Novel chi3l1 inhibitor and use thereof

Abstract

A novel CHI3L1 inhibitor provided in the present invention can efficiently inhibit or treat disease progression of nontuberculous mycobacteria-infected lung diseases by inhibiting CHI3L1. In particular, it has been identified that K284-6111 can inhibit or treat disease progression of nontuberculous mycobacteria-infected lung diseases by inhibiting CHI3L1, and by newly synthesizing derivatives of K284-6111, novel compounds having a CHI3L1 inhibitory ability superior to that of K284-6111 have been identified. Therefore, the novel CHI3L1 inhibitors provided in the present invention can inhibit or treat disease progression of nontuberculous mycobacteria-infected lung diseases by inhibiting CHI3L1.

Description

Novel CHI3L1 Inhibitor and Uses thereof

[0001] The present invention relates to the Fourth Industrial Revolution and specifically concerns innovative new drugs or next-generation biopharmaceuticals. In particular, the present invention relates to the novel synthesis of derivatives of 2-(3-[2-(1-cyclohexene-1-yl)ethyl]-6,7-dimethoxy-4-oxo-3,4-dihydro-2-quinazolinylsulfanyl)-N-(4-ethylphenyl)butanamide (K284-6111), and a method for inhibiting or treating the progression of lung disease caused by non-tuberculosis mycobacterial infection by administering the novel derivatives of K284-611 to inhibit the CHI3L1 (Chitinase 3-Like 1) protein in lung tissue.

[0002]

[0003] Species of the genus Mycobacterium are classified into two groups based on the degree of pathogenicity and infectivity to the host: the tuberculosis group, which includes obligate pathogenic bacteria such as Mycobacterium tuberculosis and Mycobacterium leprae, and nontuberculous mycobacteria (NTM), which are opportunistic species excluding these.

[0004] The aforementioned non-tuberculous mycobacteria are widely distributed in nature, and the causative power of disease varies depending on the species. Specifically, M. avium complex (MAC), M. abscessus (MAB), and M. kansasii have a relatively high causative power, while M. fortuitum has a relatively low causative power. Diseases caused by non-tuberculous mycobacteria are broadly classified into four characteristic clinical syndromes: lung disease, lymphadenitis, skin, soft tissue, and bone infections, and disseminated disease. Among these, lung disease is the most common form, accounting for more than 90% of all diseases.

[0005] Reports of lung diseases caused by non-tuberculous mycobacteria have been increasing since 2000, and currently, lung diseases caused by MAC are the most frequently reported in most developed countries. In Korea, lung diseases caused by MAC are reported to account for approximately 70-80% of all causative agents of non-tuberculous mycobacterial lung diseases, with MAB reported as the second most observed causative agent. Lung diseases caused by the aforementioned non-tuberculous mycobacteria manifest with symptoms such as cough, fever, hemoptysis, and sputum.

[0006] The present invention is a result derived as part of the research project titled "Identification of the role of Chitinase-3-like protein 1 as an exacerbation determinant of non-tuberculous mycobacterial lung disease" (Project No. 27100886119, Project No. 00405542), which was conducted as a group research support (Global Basic Research Laboratory) project funded by the Ministry of Science and ICT of the Republic of Korea through the National Research Foundation of Korea to the Industry-Academic Cooperation Foundation of Yonsei University in 2024.

[0007] The inventors not only confirmed that inhibiting the expression of the CHI3L1 protein can prevent or treat lung diseases caused by non-tuberculosis mycobacteria, but also completed the present invention by confirming the inhibitory efficacy of 2-(3-[2-(1-cyclohexene-1-yl)ethyl]-6,7-dimethoxy-4-oxo-3,4-dihydro-2-quinazolinylsulfanyl)-N-(4-ethylphenyl)butanamide (K284-6111) and a novelly synthesized K284-6111 derivative on CHI3L1 protein expression.

[0008] Accordingly, one objective of the present invention is to provide a novel CHI3L1 inhibitor and a pharmaceutical composition for the prevention or treatment of lung disease caused by non-tuberculous mycobacterial infection comprising the same as an active ingredient.

[0009] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0010] Various embodiments of the present invention are described with reference to the drawings. In the following description, for a complete understanding of the present invention, various specific details, such as specific forms, compositions, and processes, are described. However, specific embodiments may be practiced without one or more of these specific details, or in combination with other known methods and forms. In other examples, known processes and manufacturing techniques are not described as specific details so as not to make the present invention unnecessary or obscure. Reference throughout this specification to one embodiment implies that a particular feature, form, composition, or characteristic described in association with the embodiment is included in one or more embodiments of the present invention. Accordingly, the circumstances of an embodiment expressed at various locations throughout this specification do not necessarily represent the same embodiment of the present invention. Additionally, a particular feature, form, composition, or characteristic may be combined in any suitable way in one or more embodiments.

[0011] The entire contents of Korean Patent Application No. 10-2023-0134101, filed on October 10, 2023, are incorporated into the specification of the present invention by reference.

[0012] In the first embodiment of the present invention, a compound represented by the following chemical formula 1 is provided:

[0013] [Chemical Formula 1]

[0014]

[0015] In the above chemical formula,

[0016] R1 is hydrogen or C1-C6 alkyl, and

[0017] R2 is hydrogen or C1-C6 alkyl, and

[0018] R3 is hydrogen or C1-C6 alkyl, and

[0019] R4 is O or S, and

[0020] R5 is a C5-C7 cycloalkenyl C1-C3 alkyl, C1-C3 haloalkyl C6-C 10 It is a heteroaryl ring of 5 to 9 members substituted with one or more substituents selected from the group consisting of aryl C1-C3 alkyl, C1-C5 alkoxy, and hydroxy.

[0021] In this specification, the term “alkyl” means a straight-chain or branched saturated hydrocarbon group, including, for example, methyl, ethyl, propyl, isopropyl, etc. For example, C1-C3 alkyl means an alkyl group having alkyl units having 1 to 3 carbon atoms, and when C1-C3 alkyl is substituted, the number of carbon atoms of the substituent is not included.

[0022] In this specification, the term “haloalkyl” means an alkyl group substituted with a halogen, for example, C1-C3 haloalkyl means a functional group in which one or more hydrogens of an alkyl having 1 to 3 carbon atoms are substituted with a halogen.

[0023] In this specification, the term “halogen” refers to a halogen group element and includes, for example, fluoro, chloro, bromo, and iodo.

[0024] In this specification, the term “aryl” refers to a monocyclic or polycyclic carbon ring that is wholly or partially unsaturated and has aromaticity. For example, C6-C 10 Aryl means a monocyclic or polycyclic carbon ring that is wholly or partially unsaturated and aromatic, composed of 6 to 10 carbon atoms.

[0025] In this specification, the term “heteroaryl” means a heterocyclic aromatic group containing oxygen, sulfur, or nitrogen within the ring as a heteroatom. The number of heteroatoms included within the ring is 1 to 4, specifically 3 to 4.

[0026] In this specification, the term “cycloalkenyl” means an unsaturated hydrocarbon ring forming a single or multiple rings, and means a cyclic group comprising at least one carbon-carbon double bond in a cycloalkyl group. Examples include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, etc. For example, C3-C7 cycloalkenyl means an unsaturated hydrocarbon ring composed of an alkene unit having 3 to 7 carbon atoms, and when C3-C7 alkenyl is substituted, the number of carbon atoms of the substituent is not included.

[0027] In this specification, the term “alkoxy” means a radical formed by removing hydrogen from an alcohol, for example, C1-C5 alkoxy means an alkyl ether formed by removing hydrogen from an alcohol having 1 to 5 carbon atoms.

[0028] In this specification, the term “saturated hydrocarbon group of a pulverized group” means a branched alkyl (chain alkyl) rather than a straight-chain alkyl (straight alkyl).

[0029] 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.

[0030] In a second embodiment of the present invention, a compound is provided in which, in the first embodiment, R1 is a C1-C6 alkyl.

[0031] In the third embodiment of the present invention, a compound is provided in which, in the first and second embodiments, R2 is a C1-C6 alkyl.

[0032] In the fourth embodiment of the present invention, a compound is provided in which, in the first to third embodiments, R3 is hydrogen or a C1-C6 alkyl.

[0033] In the fifth embodiment of the present invention, in the first to fourth embodiments, R5 is a C5-C7 cycloalkenyl C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl C6-C 10 The present invention provides a heteroaryl compound in which one or more carbons of a 5- to 9-membered ring are substituted with nitrogen, substituted with one or more substituents selected from the group consisting of aryl C1-C3 alkyl and oxo groups.

[0034] In the sixth embodiment of the present invention, a compound is provided in which, in the first to fifth embodiments, the compound represented by Formula 1 is selected from the group consisting of compounds represented by Formulas 2 to 5 below.

[0035] [Chemical Formula 2]

[0036]

[0037] [Chemical Formula 3]

[0038]

[0039] [Chemical Formula 4]

[0040]

[0041] [Chemical Formula 5]

[0042]

[0043] In the seventh embodiment of the present invention, an antimicrobial composition is provided that includes the compounds provided in the first to sixth embodiments as active ingredients.

[0044] In the present invention, “CHI3L1 (Chitinase 3-Like 1)” is a secreted glycoprotein also known as YKL-40, belonging to the chitinase-like proteins (CLPs). This protein has a size of 40 kDa and possesses conserved characteristics as a chitin-binding protein. The three-dimensional crystal structure of CHI3L1 exhibits the typical folding structure of a chitinase family protein, but chitinase activity is not expressed due to the lack of essential amino acid residues within the enzyme domain. CHI3L1 is expressed in various cells of lung tissue (neutrophils, monocytes / macrophages, monocyte-derived dendritic cells, epithelial cells, etc.) and is closely associated with various biological activities such as tissue damage, inflammatory responses, and tissue repair. CHI3L1 is also associated with several diseases. For example, high levels of CHI3L1 protein and gene expression are observed in patients suffering from various diseases such as asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, cancer, diabetes, and arteriosclerosis. The CHI3L1 (Chitinase 3-Like 1) inhibitor provided in one embodiment of the present invention can increase resistance to acid-fast bacteria, particularly non-tuberculous mycobacteria, by regulating inflammation and immune responses caused by CHI3L1, or induce apoptosis in cells infected with acid-fast bacteria, particularly non-tuberculous mycobacteria, and can reduce tissue damage and promote recovery by alleviating inflammatory responses caused by CHI3L1.

[0045] In the eighth embodiment of the present invention, an antimicrobial composition is provided in which, in the seventh embodiment, the antimicrobial agent is an antimicrobial agent against acid-fast bacteria (Mycobacteria).

[0046] In the present invention, the term “acid-fast bacteria (Mycobacteria)” or “Mycobacteria or Mycocobacterium” refers to a genus of bacteria belonging to Gram-positive bacteria, which is divided into Mycobacterium tuberculosis complex, Mycobacterium leprae, or the nontuberculous mycobacteria (NTM) group. Additionally, they are rod-shaped bacteria that are relatively small in size and possess strong resistance to acid. They contain a long fatty acid called mycolic acid in their cell walls, so they appear like Gram-negative bacteria under Gram staining, but they have very high resistance to acid. For this reason, they are classified as acid-fast bacteria. Furthermore, they are generally aerobic bacteria, although some can grow under anaerobic conditions.

[0047] In the ninth embodiment of the present invention, in the seventh and eighth embodiments, the nontuberculous mycobacteria group (NTM) comprises Mycobacterium avium (Mav), Mycobacterium abscessus subsp. abscessus (Mabc), Mycobacterium abscessus subsp. massiliense (Mmass), and Mycobacterium abscessus subsp.bolletii), Mycobacterium Intracellurare, Mycobacterium chimaera, Mycobacterium Scrofulaceum, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium peregrinum, Mycobacterium ulcerans, Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium Genevans, Mycobacterium simiae, Mycobacterium terrae, Mycobacterium The present invention provides an antimicrobial composition comprising one or more selected from the group consisting of Mycobacterium nonchromogenicum, Mycobacterium celatum, Mycobacterium gordonae, Mycobacterium szulgai, Mycobacterium mucogenicum, Mycobacterium xenopi, and Mycobacterium aubagnens.

[0048] In the 10th embodiment of the present invention, a pharmaceutical composition for preventing or treating non-tuberculous mycobacterial infections is provided, comprising the antimicrobial composition provided in the 7th to 9th embodiments as an active ingredient.

[0049] In the 11th embodiment of the present invention, a pharmaceutical composition is provided in which, in the 10th embodiment, the non-tuberculous mycobacterial infection disease is one or more selected from the group consisting of lung disease, superficial lymphadenitis, skin, soft tissue, and bone infection, and disseminated disease, and is not particularly limited as long as it is a disease that can be caused by non-tuberculous mycobacterial infection.

[0050] In this specification, the term “prevention” means suppressing the occurrence of a disease or illness in a subject who has not been diagnosed with having such a disease or illness but is at risk of developing such a disease or illness.

[0051] 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. Accordingly, the compositions of the present invention may serve as compositions for treating these diseases in themselves, or may be administered together with other pharmacological components to be applied as therapeutic adjuvants for said diseases. Accordingly, in this specification, the terms “treatment” or “therapeutic agent” include the meanings of “therapeutic aid” or “therapeutic adjuvant.”

[0052] 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.

[0053] 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 a “preventive effective amount.”

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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).

[0058] The pharmaceutical composition of the present invention may be administered orally or parenterally, and specifically, may be administered orally, intravenously, or intraventricularly.

[0059] 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.

[0060] 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.

[0061] According to one embodiment of the present invention, the present invention provides a functional food composition for improving or preventing non-tuberculous mycobacterial infections, comprising the compound of the present invention described above or a food-grade salt thereof as an active ingredient.

[0062] 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.

[0063] 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.

[0064] The novel CHI3L1 inhibitor provided in the present invention can effectively inhibit or treat the progression of lung disease caused by non-tuberculous mycobacterial infection by inhibiting CHI3L1. In particular, it was confirmed that K284-6111 can inhibit or treat the progression of lung disease caused by non-tuberculous mycobacterial infection by inhibiting CHI3L1. Furthermore, by newly synthesizing derivatives of K284-6111, novel compounds with superior CHI3L1 inhibitory ability compared to K284-6111 were identified. Accordingly, the novel CHI3L1 inhibitors provided in the present invention can inhibit or treat the progression of lung disease caused by non-tuberculous mycobacterial infection by inhibiting CHI3L1 more effectively than K284-6111.

[0065] Figure 1 is the chemical structural formula of K284-6111.

[0066] Figure 2 shows the results of evaluating the ability of K284-6111 to inhibit CHI3L1 protein in lung tissue through ELISA.

[0067] Figure 3 is a schematic diagram showing the mouse experimental design. Female C57BL / 6 mice (WT) and Chil1 KO mice were airborne infecting them with M. aviumSMC#7, and at 2 weeks of infection, K284-6111 was administered intravenously twice a week for 8 weeks to a portion of the WT mouse group. All mice were necropsied at 10 weeks of infection.

[0068] Figure 4 shows the results of a comparative analysis of CHI3L1 protein expression levels in lung tissue via ELISA. It was confirmed that CHI3L1 protein expression, which was increased due to MAC infection, was significantly reduced by the administration of K284-6111. Lung tissue lysates from Chil1 KO mice were used as a negative control.

[0069] Figure 5A shows the results confirming that the bacterial count in lung tissue was significantly reduced when K284-6111 was administered to WT mice, Figure 5B shows the results confirming that the degree of histopathological inflammation in the lung tissue of WT mice was significantly reduced when K284-6111 was administered, and Figure 5C shows the results of visualizing the degree of inflammation by performing H&E staining on lung tissues between each group.

[0070] Figure 6 shows the chemical structural formulas of derivatives K-1, K-2, K-14, and K-15 prepared based on K284-6111.

[0071] Figure 7 shows data showing that K-1, K-2, K-14, and K-15, derivatives of K284-6111, exhibit significantly superior CHI3L1 inhibitory activity in lung tissue lysates compared to K284-6111 at the same concentration.

[0072] Figure 8 shows data showing that K-1, K-2, K-14, and K-15, derivatives of K284-6111, exhibit significantly superior CHI3L1 inhibitory activity in lung tissue lysates compared to other K284-6111 derivatives, K-12 and K-13, at the same concentration.

[0073]

[0074] The present invention will be described in more detail below through examples. These examples are intended solely to explain the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the invention.

[0075] [Example]

[0076] [Example 1] Evaluation of CHI3L1 Inhibitory Activity by K284-6111

[0077] Example 1 was performed to evaluate whether K284-6111 effectively neutralizes CHI3L1 protein in lung tissue. To this end, analysis was conducted using lung tissue lysates obtained by necropsy 10 weeks after air-infecting C57BL / 6 wild-type (WT) mice with M. avium.

[0078] [Example 1-1] Confirmation of CHI3L1 Inhibitory Ability of K284-6111 in Lung Tissue via ELISA

[0079] In Example 1-1, the expression level of CHI3L1 protein in the sample was measured using an enzyme-linked immunosorbent assay (ELISA). For the analysis, CHI3L1 protein was specifically detected using the DY2649 ELISA kit and the DY008 auxiliary reagent kit from R&D Systems. Lung tissue samples were homogenized with sterile PBS to prepare a lysate, which was then diluted 1:2500 in PBS for use in the ELISA analysis.

[0080] First, the capture antibody was diluted to the working concentration using protein-free PBS, and 100 µL was added to each 96-well microplate. The plates were then sealed and incubated overnight at 4°C. The following day, each well was washed three times with wash solution, with the solution completely removed after each wash. After the final wash, the plates were inverted onto a clean paper towel to remove any remaining wash solution, and 300 µL of assay buffer was added to each well to block the plates. The plates were incubated at room temperature for 1 hour. After incubation, the washing process was repeated three times as before. During this time, equal volumes of lung tissue lysates were treated with K284-6111 at concentrations of 10 µM, 100 µM, and 1000 µM, respectively, and then rotated and incubated at room temperature for 2 hours. Subsequently, 100 µL of a total of eight solutions (including blanks), prepared by diluting the sample under the corresponding conditions with a standard solution of known concentration at a 1:2 ratio, were added to each well. The plates were covered with adhesive strips and incubated at room temperature for 2 hours. Afterward, each well was washed by repeating the aspiration and washing process three times. Next, 100 µL of the detection antibody, diluted with analysis buffer, was added to each well, covered with an adhesive strip, and incubated at room temperature for 2 hours. After incubation, the aspiration and washing process was repeated three times. Subsequently, 100 µL of streptavidin-HRP diluted to the working concentration was added to each well. The plates were wrapped in foil to block light and incubated at room temperature for 20 minutes. Finally, the plates were washed by repeating the aspiration and washing process. In the final step, 100 µL of substrate solution was added to each well and incubated for 20 minutes under light blockage. Once incubation was complete, 50 µL of stop solution was added to each well.After thoroughly mixing the solution by gently tapping the plate, the optical density was measured at 450 nm using a microplate reader. This allowed for the quantitative analysis of the reduction of CHI3L1 protein in lung tissue lysates following K284-6111 treatment.

[0081] As a result of evaluating the inhibitory ability of K284-6111 (2-(3-[2-(1-cyclohexene-1-yl)ethyl]-6,7-dimethoxy4-oxo-3,4-dihydro-2-quinazolinylsulfanil)-N-(4-ethylphenyl)butanamide) shown in Fig. 1 on CHI3L1 protein in lung tissue, it was confirmed that K284-6111 significantly inhibited CHI3L1 protein in a concentration-dependent manner, as shown in Fig. 2.

[0082] [Example 2] Study on Acid-Fast Bacterial Infection and Effects of K284-6111 Administration in Wild-Type Mice

[0083] Example 2 was performed to evaluate whether K284-6111 could effectively neutralize CHI3L1 protein in lung tissue and inhibit disease progression. To this end, C57BL / 6 wild-type mice (wild-type; WT) and mice with the CHI3L1 gene removed (Chil1 knockout; Chil1 KO) were air-infected with MAC, and various analyses were performed.

[0084] [Example 2-1] Animal Experiment Design

[0085] WT mice and Chil1 knockout mice were air-infected with M. avium SMC#7, respectively. Starting 2 weeks after infection, K284-6111 was administered intravenously to a subgroup of WT mice twice a week for a total of 8 weeks. All mice were necropsied simultaneously at 10 weeks after infection to evaluate the experimental results.

[0086] experimental group

[0087] 1. WT mouse, non-infected (WT)

[0088] 2. WT mouse, M. avium SMC#7 infected 10 weeks (WT INF)

[0089] 3. WT mice, M. avium SMC#7 + K284-6111 administration infection 10 weeks (WT INF + K284-6111)

[0090] 4. Chil1 KO mice, 10 weeks infected with M. avium SMC#7 (Chil1 KO INF)

[0091] [Example 2-2] Confirmation of CHI3L1 expression levels in lung tissue via ELISA

[0092] The expression level of CHI3L1 in lung tissue lysates of each mouse group was measured using the DY2599 ELISA kit from R&D Systems with the same analysis method as in Example 1-1 above.

[0093] As a result, as shown in Figure 4, it was confirmed that the CHI3L1 protein expression, which was increased due to MAC infection, was significantly reduced by the administration of K284-6111.

[0094] [Examples 2-3] Analysis of Colony Forming Units (CFU) in Lung Tissue Samples from Animal Experiment Groups

[0095] In this example, lung tissue samples were homogenized using sterile PBS to quantify the colony-forming units (CFU) of microorganisms in the lung tissues of animals in each experimental group. The homogenized samples were diluted with PBS at ratios of 1 / 100, 1 / 1000, and 1 / 10000. Subsequently, 50 µL of the diluted samples were inoculated onto Middlebrook 7H10 solid medium. The medium was cultured in a microbial incubator at 37°C for approximately 10 days. After the culture period ended, the number of formed colonies was counted and expressed as CFU.

[0096] As a result, as shown in Figure 5a, it was confirmed that the bacterial count in lung tissue was significantly reduced by K284-6111 inhibiting the CHI3L1 protein expression increased by MAC infection when K284-6111 was administered.

[0097] [Examples 2-4] Evaluation of pathological changes in lung tissue within animal experiment groups

[0098] In this example, to evaluate pathological changes in the lung tissue of each experimental animal group, a lung perfusion procedure was performed using sterile PBS, and then one lobe of lung tissue from each animal was isolated. Subsequently, the isolated tissue was fixed in a 4% paraformaldehyde solution for 24 hours, followed by additional fixation with paraffin. The paraffin-fixed lung tissue was prepared into 5 µm thick sections.

[0099] Pathological changes in the prepared lung tissue sections were visualized through H&E (Hematoxylin & Eosin) staining to confirm the presence and extent of lesions within the lung tissue. For the quantitative evaluation of the lesions, the ratio of the lesion area to the total area of ​​each section was calculated and quantified as a percentage (%) of the lesion area.

[0100] As a result, as shown in Figure 5b, it was confirmed that the degree of histopathological inflammation in the lung tissue of WT mice was significantly reduced by K284-6111 inhibiting the increased CHI3L1 protein expression caused by MAC infection upon administration of K284-6111, and as shown in Figure 5c, it was confirmed through H&E staining that inflammation was significantly reduced upon administration of K284-6111.

[0101] [Example 3] Synthesis of K284-6111-based derivatives K-1, K-2, K-14, and K-15

[0102] [Example 3-1] Synthesis of Compounds K-1 and K-2

[0103] (1) Synthesis of intermediate 1, N-(4-ethylphenyl)-2-((5-methoxy-1H-imidazo[4,5-b]pyridin-2-yl)thio)butanamide

[0104] [Reaction Equation]

[0105]

[0106] 67 mg (0.37 mmol) of compound A was dissolved in 4 mL of DMF solvent, 2 mg (0.74 mmol, 2 eq) of K2CO310 was added, and the mixture was heated and stirred at 85 °C for 30 minutes. Subsequently, 110 mg (0.4 mmol, 1.1 eq) of compound B was added, and the reaction mixture was further stirred at 85 °C for 18 hours. After the reaction was complete, the mixture was cooled to room temperature, 6 mL of saturated aqueous NH4Cl solution was added, and the mixture was extracted with EtOAc. The extracted organic layer was dried with MgSO4, filtered under reduced pressure, and concentrated. The mixture obtained in this way was separated and purified by silica gel column chromatography (hexane:acetone = 4:1, volume ratio) to synthesize compound intermediate 1 (N-(4-ethylphenyl)-2-((5-methoxy-1H-imidazo[4,5-b]pyridin-2-yl)thio)butanamide) 131 mg (0.354 mmol, yield 96%).

[0107] 1 H NMR (600 MHz, Chloroform-d) δ 10.26 (s, 1H), 7.80 (d, J = 8.7 Hz, 1H), 7.55 – 7.47 (m, 2H), 7.15 – 7.08 (m, 3H), 6.71 (d, J = 8.7 Hz, 1H), 4.53 – 4.45 (m, 1H), 3.98 (s, 3H), 2.63 – 2.53 (m, 3H), 2.28 – 2.15 (m, 1H), 1.97 – 1.84 (m, 1H), 1.22 – 1.14 (m, 4H), 1.13 (t, J = 7.3 Hz, 4H).

[0108] (2) Synthesis of compounds K-1 and K-2

[0109] [Reaction Equation]

[0110]

[0111] 65 mg (0.18 mmol) of intermediate 1 was dissolved in 1.8 mL of DMF solvent, and then 14 mg (0.36 mmol, 2 eq) of Cs2CO3 and 50 mg (0.26 mmol, 1.5 eq) of 1-(2-bromoethyl)cyclohex-1-ene were added, followed by heating and stirring at 80 °C for 18 hours. The reaction mixture was extracted with EtOAc, dried with MgSO4, filtered under reduced pressure, and concentrated. The resulting mixture was separated and purified by silica gel column chromatography (hexane:acetone = 9:1, volume ratio) to obtain 32 mg (0.067 mmol, yield 38%) of compound K-1 and 24 mg (0.050 mmol, yield 29%) of K-2.

[0112] [Compound K-1]

[0113]

[0114] 2-((1-(2-(cyclohex-1-en-1-yl)ethyl)-6-methoxy-1H-benzo[d]imidazol-2-yl)thio)-N-(4-ethylphenyl)butanamide

[0115] 1H NMR (600 MHz, Methanol-d4) δ 7.84 (d, J = 8.6 Hz, 1H), 7.37 – 7.32 (m, 2H), 7.13 – 7.09 (m, 2H), 6.70 – 6.66 (m, 1H), 5.03 – 5.00 (m, 1H), 4.38 – 4.22 (m, 3H), 3.96 (s, 3H), 2.63 – 2.54 (m, 2H), 2.48 – 2.38 (m, 2H), 2.17 – 2.06 (m, 1H), 2.06 – 2.02 (m, 2H), 2.01 – 1.93 (m, 1H), 1.68 – 1.61 (m, 2H), 1.55 – 1.48 (m, 2H), 1.41 – 1.34 (m, 2H), 1.22 – 1.16 (m, 3H), 1.11 (t, J = 7.4 Hz, 3H).

[0116] [Compound K-2]

[0117]

[0118] 2-((1-(2-(cyclohex-1-en-1-yl)ethyl)-5-methoxy-1H-benzo[d]imidazol-2-yl)thio)-N-(4-ethylphenyl)butanamide

[0119] 1H NMR (600 MHz, Chloroform-d) δ 10.23 (s, 1H), 7.48 (t, J = 8.6 Hz, 3H), 7.10 – 7.05 (m, 2H), 6.66 (d, J = 8.6 Hz, 1H), 4.61 (t, 1H), 4.08 – 4.04 (m, 4H), 2.59 – 2.53 (m, 2H), 2.38 – 2.25 (m, 3H), 1.95 – 1.87 (m, 3H), 1.85 – 1.77 (m, 2H), 1.61 – 1.52 (m, 5H), 1.49 – 1.42 (m, 2H), 1.19 – 1.12 (m, 6H).

[0120] [Example 3-1] Synthesis of Compounds K-14 and K-15

[0121] (1) Synthesis of the intermediate 2,6-thioxo-5-(4-(trifluoromethyl)phenethyl)-1,5,6,7-tetrahydro-4H-pyrazolo[3,4-d]pyrimidin-4-one

[0122] [Reaction Equation]

[0123]

[0124] 200 mg (1.29 mmol) of compound C was dissolved in 1 mL of EtOH solvent and heated to 50 °C. 298 mg (1.29 mmol, 1 eq) of compound D was dissolved in 1 mL of EtOH and added to the compound C solution. Subsequently, the mixture was heated and stirred at 80 °C under nitrogen gas for 20 hours. After 20 hours, 50 mg (1.29 mmol, 1 eq) of NaOH was added, followed by heating and stirring for an additional 2 hours. 10% HCl was added to the reaction mixture to adjust the pH to neutral, and the mixture was concentrated. The resulting mixture was extracted with EtOAc, dried with MgSO4, filtered under reduced pressure, and concentrated. Without undergoing a separation and purification process, 228 mg (0.67 mmol, yield 52%) of intermediate 2 (6-thioxo-5-(4-(trifluoromethyl)phenethyl)-1,5,6,7-tetrahydro-4H-pyrazolo[3,4-d]pyrimidin-4-one) was obtained.

[0125] 1 H NMR (600 MHz, Methanol-d4) δ 8.13 – 8.08 (m, 2H), 8.06 – 8.01 (m, 1H), 7.68 – 7.62 (m, 2H), 4.68 – 4.58 (m, 2H), 3.10 – 3.02 (m, 2H).

[0126] (2) Intermediate 3, synthesis of N-(4-ethylphenyl)-2-((4-oxo-5-(4-(trifluoromethyl)phenethyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)thio)butanamide

[0127] [Reaction Equation]

[0128]

[0129] 120 mg (0.35 mmol) of intermediate 2, 95 mg (0.35 mmol, 1 eq) of compound B, and 97 mg (0.705 mmol, 2 eq) of K2CO3 were dissolved in 3.5 mL of DMF solvent and stirred at room temperature under nitrogen gas for 24 hours. The reaction mixture was extracted with water and EtOAc, dried with MgSO4, and filtered under reduced pressure. The resulting mixture was separated and purified by silica gel column chromatography (hexane:acetone = 3:2, volume ratio) to obtain 150 mg (0.28 mmol, yield 80%) of intermediate 3 (N-(4-ethylphenyl)-2-((4-oxo-5-(4-(trifluoromethyl)phenethyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)thio)butanamide).

[0130] 1 H NMR (600 MHz, Chloroform-d) δ 9.03 (s, 1H), 8.15 (s, 1H), 7.58 (d, J = 8.0 Hz, 2H), 7.41 (d, J = 7.9 Hz, 2H), 7.39 – 7.34 (m, 2H), 7.07 – 7.02 (m, 2H), 4.46 (t, J = 7.5 Hz, 1H), 4.38 – 4.23 (m, 2H), 3.15 – 3.03 (m, 2H), 2.54 (q, J = 7.6 Hz, 2H), 2.28 – 2.18 (m, 1H), 2.02 – 1.91 (m, 1H), 1.15 (q, J = 7.4 Hz, 6H).

[0131] (3) Synthesis of compounds K-14 and K-15

[0132] [Reaction Equation]

[0133]

[0134] 100 mg (0.19 mmol) of intermediate 3, 31 mg (0.23 mmol, 1.2 eq) of 1-bromo-2-methylpropane, and 52 mg (0.38 mmol, 2 eq) of K2CO3 were dissolved in 1.8 mL of DMF solvent, and then heated and stirred under nitrogen gas at 50 °C. The reaction mixture was extracted with water and EtOAc, dried with MgSO4, and filtered under reduced pressure. The obtained mixture was separated and purified by silica gel column chromatography (hexane:EtOAc = 4:1, volume ratio) to obtain 30 mg (0.05 mmol, yield 27%) of BRP-14 and 25 mg (0.04 mmol, yield 23%) of BRP-15.

[0135] [Compound K-14]

[0136]

[0137] N-(4-ethylphenyl)-2-((1-isobutyl-4-oxo-5-(4-(trifluoromethyl)phenethyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-yl)thio)butanamide

[0138] 1H NMR (600 MHz, Chloroform-d) δ 9.60 (s, 1H), 8.01 (s, 1H), 7.56 (d, J = 7.9 Hz, 2H), 7.55 – 7.47 (m, 2H), 7.41 (d, J = 7.9 Hz, 2H), 7.08 (d, J = 8.2 Hz, 2H), 4.55 – 4.46 (m, 1H), 4.33 – 4.18 (m, 2H), 4.09 (d, J = 7.2 Hz, 2H), 3.14 – 2.98 (m, 2H), 2.57 (q, J = 7.6 Hz, 2H), 2.45 – 2.31 (m, 1H), 2.35 – 2.22 (m, 1H), 1.96 – 1.82 (m, 1H), 1.21 – 1.10 (m, 6H), 1.00 (d, J = 6.7 Hz, 6H).

[0139] [Compound K-15]

[0140]

[0141] N-(4-ethylphenyl)-2-((2-isobutyl-4-oxo-5-(4-(trifluoromethyl)phenethyl)-4,5-dihydro-2H-pyrazolo[3,4-d]pyrimidin-6-yl)thio)butanamide

[0142] 1H NMR (600 MHz, Chloroform-d) δ 8.33 (s, 1H), 8.03 (s, 1H), 7.58 (d, J = 8.0 Hz, 2H), 7.45 – 7.34 (m, 4H), 7.18 – 7.08 (m, 2H), 4.44 – 4.19 (m, 3H), 4.11 – 4.01 (m, 2H), 3.16 – 3.05 (m, 2H), 2.60 (q, J = 7.6 Hz, 2H), 2.31 – 2.20 (m, 2H), 2.06 – 1.94 (m, 1H), 1.22 – 1.14 (m, 6H), 0.86 (t, J = 6.5 Hz, 6H).

[0143] [Example 4] Evaluation of CHI3L1 Inhibitory Activity by K284-6111 Derivatives (K-1, K-2, K-14, and K-15)

[0144] In this example, we evaluated whether the K284-6111 derivatives synthesized in Example 3 effectively neutralized CHI3L1 protein in lung tissue. To do this, we performed an analysis using lung tissue lysates obtained by necropsy 10 weeks after air-infecting C57BL / 6 wild-type (WT) mice with M. avium.

[0145] [Example 4-1] Confirmation of CHI3L1 Inhibitory Ability of K284-6111 Derivatives in Lung Tissue via ELISA

[0146] The expression level of CHI3L1 was measured using the DY2599 ELISA kit from R&D Systems with the same analytical method as in Example 1-1. In this experiment, samples were treated with K284-6111 and its derivatives K-1, K-2, K-14, and K-15 at concentrations of 10 μM and 100 μM, respectively, in a rotational culture at room temperature for 2 hours, and then placed in wells.

[0147] As a result, as shown in Figure 7, derivatives of K284-6111, K-1, K-2, K-14, and K-15, exhibited significantly superior CHI3L1 inhibitory activity in lung tissue lysates compared to K284-6111 at the same concentration. Therefore, by inhibiting CHI3L1 protein expression more than K284-6111, K-1, K-2, K-14, and K-15 suggest that disease progression in non-tuberculous mycobacterial lung disease can be inhibited through the inhibition of CHI3L1 (Chitinase 3-Like 1) protein in lung tissue.

[0148] [Comparative Example] Confirmation of CHI3L1 inhibitory activity of K-1, K-2, K-14, and K-15 compared with other K284-6111 derivatives (K-12 and K-13)

[0149] The expression level of CHI3L1 was measured using the DY2599 ELISA kit from R&D Systems with the same analytical method as in Example 1-1. Specifically, the inhibitory ability of CHI3L1 was to be compared by comparing K-12 and K-13 (chemical structure not shown), which were additionally synthesized during the process of synthesizing derivatives of K284-6111, with K-1, K-2, K-14, and K-15. In this experiment, samples were treated with different derivatives of K284-6111, K-12 and K-13, and K-1, K-2, K-14, and K-15, at concentrations of 10 μM and 100 μM, respectively, in a rotating culture at room temperature for 2 hours, and then placed in wells.

[0150] As a result, as shown in Figure 8, it was confirmed that while K-12 and K-13, which are other derivatives of K284-6111, have CHIL31 inhibitory activity similar to that of K284-6111, K-1, K-2, K-14, and K-15 have superior CHIL31 inhibitory activity.

[0151] 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

Compound represented by the following chemical formula 1: [Chemical Formula 1] In the above chemical formula, R1 is hydrogen or C1-C6 alkyl, and R2 is hydrogen or C1-C6 alkyl, and R3 is hydrogen or C1-C6 alkyl, and R4 is O or S, and R5 is a C1-C7 alkyl, C5-C7 cycloalkenyl, C1-C3 alkyl, C1-C3 haloalkyl C6-C 10 It is a heteroaryl ring of 5 to 9 members substituted with one or more substituents selected from the group consisting of aryl C1-C3 alkyl, C1-C5 alkoxy, and hydroxy. In paragraph 1, The above R1 is a compound that is a C1-C6 alkyl. In paragraph 1, The above R2 is a compound that is a C1-C6 alkyl. In paragraph 1, The above R3 is a compound that is hydrogen or a C1-C6 alkyl. In paragraph 1, R5 is a C1-C7 alkyl, C5-C7 cycloalkenyl, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl C6-C 10 A heteroaryl compound in which one or more carbons of a 5- to 9-membered ring are substituted with nitrogen, substituted with one or more substituents selected from the group consisting of aryl C1-C3 alkyl and oxo groups. In paragraph 1, A compound in which the compound represented by the above chemical formula 1 is selected from the group consisting of compounds represented by the following chemical formulas 2 to 5. [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] An antimicrobial composition comprising a compound of any one of claims 1 to 6 as an active ingredient. In Paragraph 7, The above antimicrobial agent is an antimicrobial agent against acid-fast bacteria (Mycobacteria), and is an antimicrobial composition. In paragraph 8, An antimicrobial composition in which the above acid-fast bacteria is one or more selected from the group consisting of Mycobacterium tuberculosiscomplex, Mycobacterium leprae, or nontuberculous mycobacteria (NTM). In Paragraph 9, The above nontuberculous mycobacteria (NTM) group includes Mycobacterium avium (Mav), Mycobacterium abscessus subsp. abscessus (Mabc), Mycobacterium abscessus subsp. massiliense (Mmass), and Mycobacterium abscessus subsp. bolletii.bolletii), Mycobacterium Intracellurare, Mycobacterium chimaera, Mycobacterium Scrofulaceum, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium peregrinum, Mycobacterium ulcerans, Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium Genevans, Mycobacterium simiae, Mycobacterium terrae, Mycobacterium An antimicrobial composition selected from the group consisting of Mycobacterium nonchromogenicum, Mycobacterium celatum, Mycobacterium gordonae, Mycobacterium szulgai, Mycobacterium mucogenicum, Mycobacterium xenopi, and Mycobacterium aubagnens. A pharmaceutical composition for the prevention or treatment of non-tuberculous mycobacterial infections, comprising a compound of any one of claims 1 to 6 as an active ingredient. In Paragraph 11, A pharmaceutical composition wherein the above-mentioned non-tuberculous mycobacterial infectious disease is one or more selected from the group consisting of lung disease, superficial lymphadenitis, skin, soft tissue, and bone infection, and disseminated disease. A method for preventing or treating non-tuberculous mycobacterial infections comprising a compound of any one of claims 1 to 6 as an active ingredient. In Paragraph 13, A method for preventing or treating a non-tuberculous mycobacterial infection, wherein the above-mentioned non-tuberculous mycobacterial infection is one or more selected from the group consisting of lung disease, superficial lymphadenitis, skin, soft tissue, and bone infection, and disseminated disease.

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