Novel compound and pharmaceutical composition containing the compound as an active ingredient for preventing or treating pulmonary fibrosis

Abstract

The present invention relates to a novel compound and a pharmaceutical composition for preventing or treating pulmonary fibrosis containing the compound as an active ingredient, specifically, a compound represented by chemical formula I, a pharmaceutical composition for preventing or treating pulmonary fibrosis containing 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 improving pulmonary fibrosis containing the compound as an active ingredient. JPEG2025516003000011.jpg36170In formula I, R is methyl or ethenyl.

Description

[Technical Field]

[0001] The present invention relates to a novel compound and a pharmaceutical composition for preventing or treating pulmonary fibrosis containing the compound as an active ingredient, specifically to a compound represented by chemical formula I, a pharmaceutical composition for preventing or treating pulmonary fibrosis containing 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 improving pulmonary fibrosis containing the compound as an active ingredient.

[0002] [ka]

[0003] In Formula I, R is methyl or ethenyl. [Background technology]

[0004] Fibrosis refers to the excessive formation of fibrous connective tissue in organs or tissues, such as during regeneration, as opposed to the normal formation of fibrous tissue in organs or tissues. Examples of fibrosis include pulmonary fibrosis, liver fibrosis, renal fibrosis, pancreatic fibrosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, massive fibrosis (lung), nephrogenic systemic fibrosis (skin), Crohn's disease, keloids, myocardial infarction, and systemic sclerosis.

[0005] Among these, pulmonary fibrosis (Idiopathic Pulmonary Fibrosis; IPF) is a typical lung disease in which repeated inflammation due to alveolar damage causes fibrosis, leading to respiratory failure in patients. Idiopathic pulmonary fibrosis is a progressive disease whose cause is still unknown. Patients experience worsening dyspnea and cough, leading to death from respiratory failure within 3 to 4 years after diagnosis, and the 5-year survival rate is approximately 30% to 40%, which is similar to that of lung cancer.

[0006] Currently, steroids and immunosuppressants, which are cytotoxic drugs, are mainly used to treat pulmonary fibrosis. Of these, steroids were used first, and currently, a combination therapy of steroids with azathioprine or cyclophosphamide is used to treat pulmonary fibrosis caused by radiation exposure (Non-Patent Document 1).

[0007] Additionally, Roche's Esbriet (active ingredient: pirfenidone) and Boehringer Ingelheim's Ofev (active ingredient: nintedanib) are known as drugs for treating or ameliorating pulmonary fibrosis. Of these, pirfenidone, approved by the FDA in 2014, primarily inhibits the action of TGF-β and is known to delay the worsening and progression of idiopathic pulmonary fibrosis as an anti-inflammatory and anti-fibrotic agent, while nintedanib exerts anti-fibrotic effects as a multiple tyrosine kinase inhibitor.

[0008] However, these two drugs have limited therapeutic effects in treating early or moderate idiopathic pulmonary fibrosis, and cause gastrointestinal side effects such as diarrhea, abdominal pain, loss of appetite, and decreased liver function, as well as photosensitivity. These drugs only alleviate the decline in pulmonary function and are unable to exert a fundamental therapeutic effect, so there is a need for the development of a therapeutic agent that can more effectively treat pulmonary fibrosis. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Ochoa et al., Journal of Medical Case Reports, 6:413.2012 Summary of the Invention [Problem to be solved by the invention]

[0010] The present inventors have conducted research efforts to develop candidate substances that can treat and improve pulmonary fibrosis more effectively than conventional methods. As a result, they have confirmed that novel sulforaphane compounds have the effect of suppressing pulmonary fibrosis by regulating the expression of genes and proteins related to pulmonary fibrosis, thereby completing the present invention. [Means for solving the problem]

[0011] The present invention provides a compound represented by formula I or a pharmaceutically acceptable salt thereof.

[0012] [ka]

[0013] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating pulmonary fibrosis, which comprises the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0014] Another object of the present invention is to provide a method for preventing or treating pulmonary fibrosis using the pharmaceutical composition.

[0015] A further object of the present invention is to provide a food composition for preventing or ameliorating pulmonary fibrosis, which contains the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0016] A further object of the present invention is to provide use of the compound or a pharmaceutically acceptable salt thereof, or a composition containing the compound or a salt thereof, for preventing, ameliorating or treating pulmonary fibrosis. [Effects of the Invention]

[0017] The composition containing the compound according to the present invention not only regulates the expression of phosphorylated proteins such as p38, AKT, smad2, and smad7, but also specifically inhibits the SRF / MRTF signaling pathway, thereby suppressing the expression of pulmonary fibrosis marker genes and proteins. Therefore, the composition containing the compound as an active ingredient is useful as an effective therapeutic agent for pulmonary fibrosis. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a diagram confirming the cytotoxicity of sulforaphane-based synthetic compounds. [Figure 2] FIG. 1 shows the inhibitory effect of treatment with 16 compounds on the expression of pulmonary fibrosis marker proteins in lung fibroblasts induced by TGF-β1. [Figure 3] FIG. 1 shows the inhibitory effect of treatment with various concentrations of Compound 1 and Compound 2, which are sulforaphane-based synthetic compounds that exhibit an inhibitory effect on pulmonary fibrosis, on the expression of pulmonary fibrosis marker proteins. [Figure 4] This figure shows the results of cytotoxicity, morphological changes, pulmonary fibrosis marker protein expression analysis, and pulmonary fibrosis marker gene expression analysis in normal lung fibroblasts (MRC-5) and diseased lung fibroblasts (DHLF-IPF) treated with compound 1 and compound 2, respectively. [Figure 5] FIG. 1 shows the inhibitory effect of treatment with Compound 1 and Compound 2 on cell migration in normal lung fibroblasts (MRC-5) and diseased lung fibroblasts (DHLF-IPF) by Transwell migration analysis. [Figure 6] FIG. 1 shows the cell migration inhibitory effect of treatment with Compound 1 and Compound 2 on normal lung fibroblasts (MRC-5) and diseased lung fibroblasts (DHLF-IPF) by wound healing analysis. [Figure 7] FIG. 1 shows the results of an analysis of the expression of proteins related to signal transduction pathways associated with TGF-β1 induction by treatment with Compound 1 in diseased lung fibroblasts (DHLF-IPF). [Figure 8]FIG. 1 shows the results of an analysis of the expression of proteins related to signal transduction pathways associated with TGF-β1 induction by treatment with Compound 2 in diseased lung fibroblasts (DHLF-IPF). [Figure 9] FIG. 1 shows the results of analyzing changes in body weight and lung weight depending on the treatment period and duration of Compound 1 and Compound 2 in an animal model of bleomycin-induced pulmonary fibrosis. [Figure 10] FIG. 1 shows the results of analyzing changes in collagen content in lung tissues by treatment with Compound 1 and Compound 2 in an animal model of bleomycin-induced pulmonary fibrosis. [Figure 11a] FIG. 1 shows the results of analyzing histological changes in inflammation- and fibrosis-related indices in lung tissues following treatment with Compound 1 in an animal model of bleomycin-induced pulmonary fibrosis. [Figure 11b] FIG. 1 shows the results of analyzing histological changes in inflammation- and fibrosis-related indices in lung tissues following treatment with Compound 2 in an animal model of bleomycin-induced pulmonary fibrosis. [Figure 12a] FIG. 1 shows the results of histological analysis of the expression of fibrosis-related protein markers in lung tissues following treatment with Compound 1 in an animal model of bleomycin-induced pulmonary fibrosis. [Figure 12b] FIG. 1 shows the results of histological analysis of the expression of fibrosis-related protein markers in lung tissues following treatment with Compound 2 in an animal model of bleomycin-induced pulmonary fibrosis. [Figure 13] FIG. 1 shows the results of analyzing the expression of fibrosis-related genes in lung tissues treated with Compound 1 and Compound 2 in an animal model of bleomycin-induced pulmonary fibrosis. DETAILED DESCRIPTION OF THE INVENTION

[0019] These will be described in detail below. Note that each description and embodiment disclosed in the present invention also applies to other descriptions and embodiments. In other words, all combinations of various elements disclosed in the present invention are included in the present invention. Furthermore, the present invention is not limited to the following specific description.

[0020] To achieve the above object, one aspect of the present invention provides a compound represented by chemical formula I or a pharmaceutically acceptable salt thereof:

[0021] [ka]

[0022] In Formula I, R is methyl or ethenyl.

[0023] Chemical Formula I may specifically be Chemical Formula 1 or Chemical Formula 2, which represent Compound 1 or Compound 2.

[0024] [ka]

[0025] [ka]

[0026] In the present invention, Chemical Formula 1 and Chemical Formula 2 are novel sulforaphane-based compounds having molecular weights of 368.5 and 380.5, respectively, which are more than twice as large as sulforaphane, which has a molecular weight of 177.3, and are characterized by their ability to inhibit the aggravation or progression of pulmonary fibrosis.

[0027] The novel compounds of the present invention can be chemically synthesized by methods known in the art and may exist in unsolvated as well as solvated forms, and may also exist in crystalline or amorphous forms, and all such physical forms are encompassed by the present invention.

[0028] The term "pharmaceutically acceptable salts" as used herein means salts commonly used in the pharmaceutical industry, and includes, for example, inorganic ion salts prepared with calcium, potassium, sodium, magnesium, etc., inorganic acid salts prepared with hydrochloric acid, nitric acid, phosphoric acid, bromic acid, iodic acid, perchloric acid, tartaric acid, sulfuric acid, etc., acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronolic acid, etc. Examples of suitable salts include organic acid salts made with carboxylic acids, vanillic acid, hydroiodic acid, etc.; sulfonates made with methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc.; amino acid salts made with glycine, arginine, lysine, etc.; and amine salts made with trimethylamine, triethylamine, ammonia, pyridine, picoline, etc., but the types of salts used in the present invention are not limited to these salts.

[0029] In order to achieve the above object, another aspect of the present invention provides a pharmaceutical composition for preventing or treating pulmonary fibrosis, which comprises the novel compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0030] In the present invention, "pulmonary fibrosis" refers to a type of chronic interstitial lung disease, also known as idiopathic pulmonary fibrosis, in which lung tissue cells transform into fibrocytes, causing symptoms such as dyspnea, cough, cyanosis, and digital clubbing. Upon tissue examination, honeycomb-like or atypical fibrocyte clusters are observed. To date, immunosuppressants including steroidal therapeutic agents, interferon-γ, acetylcysteine, pirfenidone, nintedanib, and bosentan have been used, but no formulations demonstrating specific therapeutic effects have been reported.

[0031] The pulmonary fibrosis in the present invention refers to pulmonary fibrosis selected from the group consisting of chronic obstructive pulmonary disease combined pulmonary fibrosis (COPD combined pulmonary fibrosis), combined pulmonary fibrosis and emphysema, idiopathic pulmonary fibrosis (IPF), pulmonary fibrosis caused by anticancer treatment, and pulmonary fibrosis induced by a virus, and specifically refers to idiopathic pulmonary fibrosis, but is not limited to these.

[0032] In the present invention, "prevention" refers to any action of suppressing or delaying pulmonary fibrosis by administering a pharmaceutical composition containing the compound as an active ingredient.

[0033] In the present invention, "treatment" refers to any action that improves or favorably changes the symptoms of pulmonary fibrosis by administering a pharmaceutical composition containing the compound as an active ingredient.

[0034] The "pharmaceutical composition" of the present invention may further contain a pharmaceutically acceptable carrier, excipient, or diluent commonly used in its preparation, and the carrier may be a non-naturally occurring carrier. Specific examples of the carrier, excipient, and diluent include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0035] The pharmaceutical compositions are formulated by conventional methods into any of the following forms: tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories, and are used in a variety of oral and parenteral dosage forms. When formulated, they are prepared using commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Oral solid formulations include tablets, pills, powders, granules, and capsules, and these solid formulations contain at least one excipient, such as starch, calcium carbonate, sucrose or lactose, or gelatin. In addition to commonly used excipients, lubricants, such as magnesium stearate and talc, are also used. Oral liquid preparations include suspensions, oral solutions, emulsions, syrups, etc., and in addition to the commonly used diluents of water and liquid paraffin, various excipients such as humectants, sweeteners, flavorings, and preservatives are used. Parenteral preparations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include, but are not limited to, witepsol, macrogol, Tween 61, cocoa butter, lauric butter, and glycerogelatin.

[0036] The content of the compound in the pharmaceutical composition of the present invention can be adjusted appropriately depending on the symptoms of the disease, the degree of progression, the condition of the patient, etc., and is, for example, 0.0001 to 99.9 wt % or 0.001 to 50 wt % relative to the total weight of the composition, but is not limited to these.

[0037] In one example of the present invention, it was confirmed that the expression of fibronectin and a-SMA, which was increased by TGF-β1, was reduced in a concentration-dependent manner by treatment with various concentrations of compound 1 and compound 2, respectively, and that each compound at a concentration of 20 μM or 10 μM showed a similar or superior reducing effect compared to sulforaphane (Figure 3).

[0038] In one example of the present invention, normal human lung fibroblast cells (MRC-5) and diseased human lung fibroblast cells (DHLF-IPF) were treated with sulforaphane, Compound 1, and Compound 2, and changes in cell morphology were then observed. As a result, it was confirmed that cell proliferation was suppressed in the Compound 1 and 2-treated groups compared to the sulforaphane-treated group, and the cells had an elongated cell shape and a non-compact morphology similar to the control group (Figure 4a).

[0039] Furthermore, in one example of the present invention, the effects on pulmonary fibrosis-related protein and gene expression were confirmed in MRC-5 and DHLF-IPF fibroblasts. As a result, it was confirmed that in both of the two cells, protein expression and gene expression induced by TGF-β1 were reduced by sulforaphane, Compound 1, and Compound 2, and in particular, Compound 1 and Compound 2 showed a significantly greater reducing effect than sulforaphane (Figure 4b to d).

[0040] Furthermore, in one example of the present invention, the effects on cell migration were confirmed using MRC-5 and DHLF-IPF. As a result, it was confirmed that treatment with TGF-β1 increased migration. In particular, in DHLF-IPF cells, migration was significantly increased even without TGF-β1 induction, but was significantly suppressed in a concentration-dependent manner by treatment with Compound 1 and Compound 2 (Figures 5 and 6).

[0041] In one embodiment of the present invention, the inhibitory effect of Compound 1 on pulmonary fibrosis in DHLF-IPF was confirmed by analyzing the expression of various TGF-β1-related signaling pathway proteins. The results showed that Compound 1 inhibited the phosphorylation of Smad-2, increased the phosphorylation of Smad-7, and significantly reduced the activity of p-p38MAPK and p-AKT. Furthermore, Compound 1 suppressed the expression of ROCK in the Rho-ROCK signaling pathway and the expression of MRTF and SRF in the nucleus in the MRTF-SRF signaling pathway (Figure 7).

[0042] In one embodiment of the present invention, the inhibitory effect of Compound 2 on pulmonary fibrosis in DHLF-IPF was confirmed by analyzing the expression of various TGF-β1-related signaling pathway proteins. Compound 1 was found to suppress the phosphorylation of Smad-2 and Smad-3, increase the phosphorylation of Smad-7, significantly reduce the activity of three phosphorylated proteins, p-ERK, p-JNK, and p-p38, in the MAPK signaling pathway, and significantly reduce the activity of p-AKT in the AKT signaling pathway. Furthermore, Compound 1 suppressed the expression of ROCK in the Rho-ROCK signaling pathway and the expression of MRTF and SRF in the nucleus in the MRTF-SRF signaling pathway (Figure 8).

[0043] These results suggest that the compounds of the present invention are useful for the prevention or treatment of pulmonary fibrosis.

[0044] In order to achieve the above object, yet another aspect of the present invention provides a method for preventing or treating pulmonary fibrosis, comprising the step of administering the pharmaceutical composition to an individual.

[0045] The pharmaceutical composition, prevention or treatment of pulmonary fibrosis is as described above.

[0046] In the present invention, the term "individual" refers to any animal, including humans, mice, livestock, and the like, that has developed or is at risk of developing pulmonary fibrosis. Specifically, the term "individual" refers to mammals, including not only humans but also cattle, horses, sheep, pigs, goats, camels, serows, dogs, and cats, that require prevention or treatment of symptoms similar to the above-mentioned diseases, but is not limited to these.

[0047] Furthermore, the individual may include or exclude humans.

[0048] In the present invention, "administration" means introducing the composition of the present invention into a patient by any suitable method, and the route of administration of the composition can be any common route that allows delivery to the target tissue.

[0049] The pharmaceutical compositions of the present invention are administered in pharmaceutically effective amounts.

[0050] The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level is determined by factors including the type and severity of the disease in the individual, age, sex, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors known in the medical field.

[0051] The pharmaceutical composition may be administered alone or in combination with other therapeutic agents, or may be administered sequentially or simultaneously with conventional therapeutic agents. It may also be administered singly or in multiple doses. It is important to administer an amount that provides maximum efficacy with minimal side effects, taking into consideration all of the above factors, and this can be easily determined by those skilled in the art.

[0052] The pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage may vary depending on the patient's condition and weight, the severity of the disease, the drug form, the administration route, and the time, and may be appropriately selected by those skilled in the art. Specifically, the composition is generally administered once a day or in divided doses several times a day, and the preferred dosage may be appropriately selected by those skilled in the art depending on the patient's condition and weight, the severity of the disease, the drug form, the administration route, and the time period.

[0053] In one embodiment of the present invention, the inhibitory effects of the final selected compounds 1 and 2 on pulmonary fibrosis were investigated in an animal model of bleomycin-induced pulmonary fibrosis. As a result, it was confirmed that in the groups treated with the two compounds, the hydroxyproline content in the lung tissue was significantly reduced (FIG. 10), the degree of inflammation and fibrosis in the lung tissue was reduced (FIG. 11), and the expression of fibrosis-related proteins and genes such as α-SMA, collagen, and fibronectin was also reduced (FIGS. 12 and 13).

[0054] In order to achieve the above object, yet another aspect of the present invention provides a food composition for preventing or ameliorating pulmonary fibrosis, which contains the above compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0055] The compound, pulmonary fibrosis or prevention is as described above.

[0056] "Amelioration" in the present invention means any action that at least reduces the parameters, such as the severity of symptoms, related to the condition being treated by administration of a composition containing said novel compound.

[0057] The term "food" in the present invention refers to any food in the usual sense, such as meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, soft drinks, tea, energy drinks, alcoholic beverages, vitamin complexes, functional foods, and health foods.

[0058] The term "functional food" is synonymous with "food for special health use (FoSHU)" and refers to food with high medical and therapeutic effects that is processed to efficiently exhibit bioregulatory functions in addition to providing nutrients.

[0059] Here, "functionality" means regulating nutrients for the structure and function of the human body or providing beneficial effects for health purposes, such as physiological actions. The term "health food" refers to food that has more active health maintenance and promotion effects than general foods, and the term "health supplement food" refers to food intended for health supplementation. In some cases, the terms "functional food," "health food," and "health supplement" are used interchangeably. Specifically, the term "functional food" refers to food in which the novel compound of the present invention is added to food ingredients such as beverages, teas, spices, gum, and confectioneries, or is prepared in the form of capsules, powders, suspensions, etc., and which, when ingested, provides specific health benefits.

[0060] The food of the present invention can be produced by a method commonly used in the art, and can be produced by adding raw materials and ingredients commonly added in the art.

[0061] The dosage form of the food composition may be any dosage form that is acceptable as a food.

[0062] Furthermore, the food composition may further contain a carrier that is nutrient-wise acceptable, but the type of carrier is not particularly limited, and any carrier commonly used in the art may be used.

[0063] Furthermore, the food composition may contain additional ingredients commonly used in food compositions to enhance aroma, taste, and visual appearance, such as vitamins A, C, D, E, B1, B2, B6, and B12, niacin, biotin, folate, and panthotenic acid. Additionally, the food composition may contain minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr), and amino acids such as lysine, tryptophan, cysteine, and valine.

[0064] The food compositions may further contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder, high-strength bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.), colorants (tar dyes, etc.), color formers (sodium nitrite, sodium acetate, etc.), bleaching agents (sodium sulfite), seasonings (monosodium glutamate (MSG), etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, potassium D-bitartrate, etc.), fortifiers, emulsifiers, thickeners (thickening agents), coating agents, gum bases, foam inhibitors, solvents, and improvers. The additives may be selected and used in appropriate amounts depending on the type of food.

[0065] In order to achieve the above object, still another aspect of the present invention provides a feed composition for preventing or ameliorating pulmonary fibrosis, which comprises the above compound or a pharmaceutically acceptable salt thereof as an active ingredient.

[0066] The compound, pulmonary fibrosis or prevention is as described above.

[0067] The term "feed" means any natural or artificial diet, meal, etc., or component of such a meal, intended or suitable for ingestion and digestion by an animal.

[0068] The type of feed is not particularly limited, and feeds commonly used in the art can be used. Examples of the feed include, but are not limited to, plant-based feeds such as grains, nuts, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meals, and grain by-products, and animal-based feeds such as proteins, inorganic substances, oils and fats, minerals, single-cell proteins, zooplankton, and food and beverages. These may be used alone or in combination of two or more types.

[0069] In order to achieve the above-mentioned object, yet another aspect of the present invention provides use of the above-mentioned compound or a pharmaceutically acceptable salt thereof, or a composition containing same for preventing, ameliorating, or treating pulmonary fibrosis, and further provides use of the above-mentioned compound or a pharmaceutically acceptable salt thereof, or a composition containing same for producing a drug, food, or feed for preventing, ameliorating, or treating pulmonary fibrosis.

[0070] The compound, pharmaceutically acceptable salt, and pulmonary fibrosis, prevention, amelioration, or treatment are as described above. [Example]

[0071] The present invention will be described in more detail below with reference to examples. These examples are intended to explain the present invention more specifically, but the present invention is not limited to these examples.

[0072] Production Example 1: Method for producing a novel compound

[0073] [ka]

[0074] To a 2 mL solution of N-acetyl-L-cysteine ​​ethyl ester (CAS No. 59587-09-6, 150 mg, 0.78 mmol) in ethanol, the pH was adjusted to 8 with 1N NaOH, and a 2 mL solution of sulforaphane (CAS No. 4478-93-7, 50 mg, 0.28 mmol) in ethanol was added. The reaction mixture was stirred under nitrogen at room temperature for 4 hours, the solvent was evaporated, and the residue was purified by reverse-phase column chromatography with 0.05% TFA in methanol to give compound 1 (160 mg, 55% yield). 1 H NMR (600 MHz, CDCl3) δ 9.09 (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. Hz,4H); 13 C NMR (150MHz,CDCl3) δ 196.75,170.67,170.32,61.99,53.50,53.08,46.84,38.47,35.82,26.94,23.00,20.19,14.13;ESI-MS(positive mode):m / z calculated for C 13 H 24 O4N2NaS3,[M+Na] + =391.53 found:391.40

[0075] [ka]

[0076] To a solution of Ac-Cys-OAllyl (CAS No: 616-91-1, 138 mg, 0.67 mmol) in ethanol (2 mL), the pH of the solution was adjusted to 8 with 1N NaOH, and 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 under nitrogen at room temperature for 10 hours, the solvent was evaporated, and the residue was purified by reverse-phase column chromatography with 0.05% TFA in methanol to give compound 2 (112 mg, 52% yield). 1 H NMR (600MHz,CD3OD) 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 5.2Hz,1H),3.77-3.71(m,1H),3.52(dd,J=14.2 and 8.5Hz,1H),2.96-2.76(m,3H),2.65(d,J=10.4Hz,3H),1.97(s,3H),1.91-1.76(m,4H); 13 C NMR(150MHz,CD3OD) 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 mode):m / z calculated for C 14 H 24 N2NaO4S3,[M+Na] + =403.08 found:403.21. [Example]

[0077] Cytotoxicity evaluation of sulforaphane-based synthetic compounds Cell culture and cytotoxicity assessment The normal lung fibroblast MRC-5 cell line was provided by the Korea Cell Line Bank. The cells were cultured in a DMEM medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin in a 5% CO2 incubator at 37°C. All cells were cultured at 80-90% confluency throughout the experiment.

[0078] To determine the optimal concentration of each compound synthesized from sulforaphane without toxicity, MRC-5 cells were cultured in 96-well plates at 5 × 10 3 100 μl of cells were dispensed into each well, and the cells were cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0079] After incubation, the medium was removed, and each compound was prepared at different concentrations. Each group was treated in triplicate and then incubated for 24 hours in a 37°C, 5% CO2 incubator. After incubation, 10 μl of 5 mg / ml MTT reagent was dispensed per well into the medium containing the test solution. The 96-well plate was then incubated in a 37°C, 5% CO2 incubator in the dark for 2-4 hours. After incubation, the medium containing the MTT reagent was removed, and 100 μl of DMSO (dimethyl sulfoxide) was added to dissolve the MTT-formazan crystals. Absorbance was measured at 570 nm, and cell viability was determined by comparing with the control group.

[0080]

number

[0081] As a result, as shown in Figure 1, when treated at a concentration that did not reduce cell viability to less than 80% compared to the untreated control group, most compounds maintained cell viability of 80% or more even at 40uM, and GSF-18 showed cell viability of 80% or more at 10uM. [Example]

[0082] Analysis of fibrosis-related protein expression by sulforaphane-based synthetic compounds Since overexpression of extracellular matrix components such as fibronectin and a-SMA is an indicator of tissue fibrosis, MRC-5 cells in which fibrosis was induced by TGF-β1 were treated with various concentrations of each compound, and the levels of fibronectin and a-SMA expression were compared.

[0083] Specifically, to extract fibronectin and α-SMA proteins, cells were washed once with cold PBS and then lysed in RIPA buffer (150 mM NaCl, 0.5% Triton X-100, 50 mM Tris-HCl, pH 7.4, 25 mM NaF, 20 mM EGTA) supplemented with a protease inhibitor cocktail. Proteins were then quantified using a BCA protein quantification kit. All protein samples were electrophoresed (10 μg each) on an 8-10% PAGE gel and then transferred to a 0.2 μm PVDF membrane (EMD Millipore, MA, USA). The membrane with the transferred proteins was blocked with 5% BSA or 5% skim milk for 1 hour, followed by incubation with primary antibodies overnight at 4°C. The following day, the cells were washed three times with PBST and then incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. After this process, the PVDF membrane was coated with ECL (Luminata™ Crescendo, EMD Millipore) and then exposed to light using a fluorescence reader to confirm the degree of protein expression.

[0084] As shown in Figure 2, the induction of pulmonary fibrosis by TGF-β1 significantly increased the expression of fibronectin and a-SMA, whereas treatment with sulforaphane compounds GSF-016 and GSF-018 significantly reduced the expression of the two aforementioned proteins.

[0085] Therefore, the two compounds mentioned above were named Compound 1 and Compound 2, respectively, and selected as candidate compounds for use in treating fibrosis according to the present invention. [Example]

[0086] Selection of sulforaphane prodrugs Example 3-1. Analysis of fibrosis-related protein expression using candidate compounds Western blotting was performed to compare the expression levels of the two proteins mentioned above by sulforaphane (SFN) treatment at different concentrations, along with Compound 1 and Compound 2, which significantly reduced the expression of fibronectin and a-SMA, which were increased due to pulmonary fibrosis in Example 2.

[0087] As in Example 2, specific experimental methods involved treatment with sulforaphane at a concentration of 20 μM, compound 1 at concentrations of 5 μM, 10 μM, and 20 μM, and compound 2 at concentrations of 2.5 μM, 5 μM, and 10 μM.

[0088] As a result, as shown in Figure 3, it was confirmed that the expression of fibronectin and a-SMA was reduced in a concentration-dependent manner by treatment with each concentration of compound 1 and compound 2, respectively, and that each compound at a concentration of 20uM or 10uM showed a reduction effect equivalent to or superior to sulforaphane.

[0089] Example 3-2. Cytotoxicity and morphology analysis of candidate compounds in two cell lines We investigated whether two candidate sulforaphane prodrug compounds affect cytotoxicity and morphological changes in normal lung fibroblasts MRC-5 and diseased lung fibroblasts DHLF-IPF. The diseased lung fibroblast DHLF-IPF cell line was purchased from Lonza and used in FGM TM -2 Fibroblast Growth Medium-2 BulletKit TM The cells were cultured in a 5% CO2 incubator at 37°C using the medium. All cells were cultured at 80-90% confluency during the experiment.

[0090] Both cells were cultured in a 96-well plate at 5 × 10 3 100 μl of cells were dispensed into each well and cultured in a 37°C, 5% CO2 incubator for 24 hours. After that, the medium was removed and each compound was prepared at a concentration of 10 μM. Each group was treated in triplicate and then cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0091] The detailed experimental method for cytotoxicity was the same as in Example 1.

[0092] Furthermore, changes in cell morphology due to treatment with two types of compounds (compound 1 and compound 2) in two types of lung fibroblasts induced by TGF-β1 were visually confirmed under a microscope.

[0093] As shown in Figure 4a, the two compounds showed over 80% cell viability in the two cell lines at a concentration of 10 μM. Morphological changes in the two cell lines were also examined. The TGF-β1-treated group showed increased proliferation compared to the control group, which showed an elongated morphology, and cells became tangled and dense, like fibrous tissue. In contrast, the two compounds inhibited cell proliferation compared to the sulforaphane-treated group, and the cells retained an elongated cell morphology similar to the control group, rather than the dense morphology of fibrous tissue.

[0094] Example 3-3. Fibrosis-related protein expression analysis of candidate compounds in two cell lines Normal MRC-5 and diseased DHLF-IPF cell lines, in which fibrosis was induced by TGF-β1, were treated with SFN and the two compounds mentioned above, and Western blotting was performed to compare the changes in fibrosis-related protein expression.

[0095] As in Example 2, the specific experimental method was to treat the cells with 1 ng / ml of TGF-β1 to induce fibrosis, and then treat them with 10 μM of sulforaphane and the two compounds.

[0096] As a result, as shown in FIG. 4b, the two compounds demonstrated a more significant decrease in fibronectin and α-SMA protein expression than sulforaphane.

[0097] Examples 3-4. Fibrosis-related gene expression analysis of candidate compounds in two cell lines In normal MRC-5 and diseased DHLF-IPF cells induced by TGF-β1 fibrosis, SFN and the two compounds effectively suppressed the expression of fibrosis-related proteins. To confirm whether they also had the same effect at the gene level, we examined the gene expression of FN, COL1A1, and α-SMA by qRT-PCR analysis.

[0098] RNA extraction from cells MRC-5 cells and DHLF-IPF cells were cultured in growth medium. The medium was removed and serum-free culture medium was pretreated with 10 μM of each experimental group (SFN, GSF-016, or GSF-018) for 1 hour. To induce fibrosis, 1 ng / ml TGF-β1 was added and cultured for 48 hours at 37°C and 5% CO2. The medium was then removed and washed once with PBS. Total RNA was then extracted from the cultured cells using TRIzol reagent (TaKaRa Bio Inc., Japan) for gene expression analysis. Cells were lysed and denatured with 1 ml of TRizol reagent. Then, the cells were transferred to 1.5 ml tubes, 200 μl of chloroform was added, and the mixture was thoroughly mixed by vortexing for 20 seconds. The mixture was incubated at room temperature for 15 minutes, then centrifuged at 14,000 rpm for 20 minutes to obtain the supernatant. The supernatant was then inverted with an equal volume of isopropyl alcohol and allowed to stand at room temperature for 10 minutes. The sample was centrifuged at 14,000 rpm for 15 minutes to obtain an RNA pellet. It was then washed with 70% RNA-safe ethanol by centrifugation at 14,000 rpm for 5 minutes and then dried for 5 minutes. The dried RNA sample was dissolved in 20 μl of distilled water treated with 0.1% DEPC (diethyl pyrocarbonate), and then used as a sample for cDNA synthesis. The RNA concentration and purity were measured at OD260 / 280 nm using a Nanodrop™.

[0099] cDNA synthesis First-strand cDNA synthesis was performed by mixing 1 μl of oligo-d(T) primer (100 pmol) and 10 mM dNTPs (TaKaRa Bio Inc., Japan) with 1 μg of extracted total RNA and incubating at 65°C for 5 minutes, followed by rapid cooling. To the template, 4 μl of 5x RT buffer, 0.5 μl of RNA inhibitor, and 100 units of RTase (TaKaRa Bio Inc., Japan) were added, and the total volume was adjusted to 20 μl with DEPC-treated distilled water. The sample was subjected to synthesis at 25°C for 5 minutes and 42°C for 1 hour, followed by 15 minutes at 72°C to inactivate the reverse transcriptase and terminate the reaction.

[0100] RT-PCR The level of expression of each gene was measured by real-time PCR. 5 μl of extracted cDNA from each sample was mixed with 10 μl of 2X SYBR Green MasterMix (TaKaRa Bio Inc., Japan) and 1 μl each of 10 pmol (forward and reverse) primers for each gene, and the total volume was adjusted to 20 μl with distilled water. The reaction was incubated at 95°C for 10 minutes, followed by 40 cycles of 30 seconds, followed by 30 seconds at 60°C and 30 seconds at 72°C. The specificity of the amplified products was confirmed by melting curve analysis, and the target genes were quantified and compared using GAPDH as a control gene.

[0101] As a result, as shown in Figure 4c to 4d, in both types of cells, the increased expression of FN, COL1A1, and a-SMA genes was all reduced by treatment with sulforaphane and the two compounds, and in particular, the two compound-treated group showed a significant reduction compared to the sulforaphane-treated group.

[0102] Based on these results, Compounds 1 and 2 were selected as the final compounds for use in the treatment of pulmonary fibrosis of the present invention from among the sulforaphane-based synthetic compounds. [Example]

[0103] cell migration Since myofibroblasts exhibit a characteristic that cell migration and invasion increase with increased expression of fibrosis-related proteins, migration and wound healing experiments were performed to examine the effects of the final selected compounds 1 and 2 on cell migration using normal and diseased fibroblasts.

[0104] Example 4-1. Transwell migration assay 500 μl of medium containing FBS was placed in a 24-well culture plate, and an 8 μm pore size insert was placed on top of it. MRC-5 and DHLF-IPF were added at 1 × 10 cells / well to 300 μl of medium without FBS inside the insert. 5 Cells were seeded onto the inserts and cultured for 24 hours in an incubator maintained at 37°C and 5% CO2. Afterwards, the medium was gently removed from the 8 μm pore polycarbonate membrane inserts, which were then washed with DPBS and fixed with methanol for 5 minutes. The inserts were then washed twice with triple-distilled water and then stained with Mayer's hematoxylin for 8 minutes. The stained cells were washed with DPBS, and the inside of the insert was gently wiped with a cotton swab. The insert membrane was then removed and mounted on a slide. The migrated cells were counted under a microscope at 200x magnification.

[0105] As a result, as shown in Figure 5, it was confirmed that the migration of normal MRC-5 cells was increased by treatment with TGF-β1, and that this was significantly inhibited in a concentration-dependent manner by treatment with Compound 1 and Compound 2. Furthermore, it was confirmed that the migration of diseased DHLF-IPF cells themselves was active, and, as with MRC-5, it was significantly inhibited in a concentration-dependent manner by treatment with Compound 1 and Compound 2.

[0106] Example 4-2. Wound healing assay The effects of each concentration of Compound 1 and Compound 2, which were finally selected in Example 3, on cell migration (wound healing) in MRC-5 and DHLF-IPF were examined.

[0107] Specifically, cells (1 × 10 ) were cultured to achieve a cell confluency of approximately 90%. 5 Cells (1000 cells / well) were seeded into 60 mm dishes and cultured for 24 hours. The bottom of the dish was then scraped in a straight line with a 200 μl pipette tip, followed by a single wash with Dulbecco's Phosphate-Buffered Saline (DPBS) to remove the scraped cells. Wounds were photographed under a microscope. Cells were treated with TGF-β1 alone, TGF-β1 + SFN, TGF-β1 + GSF-016, or TGF-β1 + GSF-018, and then cultured for approximately 24 hours. The wounds were then compared. In images taken after 24 hours of culture, the intercellular distance was measured using public domain software, Image J (Fiji package), and the relative mobility compared to the control group was expressed as a percentage (%).

[0108] As a result, as shown in Figure 6, in both MRC-5 and DHLF-IPF cells, treatment with TGF-β1 increased migration and narrowed the wound compared to the control group. In contrast, migration was significantly inhibited in all compound-treated groups in a concentration-dependent manner. [Example]

[0109] TGF-β1 signaling pathway-related protein expression analysis The TGF-β1 signaling pathway is activated by both Smad-dependent and Smad-independent pathways. The Smad-dependent pathway, TGF-β / Smad signaling, is an important pathway that regulates the synthesis of extracellular matrix components. The Smad-independent (TGF-β / non-Smad) pathway is influenced by PI3K / AKT / mTOR and MAPKs (JNK, ERK, p38), which are known to be involved in cell proliferation and growth. Based on this mechanism, we comparatively analyzed the expression of proteins related to the TGF-β1 signaling pathway, similar to the Western blotting performed in Example 2.

[0110] As a result, as shown in Figure 7, compound 1 was confirmed to inhibit the phosphorylation of Smad-2, increase the phosphorylation of Smad-7, and significantly reduce the activity of p-p38 and MAPK. Furthermore, the influence of MRTF / SRF on the Rho / Rack pathway was confirmed. Treatment with TGF-β1 increased the expression of Rock protein, which binds to activated Rho, and the expression of MRT / SRF, a downstream step, increased in the cytoplasm and nucleus. Treatment with compound 1 was confirmed to significantly reduce the increased protein expression.

[0111] As shown in Figure 8, compound 2 inhibited the phosphorylation of Smad-2 and Smad-3, increased the phosphorylation of Smad-7, and reduced the activity of all three MAPKs: p-ERK, p-JNK, and p-p38. Furthermore, the effects of MRTF / SRF on the Rho / Rack pathway were confirmed. TGF-β1 treatment increased the expression of Rock protein, which binds to activated Rho, and the expression of MRTF / SRF, a downstream step, increased in the cytoplasm and nucleus. Compound 2 treatment significantly reduced the increased protein expression. [Example]

[0112] Inhibitory effect on pulmonary fibrosis in animal models Example 6-1. Analysis of changes in body weight and lung weight in an animal model of pulmonary fibrosis induced by Compounds 1 and 2 The finally selected compounds 1 and 2 were examined for their inhibitory effects on pulmonary fibrosis in an animal model of bleomycin-induced pulmonary fibrosis.

[0113] Specifically, 7-week-old C57BL / 6N mice were exposed to BLM (3-5 units / kg) via the airways, and then orally administered 200µg / kg and 500µg / kg of Compound 1, and 100µg / kg and 200µg / kg of Compound 2, three times a week for three weeks, starting the day after BLM exposure. Body weight gain was measured periodically during the experiment, and lung weights were measured after the animal experiment.

[0114] As a result, as shown in Figure 9a, changes in weight gain during the experimental period were confirmed. For Compound 1, the normal group (CTL) steadily increased, while the BLM group and BLM+GSF-016_500ug / kg group showed a tendency to decrease and then gradually increase, while the BLM+GSF-016_200ug / kg group showed a tendency to steadily increase. Furthermore, lung weight was confirmed, and the BLM group showed the largest lung weight, while the normal group and the BLM+GSF-016_200ug / kg group were at similar levels.

[0115] For Compound 2, as shown in Figure 9b, the BLM group showed a significant decrease followed by a gradual increase, demonstrating a significant weight loss compared to the normal and GSF-018 groups. Meanwhile, the BLM+GSF-018_100 μg / kg and BLM+GSF-018_200 μg / kg groups showed a steady increase, though not as rapid as the normal group, and demonstrated a recovery of weight comparable to that of the normal group. Furthermore, lung weight was significantly greater in the BLM group, while the normal, BLM+GSF-018_100 μg / kg, and BLM+GSF-018_200 μg / kg groups were similarly low, demonstrating a significant difference compared to the BLM group.

[0116] Example 6-2. Quantitative analysis of hydroxyproline in lung tissue To confirm the hydroxyproline content, which indirectly indicates the collagen content in lung tissue as another index for judging fibrosis, ELISA analysis was performed using a hydroxyproline colorimetric assay kit.

[0117] Specifically, homogenized lung tissue was transferred to 1.5 ml tubes in equal amounts without dissolving. 100 μl of 12 M hydrochloric acid was added and hydrolyzed at 120°C for 3 hours. The samples were then centrifuged at 10,000 × g for 5 minutes. The supernatant was transferred to a new tube, and 10 μl of the supernatant was transferred to a 96-well plate and evaporated at 60°C. Next, 100 μl of chloramine T reagent / oxidation buffer mixture was added to each well, followed by 100 μl of para-dimethylaminobenzaldehyde reagent. The mixture was incubated at 60°C for 90 minutes. The absorbance was then measured at 540 nm, and the content was determined using a standard calibration curve developed in the same way.

[0118] As a result, as shown in Figure 10(a) and (b), compound 1 significantly increased the BLM group in which pulmonary fibrosis was induced, but decreased the BLM+GSF-016_200 μg / kg group, while compound 2 significantly decreased the BLM group in both the BLM+GSF-018_100 μg / kg and BLM+GSF-018_200 μg / kg concentration groups.

[0119] Example 6-3. Histological evaluation of lungs - 1 To observe changes in lung tissue, lung tissue specimens were stained with hematoxylin and eosin (H&E) and Masson-trichrome staining to confirm the degree of inflammation and fibrosis in the lung tissue.

[0120] Specifically, excised lung tissue was embedded in paraffin and then cut into 4-μm pieces using a tissue cutter. These tissue sections were used for H&E, MT staining, and immunohistochemistry (IHC). Each experiment was performed by deparaffinization and rehydration using xylene and ethanol. For IHC, the LSAB2 system HRP kit (DAKO, Carpinteria, CA, USA) was used. Antibody retrieval was performed using 0.1 mM citric acid (pH 6) according to the provided instructions. After blocking in blocking buffer for 1 hour at room temperature, primary antibodies (fibronectin, collagen 1, and α-SMA) were diluted and incubated overnight at 4°C. The secondary antibody was blocked for 1 hour at room temperature, then exposed to DAB solution for 10 minutes using the streptavidin-HRP system. The slides were then hematoxylene stained, dehydrated, cleared, and mounted in Canada balsam. After H&E, MT, and IHC staining, the slides were examined under a light microscope (BK51, Olympus, Japan). The staining results were analyzed using the Image J program for quantification.

[0121] As a result, as shown in Figures 11a and 11b, minimal inflammatory responses were observed in the normal group, whereas inflammation caused by lymphocytes, neutrophils, and macrophages significantly increased in the BLM group, resulting in the collapse of lung tissue structure and the severe progression of fibrosis, as confirmed morphologically.

[0122] However, in the group that received oral administration of compound 1, it was confirmed that inflammation was significantly reduced regardless of the concentration, and the analysis of the degree of fibrosis in lung tissue also confirmed that the BLM+GSF-016_200ug / kg group and the BLM+GSF-016_500ug / kg group had a significantly reduced fibrosis compared to the BLM group.

[0123] In the group orally administered Compound 2, it was confirmed that inflammation was significantly reduced at both concentrations in the BLM+GSF-016 group. Analysis of the degree of fibrosis in lung tissue also showed that the BLM group had more than three times more fibrosis than the normal group, but the BLM+GSF-018_100ug / kg group and the BLM+GSF-018_200ug / kg group had significantly reduced fibrosis.

[0124] Example 6-4. Histological evaluation of lungs - 2 To investigate the most important factor involved in fibrosis, immunohistochemistry (IHC) was performed in the same manner as in Example 6-3.

[0125] As a result, as shown in Figures 12a and 12b, the expression of α-SMA, collagen, and fibronectin was increased in the BLM group compared to the normal group, similar to the pattern in the IPF cell line, while the expression of these proteins was significantly reduced in the BLM + GSF-016 group. In particular, the low concentration of 200 μg / kg showed the most effective effect on weight recovery and the suppression of the expression of related proteins.

[0126] In the case of Compound 2, the expression of fibronectin, collagen, and α-SMA was also increased in the BLM group compared to the normal group, and it was confirmed that the expression of all of these was significantly decreased in a concentration-dependent manner in the BLM+GSF-018 group.

[0127] Example 6-5. Analysis of fibrosis-related gene expression in lung tissue In the above examples, qRT-PCR analysis was performed to confirm whether the expression of various fibrosis-related genes, including fibronectin, collagen, α-SMA, and other fibrosis-related proteins, showed the same pattern as the histological findings.

[0128] Specifically, to extract RNA from lung tissue, approximately 100 mg of pulverized lung tissue was dispensed into 1.5 ml tubes. Total RNA was extracted from the lung tissue for gene expression analysis using TRIzol reagent (TaKaRa Bio Inc., Japan). The tissue was denatured by adding 1 ml of TRizol reagent, then transferred to a 1.5 ml tube and 200 μl of chloroform was added. The mixture was then vortexed for 20 seconds to thoroughly mix. The mixture was incubated at room temperature for 15 minutes, then centrifuged at 14,000 rpm for 20 minutes to obtain the supernatant. The supernatant was then inverted with an equal volume of isopropyl alcohol and allowed to stand at room temperature for 10 minutes. The sample was centrifuged at 14,000 rpm for 15 minutes to obtain an RNA pellet. The pellet was then washed with 70% RNA-safe ethanol by centrifugation at 14,000 rpm for 5 minutes and then dried for 5 minutes. The dried RNA sample was dissolved in 20 μl of distilled water treated with 0.1% DEPC (diethyl pyrocarbonate), and then used as a sample for cDNA synthesis. The RNA concentration and purity were measured at OD260 / 280nm using a Nanodrop™.

[0129] As a result, as shown in Figure 13a and b, the expression of all five genes (FN, COL1A1, α-SMA, TGF-β, and CTGF) in the BLM group was increased by 2 to 6 times compared to the normal group when compound 1 was used, demonstrating a significant reduction effect of compound 1.

[0130] In the case of Compound 2, as in the case of Compound 1, the expression of all five genes increased 2- to 6-fold in the BLM group compared to the normal group, confirming a significant reduction effect of Compound 2.

[0131] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be embodied in other specific forms without changing the technical spirit or essential features thereof. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be construed as including all modifications and variations derived from the meaning and scope of the claims, rather than the specification, and their equivalents.

Claims

1. A compound represented by the following chemical formula I or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 In the above formula I, R is methyl or ethenyl.

2. The compound according to claim 1, characterized in that the compound inhibits the aggravation or progression of pulmonary fibrosis.

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

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

5. The pharmaceutical composition of claim 4, further comprising a pharmaceutically acceptable carrier or excipient.

6. 5. The pharmaceutical composition of claim 4, wherein the pulmonary fibrosis is at least one selected from the group consisting of chronic obstructive pulmonary disease combined pulmonary fibrosis (COPD combined pulmonary fibrosis), combined pulmonary fibrosis and emphysema, idiopathic pulmonary fibrosis (IPF), pulmonary fibrosis induced by anti-cancer therapy, and pulmonary fibrosis induced by a virus.

7. The pharmaceutical composition of claim 4, wherein the compound suppresses the expression of pulmonary fibrosis marker genes and proteins by regulating the expression of phosphorylated proteins and modulating 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 modulation of the MRTF / SRF signaling pathway reduces the expression of MRTF / SRF that is increased in the cytoplasm and nucleus due to increased expression of Rock protein, which binds to Rho activated by TGF-β1 treatment.

10. A method for preventing or treating pulmonary fibrosis, comprising the step of administering the pharmaceutical composition according to any one of claims 4 to 9 to an individual other than a human.

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

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