Use of chromone derivative as pharmaceutical composition for preventing or treating fibrosis using inhibition of TGF-beta signaling and promotion of autophagy

Abstract

The present invention provides a use of a novel chromone derivative as a preventive and therapeutic agent for fibrosis. The compound can effectively prevent, ameliorate, or treat fibrosis by inhibiting TGF-beta signaling and inducing autophagy, by means of proteasome inhibition.

Description

Use of chromone derivatives as pharmaceutical compositions for the prevention or treatment of fibrosis utilizing TGF-BETA signaling inhibitory and autophagy-promoting activities

[0001] The present invention relates to the use of chromone derivatives as pharmaceutical compositions for the prevention or treatment of fibrosis.

[0002] Fibrosis is a pathological tissue response triggered by external stimuli, chronic inflammation, and genetic factors. It is a progressive disease characterized by the excessive accumulation of abnormal fibrous connective tissue in various tissues and organs (e.g., lungs, liver, kidneys, heart, bone marrow, etc.), which impairs normal function. In particular, idiopathic pulmonary fibrosis (IPF), hepatic fibrosis, renal fibrosis, and systemic sclerosis (SSc) are classified as intractable diseases with limited current treatments and poor prognoses.

[0003] One of the major pathogenesis mechanisms of fibrosis is the activation of the TGF-β signaling pathway. TGF-β is a central cytokine that promotes fibrosis by inducing the activation of fibroblasts and the overproduction of skeletal proteins (e.g., collagen). Consequently, the development of drugs targeting the TGF-β pathway is actively underway.

[0004] Furthermore, autophagy is a mechanism that removes damaged organelles or proteins within cells and maintains homeostasis; recent studies have reported that inhibition of autophagy is involved in the progression of fibrosis, while conversely, promoting autophagy can inhibit the progression of fibrosis.

[0005] Cromone derivatives are a group of substances containing flavonoid structures with anti-inflammatory activity, and some are known to inhibit histamine secretion and reduce oxidative stress. However, the therapeutic effects of these substances on fibrosis through the inhibition of the TGF-β pathway and the promotion of autophagy have not yet been elucidated.

[0006] Accordingly, the inventors confirmed that a specific chromon derivative can effectively inhibit TGF-β signaling through proteasome inhibition and simultaneously significantly promote autophagy, thereby preventing and treating various types of fibrosis, and invented a pharmaceutical composition based on this.

[0007] The objective of the present invention is to provide a novel chromone derivative for use as a preventive or therapeutic agent for fibrosis.

[0008] Another objective of the present invention is to provide a new pharmaceutical composition for the prevention or treatment of fibrosis.

[0009] Another objective of the present invention is to provide a new food composition for the prevention or improvement of fibrosis.

[0010] Another objective of the present invention is to provide a method for effectively preventing or treating fibrosis.

[0011] To solve the above-mentioned problem, the present invention provides a pharmaceutical composition for the prevention or treatment of fibrosis comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0012] [Chemical Formula 1]

[0013]

[0014] In the above chemical formula 1, R1 is methyl, ethyl, cyclopentyl, cyclohexyl, phenyl, or benzyl; R2 is hydrogen, ethyl, acetyl, acetoxy, carboxyl, benzoyloxy, or 3,4,5-trihydroxybenzoyloxy; and R3 to R5 are each independently hydrogen, hydroxyl, methyl, methoxy, acetoxy, carboxyl, or benzoyloxy.

[0015] According to one embodiment of the present invention, in the above formula 1, R1 is methyl, R2 is hydrogen, R3 is hydrogen, hydroxyl or methoxy, R4 is hydroxyl or methoxy, and R5 is hydrogen, hydroxyl or methoxy.

[0016] According to one embodiment of the present invention, the compound may be characterized as being represented by any one of the following chemical formulas 2 to 5.

[0017] [Chemical Formula 2]

[0018]

[0019] [Chemical Formula 3]

[0020]

[0021] [Chemical Formula 4]

[0022]

[0023] [Chemical Formula 5]

[0024]

[0025] According to one embodiment of the present invention, the fibrosis may be characterized as a disease selected from the group consisting of idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, rheumatic pulmonary fibrosis (RA-ILD), asbestos-induced pulmonary fibrosis, ocular fibrosis, pancreatic fibrosis, myelofibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, and systemic sclerosis.

[0026] According to one embodiment of the present invention, the compound may be characterized by inhibiting TGF-β (Transforming Growth Factor-β) signaling by inhibiting intracellular proteasomes.

[0027] According to one embodiment of the present invention, the compound may be characterized by inducing autophagy by inhibiting an intracellular proteasome.

[0028] In addition, the present invention provides a food composition for the prevention or improvement of fibrosis comprising the above-described compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0029] According to one embodiment of the present invention, the fibrosis may be characterized as a disease selected from the group consisting of idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, rheumatic pulmonary fibrosis (RA-ILD), asbestos-induced pulmonary fibrosis, ocular fibrosis, pancreatic fibrosis, myelofibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, and systemic sclerosis.

[0030] In addition, the present invention provides the use of the above-described compound or a pharmaceutically acceptable salt thereof for the prevention or treatment of fibrosis.

[0031] In addition, the present invention provides a method for preventing or treating fibrosis, wherein the method comprises the step of administering an effective amount of the compound described above or a pharmaceutically acceptable salt thereof to a subject requiring treatment for fibrosis.

[0032] A pharmaceutical or food composition containing a novel chromone derivative according to the present invention can effectively prevent, improve, or treat the occurrence of fibrosis by inhibiting TGF-beta signaling through proteasome inhibition, and can effectively inhibit or improve the progression of fibrosis by inducing autophagy activation.

[0033] FIG. 1 shows the synthesis process of compound 1 (ONG41003) according to one embodiment of the present invention.

[0034] Figure 2 is the result of confirming 7-(benzyloxy)-5-hydroxy-2-phenyl-4H-chromen-4-one prepared during the synthesis process of compound 1 according to one embodiment of the present invention using LCMS.

[0035] Figure 3 is the result of confirming 7-(benzyloxy)-5-methoxy-2-phenyl-4H-cromen-4-one prepared during the synthesis process of compound 1 according to one embodiment of the present invention using LCMS.

[0036] Figure 4 is the result of confirming 1-(4-(benzyloxy)-2-hydroxy-6-methoxyphenyl)ethane-1-one prepared during the synthesis process of compound 1 according to one embodiment of the present invention using LCMS.

[0037] Figure 5 is the result of confirming 1-(4-(benzyloxy)-3,6-dihydroxy-2-methoxyphenyl)ethane-1-one by LCMS during the synthesis process of compound 1 according to one embodiment of the present invention.

[0038] Figure 6 is the result of confirming 1-(4-(benzyloxy)-6-hydroxy-2,3-dimethoxyphenyl)ethane-1-one by LCMS during the synthesis process of compound 1 according to one embodiment of the present invention.

[0039] Figure 7 is the result of confirming (E)-1-(4-(benzyloxy)-6-hydroxy-2,3-dimethoxyphenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one by LCMS during the synthesis process of compound 1 according to one embodiment of the present invention.

[0040] Figure 8 is the result of confirming 7-(benzyloxy)-2-(3,4-dimethoxyphenyl)-5,6-dimethoxy-4H-cromen-4-one prepared during the synthesis process of compound 1 according to one embodiment of the present invention using LCMS.

[0041] Figure 9 is the result of confirming 2-(3,4-dimethoxyphenyl)-5,7-dihydroxy-6-methoxy-4H-cromen-4-one prepared during the synthesis process of compound 1 according to one embodiment of the present invention using LCMS.

[0042] Figure 10 is the result of confirming 2-(3,4-dimethoxyphenyl)-5-hydroxy-6-methoxy-7-(2-(pyrrolidin-1-yl)ethoxy)-4H-chromen-4-one by LCMS during the synthesis process of compound 1 according to one embodiment of the present invention.

[0043] Figure 11 confirms the proteasome inhibitory activity of compound 1 according to one embodiment of the present invention.

[0044] Figure 12 is a comparative analysis of the changes in the expression of ER stress and autophagy-related signaling proteins of compound 1 (ONG41003) and comparative compound 2 (ONG41008) according to one embodiment of the present invention.

[0045] Figure 13 confirms the TGF-beta pathway inhibitory activity of compound 1 according to one embodiment of the present invention.

[0046] FIG. 14a confirms the inhibition of expression of collagen 11a1, a fibrosis-related ECM (Extracellular Matrix) constituent protein, by compound 1 according to one embodiment of the present invention.

[0047] FIG. 14b shows the change in expression of the ECM constituent protein collagen 5a1 according to each treatment group to confirm the inhibition of the expression of the fibrillation-related ECM constituent protein by compound 1 according to one embodiment of the present invention.

[0048] FIG. 14c shows the change in expression of the ECM constituent protein collagen 6a3 according to each treatment group to confirm the inhibition of the expression of the fibrosis-related ECM constituent protein by compound 1 according to one embodiment of the present invention.

[0049] Figure 15 confirms the inhibition of expression of ECM constituent proteins by compound 1 according to one embodiment of the present invention.

[0050] FIG. 16 shows that compound 1 according to one embodiment of the present invention inhibits pulmonary fibrosis in a dose-dependent manner (data are expressed as mean ± standard error (SEM); Student's t-test was used to compare the physiological saline control group vs. the bleomycin control group, p<0.001; Dunnett's test of multiple comparisons (group 2 vs. the remaining groups, excluding group 1) was performed after one-way ANOVA).

[0051] Figure 17 shows the pharmacokinetic characteristics (concentration detected in plasma) upon oral administration of comparative compound 1 (ONG21001) according to one embodiment of the present invention.

[0052] Figure 18 shows the pharmacokinetic characteristics (half-life, Cmax, AUC, bioavailability (F%), and plasma detection concentration) of comparative compound 2 (ONG41008) upon oral administration according to one embodiment of the present invention.

[0053] FIG. 19 shows the pharmacokinetic characteristics (half-life, Cmax, AUC, bioavailability (F%), and plasma detection concentration) of compound 1 (ONG41003) upon oral administration according to one embodiment of the present invention.

[0054] FIG. 20a shows the drug concentration in lung tissue upon oral administration of comparative compound 1 (ONG21001) according to one embodiment of the present invention.

[0055] FIG. 20b shows the drug concentration in lung tissue upon oral administration of comparative compound 2 (ONG41008) according to one embodiment of the present invention.

[0056] FIG. 20c shows the drug concentration in lung tissue upon oral administration of compound 1 (ONG41003) according to one embodiment of the present invention.

[0057] FIG. 21a quantifies the therapeutic effect on pulmonary fibrosis upon oral administration of Compound 1 (ONG41003) according to one embodiment of the present invention (data are expressed as mean ± standard error (SEM); Student's t-test was used to compare the physiological saline control group vs. the bleomycin control group, p<0.001; Dunnett's multiple comparison test (Group 2 vs. the remaining groups, excluding Group 1) was performed after one-way ANOVA).

[0058] FIG. 21b shows the quantified therapeutic effect on pulmonary fibrosis upon oral administration of Comparative Compound 2 (ONG41008) according to one embodiment of the present invention ("#" indicates a statistically significant difference compared to the BLM / 0.1% HEC treatment group, analyzed using the Wilcoxon Signed Rank Test with a significance level of p < 0.05. "*" indicates a statistically significant difference compared to the BLM / 0.1% HEC treatment group, analyzed using the One-tailed Mann-Whitney test with a significance level of p < 0.05).

[0059] FIG. 22a shows the cytotoxicity of compound 1 (ONG41003) according to one embodiment of the present invention on mesenchymal stem cell ONGHEPA1 (KCTC13086BP) (at the time of 24 hours).

[0060] FIG. 22b shows the cytotoxicity of comparative compound 2 (ONG41008) according to one embodiment of the present invention on mesenchymal stem cell ONGHEPA1 (KCTC13086BP) (at the time of 48 hours).

[0061] Preferred embodiments according to the present invention will be described in detail below with reference to the attached drawings.

[0062] The advantages and features of the present invention and the method for achieving them will become clear by referring to the embodiments described in detail below together with the accompanying drawings.

[0063] However, the present invention is not limited by the embodiments disclosed below but may be implemented in various different forms, and these embodiments are provided merely to make the disclosure of the present invention complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0064] Unless otherwise specifically defined, terms used in this specification have the meanings commonly used in the relevant technical field.

[0065] In addition, in describing the present invention, if it is determined that related known technologies, etc., may obscure the essence of the present invention, a detailed explanation thereof will be omitted.

[0066] The present invention will be described in detail below.

[0067] The present invention provides a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0068] [Chemical Formula 1]

[0069]

[0070] In the above chemical formula 1, R1 is methyl, ethyl, cyclopentyl, cyclohexyl, phenyl, or benzyl; R2 is hydrogen, ethyl, acetyl, acetoxy, carboxyl, benzoyloxy, or 3,4,5-trihydroxybenzoyloxy; and R3 to R5 are each independently hydrogen, hydroxyl, methyl, methoxy, acetoxy, carboxyl, or benzoyloxy.

[0071] The novel compound of the present invention or a pharmaceutically acceptable salt thereof can prevent, improve, or treat fibrosis.

[0072] The compound of the present invention or a pharmaceutically acceptable salt thereof can inhibit cellular fibrosis by inhibiting proteasome activity, which is known as a key mechanism of fibrosis, thereby regulating the expression of important factors involved in TGF-β (Transforming Growth Factor-β) and / or autophagy.

[0073] Due to these effects, the compound of the present invention or a pharmaceutically acceptable salt thereof can prevent, improve, or treat fibrosis, a disease in which cells of an organ or tissue become fibrotic due to some cause.

[0074] In particular, the compound of the present invention or its pharmaceutically acceptable salt exhibits the effect of significantly inhibiting the level of fibrosis and restoring normal cells in animal models in which fibrosis has already progressed. These results imply that the compound of the present invention or its pharmaceutically acceptable salt possesses a restorative ability to return tissues to a normal state even after fibrosis has progressed, going beyond a mere preventive effect, and thereby demonstrate that it can exert a very powerful effect in the treatment of fibrosis.

[0075] The compound of the present invention or its pharmaceutically acceptable salt has excellent bioavailability and a long half-life in the body, which is characterized by maintaining a stable drug concentration in plasma. In addition, the drug delivery efficiency into tissues is excellent, allowing effective delivery to target tissues to maximize therapeutic efficacy.

[0076] For the above-mentioned fibrosis inhibitory effect, bioavailability, half-life, and drug delivery efficiency, it is preferable that R1 in the compound of the present invention or its pharmaceutically acceptable salt is methyl, ethyl, cyclopentyl, cyclohexyl, phenyl, or benzyl. Additionally, it is preferable that R2 is hydrogen, R4 is hydroxyl or methoxy, and R3 and R5 are each independently hydrogen, hydroxyl, or methoxy. Furthermore, it is preferable that the compound of the present invention or its pharmaceutically acceptable salt is a compound represented by any one of the following chemical formulas 2 to 5 or its pharmaceutically acceptable salt.

[0077] [Chemical Formula 2]

[0078]

[0079] [Chemical Formula 3]

[0080]

[0081] [Chemical Formula 4]

[0082]

[0083] [Chemical Formula 5]

[0084]

[0085] The compound of the present invention can be prepared by including steps such as those in Reaction Scheme 1 below.

[0086] [Reaction Equation 1]

[0087]

[0088] In the above reaction formula 1, R1 to R5 correspond to R1 to R5 of the above chemical formula 1.

[0089] Specifically, the compound represented by the above chemical formula 1 (hereinafter referred to as the 'compound of the present invention') can inhibit TGF-β (Transforming Growth Factor-β) signaling and induce autophagy by inhibiting proteasome activity, which is known as a key mechanism of fibrosis.

[0090] In an in vitro test using AsPC-1 cells to confirm whether the compound of the present invention exhibits proteasome inhibitory activity, the expression of HA-tagged ubiquitin protein increased in the group treated with the compound of the present invention, similar to the positive control (MG132), which means that the ubiquitinated protein was not degraded and accumulated within the cell. These results indicate that the compound of the present invention can effectively inhibit proteasome activity (Fig. 11).

[0091] In an evaluation to determine whether the proteasome inhibitory activity of the compound of the present invention induces ER stress and autophagy, cells were treated with the compound of the present invention at a concentration of 20 μM and observed for 48 hours. As a result, ER stress markers (P-IRE1α, ATF6, GRP78) and autophagy markers (LC3-I / II) gradually increased from 3h and significantly increased until 36h, while another autophagy marker (P62) was confirmed to decrease from 48h. These results indicate that the compound of the present invention activates the autophagy pathway along with inducing ER stress through proteasome inhibition (Fig. 12).

[0092] In an analysis to confirm the effect of the proteasome inhibitory activity of the compound of the present invention on the TGF-β signaling pathway, AsPC-1 cells were treated with TGF-β followed by additional treatment with the compound of the present invention at various concentrations. As a result, the expression of p-SMAD3 protein, a downstream factor of TGF-β, began to decrease at concentrations of 5 μM or higher, and a distinct inhibitory effect was confirmed in the 25 μM treatment group. This confirmed that the compound of the present invention can inhibit the TGF-β signaling pathway in a dose-dependent manner (Fig. 13).

[0093] In an experiment to determine whether the compound of the present invention inhibits the expression of ECM constituent proteins associated with fibrotic diseases, analysis using lung fibroblasts (DHLF) derived from IPF patients revealed that the expression of collagen 11a1, 5a1, and 6a3 induced by TGF-β stimulation was inhibited upon treatment with the compound of the present invention, and the expression of Fibronectin, an ECM protein, was also confirmed to decrease in a concentration-dependent manner. Thus, it was confirmed that the compound of the present invention can inhibit the progression of fibrosis by inhibiting ECM accumulation (Figs. 14a to 14c, Fig. 15).

[0094] Through these series of results, it was confirmed that the compound of the present invention can inhibit the progression of fibrosis by inducing ER stress and autophagy, inhibiting the TGF-β signaling pathway, and inhibiting the expression of ECM constituent proteins. Based on the above mechanism, the therapeutic effect of the compound on fibrosis was subsequently evaluated using an animal model of pulmonary fibrosis induction.

[0095] To this end, the therapeutic effect of the compound of the present invention was evaluated in a mouse model with induced pulmonary fibrosis. As a result, when the compound was orally administered at doses of 25 and 50 mg / kg, fibrotic lesions were significantly reduced. In particular, in the 50 mg / kg group, an inhibitory effect on fibrosis equivalent to or superior to that of the positive controls, Pirfenidone and Nintedanib, was confirmed. These results demonstrate that the compound of the present invention can inhibit the progression of pulmonary fibrosis in a dose-dependent manner, suggesting efficacy and potential as a fibrosis treatment (Fig. 16).

[0096] In addition, the pharmacokinetic characteristics of the compounds of the present invention in plasma were evaluated, and it was confirmed that they exhibited superior bioavailability and an extended half-life compared to the comparative compounds upon oral administration, enabling the stable maintenance of blood concentrations. On the other hand, the blood drug concentration of Comparative Compound 1 was barely detectable, and Comparative Compound 2 showed limitations as an oral formulation due to a bioavailability of less than 20% and a very short half-life of less than 1 hour. From this, it is suggested that the compounds of the present invention are suitable for the development of oral formulations and enable effective drug delivery with low doses and fewer administrations (Figs. 17 to 19).

[0097] In addition, in a comparative evaluation of drug concentrations of the compound of the present invention within lung tissue, it was confirmed that the concentration of the compound of the present invention within lung tissue was maintained at a level more than 100 times higher than that of the comparative compound under the same conditions. In particular, the compound exhibited a high level of tissue accumulation even at low doses, which is a result proving that it has significantly superior delivery efficiency to lung tissue and possesses pharmacokinetic characteristics highly suitable for the development of targeted lung therapies (Figs. 20a to 20c).

[0098] The pharmacokinetic characteristics of the compound of the present invention described above in plasma and lung tissue confirmed its advantages for drug development. Based on this, an evaluation of therapeutic effects was performed using an animal model of pulmonary fibrosis induction. As a result, the compound of the present invention exhibited a fibrosis inhibitory effect equivalent to that of Pirfenidone and Nintedanib, particularly at a dose of 50 mpk. Furthermore, in the same disease model, it showed superior therapeutic effects despite the total dosage being approximately one-quarter that of Comparative Compound 2, proving that the compound possesses superior competitiveness over existing treatments in terms of efficacy and drug efficiency (Figs. 21a, 21b).

[0099] In addition, the results of the cytotoxicity evaluation of the compound of the present invention using hepatic stellate-derived ONGHEPA1 cells showed that toxicity was limited at concentrations of 25 μM or less, but exhibited strong toxicity at 50 μM or more, confirming that an appropriate concentration range must be established for therapeutic application (Figs. 22a, 22b).

[0100] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of fibrosis containing the above compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0101] The above fibrosis is a disease caused by fibrosis of an organ or tissue of the body, and specifically, it may be a disease selected from the group consisting of idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, rheumatic pulmonary fibrosis (RA-ILD), asbestos-induced pulmonary fibrosis, ocular fibrosis, pancreatic fibrosis, myelofibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, and systemic sclerosis, but is not limited thereto.

[0102] The pharmaceutical composition of the present invention may be the compound of the present invention or its pharmaceutically acceptable salt itself, or a composition mixed with a pharmaceutically acceptable carrier.

[0103] It is determined that the pharmaceutical composition of the present invention may contain 0.0001 to 100% by weight of the compound of the present invention or a pharmaceutically acceptable salt thereof based on the weight of the total composition.

[0104] The pharmaceutical composition of the present invention can be administered orally or parenterally during clinical administration, and when administered parenterally, it can be administered by intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dura injection, intracerebrovascular injection, or intrathoracic injection, and it is considered that it can be used in the form of a general pharmaceutical preparation.

[0105] It is determined that the pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

[0106] The daily dosage of the pharmaceutical composition of the present invention may be about 0.0001 to 100 mg, preferably 0.001 to 10 mg per kg of body weight, based on the compound of the present invention or a pharmaceutically acceptable salt thereof contained in the composition, and may be administered once or several times a day, but the range may vary depending on the patient's body weight, age, gender, health condition, diet, time of administration, method of administration, excretion rate, and severity of the disease.

[0107] When administered clinically, it can be formulated into various forms of oral or parenteral dosage forms, and can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0108] In addition, the present invention provides a food composition for the prevention or improvement of fibrosis containing the above compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0109] The above fibrosis is a disease caused by fibrosis of an organ or tissue of the body, and specifically, it may be a disease selected from the group consisting of idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, rheumatic pulmonary fibrosis (RA-ILD), asbestos-induced pulmonary fibrosis, ocular fibrosis, pancreatic fibrosis, myelofibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, and systemic sclerosis, but is not limited thereto.

[0110] The food composition of the present invention may be a composition mixed with the compound of the present invention or its pharmaceutically acceptable salt itself, or with a food-acceptable carrier. In this case, the content of the compound of the present invention or its pharmaceutically acceptable salt may be appropriately adjusted according to conventional methods based on the content and dosage in the said pharmaceutical composition. It is determined that the food composition of the present invention may take the form of processed meat products, fish products, tofu, jelly, porridge, noodles such as ramen or noodles, seasoning products such as soy sauce, soybean paste, red pepper paste, mixed sauce, sauces, snacks, dairy products such as fermented milk or cheese, pickled foods such as kimchi or pickled vegetables, fruits, vegetables, soy milk, fermented beverages, and beverages. Additionally, the said pharmaceutically acceptable carrier may also be used as the food-acceptable carrier.

[0111] The present invention provides a use of the above compound or a pharmaceutically acceptable salt thereof for the prevention or treatment of fibrosis.

[0112] As previously described, the above compound blocks the TGF-β (Transforming Growth Factor-β) signaling pathway by inhibiting intracellular proteasome activity, thereby inhibiting the activation of fibroblasts and the induction of collagen synthesis. Furthermore, the compound of the present invention effectively inhibits fibrosis by inducing autophagy activation to promote the removal of damaged intracellular proteins and fibrous accumulations.

[0113] The above fibrosis is a disease caused by fibrosis of an organ or tissue of the body, and specifically, it may be a disease selected from the group consisting of idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, rheumatic pulmonary fibrosis (RA-ILD), asbestos-induced pulmonary fibrosis, ocular fibrosis, pancreatic fibrosis, myelofibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, and systemic sclerosis, but is not limited thereto.

[0114] The present invention provides a method for preventing or treating fibrosis, wherein the method comprises the step of administering an effective amount of the compound or a pharmaceutically acceptable salt thereof to a subject requiring treatment for fibrosis.

[0115] As previously described, the above compound blocks the TGF-β (Transforming Growth Factor-β) signaling pathway by inhibiting intracellular proteasome activity, thereby inhibiting the activation of fibroblasts and the induction of collagen synthesis. Furthermore, the compound of the present invention effectively inhibits fibrosis by inducing autophagy activation to promote the removal of damaged intracellular proteins and fibrous accumulations.

[0116] The above fibrosis is a disease caused by fibrosis of an organ or tissue of the body, and specifically, it may be a disease selected from the group consisting of idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, rheumatic pulmonary fibrosis (RA-ILD), asbestos-induced pulmonary fibrosis, ocular fibrosis, pancreatic fibrosis, myelofibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, and systemic sclerosis, but is not limited thereto.

[0117] The present invention will be explained in more detail below through examples, but the following examples are not intended to limit the scope of the invention and should be interpreted as being for the purpose of aiding understanding of the invention.

[0118] Example 1: Preparation of Compound 1 of the present invention

[0119] 1-1. Step 1: Preparation of 7-(benzyloxy)-5-hydroxy-2-phenyl-4H-chromen-4-one (precursor compound 2)

[0120] This step is specifically described as the step of performing Step-1 of Fig. 1 as follows.

[0121] Potassium carbonate (680 g; 4.917 mol; 2.5 equivalents) was added at room temperature to a yellow suspension of chrysine 1 (500 g; 1.967 mol; 1 equivalent) dissolved in DMF (2.5 L) and THF (2.5 L). The reaction mixture turned a deep yellow, and benzyl bromide (404 g (280 mL); 2.36 mol; 1.2 equivalents) was added dropwise at room temperature over approximately 45-60 minutes. The reaction mixture was stirred at room temperature for 12 hours. Completion of the reaction was confirmed by TLC (1:1 / PE:EtOAc; Rf ~ 0.5). The reaction mixture was diluted with ice water, and the pH was adjusted to ~3 using concentrated hydrochloric acid. The generated yellow solid was filtered, washed with water, suction-dried for 12 hours, and further azeotropically mixed with toluene (2L) to obtain precursor compound 2 of the yellow solid.

[0122] The obtained 7-(benzyloxy)-5-hydroxy-2-phenyl-4H-cromen-4-one (precursor compound 2) was confirmed by LCMS analysis under the following conditions, and the results are shown in Figure 2.

[0123] - Yield: 630 g; 88%

[0124] - Note: Two reactions were performed on a 500g scale following the same method as above, and as a result, a total of 1.255kg (yield 91%) of precursor compound 2 was obtained in the form of a yellow solid.

[0125] - LCMS: Mass found; (345.2; M+1)

[0126] - Column: X-Bridge BEH C18 (4.6x50mm) 2.5um

[0127] - Method: B: 5mM NH4HCO3in water, A: ACN, Flow Rate: 2.2 mL / min

[0128] - Rt (min): 2.41; Area% - 94.774

[0129] 1-2. Step 2: Preparation of 7-(benzyloxy)-5-methoxy-2-phenyl-4H-chromen-4-one (precursor compound 3)

[0130] This step is specifically described as the step of performing Step-2 of Fig. 1 as follows.

[0131] Precursor compound 2 (630 g; 1.829 mol; 1 equivalent) was dissolved in acetone (10 L; 16 Vol) and stirred to obtain a yellow suspension to which solid KOH (308 g; 5.488 mol; 3 equivalents) was added at room temperature. After stirring for 10-20 minutes, the reaction mixture was heated to 60°C, and dimethyl sulfate (260.0 mL; 2.744 mol; 1.5 equivalents) was added dropwise over 45 minutes. After the addition was complete, the yellow reaction mixture was stirred at 60°C for 16 hours. The completion of the reaction was confirmed by TLC (1:1 / PE:EtOAc; Rf ~ 0.2). After cooling the mixture to room temperature, the solvent was concentrated and removed, the residue was diluted with water, and the pH was adjusted to 2 with concentrated hydrochloric acid. After filtering the precipitated solid and washing it with water, the precursor compound 3 was dried under vacuum for 24 hours to obtain a yellow solid.

[0132] The obtained 7-(benzyloxy)-5-methoxy-2-phenyl-4H-cromen-4-one (precursor compound 3) was confirmed by LCMS analysis under the following conditions, and the results are shown in Figure 3.

[0133] - Yield: 620 g; 93%

[0134] - Note: Reactions were carried out on scales of 620g and 635g following the same method as above, and as a result, a total of 1.255kg (yield 96%) of precursor compound 3 was obtained in the form of a yellow solid.

[0135] - LCMS: Mass found; (359.1; M+1)

[0136] - Method: A: 0.1% HCOOH in H2O, B: 0.05% HCOOH in ACN; Flow Rate: 0.8 mL / min; +ve mode

[0137] - Column: ACQUITY UPLC BEH C18 (2.1x50mm) 1.7 μm

[0138] - Rt (min): 2.167; Area% - 97.977

[0139] 1-3. Step 3: Preparation of 1-(4-(benzyloxy)-2-hydroxy-6-methoxyphenyl)ethane-1-one (precursor compound 4)

[0140] This step is specifically described as the step of performing Step-3 of Fig. 1 as follows.

[0141] Precursor compound 3 (200 g; 0.558 mol; 1 equivalent) was dissolved in an aqueous sodium hydroxide solution (50%; 1.4 L) and stirred to obtain a yellow suspension to which pyridine (428 mL; 0.531 mol; 9.5 equivalents) was added at room temperature. The dark brown mixture was vigorously stirred, and diethylene glycol (452 ​​mL; 0.502 mol; 9 equivalents) was added dropwise at room temperature. The reaction was exothermic, and the internal temperature rose by approximately 25–40°C during the addition. After the addition was complete, the dark brown mixture was heated to 110°C and stirred for 5 hours. At this point, the reaction mixture became a clear, dark solution. After confirming the completion of the reaction by TLC (1:1 / PE:EtOAc; Rf ~ 0.5), the mixture was cooled to 0°C and the pH was adjusted to 1 with concentrated hydrochloric acid. The aqueous solution was extracted with ethyl acetate (2 x 2500 mL). The combined organic phase was washed with a saturated sodium bicarbonate aqueous solution, water, and salt water. The organic layer was dried with sodium sulfate, and the solvent was removed under reduced pressure. The resulting dark brown solid was ground with methanol (1000 mL), filtered, and dried under vacuum to obtain precursor compound 4 as a grayish-white solid.

[0142] The obtained 1-(4-(benzyloxy)-2-hydroxy-6-methoxyphenyl)ethane-1-one (precursor compound 4) was confirmed by LCMS analysis under the following conditions, and the results are shown in Figure 4.

[0143] - Yield: 90.2 g; 58.9 %

[0144] - Note: Two reactions were performed on a 500g scale under similar conditions, and as a result, 448g (yield 58.7%) of the precursor compound 4 of the title was obtained in the form of a light gray solid.

[0145] - LCMS: Mass found; (273.2; M+1)

[0146] - Method: A: 0.1% HCOOH in H2O, B: 0.05% Formic Acid in ACN; Flow Rate: 0.8 mL / min; +ve mode

[0147] - Column: ACQUITY UPLC BEH C18 (2.1x50mm) 1.7 μm

[0148] - Rt (min): 2.278; Area% - 99.15

[0149] 1-4. Step 4: Preparation of 1-(4-(benzyloxy)-3,6-dihydroxy-2-methoxyphenyl)ethane-1-one (precursor compound 5)

[0150] This step is specifically described as the step of performing Step-4 of Fig. 1 as follows.

[0151] Pyridine (234 mL; 2.91 mol; 7.9 equivalents) was added dropwise at room temperature to a stirred suspension of precursor compound 4 (100 g; 0.367 mol; 1 equivalent) dissolved in an aqueous solution of tetraethylammonium hydroxide (35%; 2059 mL; 4.99 mol; 13.6 equivalents). The reaction mixture became a clear, dark solution. A solution of potassium persulfate (169 g; 0.624 mol; 1.7 equivalents) dissolved in water (3.5 L) was placed in a separate flask, and the reaction mixture was added dropwise to this solution at room temperature through an addition funnel and stirred continuously for 24 to 48 hours.

[0152] The reaction process was monitored using LCMS. The reaction mixture was cooled to 10–15°C, and concentrated HCl was added to the mixture to adjust the pH to 1–2. The resulting brown solid was filtered and collected, and the aqueous filtrate was used in the next step.

[0153] Sodium sulfite (37 g; 0.294 mol; 0.8 equivalents), concentrated HCl (349 mL, 10.64 mol), and toluene (300 mL) were added to the aqueous filtrate and refluxed at 100°C for 16 hours. Then, the reaction mixture was cooled to room temperature and extracted with ethyl acetate (2000 mL x 2). The combined organic layer was washed with water (1000 mL) and brine (1000 mL), dried with sodium sulfate, and concentrated to obtain a crude product (61.3 g). The crude oil was purified by column chromatography (silica gel 60, 120 mesh, eluent: ethyl acetate 10-12% in petroleum ether) to obtain a precursor compound 5 in the form of a yellow solid.

[0154] The obtained 1-(4-(benzyloxy)-3,6-dihydroxy-2-methoxyphenyl)ethane-1-one (precursor compound 5) was confirmed by LCMS analysis under the following conditions, and the results are shown in Figure 5.

[0155] - Yield: 35.5 g; 32.2%

[0156] - Note: Four individual reactions were performed on a 100g scale following the same method as above, and as a result, 128.5g (yield 31.1%) of the precursor compound 5 of the title was obtained.

[0157] - LCMS: Mass found; (289.2; M+1)

[0158] - Method: A: 0.1% HCOOH in H2O, B: 0.05% Formic Acid in ACN; Flow Rate: 0.8 mL / min; +ve mode

[0159] - Column: ACQUITY UPLC BEH C18 (2.1x50mm) 1.7 μm

[0160] - Rt (min): 1.955; Area% - 96.09

[0161] 1-5. Step 5: Preparation of 1-(4-(benzyloxy)-6-hydroxy-2,3-dimethoxyphenyl)ethane-1-one (precursor compound 6)

[0162] This step is specifically described as the step of performing Step-5 of Fig. 1 as follows.

[0163] Precursor compound 5 (50 g; 0.173 mol; 1 equivalent) was dissolved in acetone (1000 mL) and stirred to a suspension. To this, K2CO3 (38.3 g; 0.277 mol; 1.6 equivalents) was added at room temperature, followed by the dropwise addition of dimethyl sulfate (32.9 mL; 0.347 mol; 2 equivalents). The reaction mixture was stirred at the same temperature for 16 hours. The reaction was not completed by TLC. The solvent was concentrated and removed, the residue was diluted with water, extracted with ethyl acetate (1000 mL x 2), washed with water and brine, dried with sodium sulfate, and concentrated to obtain the residue. Precursor compound 6 was obtained in the form of a yellow solid by purification using column chromatography with silica gel (60-120 mesh, 10-12% ethyl acetate in petroleum ether) as the eluent. 5 (~10 g) of unreacted material was recovered using 15-25% ethyl acetate as the eluent in petroleum ether.

[0164] The obtained 1-(4-(benzyloxy)-6-hydroxy-2,3-dimethoxyphenyl)ethane-1-one (precursor compound 6) was confirmed by LCMS analysis under the following conditions, and the results are shown in Figure 6.

[0165] - Yield: 26.5 g; 63.2%

[0166] - Note: Two reactions were performed on a 50g scale following the same method as above, and as a result, 60.5g (yield 72%) of the precursor compound 6 of the title was obtained in the form of a yellow solid.

[0167] - LCMS: Mass found; (303.0; M+1)

[0168] - Method: A: 0.1% HCOOH in H2O, B: 0.05% Formic Acid in ACN; Flow Rate: 0.8 mL / min; +ve mode

[0169] - Column: ACQUITY UPLC BEH C18 (2.1x50mm) 1.7 μm

[0170] - Rt (min): 2.222; Area% - 97.30

[0171] 1-6. Step 6: Preparation of (E)-1-(4-(benzyloxy)-6-hydroxy-2,3-dimethoxyphenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (precursor compound 7)

[0172] This step is specifically described as the step of performing Step-6 of Fig. 1 as follows.

[0173] Precursor compound 6 (50 g; 0.1653 mol; 1.0 equivalent) and 3,4-dimethoxybenzaldehyde (36.3 g; 0.218 mol; 1.2 equivalent) were dissolved in ethanol (350 mL), to which KOH (22.5 g; 0.401 mol; 2.2 equivalents) dissolved in water (50 mL) was added. The reaction mixture was heated at 50°C. The progress of the reaction was monitored by TLC. After 16 hours, the reaction mixture was cooled to room temperature and concentrated under reduced pressure to remove the ethanol. The residue was diluted with ice water and acidified with concentrated HCl until the pH reached 3. The resulting yellow solid was filtered by suction, washed with water, and completely dried under vacuum. The solid was further ground with MTBE (100 mL), filtered, and dried under vacuum to obtain the yellow solid precursor compound 7.

[0174] The obtained (E)-1-(4-(benzyloxy)-6-hydroxy-2,3-dimethoxyphenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (precursor compound 7) was confirmed by LCMS analysis under the following conditions, and the results are shown in Figure 7.

[0175] - Yield: 45 g; 60.4%

[0176] - Note: One reaction was performed on a scale of 37g following the same method as above, and as a result, 30g (yield 54%) of precursor compound 7 of the title was obtained in the form of a yellow solid.

[0177] - Method: A-0.1% HCOOH in H2O, B: 0.05% Formic Acid in ACN; Flow Rate: 0.8 mL / min; +ve mode

[0178] - Column: ACQUITY UPLC BEH C18 (2.1x50mm) 1.7 μm

[0179] - Rt (min): 2.438; Area% - 89.007

[0180] 1-7. Step 7: Preparation of 7-(benzyloxy)-2-(3,4-dimethoxyphenyl)-5,6-dimethoxy-4H-cromen-4-one (precursor compound 8)

[0181] This step is specifically described as the step of performing Step-7 of Fig. 1 as follows.

[0182] Iodine (1 g; 0.004 mol; 0.025 equivalents) was added to a suspension of precursor compound 7 (75 g; 0.166 mol; 1 equivalent) dissolved in DMSO (750 mL), and the reaction mixture was heated to 110°C. The progress of the reaction was monitored by TLC. After 6 hours, the reaction mixture was cooled to room temperature and slowly poured into a 10% sodium sulfite solution cooled with ice while stirring vigorously. The precipitated solid was extracted twice (1000 mL x 2) with ethyl acetate. The combined organic layer was washed with water (500 mL) and a brine solution (500 mL), dried with sodium sulfate, and concentrated to obtain a yellow solid. The obtained solid was washed with 10% ethyl acetate dissolved in MTBE, filtered, and dried to obtain precursor compound 8 of the yellow solid.

[0183] The obtained 7-(benzyloxy)-2-(3,4-dimethoxyphenyl)-5,6-dimethoxy-4H-cromen-4-one (precursor compound 8) was confirmed by LCMS analysis under the following conditions, and the results are shown in Figure 8.

[0184] - Yield: 65 g; 87%

[0185] - LCMS: Mass found; (449.2; M+1)

[0186] - Method: A: 0.1% HCOOH in H2O, B: 0.05% HCOOH in ACN; Flow Rate: 0.8 mL / min; +ve mode

[0187] - Column: BEHC18 (50*2.1) 1.7μ

[0188] - Rt (min): 2.095; Area% - 96.7

[0189] Step 1-8.8: Preparation of 2-(3,4-dimethoxyphenyl)-5,7-dihydroxy-6-methoxy-4H-chromen-4-one (precursor compound 9)

[0190] This step is specifically described as the step of performing Step-8 of Fig. 1 as follows.

[0191] Precursor compound 8 (30 g, 0.066 mol, 1 eq) was dissolved in acetic acid (210 mL, 7 Vol), to which Con. HCl (90 mL, 3 Vol) was added, and the reaction mixture was heated at 85 °C. The reaction progress was monitored by UPLC. After 16 hours, the reaction mixture was cooled to room temperature and concentrated under vacuum. The residue was diluted with water and extracted with 10% methanol in dichloromethane (2 x 500 mL). The organic layer was dried with sodium sulfate and concentrated under vacuum to obtain 70 g in the form of a dark brown gum. The residue was ground with acetonitrile (150 mL) and filtered. The obtained yellow solid was further ground with acetone (100 mL) at 50 °C and stirred for 15 minutes. The hot solution was filtered, and the solid was washed with cold acetone. The solid was dried under vacuum to obtain precursor compound 9, a yellow solid.

[0192] The obtained 2-(3,4-dimethoxyphenyl)-5,7-dihydroxy-6-methoxy-4H-cromen-4-one (precursor compound 9) was confirmed by LCMS analysis under the following conditions, and the results are shown in Figure 9.

[0193] Yield: 9.0 g; 33%

[0194] LCMS: Mass found; (345.0; M+1)

[0195] Method: A: 0.1% HCOOH in H2O, B: 0.05% Formic Acid in ACN; Flow Rate: 0.8 mL / min; +ve mode

[0196] Column: ACQUITY UPLC BEH C18 (2.1x50mm) 1.7 μm

[0197] Rt (min): 1.776 ; Area% - 83.6

[0198] Note: One reaction was performed on a scale of 30 g following the same method as above, and as a result, 9.2 g (yield 34%) of precursor compound 9 was obtained in the form of a yellow solid.

[0199] 1-9. Step 9: Preparation of 2-(3,4-dimethoxyphenyl)-5-hydroxy-6-methoxy-7-(2-(pyrrolidin-1-yl)ethoxy)-4H-chromen-4-one

[0200] This step is specifically described as the step of performing Step-9 of Fig. 1 as follows.

[0201] 1-(2-chloroethyl)pyrrolidine hydrochloride (11.47 g, 0.674 mol, 1.5 eq) was added to a solution of precursor compound 9 (18 g, 0.045 mol, 1 eq) and potassium carbonate (18.65 g, 0.135 mol, 3 eq) dissolved in DMF (180 mL), and the mixture was heated at 80°C for 16 hours. The reaction was observed by TLC. The reaction mixture was diluted with water and extracted twice with ethyl acetate (500 mL x 2). The combined organic layer was washed with water-saline solution, dried with sodium sulfate, and concentrated to obtain a crude solid. The solid was stirred with 20% methanol dissolved in ethyl acetate, filtered, and dried to obtain a yellow solid. The solid was further stirred with acetonitrile (50 mL) and filtered to remove polar impurities (repeated 3 times). In addition, the compound was stirred with methanol at 50°C for 5 minutes, filtered while hot, washed with methanol, and dried to obtain 2-(3,4-dimethoxyphenyl)-5-hydroxy-6-methoxy-7-(2-(pyrrolidin-1-yl)ethoxy)-4H-cromen-4-one as a yellow solid.

[0202] The obtained 2-(3,4-dimethoxyphenyl)-5-hydroxy-6-methoxy-7-(2-(pyrrolidin-1-yl)ethoxy)-4H-cromen-4-one was confirmed by LCMS analysis under the following conditions, and the results are shown in Figure 10.

[0203] - Yield: 8.2 g; 44%

[0204] - LCMS: Mass found; (442.2; M+1)

[0205] - Method: A: 0.1% HCOOH in H2O, B: 0.05% Formic Acid in ACN; Flow Rate: 0.8 mL / min; +ve mode

[0206] - Column: XBridge BEH C18 (2.1x50mm) 2.5 μm

[0207] - Rt (min): 1.426 ; Area% - 98.675

[0208] - HPLC: 98.675%

[0209] - Mobile Phase: A: 0.1% TFA in water Mobile phase B: 100% ACN; Flow Rate: 1.2 mL / min

[0210] - Column: Atlantis T3 (150*4.6)mm, 3µm

[0211] - Rt (min): 8.304; Area% - 98.675

[0212] - Melting range: 139.1 °C - 140.5 °C

[0213] - 1H-NMR (400 MHz, DMSO-d6): δ 12.89 (s, 1H), 7.74 (d, J = 2.00 Hz, 1H), 7.61 (s, 1H), 7.15 (d, J = 8.80 Hz, 1H), 7.04 (d, J = 11.60 Hz, 2H), 4.25 (t, J = 6.00 Hz, 2H), 3.90 (s, 3H), 3.87 (s, 3H), 3.75 (s, 3H), 2.88 (t, J = 5.60 Hz, 2H), 2.59-2.56 (m, 4H), 1.72-1.69 (m, 4H).

[0214] The chemical formula of the compound 1 of the present invention (hereinafter referred to as 'ONG41003') prepared as described above is as follows.

[0215]

[0216] - Chemical Formula: C 24 H 27 NO7

[0217] - Molecular Weight: 441.48

[0218] In addition, the chemical formulas and preparation methods of comparative compounds 1 and 2 used in the following examples are as follows.

[0219] 1) Comparative compound 1 (hereinafter referred to as 'ONG21001')

[0220]

[0221] Comparative compound 1 was prepared as described in registered patent No. 10-1780456.

[0222] 2) Comparative compound 2 (hereinafter referred to as 'ONG41008')

[0223]

[0224] Comparative compound 2 was prepared as described in registered patent No. 10-1871166.

[0225] Example 2: Confirmation of proteasome inhibition and autophagy promotion effects

[0226] 2-1. Evaluation of Proteasome Inhibitory Activity

[0227] The proteasome inhibitory activity of compound 1 of the present invention (ONG41003) was evaluated on AsPC-1, a pancreatic cancer cell line. Cells were inoculated into a 6-well plate and stabilized for 24 hours, after which they were treated with the proteasome inhibitors MG132 (control) and ONG41003 at concentrations of 10 μM and 20 μM, respectively. Six hours after treatment, proteins were extracted from the cells, and the expression level of HA-tagged ubiquitin (HA-UB) was confirmed through Western blot analysis.

[0228] As a result of the analysis, it was observed that the expression of HA-UB was significantly increased in the ONG41003-treated group, just as in the MG132-treated group. This means that the ubiquitinated protein accumulated without being degraded by the proteasome, suggesting that compound 1 of the present invention exhibits a proteasome inhibitory effect in AsPC-1 cells (see Fig. 11).

[0229] 2-2. Analysis of ER Stress and Autophagy Induction Effects

[0230] From the results of Example 2-1 above, it was confirmed that compound 1 of the present invention (ONG41003) exhibits proteasome inhibitory activity in AsPC-1 cells. Accordingly, the possibility that untreated proteins accumulate in the endoplasmic reticulum (ER) and induce ER stress was further examined below. In addition, the possibility that autophagy may be activated through the intracellular Unfolded Protein Response (UPR) pathway when ER stress is induced above a certain level was also evaluated.

[0231] To this end, AsPC-1 cells were treated with ONG41003 and ONG410008 at a concentration of 20 μM, and changes in the expression of ER stress and autophagy-related signaling proteins were evaluated at 3 h, 6 h, 24 h, 36 h, and 48 h, respectively, through Western blot analysis.

[0232] As a result, the expression of ER stress markers P-IRE1α, ATF6, GRP78, and P-ERK proteins gradually increased from 3h and showed a significant increase by 24h or 36h, while the autophagy marker protein LC3-I / II continuously increased from 3h and reached a significantly high level after 24h. On the other hand, another autophagy marker protein, P62, gradually increased after 3h but decreased at 48h. Through this, it was confirmed that the compound of the present invention increases ER stress in cells and induces autophagy (see Fig. 12).

[0233] Example 3: Analysis of TGF-β signaling inhibitory effect

[0234] Through Example 2-1 above, it was confirmed that the compound of the present invention exhibits proteasome inhibitory activity in the human pancreatic cancer cell line AsPC-1. According to prior studies, proteasome inhibitors induce the accumulation of SMAD7 protein within cells by inhibiting its degradation, which is reported to have the effect of negatively regulating the TGF-β (Transforming Growth Factor-beta) signaling pathway (Reference: Y. Wang et al., Basic & Clinical Medicine, vol. 36, no. 2, 2016, pp. 156–160).

[0235] Accordingly, the effect of compound 1 of the present invention (ONG41003) on the TGF-β signaling pathway was analyzed using AsPC-1 cells. Specifically, AsPC-1 cells were treated with TGF-β (2 ng / mL) and cultured for 24 hours, followed by additional treatment with ONG41003 at concentrations of 1, 5, 10, and 25 μM, respectively. Subsequently, proteins were extracted from the cells 48 hours after drug treatment, and changes in p-SMAD3 protein expression were confirmed through Western blot analysis.

[0236] As a result of the analysis, ONG41003 began to reduce the expression of p-SMAD3 protein starting from a concentration of 5 μM or higher, and a distinct level of expression inhibition was observed at a concentration of 25 μM (see Fig. 13). These results demonstrate that the compound of the present invention inhibits the activation of p-SMAD3, a downstream signaling factor of the TGF-β signaling pathway, in a dose-dependent manner.

[0237] Therefore, the compound of the present invention exhibits an inhibitory effect on the TGF-β pathway in AsPC-1 cells, which can be utilized as an effective mechanism for regulating cancer cell growth and the tumor microenvironment.

[0238] Example 4: Confirmation of Fibrosis Inhibitory Effect

[0239] 4-1. TGF-β-induced inhibitory effect on ECM constituent protein expression in DHLF cell line

[0240] In this embodiment, experiments were conducted using diseased human lung fibroblasts (DHLF) derived from patients with idiopathic pulmonary fibrosis (IPF) to confirm whether the compound of the present invention inhibits the expression of extracellular matrix (ECM) constituent proteins associated with fibrotic diseases.

[0241] First, DHLF cells were treated with TGF-β (2.5 ng / ml) and cultured for 72 hours, after which changes in the expression of ECM constituent proteins collagen 11a1, collagen 5a1, and collagen 6a3 were measured via RT-PCR analysis. As a result, it was confirmed that the expression of the corresponding genes significantly increased in the group treated with TGF-β alone compared to the untreated group, whereas the expression levels of the corresponding genes were suppressed in the groups simultaneously treated with TGF-β 2.5 ng / ml and ONG41003 at concentrations of 3 µM and 6 µM, respectively (see Figures 14a–14c).

[0242] In addition, DHLF was treated with TGF-β (2.5 ng / ml) and cultured for 72 hours, and immunocytochemistry (ICC) was performed on Fibronectin, an ECM protein. As a result, the fluorescence signal (green) of Fibronectin increased in the TGF-β-treated group compared to the untreated group, but in the group treated with ONG41003, a concentration-dependent decrease in the fluorescence expression area of ​​the corresponding protein was observed (see Fig. 15).

[0243] In summary, the compound of the present invention exhibited an effect of inhibiting the expression of ECM constituent proteins induced by TGF-β stimulation in lung fibroblasts derived from IPF patients, thereby suggesting that it may have efficacy as a therapeutic agent for fibrotic diseases.

[0244] 4-2. Therapeutic Effects in an Animal Model of Pulmonary Fibrosis

[0245] From the results of Examples 2-1 and 3-1, it was confirmed that the compound of the present invention regulates the TGF-β signaling pathway through proteasome inhibition, and thereby exhibits an inhibitory effect on fibrosis by regulating the expression of ECM-related genes and proteins. Based on these mechanistic characteristics, the therapeutic effect of the compound 1 of the present invention (ONG41003) on fibrosis was evaluated in this example using an animal model with induced pulmonary fibrosis.

[0246] 1) Experimental design: 14-week-old male C57BL / 6J mice were assigned to each test group, with 11 mice per group. The experimental substance was set at three doses of ONG41003: 10, 25, and 50 mpk (mg / kg), and Pirfenidone (100 mpk) and Nintedanib (60 mpk), which are used as treatments for idiopathic pulmonary fibrosis (IPF), were included as positive controls (see Fig. 7).

[0247] 2) Method of inducing pulmonary fibrosis: Pulmonary fibrosis induction was performed based on the intratracheal instillation (IT) method as reported by Kremer, Laxer, and Berkman et al. 0.03 U of bleomycin dissolved in 50 μL of physiological saline was injected once through the trachea of ​​anesthetized experimental animals on Day 0 of the experiment, and a total fibrosis induction period of 7 days was established based on this.

[0248] 3) Administration and Analysis: From day 7 to day 21, for a total of 14 days, ONG41003 was administered orally once daily (morning) at a set dose. Positive controls, Pirfenidone and Nintedanib, were also administered orally once daily (morning) at their respective doses during the same period. Subsequently, on day 21, lung tissues were collected from mice in each group for pathological evaluation. The degree of fibrosis was quantitatively analyzed based on the Modified Ashcroft score system (Reference: Hubner et al., Biotech. 44: 507–517, 2008).

[0249] 4) Experimental Results: It was confirmed that pulmonary fibrosis was significantly induced in the group treated with bleomycin alone. In contrast, the level of fibrosis was significantly reduced in the groups administered ONG41003 at doses of 25 mpk and 50 mpk. In particular, the 50 mpk group showed an inhibitory effect on fibrosis equivalent to or superior to that of the positive controls (Pirfenidone and Nintedanib), and a stronger inhibitory effect compared to the 25 mpk group.

[0250] These results suggest that the compound of the present invention inhibits pulmonary fibrosis in a dose-dependent manner, serving as evidence of its efficacy as a fibrosis treatment (see Fig. 16).

[0251] Example 5: Comparative evaluation of pharmacokinetic properties with a comparative compound

[0252] 5-1. Comparative Evaluation of Pharmacokinetic Characteristics in Plasma

[0253] In this example, in order to confirm the suitability for oral administration and the superior pharmacokinetics of Compound 1 of the present invention, a comparative evaluation was performed focusing on bioavailability and half-life with comparative compounds ONG21001 and ONG41008.

[0254] Pharmacokinetic characteristics were evaluated after administering Comparative Compound 1 (ONG21001) orally once to male C57BL / 6 mice as shown in Table 1 below.

[0255] Experimental Group Dosage and Method Time to End Sample Size (n) Negative Control (Sham) -- 3 ONG21001 5 min ONG21001, 100 mg / kg, Oral Administration (PO) 5 min 3 ONG21001 15 min ONG21001, 100 mg / kg, Oral Administration (PO) 15 min 3 ONG21001 30 min ONG21001, 100 mg / kg, Oral Administration (PO) 30 min 3 ONG21001 1 hour ONG21001, 100 mg / kg, Oral Administration (PO) 1 hour 3 ONG21001 2 hours ONG21001, 100 mg / kg, Oral Administration (PO) 2 hours 3

[0256] Pharmacokinetic characteristics were evaluated after administering Comparative Compound 2 (ONG41008) once to male C57BL / 6 mice as shown in Table 2 below.

[0257] Experimental Group Dosage and Method Solvent Number of Samples (n) G1-ONG41008(IV)ONG41008, 25 mg / kg, Intravenous Administration (IV) DMSO:PEG400:Saline (20:55;25)6 G2-ONG41008(PO)ONG41008, 50 mg / kg, Oral Administration (PO) 30% HPβCD6 G3-ONG41008(PO)ONG41008, 25 mg / kg, Oral Administration (PO) 2.5 citric acid in DW + 7% gelucire 44 / 146 G4-ONG41008(PO)ONG41008, 50 mg / kg, Oral Administration (PO) 2.5 citric acid in DW + 7% gelucire 44 / 146 G5-ONG41008(PO)ONG41008, 100 mg / kg, oral administration (PO) 2.5 citric acid in DW + 7% gelatin 44 / 146

[0258] Pharmacokinetic characteristics were evaluated after administering compound 1 of the present invention (ONG41003) once to male C57BL / 6 mice as shown in Table 3 below.

[0259] Experimental Group Dosage and Administration Method Solvent Number of Samples (n) G1-ONG41003 (IV) ONG41003, 12.5 mg / kg, Intravenous Administration (IV) DMSO:PEG400:Saline (20:55;25) 6 G2-ONG41003 (PO) ONG41003, 50 mg / kg, Oral Administration (PO) 30% HPβCD 6 G3-ONG41003 (PO) ONG41003, 10 mg / kg, Oral Administration (PO) 2.5 citric acid in DW + 7% gelucire 44 / 146 G4-ONG41003 (PO) ONG41003, 25 mg / kg, Oral Administration (PO) 2.5 citric acid in DW + 7% gelucire 44 / 146 G5-ONG41003 (PO) ONG41003, 50 mg / kg, oral administration (PO) 2.5 citric acid in DW + 7% gelucire 44 / 146G6-ONG41003(PO)ONG41003, 100 mg / kg, oral administration (PO) 2.5 citric acid in DW + 7% gelucire 44 / 146

[0260] Evaluation of Comparative Compound 1 (ONG21001) confirmed that in the test group orally administered ONG21001 at a dose of 100 mpk (mg / kg), the plasma concentration was almost undetectable. This result implies that the compound has a low absorption rate that prevents it from reaching systemic circulation upon oral administration, suggesting that it exhibits characteristics unsuitable for development as an oral drug formulation (see Fig. 17).

[0261] As a result of the evaluation of Comparative Compound 2 (ONG41008), the bioavailability (F%) in the test groups orally administered ONG41008 at doses of 25, 50, and 100 mpk was confirmed to be at a low level (<20%), ranging from 12 to 16%. In addition, the half-life (t 1 / 2The drug showed a tendency to be rapidly eliminated from the body after administration, with a duration of 0.75 to 0.95 hours. In particular, it was confirmed that the plasma drug concentration decreased sharply starting 4 hours after administration, suggesting that multiple administrations of three or more times a day would be necessary when developing an oral formulation (see Fig. 18).

[0262] As a result of evaluating Compound 1 of the present invention (ONG41003), in the test groups orally administered ONG41003 at doses of 10, 25, 50, and 100 mpk, the bioavailability (F%) was found to be 27–53%, which was evaluated as an intermediate level (20–50%) or high level (>50%) of bioavailability. In addition, the half-life (t 1 / 2 The duration was maintained for 3.25 to 4.2 hours, which was significantly longer than that of the comparative compound, and this was confirmed to be a characteristic that enables the stable maintenance of plasma drug concentration after oral administration (see Fig. 19).

[0263] As a result of this comparative evaluation, it was confirmed that the compound of the present invention has significantly improved bioavailability compared to Comparative Compound 1 and Comparative Compound 2, and that stable maintenance of drug concentration after administration is possible through the extension of the half-life. Accordingly, the compound of the present invention suggests that in the development of oral formulations, effective drug delivery is possible with a smaller dose, and the frequency of daily administration can be reduced, which is advantageous in terms of improving patient compliance and ensuring economic efficiency.

[0264] 5-2. Comparative Evaluation of Pharmacokinetics in Lung Tissue in a Pulmonary Fibrosis-Induced Mouse Model

[0265] In this embodiment, using a mouse model in which pulmonary fibrosis was induced by bleomycin, the drug delivery characteristics and accumulation efficiency for the target organ, lung tissue, were quantitatively evaluated by comparing the drug concentrations in lung tissue between Compound 1 (ONG41003) of the present invention, Comparative Compound 1 (ONG21001), and Comparative Compound 2 (ONG41008).

[0266] The experimental method is as follows. A pulmonary fibrosis model was induced in 10–14 week old male C57BL / 6 mice by administering a single dose of bleomycin (0.03 U / 50 µL saline) on Day 0 using intratracheal instillation (IT) according to the literature by Kremer et al. Subsequently, the following compounds were orally administered for 14 days from Day 7 to Day 21.

[0267] - Compound of the present invention 1 (ONG41003)

[0268] - Comparative compound 1 (ONG21001, Eupatilin)

[0269] - Comparative Compound 2 (ONG41008)

[0270] Each compound was composed of multiple dose groups (10–200 mpk), and lung tissue was collected on day 21 after 14 days of administration, and the concentration of each compound in the tissue was evaluated using quantitative analysis methods such as LC-MS / MS.

[0271] As a result of the experiment, for Comparative Compound 1 (ONG21001, Eupatilin), the concentration in lung tissue was found to be between 10 and 172 ng / mL at administered doses of 10 to 200 mpk (see Fig. 20a). For Comparative Compound 2 (ONG41008), the concentration in lung tissue was found to be between 42 and 399 ng / mL at administered doses of 10 to 50 mpk (see Fig. 20b). On the other hand, it was confirmed that the compound of the present invention (ONG41003) reached a maximum concentration of 47,200 ng / mL in lung tissue under the same administration conditions (10 to 50 mpk) (see Fig. 20c).

[0272] The compound of the present invention exhibited the characteristic of maintaining a drug concentration in lung tissue that is more than 100 times higher than that of comparative compounds 1 and 2. This implies that the drug delivery efficiency to lung tissue after oral administration is significantly superior, and demonstrates that it possesses pharmacokinetic characteristics that are highly advantageous for the development of therapeutic agents targeting the lungs. In particular, the compound of the present invention is capable of maintaining a high concentration in lung tissue even at a low dose, suggesting that it is highly competitive in terms of sustained efficacy and therapeutic efficiency.

[0273] Example 6: Comparative evaluation of therapeutic effects on pulmonary fibrosis in a mouse model of pulmonary fibrosis induction

[0274] From the results of Examples 5-1 and 5-2, it was confirmed that the pharmacokinetic properties of Compound 1 of the present invention (ONG41003) in plasma and lung tissue are more favorable for drug development compared to Comparative Compound 2 (ONG41008). Based on this, in this example, the therapeutic effects of Compound 1 of the present invention and Comparative Compound 2 on pulmonary fibrosis were compared and evaluated using a mouse model in which pulmonary fibrosis was induced by bleomycin. The evaluation of Compound 1 of the present invention (ONG41003) was conducted by Syngene International, an Indian CRO, and the evaluation of Comparative Compound 2 (ONG41008) was conducted by Selvita Doo, a Polish CRO.

[0275] The experimental method is as follows. A pulmonary fibrosis model was induced in 10–14 week old male C57BL / 6 mice by administering a single dose of bleomycin (0.03 U / 50 µl saline) on Day 0 using intratracheal instillation (IT) according to the literature by Kremer et al. Subsequently, the following compounds were orally administered for each experiment for 14 days from Day 7 to Day 21.

[0276] - ONG41003 Test Evaluation: ONG41003, Pirfenidone, Nintedanib

[0277] - ONG41008 Test Evaluation: ONG41008, Nintedanib

[0278] Each compound was composed of multiple dose groups (10–200 mpk), and lung tissues were collected from mice in each group on day 21 after 14 days of administration for pathological evaluation. The degree of fibrosis was quantitatively analyzed based on the Modified Ashcroft score system (Reference: Hubner et al., Biotech. 44: 507–517, 2008).

[0279] As a result of the ONG41003 test evaluation, it was confirmed that pulmonary fibrosis was significantly induced in the bleomycin-alone treatment group. On the other hand, the level of fibrosis was significantly reduced in the test groups administered ONG41003 once a day at doses of 25 mpk and 50 mpk. In particular, a dose-dependent response was confirmed, with the effect being superior in the test group administered 50 mpk compared to the test group administered 25 mpk, and in the 50 mpk group, a fibrosis inhibitory effect equivalent to that of the two positive control drugs (Pirfenidone and Nintedanib), which are standard IPF drugs, was observed (see Fig. 21a).

[0280] The results of the ONG41008 study confirmed that pulmonary fibrosis was significantly induced in the bleomycin monotherapy group. In contrast, while the level of fibrosis was significantly reduced in the test group administered ONG41008 at a dose of 100 mpk twice daily, the effect was less pronounced compared to the positive control group administered Nintednib. In comparison, there was no significant therapeutic effect on fibrosis in the test groups administered ONG41008 at doses of 50 mpk or 200 mpk twice daily (see Fig. 21b).

[0281] In experiments conducted using a mouse animal model in which pulmonary fibrosis was induced by bleomycin, it was confirmed that the therapeutic effect on pulmonary fibrosis in the test group administered compound 1 of the present invention orally at a dose of 50 mpk (mg / kg) once daily was superior to that of the test group administered comparative compound 2 at a dose of 100 mpk twice daily. This indicates that, in the same disease model, compound 1 of the present invention exhibits a superior therapeutic effect even at a level of one-quarter of the total dose compared to comparative compound 2, suggesting that the compound of the present invention possesses superior pharmacological characteristics compared to comparative compound 2 in terms of drug efficiency and efficacy.

[0282] In addition, when combining the results of the preceding Examples 5-1 and 5-2, it was confirmed that the compound of the present invention exhibits superior characteristics compared to Comparative Compound 2 in terms of pharmacokinetic properties (PK profile), which suggests that the compound of the present invention has significantly superior competitiveness as a treatment for fibrosis compared to Comparative Compound 2.

[0283] Example 7: Comparative evaluation of cytotoxicity against ONGHEPA1 cells

[0284] Hepatic stellate cells (HSCs) are known to play a central role in the pathological process of liver fibrosis, and accordingly, they are widely used as a major cell model for evaluating the efficacy of anti-fibrotic drugs. In this embodiment, the cytotoxicity of Compound 1 of the present invention (ONG41003) and Comparative Compound 2 (ONG41008) was evaluated against ONGHEPA1 (KCTC13086BP), a mesenchymal stem cell (MSC) derived from mouse hepatic stellate cells (HSCs) capable of infinite proliferation.

[0285] The experimental method involved treating each compound at various concentrations (e.g., 6.25, 12.5, 25, 50 μM, etc.) and evaluating cytotoxicity by measuring cell viability using the CCK-8 assay at 24 and 48 hours from the time of treatment, respectively.

[0286] As a result of the experiment, Compound 1 of the present invention did not significantly affect cell viability up to a concentration of 25 μM, and no significant cytotoxicity was observed. However, at a concentration of 50 μM, most ONGHEPA1 cells died, confirming that cytotoxicity is induced at high concentrations. On the other hand, Comparative Compound 2 showed mild cytotoxicity starting from a concentration of 6.25 μM or higher, but exhibited relatively lower cytotoxicity compared to Compound 1 of the present invention at a concentration of 50 μM (see Figs. 22a and 22b).

[0287] When summarizing the above results, it was confirmed that the compound of the present invention exhibits limited cytotoxicity to ONGHEPA1 cells at concentrations below a certain level, but strong cytotoxicity at concentrations of 50 μM or higher, and therefore it is important to establish an effective and safe concentration range when used for purposes such as antifibrotic therapeutic agents.

[0288] 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. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

[0289] Since the present invention allows for the use of novel chromone derivatives for the prevention or treatment of fibrosis, there is great potential for industrial application in the fields of pharmaceuticals, food, and bio-industry.

Claims

1. A pharmaceutical composition for the prevention or treatment of fibrosis comprising, as an active ingredient, a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof. [Chemical Formula 1] In the above chemical formula 1, R1 is methyl, ethyl, cyclopentyl, cyclohexyl, phenyl, or benzyl, and R2 is hydrogen, ethyl, acetyl, acetoxy, carboxy, benzoyloxy, or 3,4,5-trihydroxybenzoyloxy, and R3 to R5 are each independently hydrogen, hydroxyl, methyl, methoxy, acetoxy, carboxyl, or benzoyloxy.

2. In Paragraph 1, R1 is methyl, and R2 is hydrogen, and R3 is hydrogen, hydroxy, or methoxy, and R4 is hydroxyl or methoxy, and A pharmaceutical composition for the prevention or treatment of fibrosis, characterized in that R5 is hydrogen, hydroxy, or methoxy.

3. In Paragraph 1, A pharmaceutical composition for the prevention or treatment of fibrosis, characterized in that the above compound is represented by any one of the following chemical formulas 2 to 5. [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] 4. In Paragraph 1, A pharmaceutical composition for the prevention or treatment of fibrosis, characterized in that the above-mentioned fibrosis is a disease selected from the group consisting of idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, rheumatic pulmonary fibrosis (RA-ILD), asbestos-induced pulmonary fibrosis, ocular fibrosis, pancreatic fibrosis, myelofibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, and systemic sclerosis.

5. In Paragraph 1, A pharmaceutical composition for the prevention or treatment of fibrosis characterized by the above compound inhibiting TGF-β (Transforming Growth Factor-β) signaling by inhibiting intracellular proteasomes.

6. In Paragraph 1, A pharmaceutical composition for the prevention or treatment of fibrosis characterized by the above compound inducing autophagy by inhibiting intracellular proteasomes.

7. A food composition for the prevention or improvement of fibrosis comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

8. In Paragraph 7, A food composition for the prevention or improvement of fibrosis, characterized in that the above-mentioned fibrosis is a disease selected from the group consisting of idiopathic pulmonary fibrosis, radiation-induced pulmonary fibrosis, rheumatic pulmonary fibrosis (RA-ILD), asbestos-induced pulmonary fibrosis, ocular fibrosis, pancreatic fibrosis, myelofibrosis, liver fibrosis, kidney fibrosis, cardiac fibrosis, and systemic sclerosis.

9. Use of the compound of paragraph 1 or its pharmaceutically acceptable salt for the prevention or treatment of fibrosis.

10. In methods for preventing or treating fibrosis, A method for the prevention or treatment of fibrosis, characterized by comprising the step of administering an effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof to a subject requiring treatment of fibrosis.

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