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

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, 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 an organ or tissue, such as during regeneration, as opposed to the normal formation of fibrous tissue in an organ or tissue. Examples of fibrosis include pulmonary fibrosis, hepatic 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 (Pulmonary Fibrosis) or idiopathic pulmonary fibrosis (IPF) is a representative lung disease in which repeated inflammation due to alveolar damage causes fibrosis, leading to respiratory failure in patients. Among pulmonary fibrosis, idiopathic pulmonary fibrosis is a progressive disease whose cause has not yet been clarified, and patients die from respiratory failure within 3 to 4 years after diagnosis due to worsening dyspnea and cough, and the 5-year survival rate is about 30% to about 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 steroids and cytotoxic drugs, steroids were used first, and currently, a combination therapy of steroids and azathioprine or cyclophosphamide is used as a treatment for pulmonary fibrosis caused by radiation exposure (Non-Patent Document 1).

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

[0008] However, these two drugs both show limited therapeutic effects as therapeutic agents for 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 decrease in pulmonary function and cannot exert a fundamental therapeutic effect, so there is a demand 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 inventors have conducted research and efforts to develop a candidate substance that can treat and improve pulmonary fibrosis more effectively than conventional methods. As a result, they have confirmed that a novel sulforaphane compound has 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 pharma- ceutically 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 contains the compound or a pharma- ceutically acceptable salt thereof as an active ingredient.

[0014] A further 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 pharma- ceutical acceptable salt thereof as an active ingredient.

[0016] A further object of the present invention is to provide use of the compound or a pharma- ceutically acceptable salt thereof, or a composition containing same, for preventing, ameliorating or treating pulmonary fibrosis. Effect 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 description of the drawings]

[0018] [Figure 1] FIG. 1 is a graph confirming the cytotoxicity of a sulforaphane-based synthetic compound. [Diagram 2] FIG. 1 shows the inhibitory effect of treatment with 16 types of compounds on the expression of pulmonary fibrosis marker proteins in pulmonary fibroblasts induced by TGF-β1. [Diagram 3] FIG. 1 shows the inhibitory effect of pulmonary fibrosis marker protein expression by treatment with various concentrations of Compound 1 and Compound 2, which are sulforaphane-based synthetic compounds and exhibit an inhibitory effect on pulmonary fibrosis. [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. [Diagram 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 in normal lung fibroblasts (MRC-5) and diseased lung fibroblasts (DHLF-IPF) as determined 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 of 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 of diseased lung fibroblasts (DHLF-IPF). [Figure 9] FIG. 2 shows the results of analysis of changes in body weight and lung weight depending on the treatment period and concentration 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 an analysis of 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 an analysis of histological changes in inflammation- and fibrosis-related indices in lung tissues by 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-associated 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-associated 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 fibrosis-related gene expression analysis in lung tissues following treatment with Compound 1 and Compound 2 in an animal model of bleomycin-induced pulmonary fibrosis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0020] In order to achieve the above object, one aspect of the present invention provides a compound represented by chemical formula I or a pharma- ceutically 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 having 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 may 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 form, all of which physical forms are encompassed by the present invention.

[0028] The term "pharmacologically acceptable salt" as used herein means a salt 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, and the like. Examples of suitable salts include organic acid salts made with carboxylic acid, vanillic acid, hydroiodic acid, etc.; sulfonate salts 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 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 pharma- ceutically 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 change into fibrocytes, causing dyspnea, cough, cyanosis, clubbing, and the like. When tissue examination is performed, honeycomb-shaped or atypical fibrocyte clusters are observed. To date, immunosuppressants including steroid-based therapeutic agents, interferon gamma, acetylcysteine, pirfenidone, nintedanib, bosentan, and the like have been used, but no preparations showing specific therapeutic effects have been reported.

[0031] The pulmonary fibrosis in the present invention refers to 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 due to anticancer treatment, and pulmonary fibrosis induced by a virus, and specifically refers to idiopathic pulmonary fibrosis, but is not limited thereto.

[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 pharma- ceutically acceptable carrier, excipient, or diluent that is commonly used in the preparation thereof, 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, gum acacia, 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 in a conventional manner into any one of the following forms: tablets, pills, powders, granules, capsules, suspensions, liquids for internal use, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories, and are used in various oral or parenteral dosage forms. When formulated, they are prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are commonly used. Oral solid preparations include tablets, pills, powders, granules, capsules, and the like, and these solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to the conventional excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, liquids for internal use, emulsions, syrups, etc., and in addition to the usual diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavorings, and preservatives are used. Parenteral preparations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried 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, cacao butter, laurin butter, and glycerogelatin.

[0036] The content of the compound in the pharmaceutical composition of the present invention can be appropriately adjusted 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% by weight, or 0.001 to 50% by weight relative to the total weight of the composition, but is not limited thereto.

[0037] In one embodiment 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 each concentration of compound 1 and compound 2, respectively, and that each compound at a concentration of 20 uM or 10 uM showed the same or better reducing effect than sulforaphane (Figure 3).

[0038] In one embodiment of the present invention, normal human lung fibroblast cells (MRC-5) and diseased human lung fibroblasts-idiopathic pulmonary fibrosis cells (DHLF-IPF) were treated with sulforaphane, compound 1, and compound 2, and changes in cell morphology were then examined. As a result, it was confirmed that cell proliferation was suppressed in the compound 1 and 2 treatment groups compared to the sulforaphane treatment 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 embodiment of the present invention, the effects on pulmonary fibrosis-related protein and gene expression in MRC-5 and DHLF-IPF fibroblasts were examined. 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 significant reducing effect compared to sulforaphane (Figures 4b to 4d).

[0040] Furthermore, in one embodiment of the present invention, the effects on cell migration were confirmed using MRC-5 and DHLF-IPF, and it was confirmed that migration was increased by treatment with TGF-β1. 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] Furthermore, in one embodiment of the present invention, as a result of confirming the effect of suppressing pulmonary fibrosis by treating compound 1 in DHLF-IPF through the expression analysis of various signal transduction pathway proteins related to TGF-β1, it was confirmed that compound 1 suppresses the phosphorylation of smad-2, increases the phosphorylation of smad-7, and significantly reduces the activities of p-p38MAPK and p-AKT. Furthermore, it was confirmed that the expression of ROCK is suppressed in the Rho-ROCK signal transduction pathway, and the expressions of nuclear MRTF and SRF are suppressed in the MRTF-SRF signal transduction pathway (Figure 7).

[0042] Furthermore, in one embodiment of the present invention, as a result of confirming the effect of suppressing pulmonary fibrosis by treating compound 2 in DHLF-IPF through the expression analysis of various signal transduction pathway proteins related to TGF-β1, it was confirmed that compound 1 suppresses the phosphorylation of smad-2 and smad-3, increases the phosphorylation of smad-7, significantly reduces the activities of three phosphorylated proteins, p-ERK, p-JNK, and p-p38, in the MAPK signal transduction pathway, and significantly reduces the activity of p-AKT in the AKT signal transduction pathway. Furthermore, it was confirmed that the expression of ROCK is suppressed in the Rho-ROCK signal transduction pathway, and the expressions of nuclear MRTF and SRF are suppressed in the MRTF-SRF signal transduction 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] Still another aspect of the present invention for achieving the above object provides a method for preventing or treating pulmonary fibrosis, which includes the step of administering the pharmaceutical composition to an individual.

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

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

[0047] Moreover, the individual may include humans or may be non-human.

[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 can deliver the composition to the target tissue.

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

[0050] The above-mentioned "pharmacologically 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, age, sex, drug activity, sensitivity to the drug, administration time, administration route and excretion rate, treatment duration, 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, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may also be administered singly or in multiple doses. Taking into consideration all of the above factors, it is important to administer an amount that provides maximum efficacy at a minimum amount without side effects, and this can be easily determined by those skilled in the art.

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

[0053] In one embodiment of the present invention, the inhibitory effects of the final selected compounds 1 and 2 on pulmonary fibrosis in an animal model of pulmonary fibrosis induced by bleomycin were examined. As a result, it was confirmed that the hydroxyproline content in the lung tissue was significantly decreased in all of the groups treated with the two compounds (FIG. 10), the degree of inflammation and fibrosis in the lung tissue was reduced (FIG. 11), and the expression of fibrogenesis-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, still another aspect of the present invention provides a food composition for preventing or ameliorating pulmonary fibrosis, which contains the above compound or a pharma- ceutically acceptable salt thereof as an active ingredient.

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

[0056] By "amelioration" in the present invention is meant any action that at least reduces a parameter related to the condition being treated by administration of a composition comprising said novel compound, such as the severity of the symptoms.

[0057] In the present invention, the term "food" refers to any food in the usual sense, such as meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gums, 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 nutrition.

[0059] Here, "functionality" means that it regulates nutrients for the structure and function of the human body or provides a useful effect for health applications such as physiological action. The health food means a food that has a more active effect on maintaining or improving health than general foods, and the health supplement food means a food for the purpose of supplementing health. In some cases, functional food, health food, and health supplement are used interchangeably. Specifically, the functional food means a food in which the novel compound of the present invention is added to food ingredients such as beverages, teas, spices, gums, and confectioneries, or is manufactured into capsules, powders, suspensions, etc., and which brings about a specific effect on health when ingested.

[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] Furthermore, 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-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 improve aroma, taste, and visual appearance, such as vitamins A, C, D, E, B1, B2, B6, and B12, niacin, biotin, folate, and panthotenic acid, as well as 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] In addition, the food composition may contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder, highly bleached powder, sodium hypochlorite, etc.), antioxidants (butyl hydroxyanisole (BHA), butyl hydroxytoluene (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.), flavors (vanillin, lactones, etc.), leavening agents (alum, potassium D-hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (thickening agents), coating agents, gum bases, foam inhibitors, solvents, and improvers. The additives are selected and used in appropriate amounts according to 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 pharma- ceutical 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, or component of such a meal, intended or suitable for consumption, ingestion and digestion by an animal.

[0068] The type of the feed is not particularly limited, and feeds commonly used in the technical field can be used. Examples of the feed include vegetable feeds such as grains, nuts, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, meals, and grain by-products, and animal feeds such as proteins, inorganic substances, oils and fats, minerals, single-cell proteins, animal plankton, and food and drink, but are not limited thereto. These may be used alone or in combination of two or more.

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

[0070] The compound, the pharma- ceutically acceptable salt, and the prevention, amelioration or treatment of pulmonary fibrosis are as described above. EXAMPLES

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

[0072] Production Example 1: Method for producing new compounds

[0073] [ka]

[0074] To a solution (2 ml) of N-acetyl-L-cysteine ​​ethyl ester (CAS No. 59587-09-6, 150 mg, 0.78 mmol) in ethanol, 1N NaOH was added to adjust the pH of the solution to 8, and a solution (2 ml) of sulforaphane (CAS No: 4478-93-7, 50 mg, 0.28 mmol) in ethanol was added. The reaction mixture was stirred at room temperature under nitrogen for 4 h, the solvent was evaporated, and the residue was then 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, CDCl 3 ) 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, CDCl 3 ) 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 O 4 N 2 NaS 3 ,[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 at room temperature under nitrogen for 10 h, the solvent was evaporated, and the residue was then purified by reverse phase column chromatography with 0.05% TFA in methanol to give compound 2 (112 mg, 52% yield). 1 H NMR (600MHz, CD 3 OD) 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, 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 mode):m / z calculated for C 14 H 24 N 2 NaO 4 S 3 ,[M+Na] + =403.08 found:403.21. EXAMPLES

[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 medium containing DMEM, 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin at 37°C in 5% CO 2 The cells were cultured in an incubator 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 with 5 × 10 3 Dispense 100 ul of cells per well and incubate at 37℃ in 5% CO 2 The cells were cultured in an incubator for 24 hours.

[0079] After incubation, the medium was removed and each compound was added at different concentrations. Each group was treated in triplicate. Then, the cells were incubated at 37°C in 5% CO 2 After incubation in an incubator for 24 hours, 10 ul of 5 mg / ml MTT reagent was dispensed per well into the medium containing the test solution, and the 96-well plate was then placed in a dark, dark place at 37°C in 5% CO 2 The cells were cultured in an incubator for 2 to 4 hours. After the above culture was completed, the medium containing the MTT reagent was removed, and 100 ul of DMSO (dimethyl sulfoxide) was added to dissolve the MTT-formazan crystals. The absorbance was measured at 570 nm, and the cell viability was measured 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 40 uM, and GSF-18 showed cell viability of 80% or more at 10 uM. EXAMPLES

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

[0083] Specifically, to extract fibronectin and α-SMA proteins, cells were washed once with cold PBS, 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, and protein was quantified using a BCA protein quantification kit. All protein samples were electrophoresed on 8-10% PAGE gels at 10 ug each, and then transferred to 0.2 μm PVDF membranes (EMD Millipore, MA, USA). The membranes with transferred proteins were blocked with 5% BSA or 5% skim milk for 1 hour, and then reacted with primary antibodies at 4°C overnight. The next day, the cells were washed three times with PBST, and then reacted with HRP-conjugated secondary antibodies at room temperature for 1 hour. The PVDF membrane that had undergone this process 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 a result, as shown in Figure 2, it was confirmed that when pulmonary fibrosis was induced by TGF-β1, the expression of fibronectin and a-SMA was significantly increased, and that when treated with sulforaphane compounds GSF-016 and GSF-018, the expression of the two aforementioned proteins was significantly decreased.

[0085] Therefore, the above-mentioned two compounds were named Compound 1 and Compound 2, respectively, and selected as candidate compounds for the treatment of fibrosis of the present invention. EXAMPLES

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

[0087] As a specific experimental method, similar to that of Example 2 described above, the cells were treated with sulforaphane at a concentration of 20 uM, compound 1 at concentrations of 5 uM, 10 uM, and 20 uM, and compound 2 at concentrations of 2.5 uM, 5 uM, and 10 uM.

[0088] As a result, as shown in FIG. 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 20 uM or 10 uM showed the same or superior reducing effect as sulforaphane.

[0089] Example 3-2. Cytotoxicity and morphology analysis of candidate compounds in two cell lines We examined whether two candidate compounds of sulforaphane prodrugs affect cytotoxicity and morphological changes in normal lung fibroblasts MRC-5 and diseased lung fibroblasts DHLF-IPF. Diseased lung fibroblasts DHLF-IPF cell lines were purchased from Lonza, Inc., and FGM TM -2 Fibroblast Growth Medium-2 BulletKit TM Culture medium at 37 °C and 5% CO 2It was cultured in an incubator. All cells in the experimental process were experimented at a density of 80 - 90%.

[0090] Both of the two types of cells were dispensed with 100 μl each into a 96-well plate at a density of 5×10 3 cells / well, and cultured in a 5% CO 2 incubator at 37°C for 24 hours. Then, the medium was removed, each compound was prepared at a concentration of 10 μM, each group was treated in triplicate, and then cultured in a 5% CO 2 incubator at 37°C for 24 hours.

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

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

[0093] As a result, as shown in Fig. 4a, the two compounds showed a cell survival rate of over 80% in the two cell lines at a concentration of 10 μM. As a result of confirming the changes in morphology in the two cell lines, it was found that the TGF-β1 treatment group had more activated proliferation compared to the control group with an elongated shape, and the cells were intertwined and densely induced like fibrous tissue. In contrast, the two compounds were confirmed to have suppressed cell proliferation compared to the sulforaphane treatment group, and had an elongated cell shape similar to the control group, rather than a dense form like fibrous tissue.

[0094] Example 3 - 3.2 Analysis of the expression of fibrosis-related proteins of candidate compounds in two cell lines Normal MRC-5 and diseased DHLF-IPF cell lines induced to fibrosis by TGF-β1 were treated with SFN and the two aforementioned compounds, and Western blotting was performed to compare the changes in the expression of fibrosis-related proteins.

[0095] The specific experimental method was the same as in Example 2 described above, in which fibrosis was induced by treatment with 1 ng / ml TGF-β1, and then treatment with 10 uM sulforaphane and each of the two compounds.

[0096] As a result, as shown in FIG. 4b, a more significant decrease in fibronectin and α-SMA protein expression was confirmed with the two compounds compared to 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 in which fibrosis was induced by TGF-β1, SFN and the two compounds effectively suppressed the expression of fibrosis-related proteins. To confirm whether they 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, and the medium was removed. First, serum-free culture medium was pretreated with the experimental group (SFN, GSF-016, or GSF-018) at a concentration of 10 μM for 1 hour, and then 1 ng / ml TGF-β1 was added to induce fibrosis. Then, the cells were incubated at 37°C, 5% CO 2The cells were cultured for 48 hours under the conditions of 100%. The medium was then removed and washed once with PBS, after which total RNA in the cells was extracted from the cultured cells using TRIzol reagent (TaKaRa Bio Inc., Japan) for gene expression analysis. 1 ml of TRizol reagent was added to dissolve the cells and denature the tissue, then transferred to 1.5 ml tubes and 200 μl of chloroform was added, followed by vortexing for 20 seconds to thoroughly mix. The cells were reacted for 15 minutes at room temperature, then centrifuged at 14,000 rpm for 20 minutes to obtain the supernatant, which was inverted by adding the same amount of isopropyl alcohol, and then left to stand at room temperature for 10 minutes. The samples were centrifuged at 14,000 rpm for 15 minutes to obtain RNA pellets, which were washed by centrifugation at 14,000 rpm for 5 minutes using 70% RNA-grade ethanol, and then dried for 5 minutes. The dried RNA samples were dissolved in 20 μl of distilled water treated with 0.1% DEPC (diethyl pyrocarbonate) and then used as samples for cDNA synthesis. The RNA concentration and purity were measured at OD260 / 280nm using Nanodrop for 1 μl of each sample.

[0099] Synthesis of cDNA For single-stranded cDNA synthesis, 1 μl of oligo-d(T) primer (100 pmol) and 10 mM dNTP (TaKaRa Bio Inc., Japan) were mixed with 1 μg of extracted total RNA and reacted at 65°C for 5 minutes, then rapidly cooled. 4 μl of 5×RT buffer, 0.5 μl of RNA inhibitor, and 100 units of RTase (TaKaRa Bio Inc., Japan) were added to the template, and the total volume was adjusted to 20 μl using distilled water treated with DEPC. The sample was subjected to synthesis reaction at 25°C for 5 minutes and 42°C for 1 hour, and then terminated by inactivating the reverse transcriptase by reaction at 72°C for 15 minutes.

[0100] RT-PCR The level of expression of each gene was measured by real-time PCR. 10 μl of 2X SYBR Green MasterMix (TaKaRa Bio Inc., Japan) and 1 μl each of 10 pmol (forward, reverse) primers for each gene were added to 5 μl of extracted cDNA from each sample, and then the total volume was adjusted to 20 μl using distilled water. The reaction was performed at 95°C for 10 minutes, followed by 40 cycles of 30 seconds, followed by 60°C for 30 seconds and 72°C for 30 seconds. 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 d, 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, it was confirmed that the expression levels in the two compound-treated groups were significantly reduced compared to the sulforaphane-treated group.

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

[0103] cell migration Since myofibroblasts show increased cell migration and invasion as the expression of fibrosis-related proteins increases, we performed migration and wound healing experiments to examine the effect 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 then added at 1 × 10 5 The cells were seeded at 37℃, 5% CO 2 The cells were cultured for 24 hours in an incubator where the concentration of 100 μm per well was maintained, after which the medium was gently removed from the 8 μm pore polycarbonate membrane insert, which was then washed with DPBS and fixed with methanol for 5 minutes. The cells were then washed twice with triple distilled water, and then stained with Mayer's Haematoxylin for 8 minutes. The stained cells were washed with DPBS, the inside of the insert was gently wiped with a cotton swab, and only the insert membrane was cut out and fixed on a slide. All migrated cells were counted under a microscope at 200x magnification.

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

[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 5The wounds were photographed under a microscope, and the cells were treated with TGF-β1 alone, TGF-β1 + SFN, TGF-β1 + GSF-016, and TGF-β1 + GSF-018, then cultured for about 24 hours, after which the wounds were compared again. In the images taken after 24 hours of culture, the distance between the cells was measured using the public domain software Image J (Fiji package), and the relative mobility 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, whereas migration was significantly inhibited in all compound-treated groups in a concentration-dependent manner. EXAMPLES

[0109] TGF-β1 signaling pathway-related protein expression analysis The TGF-β1 signaling pathway is activated by both Smad-dependent and Smad-independent pathways, and the Smad-dependent pathway, TGF-β / Smad signaling, is an important pathway that regulates the synthesis of extracellular matrix components, while the Smad-independent (TGF-β / non-Smad) pathway is influenced by PI3K / AKT / mTOR and MAPK (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, as with the western blotting performed in Example 2.

[0110] As a result, as shown in Figure 7, it was confirmed that compound 1 inhibited phosphorylation of smad-2, increased phosphorylation of smad-7, and significantly reduced the activity of p-p38 and MAPK. In addition, as a result of confirming the influence of MRTF / SRF on the Rho / Rack pathway, it was confirmed that the expression of Rock protein that binds to activated Rho was increased by treatment with TGF-β1, and the expression of MRT / SRF, which is a lower step, was increased in the cytoplasm and nucleus, but that the increased protein expression was significantly decreased by treatment with compound 1.

[0111] In addition, as shown in Figure 8, it was confirmed that compound 2 inhibited phosphorylation of smad-2 and smad-3, increased phosphorylation of smad-7, and reduced the activity of all three MAPKs, p-ERK, p-JNK, and p-p38. Furthermore, as a result of confirming the influence of MRTF / SRF on the Rho / Rack pathway, it was confirmed that the expression of Rock protein that binds to activated Rho by treatment with TGF-β1 increased, and the expression of MRT / SRF, which is a lower step, increased in the cytoplasm and nucleus, but the increased protein expression was significantly reduced by treatment with compound 2. EXAMPLES

[0112] Inhibitory effect on pulmonary fibrosis in animal models Example 6-1. Analysis of changes in body weight and lung weight in pulmonary fibrosis animal models 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 pulmonary fibrosis induced by bleomycin.

[0113] Specifically, 7-week-old C57BL / 6N mice were exposed to BLM (3-5 units / kg) in the airways, and drug administration was orally administered 200ug / kg and 500ug / kg of compound 1, and 100ug / kg and 200ug / 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 weight was measured after the animal experiment was completed.

[0114] As a result, as shown in Figure 9a, the change in weight gain during the experiment was confirmed, and in Compound 1, the normal group (CTL) steadily increased, while the BLM group and the BLM+GSF-016_500ug / kg group showed a tendency to gradually increase after decreasing, whereas the BLM+GSF-016_200ug / kg group showed a tendency to steadily increase. In addition, the lung weight was confirmed, and the BLM group was the largest, while the normal group and the BLM+GSF-016_200ug / kg group were at the same level.

[0115] In the case of compound 2, as shown in FIG. 9b, the BLM group gradually increased after a large decrease, but it was confirmed that the weight loss was significant compared to the normal group and the GSF-018 group, whereas the BLM+GSF-018_100ug / kg and BLM+GSF-018_200ug / kg groups increased steadily, although not as much as the normal group, and it was confirmed that the weight recovered to the same extent as the normal group. In addition, as a result of checking the lung weight, it was confirmed that the BLM group was significantly larger, while the normal group, the BLM+GSF-018_100ug / kg group, and the BLM+GSF-018_200ug / kg group were low at the same level, showing 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 placed in an equal amount in a 1.5 ml tube, transferred without dissolving, and 100 ul of 12 M hydrochloric acid was added and hydrolyzed at 120°C for 3 hours. The sample was then centrifuged at 10,000×g for 5 minutes, the supernatant was transferred to a new tube, 10 ul of which was transferred to a 96 well plate, and then evaporated at 60°C. Next, 100 ul of chloramine T reagent / oxidation buffer mixture was added to each well, and finally 100 ul of para-dimethylaminobenzaldehyde reagent was added and reacted at 60°C for 90 minutes. The absorbance was then measured at 540 nm, and the content was calculated using a standard calibration curve that was similarly colored.

[0118] As a result, as shown in Figure 10(a) and (b), it was confirmed that compound 1 significantly increased in the BLM group in which pulmonary fibrosis was induced, and decreased in the BLM+GSF-016_200ug / kg group, while compound 2 significantly decreased in both the BLM+GSF-018_100ug / kg and BLM+GSF-018_200ug / kg concentration groups compared to the BLM group.

[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, the excised lung tissue was embedded in paraffin and then cut into 4-um pieces using a tissue cutter. H&E, MT stain, and immunohisochemistry (IHC) were performed using these tissue sections. Each experiment was performed by deparaffinization and rehydration using xylene and ethanol, and the LSAB2 system HRP (DAKO, Carpinteria, CA, USA) kit was used for IHC. Antibody retrieval was performed with 0.1 mM citric acid (pH 6), and the experiment was performed according to the instructions provided. The sections were blocked for 1 hour at room temperature with blocking buffer, and primary antibodies (fibronectin, collagen 1, a-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, stained with hematoxylene, dehydrated, cleared, and mounted (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 to quantify the results.

[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 was significantly increased in the BLM group, and it was morphologically confirmed that the structure of lung tissue had collapsed and fibrosis had progressed significantly.

[0122] However, in the group that received oral administration of compound 1, inflammation was significantly reduced regardless of the concentration, and analysis of the degree of pulmonary fibrosis also confirmed that the BLM+GSF-016_200ug / kg group and the BLM+GSF-016_500ug / kg group showed a significant reduction 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, and the analysis of the degree of fibrosis in lung tissue 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 In order to investigate what is most importantly 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, in Compound 1, the expression of α-SMA, collagen and fibronectin was increased in the BLM group compared to the normal group, similar to the pattern of the IPF cell line, and all were significantly decreased in the BLM + GSF-016 group. In particular, the low concentration of 200ug / kg showed the best effect on weight recovery and inhibition of the expression of related proteins.

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

[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 fibrogenesis-related proteins such as fibronectin, collagen, and α-SMA, showed the same pattern as the histological viewpoint.

[0128] Specifically, to extract RNA from lung tissue, approximately 100 mg of crushed lung tissue was dispensed into 1.5 ml tubes, and total RNA in the tissue was extracted from the lung tissue for gene expression analysis using TRIzol reagent (TaKaRa Bio Inc., Japan). 1 ml of TRizol reagent was added to denature the tissue, and then the tissue was transferred to a 1.5 ml tube and 200 μl of chloroform was added, followed by vortexing for 20 seconds to thoroughly mix. The mixture was allowed to react for 15 minutes at room temperature, then centrifuged at 14,000 rpm for 20 minutes to obtain the supernatant, which was inverted by adding the same amount of isopropyl alcohol, and then left to stand 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 by centrifugation at 14,000 rpm for 5 minutes using 70% RNA-grade ethanol, and then dried for 5 minutes. The dried RNA samples were dissolved in 20 μl of distilled water treated with 0.1% DEPC (diethyl pyrocarbonate) and then used as samples for cDNA synthesis. The RNA concentration and purity were measured at OD260 / 280nm using Nanodrop for 1 μl of each sample.

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

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

[0131] From the above description, a person skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. A compound represented by the following formula I or a pharma- ceutically 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 of the sulforaphane family.

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

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

6. 5. The pharmaceutical composition according to 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 due to anticancer treatment, and pulmonary fibrosis induced by a virus.

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

8. The pharmaceutical composition of 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 MRTF / SRF expression that is increased in the cytoplasm and nucleus due to increased expression of Rock protein that binds to Rho activated by TGF-β1 treatment.

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

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

Images