Novel compound and pharmaceutical composition for preventing or treating pulmonary fibrosis comprising same as active ingredient

Abstract

The present invention relates to a novel compound and a pharmaceutical composition for preventing or treating pulmonary fibrosis comprising the same as an active ingredient. Specifically, the invention relates to a compound represented by the following chemical formula I; a pharmaceutical composition for preventing or treating pulmonary fibrosis comprising the compound as an active ingredient; a method for preventing or treating pulmonary fibrosis using the pharmaceutical composition; and a food composition for preventing or alleviating pulmonary fibrosis comprising the compound as an active ingredient. In the chemical formula I, R is methyl or vinyl. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to novel compounds and pharmaceutical compositions for preventing or treating pulmonary fibrosis containing the compounds as active ingredients. Specifically, the present invention relates to compounds represented by Formula I; pharmaceutical compositions for preventing or treating pulmonary fibrosis, which contain the compounds as active ingredients; methods for preventing or treating pulmonary fibrosis using the pharmaceutical compositions; and food compositions for preventing or treating pulmonary fibrosis, which contain the compounds as active ingredients.

[0002] [Formula I]

[0003]

[0004] Wherein in the above Formula I,

[0005] R is methyl or vinyl. Background Art

[0006] Fibrosis refers to the formation of excessive fibrous connective tissue in an organ or tissue during a regeneration process or the like, which is contrary to the normal fibrous tissue formed in the organ or tissue. Examples of fibrosis include pulmonary fibrosis, liver fibrosis, renal fibrosis, pancreatic fibrosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, necrotizing fibrosis (lung), nephrogenic systemic fibrosis (skin), Crohn's disease, keloid, myocardial infarction, systemic scleroderma, etc.

[0007] Among them, pulmonary fibrosis or idiopathic pulmonary fibrosis (IPF) is a representative pulmonary disease in which repeated inflammation induced by alveolar damage in a patient induces fibrosis and leads to respiratory failure. In pulmonary fibrosis, IPF is a progressive disease, the cause of which has not been determined, and dyspnea and cough deteriorate within three to four years after diagnosis and death results from respiratory failure, and the five-year survival rate is about 30% to 40%, similar to lung cancer.

[0008] Current treatments for pulmonary fibrosis mainly involve the use of steroids or immunosuppressants, which are cytotoxic drugs. Between steroids and cytotoxic drugs, steroids are first used, and a therapy combining steroids with azathioprine or cyclophosphamide is currently used as a treatment for radiation-induced pulmonary fibrosis (Ochoa et al., Journal of Medical Case Reports, 6:413.2012).

[0009] In addition, Esbriet (active ingredient: pirfenidone) from Roche and Ofev (active ingredient: nintedanib) from Boehringer Ingelheim are known to be drugs for treating or improving pulmonary fibrosis. Among these drugs, pirfenidone, which was approved by the FDA in 2014, is known to mainly inhibit the action of TGF-β, thereby delaying the deterioration and progression of IPF as an anti-inflammatory and anti-fibrotic agent, while nintedanib exhibits an anti-fibrotic effect as a multi-tyrosine kinase inhibitor.

[0010] However, these two drugs show limited therapeutic effects as treatments for early or moderate IPF and may cause gastrointestinal side effects such as diarrhea, abdominal pain, loss of appetite, impaired liver function, and photosensitivity. Since these drugs only relieve the decline in lung function and do not exhibit a fundamental therapeutic effect, there is a need to develop a more effective treatment for pulmonary fibrosis. Summary of the Invention

[0011] Technical Problem

[0012] Research was conducted to develop candidate substances that are more effective than existing substances in treating and improving pulmonary fibrosis. As a result, the present inventors confirmed that a new sulforaphane-based compound exhibits an inhibitory effect on pulmonary fibrosis by regulating the expression of genes and proteins related to pulmonary fibrosis, and completed the present invention.

[0013] Technical Solution

[0014] An object of the present invention is to provide a compound represented by the following formula I or a pharmaceutically acceptable salt thereof.

[0015] [Formula I]

[0016]

[0017] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating pulmonary fibrosis, which contains the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0018] Another object of the present invention is to provide a method for preventing or treating pulmonary fibrosis using the pharmaceutical composition.

[0019] Another object of the present invention is to provide a food composition for preventing or improving pulmonary fibrosis, which contains the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0020] Another object of the present invention is to provide the use of the compound or a pharmaceutically acceptable salt thereof or a composition containing them in preventing, improving, or treating pulmonary fibrosis.

[0021] Advantageous Effects

[0022] The composition containing the compound of the present invention not only regulates the expression of phosphorylated proteins such as p38, AKT, smad2 and smad7, but also inhibits the expression of pulmonary fibrosis marker genes and proteins by specifically inhibiting the SRF / MRTF signaling pathway; therefore, the composition containing the compound as an active ingredient can be effectively used as an effective treatment for pulmonary fibrosis. Brief Description of the Drawings

[0023] Figure 1 The cytotoxicity of sulforaphane-based synthetic compounds was confirmed.

[0024] Figure 2 The inhibitory effects of treatment with 16 compounds on the expression of pulmonary fibrosis marker proteins in TGF-β1-induced lung fibroblasts are shown.

[0025] Figure 3 The inhibitory effects of treatment with different concentrations of Compound 1 and Compound 2, which are sulforaphane-based synthetic compounds showing inhibitory effects on pulmonary fibrosis, on the expression of pulmonary fibrosis marker proteins are shown.

[0026] Figure 4 a to Figure 4 d show the observations of each result of cytotoxicity and morphological changes, analysis of pulmonary fibrosis marker protein expression, and analysis of pulmonary fibrosis marker gene expression in normal lung fibroblasts (MRC-5) and diseased lung fibroblasts (DHLF-IPF) according to the treatment with Compound 1 and Compound 2.

[0027] Figure 5 The inhibitory effects of treatment with Compound 1 and Compound 2 on cell migration in normal lung fibroblasts (MRC-5) and diseased lung fibroblasts (DHLF-IPF) are shown by transwell migration assay.

[0028] Figure 6 The inhibitory effects of treatment with Compound 1 and Compound 2 on cell migration in normal lung fibroblasts (MRC-5) and diseased lung fibroblasts (DHLF-IPF) are shown by wound healing assay.

[0029] Figure 7 The analysis results of the expression of proteins involved in the TGF-β1-induced signaling pathway in diseased lung fibroblasts (DHLF-IPF) treated with Compound 1 are shown.

[0030] Figure 8Shows the analysis results of the expression of proteins involved in the TGF-β1-induced signaling pathway in diseased lung fibroblasts (DHLF-IPF) treated with Compound 2.

[0031] Figure 9 Figures 9a and 9b show the analysis results of the changes in body weight and lung weight over time in a bleomycin-induced pulmonary fibrosis animal model treated with Compound 1 and Compound 2.

[0032] Figure 10 Figures 10a and 10b show the analysis results of the changes in collagen content in the lung tissue of a bleomycin-induced pulmonary fibrosis animal model treated with Compound 1 and Compound 2.

[0033] Figure 11a and 11b Shows the analysis results of the histological changes of inflammation and fibrosis markers in the lung tissue of a bleomycin-induced pulmonary fibrosis animal model treated with Compound 1 and Compound 2.

[0034] Figure 12a and 12b Shows the histological analysis results of the expression of fibrosis-related protein markers in the lung tissue of a bleomycin-induced pulmonary fibrosis animal model treated with Compound 1 and Compound 2.

[0035] Figure 13 Figures 13a and 13b show the analysis results of the expression of fibrosis-related genes in the lung tissue of a bleomycin-induced pulmonary fibrosis animal model treated with Compound 1 and Compound 2. Detailed Description of the Invention

[0036] The present invention will be described in detail below. At the same time, each description and embodiment disclosed in the present invention can also be applied to other descriptions and embodiments. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present disclosure. In addition, the scope of the present invention is not limited by the specific descriptions described below.

[0037] One aspect of the present invention for achieving the above object provides a compound represented by the following formula I or a pharmaceutically acceptable salt thereof.

[0038] [Formula I]

[0039]

[0040] Wherein, in the above formula I,

[0041] R is methyl or vinyl.

[0042] Formula I can specifically refer to Compound 1 or Compound 2 represented by the following Formula 1 or Formula 2, respectively.

[0043] [Formula 1]

[0044]

[0045] [Formula 2]

[0046]

[0047] In the present invention, Formula 1 and Formula 2 are novel sulforaphane-based compounds, which have molecular weights of 368.5 and 380.5 respectively, which are greater than twice the molecular weight of sulforaphane having a molecular weight of 177.3. These compounds are characterized by inhibiting the deterioration or progression of pulmonary fibrosis.

[0048] The novel compounds of the present invention can be chemically synthesized by methods known in the art and can exist in unsolvated as well as solvated forms. Additionally, the compounds can exist in crystalline or amorphous forms, and all such physical forms are included within the scope of the present invention.

[0049] In the present invention, the term "pharmaceutically acceptable salt" refers to salts commonly used in the pharmaceutical industry. Examples can include inorganic salts made from metal ions such as calcium, potassium, sodium, magnesium, etc.; inorganic salts made from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, bromic acid, iodic acid, perchloric acid, tartaric acid, sulfuric acid, etc.; organic salts made from organic acids such as acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, etc.; sulfonates made from methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc.; amino acid salts made from glycine, arginine, lysine, etc.; and amine salts made from trimethylamine, triethylamine, ammonia, pyridine, methylpyridine, etc. However, the types of salts involved in the present invention are not limited to these listed examples.

[0050] Another aspect of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating pulmonary fibrosis, which contains the novel compound or its pharmaceutically acceptable salt as an active ingredient.

[0051] In the present invention, the term "pulmonary fibrosis" is a type of chronic interstitial lung disease, also known as idiopathic pulmonary fibrosis (IPF), and refers to a disease in which lung tissue cells are transformed into fibrotic cells, causing symptoms such as dyspnea, cough, cyanosis, and clubbing. During a tissue biopsy, honeycombing or atypical clusters of fibrotic cells are observed. Current treatments include the use of immunosuppressants, including steroid-based therapies, interferon γ, acetylcysteine, pirfenidone, nintedanib, bosentan, etc., but there are no reports of agents showing specific therapeutic effects.

[0052] In the present invention, pulmonary fibrosis can refer to one selected from the group consisting of: chronic obstructive pulmonary disease with pulmonary fibrosis (COPD with pulmonary fibrosis), pulmonary fibrosis with emphysema, idiopathic pulmonary fibrosis (IPF), pulmonary fibrosis caused by anticancer treatment, and pulmonary fibrosis caused by a virus, particularly IPF, but not limited thereto.

[0053] In the present invention, the term "prevention" refers to all actions of inhibiting or delaying the progression of pulmonary fibrosis by administering a pharmaceutical composition containing the compound as an active ingredient.

[0054] In the present invention, the term "treatment" refers to all actions of improving or beneficially altering the symptoms of pulmonary fibrosis by administering a pharmaceutical composition containing the compound as an active ingredient.

[0055] In the present invention, the term "pharmaceutical composition" may additionally include pharmaceutically acceptable carriers, excipients, or diluents commonly used for preparing pharmaceutical compositions, and the carrier may include non-naturally occurring carriers. Specific examples of carriers, excipients, and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl paraben, propyl paraben, talc, magnesium stearate, mineral oil, etc., but not limited thereto.

[0056] In addition, the pharmaceutical composition can be in any form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions for internal use, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories, and can be in various oral or parenteral forms. When formulated, the pharmaceutical composition is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, or surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and the solid preparations can contain one or more excipients such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, solutions for internal use, emulsions, and syrups, which can contain commonly used simple diluents such as water and liquid paraffin, and various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. To prepare preparations for parenteral administration, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc. can be used. For non-aqueous solutions and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate can be used. For the matrix of suppositories, witepsol, macrogol, Tween 61, cocoa butter, glyceryl laurate, glycerogelatin, etc. can be used, but the additives to be added are not limited thereto.

[0057] The content of the compound in the pharmaceutical composition of the present invention can be appropriately adjusted according to the patient's symptoms, progression, condition, etc. For example, based on the total weight of the composition, it can be 0.0001% by weight to 99.9% by weight or 0.001% by weight to 50% by weight, but is not limited to these ranges.

[0058] In one embodiment of the present invention, it was confirmed that the expression levels of fibronectin and α-SMA increased by TGF-β1 treatment were decreased in a dose-dependent manner by treating with different concentrations of Compound 1 and Compound 2, respectively, and it was confirmed that the effect of reducing the expression at a concentration of 20 μM or 10 μM of each compound was similar to or better than that of sulforaphane ( Figure 3 ).

[0059] In addition, in one embodiment of the present invention, MRC-5 (i.e., normal human lung fibroblasts) and diseased human lung fibroblasts - idiopathic pulmonary fibrosis (DHLF-IPF) (i.e., diseased lung fibroblasts) were treated with sulforaphane, Compound 1, and Compound 2, and the morphological changes of the cells were examined. The results confirmed that compared with the group treated with sulforaphane, the inhibition of cell proliferation in the groups treated with Compound 1 and Compound 2 was more significant, and the cells showed an elongated shape similar to that of the control group, while not showing a densely packed structure (Figure 4 a).

[0060] In addition, in one embodiment of the present invention, the effects on fibrotic-related proteins and gene expression in MRC-5 and DHLF-IPF fibroblasts were examined. The results confirmed that sulforaphane, compound 1, and compound 2 decreased the expression levels of proteins and genes induced by TGF-β1 in both cell types. In particular, compared with sulforaphane, compound 1 and compound 2 showed significant effects in reducing the expression levels ( Figure 4 b to Figure 4 d).

[0061] In addition, in one embodiment of the present invention, the effects on cell migration in MRC-5 and DHLF-IPF were examined. The results confirmed that cell migration was increased by TGF-β1 treatment, especially in DHLF-IPF cells, where cell migration was significantly increased even without TGF-β1 induction. However, treatment with compound 1 and compound 2 significantly inhibited it in a dose-dependent manner ( Figure 5 and Figure 6 ).

[0062] In addition, in one embodiment of the present invention, the inhibitory effect of compound 1 treatment on pulmonary fibrosis in DHLF-IPF was examined by analyzing the expression of various signal transduction pathway proteins related to TGF-β1. The results confirmed that compound 1 inhibited the phosphorylation of smad-2, while increasing the phosphorylation of smad-7, and significantly decreased the activities of p-p38 MAPK and p-AKT. In addition, it was confirmed that compound 1 inhibited the expression of ROCK in the Rho-ROCK signal transduction pathway and the expression of both nuclear MRTF and SRF in the MRTF-SRF signal transduction pathway ( Figure 7 ).

[0063] In addition, in one embodiment of the present invention, the inhibitory effect of compound 2 treatment on pulmonary fibrosis in DHLF-IPF was examined by analyzing the expression of various signal transduction pathway proteins related to TGF-β1. The results confirmed that compound 2 inhibited the phosphorylation of smad-2 and smad-3, while increasing the phosphorylation of smad-7, and significantly decreased the activities of three phosphorylated proteins (i.e., p-ERK, p-JNK, and p-p38) in the MAPK signal transduction pathway, and significantly decreased the activity of p-AKT in the AKT signal transduction pathway. In addition, it was confirmed that compound 2 inhibited the expression of ROCK in the Rho-ROCK signal transduction pathway and the expression of both nuclear MRTF and SRF in the MRTF-SRF signal transduction pathway ( Figure 8 ).

[0064] These results indicate that the compounds of the present invention can be effectively used for preventing or treating pulmonary fibrosis.

[0065] Another aspect of the present invention for achieving the above object provides a method for preventing or treating pulmonary fibrosis, which comprises administering the pharmaceutical composition to a subject.

[0066] The pharmaceutical composition, pulmonary fibrosis, prevention and treatment are as described above.

[0067] In the present invention, the term "subject" refers to humans and all animals in which pulmonary fibrosis has occurred or may occur, including mice and domestic animals. Specifically, the subject can be a mammal, including cows, horses, sheep, pigs, goats, camels, antelopes, dogs, cats, etc., and humans who need to prevent or treat symptoms similar to those of the disease, but are not limited thereto.

[0068] In addition, the subject may or may not include humans.

[0069] In the present invention, the term "administer" means introducing the composition of the present invention into a patient by an appropriate method. The administration route of the composition can be administered by any conventional route as long as it can reach the target tissue.

[0070] The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount.

[0071] The term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit-risk ratio applicable to medical treatment, and the effective dose level can be based on factors including the type, age and sex of the subject, the severity of the disease, the activity of the drug, the sensitivity to the drug, the administration time, the administration route and the excretion rate, the duration of treatment and the concomitant medications, and other factors well known in the medical field.

[0072] The pharmaceutical composition of the present invention can be administered as a single agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. In addition, the pharmaceutical composition can also be administered in a single dose or multiple doses. Considering all these factors, it is important to administer an amount that can maximize the effect while minimizing the side effects, which can be easily determined by those skilled in the art.

[0073] In addition, the pharmaceutical composition can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically). The dose varies depending on the condition and weight of the patient, the severity of the disease, the form of the drug, and the route and time of administration, but can be appropriately selected by those skilled in the art. In a specific example, the pharmaceutical composition can usually be administered once or more times a day, but the preferred dose can be appropriately selected by those skilled in the art according to the condition and weight of the subject, the severity of the disease, the form of the drug, and the route and time of administration.

[0074] In one embodiment of the present invention, in a bleomycin-induced pulmonary fibrosis animal model, the inhibitory effects of the finally selected Compound 1 and Compound 2 on pulmonary fibrosis were examined. The results confirmed that in the two compound treatment groups, the hydroxyproline content in the lung tissue was significantly reduced ( Figure 10 ), the degree of inflammation and fibrosis in the lung tissue was reduced (Figure 11), and the expression levels of proteins and genes related to the occurrence of fibrosis such as α-SMA, collagen, and fibronectin were also reduced (Figures 12 and 13).

[0075] Another aspect of the present invention for achieving the above object provides a food composition for preventing or improving pulmonary fibrosis, which contains the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0076] The compound, pulmonary fibrosis, and prevention are as described above.

[0077] In the present invention, the term "improve" refers to all effects of reducing parameters related to a disease that can be treated by administering a composition containing a novel compound, such as the severity of symptoms.

[0078] In the present invention, the term "food" can be meat, sausage, bread, chocolate, candy, snacks, desserts, pizza, ramen, other noodles, chewing gum, dairy products, including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, health functional foods, health foods, etc., and includes all foods in the conventional sense.

[0079] The term health functional food is a synonym for foods for specified health uses (FoSHU), and refers to foods with high medical and therapeutic effects, which, after processing, can effectively exhibit biological regulatory functions in addition to providing nutrition.

[0080] In this article, "function / functionality" refers to obtaining effects useful for health purposes, such as nutritional regulation and physiological effects on the structure and function of the human body. A health food refers to a food that has a positive effect of maintaining or promoting health compared to general foods, and a health care food refers to a food for health care supplement purposes. Depending on the context, the terms health functional food, health food, and health care food can be used interchangeably. Specifically, a health functional food refers to a food prepared by adding the novel compound of the present invention to food materials such as beverages, tea, spices, gums, and desserts, or a food prepared in the form of capsules, powders, or suspensions, and can refer to a food that can provide specific health effects when consumed.

[0081] The food of the present invention can be prepared by methods conventionally used in the art and by adding raw materials and components conventionally added in the art.

[0082] In addition, the food composition can be prepared into various types of preparations without limitation, as long as the preparation is recognized as food.

[0083] In addition, the food composition may further comprise a nutritionally (sitologically) acceptable carrier, and the type of the carrier is not particularly limited and can be any carrier conventionally used in the art.

[0084] In addition, the food composition may contain additional ingredients conventionally used in food compositions that can improve the odor, taste, and appearance. Examples may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folic acid (salt), pantothenic acid, etc. In addition, it may also include minerals such as zinc (Zn), iron (Fe), calcium (Ca), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr); and amino acids such as lysine, tryptophan, cysteine, and valine.

[0085] In addition, the food composition may include food additives such as preservatives (e.g., potassium sorbate, sodium benzoate, salicylic acid, and sodium dehydroacetate), bactericides (e.g., bleaching powder, high-performance bleaching powder, and sodium hypochlorite), antioxidants (e.g., butylated hydroxyanisole (BHA) and dibutylhydroxytoluene (BHT)), colorants (e.g., tar pigments), color fixatives (e.g., sodium nitrite and sodium nitrate), bleaching agents (e.g., sodium sulfite), flavoring agents (e.g., monosodium glutamate (MSG)), sweeteners (p-ethoxyphenylurea, cyclamate, saccharin, and sodium), flavor correctives (e.g., vanillin and lactones), leavening agents (e.g., alum and potassium bitartrate), fortifiers, emulsifiers, thickeners (pastes), coating agents, gum bases, defoaming agents, solvents, and modifiers. These additives can be selected according to the type of food and used in appropriate amounts.

[0086] Another aspect of the present invention for achieving the above object provides a feed composition for preventing or improving pulmonary fibrosis, which comprises the compound or its pharmaceutically acceptable salt as an active ingredient.

[0087] The compound, pulmonary fibrosis, and prevention are as described above.

[0088] The term "feed" refers to any natural or artificially formulated diet, meal, etc., or a component of a meal that is used for or suitable for consumption, ingestion, and digestion by animals.

[0089] The type of feed is not particularly limited, and any feed conventionally used in the art can be used. Non-limiting examples of feeds can include plant-based feeds such as grains, nuts, food processing by-products, algae, fibers, pharmaceutical by-products, oils, starches, cakes or grain by-products; and animal-based feeds such as proteins, minerals, inorganic substances, fats, minerals, oils, single-cell proteins, zooplankton or foods. These can be used alone or in combination of two or more.

[0090] Another aspect of the present invention for achieving the above object provides the use of the compound or its pharmaceutically acceptable salt or a composition containing the same in preventing, improving or treating the composition. Additionally, it provides the use of the compound or its pharmaceutically acceptable salt or a composition containing the same in the preparation of a drug, food or feed for preventing, improving or treating pulmonary fibrosis.

[0091] The compound, pharmaceutically acceptable salt, pulmonary fibrosis, prevention, improvement and treatment are as described above.

[0092] Modes of implementing the present invention

[0093] The present invention will be described in detail below by way of examples. However, these examples are given for illustrative purposes only, and the scope of the present invention is not intended to be limited by these examples.

[0094] Synthesis Example 1: Synthesis of a novel compound

[0095] [Compound 1]

[0096]

[0097] To an ethanol solution (2 mL) of N-acetyl-L-cysteine ethyl ester (CAS No.: 59587-09-6, 150 mg, 0.78 mmol) was added 1N NaOH to adjust the pH of the solution to 8. To this was added an ethanol solution (2 mL) of sulforaphane (CAS No.: 4478-93-7, 50 mg, 0.28 mmol). The reaction mixture was stirred at room temperature under nitrogen for 4 hours. After evaporating the solvent, the residue was purified by reverse-phase column chromatography using a methanol solution containing 0.05% TFA to obtain Compound 1 (160 mg, yield: 55%). 1 H NMR(600MHz,CDCl 3)δ9.09(s,1H),7.02(d,J=7.4Hz,1H),4.83-4.59(m,1H),4.16(q,J=7.1Hz,3H),3.80-3.68(m,3H),3.67-3.49(m,1H),3.07-2.81(m,1H),2.78-2.67(m,1H),2.56(d,J=1.2Hz,3H),2.01(d,J=8.1Hz,1H),1.95(s,3H),1.81(s,1H),1.24(t,J=7.1Hz,4H); 13 C NMR(150MHz,CDCl 3 )δ196.75,170.67,170.32,61.99,53.50,53.08,46.84,38.47,35.82,26.94,23.00,20.19,14.13;ESI-MS(positive ion mode):C 13 H 24 O 4 N 2 NaS 3 Calculated m / z for [M+Na] + =391.53,found:391.40.

[0098] [Compound 2]

[0099]

[0100] To a solution of Ac-Cys-OAllyl (CAS No.: 616-91-1, 138 mg, 0.67 mmol) in ethanol (2 mL) was added 1 N NaOH to adjust the pH of the solution to 8. Then a solution of sulforaphane (CAS No.: 4478-93-7, 100 mg, 0.56 mmol) in ethanol (2 mL) was added. The reaction mixture was stirred at room temperature under nitrogen for 10 h. After evaporation of the solvent, the residue was purified by reverse-phase column chromatography using a methanol solution containing 0.05% TFA to give Compound 2 (112 mg, yield: 52%). 1 H NMR(600MHz,CD 3OD) δ 6.09 - 5.90 (m, 1H), 5.42 - 5.31 (m, 1H), 5.31 - 5.11 (m, 1H), 4.76 - 4.68 (m, 1H), 4.68 - 4.59 (m, 2H), 3.95 (dd, J = 14.2 and 5.2 Hz, 1H), 3.77 - 3.71 (m, 1H), 3.52 (dd, J = 14.2 and 8.5 Hz, 1H), 2.96 - 2.76 (m, 3H), 2.65 (d, J = 10.4 Hz, 3H), 1.97 (s, 3H), 1.91 - 1.76 (m, 4H); 13 C NMR (150 MHz, CD 3 OD) δ 197.75, 173.31, 171.59, 133.15, 118.59, 67.02, 54.26, 53.89, 45.48, 38.14, 34.74, 28.09, 22.36, 21.09; ESI-MS (positive ion mode): C 14 H 24 N 2 NaO 4 S 3 Calculated m / z for [M + Na] + = 403.08, found 403.21.

[0101] Example 1. Evaluation of the Cytotoxicity of Synthetic Compounds Based on Sulforaphane

[0102] Cell culture and cytotoxicity evaluation

[0103] The normal lung fibroblast MRC-5 cell line was obtained from the Korean Cell Line Bank. These cells were incubated in DMEM supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / mL streptomycin at 37 °C in a 5% CO 2 incubator. All cells used during the experiment were tested at 80% to 90% confluence.

[0104] To determine the optimal concentration of non-toxicity for each compound synthesized from sulforaphane, MRC-5 cells were seeded in a 96-well plate at a density of 5 × 10 3 cells / well in 100 μL of DMEM containing 10% FBS and cultured at 37 °C in a 5% CO 2 incubator for 24 hours.

[0105] After incubation, the medium was removed, various concentrations of each compound were prepared and added to the cells in triplicate, and the cells were incubated at 37 °C in a 5% CO 2Incubate in an incubator for 24 hours. After incubation, add 10 μL of 5 mg / mL MTT reagent to the medium containing the test solution in each well, and place the plate in a 5% CO 2 incubator and incubate in the dark for 2 to 4 hours. After completion of incubation, remove the medium containing the MTT reagent, and add 100 μL of DMSO (dimethyl sulfoxide) thereto to dissolve the MTT-formazan crystals. Measure the absorbance of the cells at 570 nm to determine the cell viability compared to the control group.

[0106] Cell viability (%) = (Absorbance of the control group / Absorbance of the experimental group) × 100

[0107] As a result, as Figure 1 shown, use a concentration that does not reduce the cell viability to less than 80% compared to the untreated control group, and most compounds maintain a cell viability of more than 80% even at 40 μM, and GSF-18 shows a cell viability of more than 80% at 10 μM.

[0108] Example 2. Analysis of the expression of fibrosis-related proteins by sulforaphane-based synthetic compounds

[0109] Since the overexpression of extracellular matrix components such as fibronectin and α-SMA serves as an indicator of fibrotic changes in tissues, MRC-5 cells undergoing fibrosis induced by TGF-β1 were treated with each compound at different concentrations to compare the expression levels of fibronectin and α-SMA.

[0110] Specifically, to extract fibronectin and α-SMA proteins, the cells were washed once with cold PBS, and then RIPA buffer (150 mM NaCl, 0.5% Triton X-100, 50 mM Tris-HCl, pH 7.4, 25 mM NaF, 20 mM EGTA) containing a protease inhibitor mixture was added thereto to lyse the cells. Then, the proteins were quantified using a BCA protein assay kit. All protein samples were electrophoresed at 10 μg on an 8% to 10% PAGE gel, and then transferred onto a 0.2 μm PVDF membrane (EMD Millipore, MA, USA). The membrane onto which the proteins were transferred was blocked with 5% BSA or 5% skim milk for 1 hour, and then a primary antibody was added and incubated overnight at 4°C. The next day, the membrane was washed three times with PBST, and then an HRP-conjugated secondary antibody was added and reacted at room temperature for 1 hour. The PVDF membrane prepared by such a procedure was smeared with ECL (Luminata TM Crescendo, EMD Millipore) and exposed to a fluorescence reader to confirm the protein expression level.

[0111] As a result, as Figure 2 shown, when lung fibrosis was induced by TGF-β1, the expression of fibronectin and α-SMA increased significantly, and when treated with GSF-016 and GSF-018 in the sulforaphane compounds, the expression levels of these two proteins decreased significantly.

[0112] Therefore, these two compounds were named Compound 1 and Compound 2 respectively and selected as candidate compounds for the treatment of fibrosis in the present invention.

[0113] Example 3. Selection of sulforaphane prodrugs

[0114] Example 3-1. Analysis of the effects of candidate compounds on the expression of fibrosis-related proteins

[0115] To compare the expression levels of fibronectin and α-SMA at concentrations of sulforaphane (SFN) and Compound 1 and Compound 2 that significantly reduced the expression of these proteins increased due to lung fibrosis in Example 2, Western blotting was performed.

[0116] The specific experimental method was the same as in Example 2, and the cells were treated with sulforaphane at a concentration of 20 μM, with Compound 1 at concentrations of 5 μM, 10 μM, and 20 μM, and with Compound 2 at concentrations of 2.5 μM, 5 μM, and 10 μM.

[0117] As a result, as Figure 3 shown, it was confirmed that the expression of fibronectin and α-SMA treated with various concentrations of Compound 1 and Compound 2 decreased in a concentration-dependent manner, and these compounds showed similar or better effects of reducing expression than sulforaphane at a concentration of 20 μM or 10 μM.

[0118] Example 3-2. Analysis of the cytotoxicity and morphology of candidate compounds in two cell lines

[0119] The effects of two candidate compounds of sulforaphane prodrugs on the cytotoxicity and morphological changes of normal lung fibroblasts MRC-5 cells and diseased lung fibroblasts DHLF-IPF cells were examined. The diseased lung fibroblast DHLF-IPF cell line was purchased from Lonza and cultured in FGM 2 -2 Fibroblast Growth Medium-2 BulletKit TM at 37 °C in a 5% CO TM incubator. All cells used in the experiment were tested at a confluence of 80% to 90%.

[0120] The two types of cells were seeded at 5×10 3Cells were each dispensed at 100 μL per well in a 96-well plate and cultured in a 5% CO 2 incubator at 37 °C for 24 hours. Then, the medium was removed, and each compound was prepared in triplicate for each group at a concentration of 10 μM, and then incubated in a 5% CO 2 incubator at 37 °C for 24 hours.

[0121] The detailed experimental method for cytotoxicity was the same as in Example 1.

[0122] In addition, changes in the cell morphology of two types of lung fibroblasts induced by TGF-β1 treated with Compound 1 and Compound 2 were examined by microscopy with the naked eye.

[0123] As a result, as Figure 4 shown in a, at a concentration of 10 μM, both compounds showed a cell viability of over 80% in both cell lines. When examining the morphological changes in the two cell lines, it was found that compared with the cells with an elongated shape in the control group, cell proliferation in the TGF-β1 treatment group was more activated, inducing the cells to intertwine and densely pack like fibrous tissue. Conversely, it was confirmed that both compounds inhibited cell proliferation more effectively than the sulforaphane treatment group, showing an elongated cell shape similar to the control group and not as dense as fibrous tissue.

[0124] Example 3-3: Analysis of the effects of candidate compounds on the expression of fibrosis-related proteins in two cell lines

[0125] To compare the changes in the expression of fibrosis-related proteins in normal MRC-5 and the diseased DHLF-IPF cell line of pulmonary fibrosis induced by TGF-β1 treated with SFN and two candidate compounds, Western blotting was performed.

[0126] The specific experimental method was the same as in Example 2. TGF-β1 was treated at 1 ng / mL to induce fibrosis, and sulforaphane and the two compounds were each treated at a concentration of 10 μM.

[0127] As a result, as Figure 4 shown in b, compared with sulforaphane, the expression levels of fibronectin and α-SMA proteins were significantly reduced by the treatment with the two compounds.

[0128] Example 3-4: Analysis of the effects of candidate compounds on the expression of fibrosis-related genes in two cell lines

[0129] To confirm whether two compounds that can effectively inhibit the expression of fibrosis-related proteins in normal MRC-5 and TGF-β1-induced fibrotic DHLF-IPF cells also have the same effect at the gene level, the gene expressions of FN, COL1A1, and α-SMA were confirmed by qRT-PCR analysis.

[0130] RNA extraction from cells

[0131] MRC-5 and DHLF-IPF cells were each cultured in growth medium, and the medium was removed. The experimental groups (SFN, GSF-016, and GSF-018) were pretreated with serum-free medium at a concentration of 10 μM for 1 hour, 1 ng / mL TGF-β1 was added to induce fibrosis, and the cells were incubated at 37 °C in 5% CO 2 incubator for 48 hours. Then the medium was removed, and the cells were washed once with PBS. Total intracellular RNA was extracted from the cultured cells using TRIzol reagent (TaKaRa BioInc., Japan) for gene expression analysis. 1 mL of TRIzol reagent was added to lyse the cells and denature the tissue. The lysate was transferred to each 1.5 mL tube, and 200 μL of chloroform was added thereto, followed by vortexing for 20 seconds to ensure complete mixing. After incubation at room temperature for 15 minutes, the mixture was centrifuged at 14,000 rpm for 20 minutes to obtain the supernatant. After adding an equal volume of isopropanol, the tube was inverted and incubated at room temperature for 10 minutes. The sample was centrifuged at 14,000 rpm for 15 minutes to obtain an RNA pellet, which was washed with 70% ethanol for RNA at 14,000 rpm for 5 minutes and dried for 5 minutes. 20 μL of distilled water treated with 0.1% diethyl pyrocarbonate (DEPC) was added to the dried RNA sample to dissolve the pellet, and the resulting product was used as a sample for cDNA synthesis. Using 1 μL of the resulting product, the RNA concentration and purity were measured at O.D. 260 / 280 nm using a Nanodrop.

[0132] cDNA synthesis

[0133] Single-stranded cDNA synthesis was performed by mixing 1 μg of the extracted total RNA with 1 μL of oligo-d(T) primer (100 pmol) and 10 mM dNTP (TaKaRa Bio Inc., Japan), reacting at 65 °C for 5 minutes, and then rapidly cooling. 4 μL of 5x RT buffer, 0.5 μL of RNA inhibitor, and 100 units of Rtase (TaKaRa Bio Inc., Japan) were added to this template, and the total volume was adjusted to 20 μL using DEPC-treated distilled water. The sample was synthesized at 25 °C for 5 minutes, at 42 °C for 1 hour, and then reacted at 72 °C for 15 minutes to inactivate the reverse transcriptase and terminate the reaction.

[0134] RT-PCR

[0135] The expression levels of each gene were measured using real-time PCR. To 5 μL of the extracted cDNA of each sample, 10 μL of 2x SYBR Green MasterMix (TaKaRa Bio Inc., Japan) and 1 μL of 10 pmol (forward, reverse) primers were added for each gene, and the final volume was adjusted to 20 μL using distilled water. PCR was performed at 95 °C for 10 minutes (30 seconds starting from the second cycle), at 60 °C for 30 seconds, at 72 °C for 30 seconds, and then for 40 cycles. The specificity of the amplification product was confirmed by melting curve analysis, and the target gene was quantified and compared using GAPDH as a control gene.

[0136] As Figure 4 As shown in 4c and 4d, by treating with SFN and the two compounds, the increased gene expression levels of FN, COL1A1, and α-SMA in the two cell lines were reduced. In particular, it was confirmed that the group treated with the two compounds showed a more significant reduction in the expression level compared to the SFN-treated group.

[0137] Based on these results, among the sulforaphane-based synthetic compounds, Compound 1 and Compound 2 were selected as the final compounds for the treatment of pulmonary fibrosis in the present invention.

[0138] Example 4. Cell Migration

[0139] According to the characteristics of myofibroblasts, where the increased expression of proteins related to the occurrence of fibrosis leads to an increase in cell migration and invasion, normal and diseased fibroblasts were used for migration and wound healing assays to examine the effects of the finally selected Compound 1 and Compound 2 on cell migration.

[0140] Example 4-1. Transwell Migration Assay

[0141] Prepare a 24-well culture plate with 500 μL of medium containing FBS and place an 8-μm pore size insert on top. Inside the insert, add 300 μL of medium without FBS and seed MRC-5 and DHLF-IPF cells at a density of 1×10 5 cells. Incubate the cells at 37 °C in a 5% CO 2 incubator for 24 hours. After incubation, carefully remove the medium inside the 8-μm pore polycarbonate membrane insert, wash with DPBS, and fix in methanol for 5 minutes. Then, after washing twice with triple distilled water, stain the cells with Mayer's hematoxylin for 8 minutes. Wash the stained cells with DPBS, gently wipe the inside of the insert with a cotton swab, cut the membrane and fix it on a glass slide. Count the migrated cells at 200× magnification under a microscope.

[0142] As Figure 5 shown, it was confirmed that the migration of normal MRC-5 cells increased with TGF-β1 treatment and was significantly inhibited by Compound 1 and Compound 2 in a dose-dependent manner. Similarly, inherent high migration was observed in diseased DHLF-IPF cells and was significantly inhibited by Compound 1 and Compound 2 in a dose-dependent manner, similar to MRC-5 cells.

[0143] Example 4-2. Wound Healing Assay

[0144] Examine the effects of Compound 1 and Compound 2 selected in Example 3 on cell migration (wound healing) in MRC-5 and DHLF-IPF cells.

[0145] Specifically, seed cells (1×10 5 cells / well) in a 60-mm culture dish to achieve approximately 90% confluence and culture for 24 hours. Subsequently, draw a straight line at the bottom with a 200-μL pipette tip and remove the detached cells by washing once with Dulbecco's phosphate-buffered saline (DPBS). Take a picture of the wound under a microscope, apply treatments of TGF-β1 alone, TGF-β1 + SFN, TGF-β1 + GSF-016, and TGF-β1 + GSF-018 and culture for approximately 24 hours, then take a picture of the wound again. Analyze the images taken at 24 hours of culture using Image J (Fiji package) software to measure the distance between cells, and represent the relative migration as a percentage (%) compared to the control group.

[0146] As Figure 6As shown, the migration of both MRC-5 and DHLF-IPF cells increased with TGF-β1 treatment, resulting in a narrower wound compared to the control group, while all compound treatment groups showed a significant dose-dependent inhibition of migration.

[0147] Example 5. Analysis of the Expression of Proteins Related to the TGF-β1 Signaling Pathway

[0148] The TGF-β1 signaling pathway can be activated through both Smad-dependent and Smad-independent pathways. TGF-β / Smad signaling is the Smad-dependent pathway and is a key pathway regulating the synthesis of extracellular matrix components. The Smad-independent (TGF-β / non-Smad) pathway is affected by PI3K / AKT / mTOR and MAPK (JNK, ERK, p38) which are known to be involved in cell proliferation and growth. Based on this mechanism, the protein expression related to the TGF-β1 signaling pathway was comparatively analyzed using the same Western blotting method as performed in Example 2.

[0149] As Figure 7 shown, Compound 1 inhibited the phosphorylation of Smad-2, increased the phosphorylation of Smad-7, and significantly reduced the activities of p-p38 and MAPK. In addition, the influence of MRTF / SRF via the Rho / Rock pathway was confirmed. The expression level of Rock protein bound to Rho activated by TGF-β1 treatment increased, and the expression levels of MRT / SRF in the cytoplasm and nucleus also increased. However, the increased protein expression levels were significantly reduced by treatment with Compound 1.

[0150] In addition, as Figure 8 shown, Compound 2 inhibited the phosphorylation of Smad-2 and Smad-3 and increased the phosphorylation of Smad-7, thereby reducing the activities of three MAPKs: p-ERK, p-JNK, and p-p38. In addition, the influence of MRTF / SRF via the Rho / Rock pathway was confirmed. The expression level of Rock protein bound to Rho activated by TGF-β1 treatment increased, and the expression levels of MRT / SRF in the cytoplasm and nucleus also increased. However, the increased protein expression was significantly reduced by treatment with Compound 2.

[0151] Example 6. Inhibitory Effect on Pulmonary Fibrosis in an Animal Model

[0152] Example 6-1. Analysis of Changes in Body Weight and Lung Weight in an Animal Model of Pulmonary Fibrosis Treated with Compound 1 and Compound 2

[0153] The inhibitory effects of the finally selected Compound 1 and Compound 2 on pulmonary fibrosis were examined in a bleomycin (BLM)-induced animal model of pulmonary fibrosis.

[0154] Specifically, 7-week-old C57BL / 6N mice were exposed to BLM (3 - 5 units / kg) via the airway. For Compound 1, it was orally administered at doses of 200 μg / kg and 500 μg / kg, and for Compound 2, at doses of 100 μg / kg and 200 μg / kg, three times a week for three weeks starting from the day after BLM exposure. Body weight gain was regularly measured during the experimental period, and lung weight was measured at the end of the experiment.

[0155] As Figure 9 shown in a, the body weight gain during the experimental period showed a steady increase in the normal group (CTL), while the BLM group and the BLM + GSF - 016_500 μg / kg group initially decreased and then gradually increased. In contrast, the BLM + GSF - 016_200 μg / kg group showed a stable increase. Lung weight measurement revealed that the BLM group had the highest lung weight, while the normal group and the BLM + GSF - 016_200 μg / kg group had similar levels.

[0156] For Compound 2, as Figure 9 shown in b, compared with the normal group and the GSF - 018 group, the BLM group showed a significant decrease in body weight and then gradually increased, but still remained significantly lower. In contrast, the BLM + GSF - 018_100 μg / kg and BLM + GSF - 018_200 μg / kg groups showed a stable increase in body weight, approaching levels similar to the normal group. Lung weight measurement indicated that the BLM group had a significantly higher lung weight, while the normal group, the BLM + GSF - 018_100 μg / kg group, and the BLM + GSF - 018_200 μg / kg group had similar lower levels, showing a significant difference compared with the BLM group.

[0157] Example 6 - 2. Quantitative analysis of hydroxyproline in lung tissue

[0158] To determine another index of fibrosis, the hydroxyproline content was measured by ELISA analysis using a hydroxyproline colorimetric assay kit, which indirectly shows the collagen content in lung tissue.

[0159] Specifically, the homogenized lung tissue was placed in 1.5 mL tubes in similar amounts to prevent thawing, and 100 μL of 12 M hydrochloric acid was added and hydrolyzed at 120 °C for 3 hours. After centrifugation at 10,000×g for 5 minutes, the supernatant was transferred to a new tube, and 10 μL of the sample was transferred to a 96-well plate and evaporated at 60 °C. Subsequently, 100 μL of the chloramine T reagent / oxidation buffer mixture was added to each well, then 100 μL of the p-dimethylaminobenzaldehyde reagent was added, and the reaction was carried out at 60 °C for 90 minutes. The absorbance was measured at 540 nm, and the content was determined using a standard calibration curve prepared in the same manner.

[0160] As Figure 10 As shown in FIGS. 10a and 10b, compound 1 significantly increased the hydroxyproline content in the BLM group, while in the BLM+GSF-016_200 μg / kg group, the hydroxyproline content decreased. For compound 2, compared with the BLM group, both the BLM+GSF-018_100 μg / kg and BLM+GSF-018_200 μg / kg groups showed a significant decrease in the hydroxyproline content.

[0161] Example 6-3. Histological Evaluation of the Lung - 1

[0162] To observe the changes in the lung tissue, the lung tissue samples were stained with hematoxylin and eosin (H&E) and Masson's trichrome stain to evaluate the degree of inflammation and fibrosis.

[0163] Specifically, the excised lung tissue was embedded in paraffin and cut into 4 μm thin sections using a microtome. These tissue sections were used for H&E, MT staining, and immunohistochemistry (IHC). Each experiment was carried out by dewaxing and rehydrating the sections using xylene and ethanol. For IHC, an LSAB2 system HRP (DAKO, Carpinteria, CA, USA) kit was used. Antibody retrieval was carried out in 0.1 mM citrate (pH 6), and the experiment was carried out according to the provided instructions. The sections were blocked with a blocking buffer at room temperature for 1 hour, then incubated with primary antibodies (fibronectin, collagen 1, α-SMA) overnight at 4 °C. After blocking with a secondary antibody at room temperature for 1 hour, the streptavidin-HRP system was used for 10 minutes, then exposed to the DAB solution, stained with hematoxylin, dehydrated, cleared, and fixed with Canada balsam. The stained slides were examined under an optical microscope (BK51, Olympus, Japan). The staining results were quantified using the Image J program.

[0164] As Figure 11a and 11bAs shown, the normal group showed the least inflammatory response, while the BLM group showed a significant increase in inflammation due to lymphocytes, neutrophils, and macrophages, severe fibrosis of the lung tissue, and structural collapse.

[0165] However, compared with the BLM group, regardless of the concentration, the groups treated with Compound 1 showed a significant reduction in inflammation, and in the BLM+GSF-016_200 μg / kg and BLM+GSF-016_500 μg / kg groups, the degree of fibrosis in the lung tissue was significantly reduced.

[0166] Similarly, compared with the BLM group, the groups treated with Compound 2 showed a significant reduction in inflammation at both concentrations, and in the BLM+GSF-018_100 μg / kg and BLM+GSF-018_200 μg / kg groups, the degree of fibrosis in the lung tissue was significantly reduced.

[0167] Example 6-4. Histological Evaluation of the Lung - 2

[0168] To examine the key factors involved in fibrosis, immunohistochemistry (IHC) was performed using the same method as in Example 6-3.

[0169] As Figure 12a and 12b shown, compared with the normal group, Compound 1 showed increased expression of α-SMA, collagen, and fibronectin in the BLM group, similar to the pattern observed in the IPF cell line. The BLM+GSF-016 group showed a significant reduction in these proteins, and the best inhibitory effect on body weight recovery and related protein expression was observed at the lower concentration of 200 μg / kg.

[0170] Compared with the normal group, Compound 2 also showed increased expression of fibronectin, collagen, and α-SMA in the BLM group, which was significantly reduced in a dose-dependent manner in the BLM+GSF-018 group.

[0171] Example 6-5. Analysis of Fibrosis-Related Gene Expression in Lung Tissue

[0172] To confirm whether the expression of various fibrosis-related genes including fibronectin, collagen, and α-SMA showed the same pattern as the histological observations in the previous examples, qRT-PCR analysis was performed.

[0173] Specifically, to extract RNA from lung tissue, approximately 100 mg of pulverized lung tissue was dispensed into a 1.5 mL tube, and total RNA was extracted using TRIzol reagent (TaKaRa Bio Inc., Japan). After adding 1 mL of TRIzol reagent to denature the tissue, the sample was transferred to a 1.5 ml tube, and 200 μl of chloroform was added, followed by vortexing for 20 seconds to mix thoroughly. After reacting at room temperature for 15 minutes, the sample was centrifuged at 14,000 rpm for 20 minutes to obtain the supernatant. An equal volume of isopropanol was added, and the tube was inverted and left standing at room temperature for 10 minutes. The sample was centrifuged at 14,000 rpm for 15 minutes to obtain an RNA pellet, washed with 70% ethanol at 14,000 rpm for 5 minutes, and dried for 5 minutes. The dried RNA sample was dissolved in 20 μL of DEPC-treated distilled water and used for cDNA synthesis. The RNA concentration and purity were measured using Nanodrop at OD 260 / 280 nm.

[0174] As Figure 13 shown in FIGS. 5a and 13b, compared with the normal group, Compound 1 significantly increased the expression of five genes (FN, COL1A1, α-SMA, TGF-β, and CTGF) in the BLM group, and a significant decrease was observed with treatment with Compound 1. Similarly, compared with the normal group, Compound 2 also significantly increased the expression of these five genes in the BLM group, and a significant decrease was observed with treatment with Compound 2.

[0175] Those of ordinary skill in the art will recognize that the present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. Thus, the scope of the present disclosure is indicated by the appended claims rather than by the foregoing description. All changes that come within the meaning and range of equivalency of the claims are to be embraced within the scope of the present disclosure.

Claims

1. A compound represented by Formula I or a pharmaceutically acceptable salt thereof: [Formula I] wherein, in the above Formula I, R is methyl or vinyl.

2. The compound according to claim 1, wherein the compound inhibits the exacerbation or progression of pulmonary fibrosis.

3. The compound according to claim 1, wherein the compound is a sulforaphane-based compound.

4. A pharmaceutical composition for preventing or treating pulmonary fibrosis, comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

5. The pharmaceutical composition according to claim 4, wherein the composition further comprises a pharmaceutically acceptable carrier or excipient.

6. The pharmaceutical composition according to claim 4, wherein the pulmonary fibrosis is one or more selected from the group consisting of: chronic obstructive pulmonary disease complicated with pulmonary fibrosis (COPD complicated with pulmonary fibrosis), pulmonary fibrosis complicated with emphysema, idiopathic pulmonary fibrosis (IPF), pulmonary fibrosis caused by cancer treatment, and pulmonary fibrosis caused by a virus.

7. The pharmaceutical composition according to claim 4, wherein the compound inhibits the expression of pulmonary fibrosis marker genes and proteins by regulating the expression of phosphorylated proteins and the MRTF / SRF signaling pathway.

8. The pharmaceutical composition according to claim 7, wherein the phosphorylated protein is p38, AKT, smad2 or smad7.

9. The pharmaceutical composition according to claim 7, wherein the regulation of the MRTF / SRF signaling pathway reduces the increased expression of MRTF / SRF in the cytoplasm and nucleus caused by the increased expression of Rock protein bound to Rho activated by treatment with TGF-β1.

10. A method for preventing or treating pulmonary fibrosis, comprising administering the pharmaceutical composition according to any one of claims 4 to 9 to a subject.

11. A food composition for preventing or improving pulmonary fibrosis, comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

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