then 200 ㎕ of 0.05% Triton X-100 was added to each well and cultured for 10 minutes to sufficiently lyse the membrane of the macrophages. Thereafter, the lysate released from the macrophages was diluted by a factor of 1 / 100 or 1 / 1000, and 50 μl of the diluted lysate was dispensed into each petri dish of the 7H10 solid medium of Preparation Example 4, and cultured in a microbiological incubator for 2 weeks. After the culture was completed, the number of bacteria (CFU) generated in the petri dish was measured, and the results are shown in Fig. 2.

[0132] As a result of evaluating the intracellular activity according to the concentration of each derivative, although it was confirmed that there was no growth inhibitory effect on the two strains in the macrophages treated with febuxostat, but rather growth was induced, as shown in Fig. 2, compared to the control group (pi_72h; CTL), among the seven derivatives administered (FT017_1μM, FT033_80μM, FT041_80μM, FT046_80μM, FT060_20μM, FT064_30μM, FT065_80μM), three derivatives (FT046, FT060, FT65) reduced the growth of M. kansasii, and four derivatives (FT017, FT060, FT64, FT065) significantly reduced the growth of M. shinjukuense. These results confirmed that the febuxostat-based derivatives of the present invention can very effectively inhibit the growth of strains infected with the host through enhanced antibacterial action even when used at low concentrations in macrophages.

[0133]

[0134] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

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

2. A compound characterized in that in claim 1, R1 is C2-C5 alkyl; and R2 is hydrogen or C1-C3 alkyl.

3. A compound characterized in that in paragraph 1, R3 and R4 are each independently hydrogen or C2-C5 alkyl, and R3 and R4 are not hydrogen at the same time.

4. A compound according to claim 1, characterized in that the heteroaryl of the 5-membered-9-membered ring is selected from the group consisting of thiophene, indole, benzimidazole, and imidazopyridine.

5. A compound characterized in that in the fourth paragraph, when the heteroaryl of the 5-membered-9-membered ring is benzimidazole, R2 is C1-C3 alkyl.

6. In the first paragraph, the compound represented by the chemical formula 1 is a compound characterized in that it is selected from the group consisting of compounds represented by the following chemical formulas 2 to 8: Chemical Formula 2 Chemical Formula 3 Chemical Formula 4 Chemical Formula 5 Chemical Formula 6 Chemical Formula 7 Chemical formula 8 .

7. A composition for preventing or treating a nontuberculous mycobacterial infection disease, comprising a compound of any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof as an active ingredient.

8. In paragraph 7, the nontuberculous mycobacteria are Mycobacterium shinjukuense, Mycobacterium kansasii, Mycobacterium avium, Mycobacterium abscessus, Mycobacterium abscessus subsp. massiliense, Mycobacterium Intracellurare, Mycobacterium chimaera, Mycobacterium Scrofulaceum, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium A composition characterized in that the composition is selected from the group consisting of Mycobacterium peregrinum, Mycobacterium ulcerans, Mycobacterium marinum, Mycobacterium Genevans, Mycobacterium simiae, Mycobacterium terrae, Mycobacterium nonchromogenicum, Mycobacterium celatum, Mycobacterium gordonae, Mycobacterium szulgai, Mycobacterium mucogenicum and Mycobacterium aubagnens.

9. A composition according to claim 8, characterized in that the nontuberculous mycobacterium is Mycobacterium shinjukuense or Mycobacterium kansasii.

10. A functional food composition for improving or preventing nontuberculous mycobacterial infections, comprising a compound of any one of claims 1 to 6 or a food-wise acceptable salt thereof as an active ingredient.

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