Novel flavonoid derivative and application thereof in improvement of pulmonary fibrosis

Abstract

The invention discloses a novel flavonoid derivative and a composition for improving pulmonary fibrosis prepared by using the same, the flavonoid derivative has an effect of inhibiting EMT by inhibiting a TGF-beta1 signal transduction pathway, and has no special cytotoxicity to A549 cells derived from lung cancer, Hulec-5a cells derived from pulmonary blood vessels and human lung fibroblasts (HLF). The expression of pulmonary fibrosis related factors in A549 cells and Hulec-5a cells which are treated by EGF (Epidermal Growth Factor) or bleomycin to cause pulmonary fibrosis can be regulated.

Description

Technical Field

[0001] The present invention relates to a novel flavonoid derivative and application thereof in improving pulmonary fibrosis. Background Art

[0002] Pulmonary fibrosis, or idiopathic pulmonary fibrosis (IPF), is a disease characterized by interstitial fibrosis in the lungs, in which connective tissue, particularly collagen, overgrows in the alveolar walls. Pulmonary fibrosis is a fatal disease characterized by a gradual decline in lung function, ultimately leading to respiratory failure, with an average survival of less than 2-3 years after diagnosis. Reports of the prevalence of pulmonary fibrosis vary, but it is known to affect approximately 2-29 people per 100,000. In South Korea, it is designated a rare and intractable disease.

[0003] Pulmonary fibrosis is a disease that occurs in older adults, and its incidence is expected to gradually increase as the population ages. Recent research results suggest that the previously reported prevalence of pulmonary fibrosis is underestimated, with approximately 7-8% of smokers and the general population suffering from the disease. Therefore, a considerable number of elderly people (currently 1 in 450 men over the age of 70) are expected to develop this disease, but there is currently no effective treatment.

[0004] As for lung transplantation, the number of patients seeking a transplant far exceeds the number of donors, leading to long wait times (during which the patient's condition may worsen and lead to death). Even if a transplant is finally obtained, many patients die from post-transplant complications (the 5-year survival rate is approximately 50%). Therefore, more effective and safer treatments are urgently needed.

[0005] The causes of pulmonary fibrosis are diverse, including lung injury, exposure to toxic substances or toxic environments (such as fine dust or ultrafine dust), anticancer drugs, autoimmune diseases, idiopathic interstitial pneumonia, etc. (Proc Am Thorac Soc 2006,3:285-92; Am J Respir Crit Care Med 2002, 165:277-304).

[0006] The basic histological changes of pulmonary fibrosis are inflammatory changes such as cell infiltration, edema, and exudation in the alveolar septa, as well as infiltration and fibrosis of extracellular matrices such as collagen, proteoglycan, fibronectin, and glycoproteins, leading to destruction of the lung parenchyma (Am J Respir Crit Care Med 2002, 165:277-304; N Engl J Med 2001, 345:517-25).

[0007] In the past, inflammation was considered the cause of pulmonary fibrosis, and anti-inflammatory treatments were tried, but with little success (Annals of Internal Medicine 2001, 134:136-51; Chest 1996, 110:1058-67). Consequently, in recent years, epithelial-mesenchymal transition (EMT) or the TGF-β1 signaling pathway that induces EMT, rather than inflammation, has been identified as a new etiology and therapeutic target (Annals of Internal Medicine 2001, 134:136-51; Chest 2007, 132:1311-21; The Journal of Clinical Investigation 2009, 119:213-24).

[0008] The primary factor inducing EMT is TGF-β1. During EMT, TGF-β1 induces the de novo synthesis of α-SMA (α-smooth muscle actin). De novo synthesis of α-SMA causes lung fibroblasts to transform into myofibroblasts.

[0009] Because these myofibroblasts are upregulated and persist in areas of progressive pulmonary fibrosis, they are thought to play an important role in the development and progression of pulmonary fibrosis (Chest 2002, 122: 286S-9S).

[0010] In particular, it is known that the expression of α-SMA, vimentin, fibronectin, SMA2 / 3, etc. increases in association with the TGF-β pathway, and that E-cadherin is converted to N-cadherin, thereby promoting EMT and causing pulmonary fibrosis (The Journal of Clinical Investigation 2009, 119: 213-24; Nat Rev Mol Cell Biol 2006, 7: 131-42; Cell Biol Int 2002, 26: 463-76). In addition, it is known that TGF-β1-induced cell activation involves Smad-dependent pathways and Smad-independent MAPK (mitogen-activated protein kinases) pathways (J Am Soc Nephrol 2002, 13: 1464-72; Proc Natl Acad Sci US A2001, 98: 6686-91; Cancer Res 2001; 61: 4222-8). NOX (NADPH oxidases) such as NOX2 and NOX4 are also known to be involved in TGF-β1-induced cell activation (Thorax. 2010 Aug; 65(8): 733-8).

[0011] Oxidative stress is also a factor that contributes to the progression of pulmonary fibrosis. Bleomycin has been shown to increase the synthesis of nitric oxide (NO) in the lungs, thereby enhancing oxidative stress and increasing the expression of NOX through phosphorylation of MAPK signaling pathway factors (Scientific Reports 7(1):2252, 2017).

[0012] In addition, HIF-1α (Hypoxia-Inducible Factor-1α) and EGFR (epidermal growth factor receptor) signaling are also considered to be involved in pulmonary fibrosis, and they have been proposed as targets for pulmonary fibrosis treatment (Am J Respir Cell Mol Biol. 2018 Feb;58(2):216-231; Am J Physiol Lung Cell Mol Physiol. 2019 Jun 1;316(6):L1025-L1034).

[0013] Especially in vascular smooth muscle cells, EGFR increases the production of NO and the expression of NOX by increasing the expression of ERK (extracellular signal-regulated kinase) and AKT (serine / threonine protein kinase) and the phosphorylation of their downstream signal transducers (Antioxidants & Redox Signaling 22 (1): 29-47, 2015), and EGFR phosphorylation is known to accelerate fibrosis by activating MAPK signaling pathways such as ERK1 / 2, P38 and JNK (c-Jun N-terminal kinases) (Journal of Respiratory Cell and Molecular Biology, 50 (4): 723-736, 2014).

[0014] Drugs such as gefitinib are EGFR tyrosine kinase inhibitors that inhibit fibroblast proliferation and extracellular collagen deposition by inhibiting EGFR phosphorylation (Am J Respir Cell Mol Biol. 2006, 174(5):550-556; Am J Physiol Lung Cell Mol Physiol. 2008., 294(6):L1217-L1225).

[0015] Steroids (glucocorticoids), immunosuppressants, and antiviral cytokines are representative drugs for the treatment of pulmonary fibrosis. However, according to the 2011 ATS / ERS idiopathic pulmonary fibrosis guidelines, combined treatment with steroids and the immunosuppressant azathioprine actually increases mortality.

[0016] In pulmonary fibrosis, alveolar epithelial cells and fibroblasts or myofibroblasts are considered the main factors in the pathogenesis, and new drug research targeting these cells is ongoing. In addition, due to the diverse causes of pulmonary fibrosis, the development of new drugs is not limited to a single pathway, but rather targets various pathways or upstream signaling systems that lead to fibrosis.

[0017] The present invention discloses a novel flavonoid derivative having EMT inhibitory activity by inhibiting the TGF-β1 signaling pathway and use thereof in improving pulmonary fibrosis. Summary of the Invention

[0018] One object of the present invention is to provide a novel flavonoid derivative.

[0019] Another object of the present invention is to provide a composition for improving pulmonary fibrosis prepared by using novel flavonoid derivatives.

[0020] Other or specific objects of the present invention will be described below.

[0021] As demonstrated in the following examples and experimental examples, the present invention is completed by demonstrating that the novel flavonoid derivatives of Chemical Formulas 1 to 3 can inhibit the TGF-β1 signaling pathway, and do not show specific cytotoxicity to A549 cells derived from lung cancer, Hulec-5a cells derived from pulmonary blood vessels, and human lung fibroblasts (HLFs), while inhibiting epithelial-mesenchymal transition (EMT), and also showing the effect of regulating the expression of pulmonary fibrosis-related factors (fibronectin, α-SMA, HIF-1α, NOX2, NOX4, etc.) in A549 cells and Hulec-5a cells that lead to pulmonary fibrosis treated with epidermal growth factor (EGF) or bleomycin.

[0022] <Chemical Formula 1>

[0023]

[0024] <Chemical Formula 2>

[0025]

[0026] <Chemical Formula 3>

[0027]

[0028] The present invention is proposed based on these experimental results. In one aspect, the present invention can be understood as a flavonoid derivative or a hydrate or a solvate thereof shown in the following <Chemical Formula 4>, and can be understood as a composition for improving pulmonary fibrosis comprising a flavonoid derivative or a hydrate or a solvate thereof shown in the following <Chemical Formula 4> as an active ingredient.

[0029] <Chemical Formula 4>

[0030]

[0031] wherein R1, R2, R3 and R4 are each independently hydrogen (H), hydroxyl (-OH), methyl (-CH3) or methoxy (-OCH3).

[0032] As used herein, "hydrate" refers to a compound combined with water, including inclusion compounds in which there is no chemical bond between water and the compound.

[0033] Furthermore, "solvate" as used herein refers to a compound formed between solute molecules or ions and solvent molecules or ions.

[0034] Furthermore, the "active ingredient" used herein refers to a component that exerts the desired activity alone or together with a carrier that is inactive itself.

[0035] In the composition of the present invention, as long as it can exert the effect of improving pulmonary fibrosis, etc., the content of the active ingredient (effective amount) can be arbitrarily selected according to the purpose, dosage form, etc., and the typical effective amount will be determined in the range of 0.001 weight% to 99 weight% based on the total weight of the composition.

[0036] Here, "effective amount" refers to the amount of the active ingredient contained in the composition of the present invention that can exert the intended medical and pharmacological effect, such as the effect of improving pulmonary fibrosis, when the composition of the present invention is administered to a mammal, preferably a human, as the application target within the administration period recommended by a medical professional.

[0037] Such effective amounts can be determined experimentally within the ordinary capabilities of those skilled in the art.

[0038] The composition of the present invention can be understood in certain aspects as a food composition.

[0039] The food composition of the present invention can be made into any shape, for example, beverages such as tea, juice, carbonated drinks, electrolyte drinks, processed dairy products such as milk and yogurt, foods such as chewing gum, rice cakes, Korean traditional candies, bread, candies, noodles, and health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, and bars.

[0040] Furthermore, the food composition of the present invention may be categorized as any product category, as long as it complies with applicable laws and regulations regarding legal and functional classification during production and distribution. For example, it may be a health functional food under the Korean Health Functional Food Act; and it may be categorized as candy, beans, tea, beverages, or special purpose foods under the Korean Food Sanitation Act (Food Standards and Specifications issued by the Ministry of Food and Drug Safety).

[0041] The food composition of the present invention may also contain food additives in addition to its active ingredient. Food additives are generally understood to be substances that are added, mixed, or incorporated into food during food production, processing, or preservation. Since these additives are consumed daily and over a long period of time, their safety must be ensured.

[0042] The Food Additive Codex is formulated based on the laws of each country that regulate food manufacturing and distribution (in Korea, it is the Food Sanitation Act), and it makes limited provisions for food additives that have guaranteed safety in terms of ingredients or functions.

[0043] In the Korean Food Additive Code (Ministry of Food and Drug Safety's "Food Additive Standards and Specifications"), food additives are divided into chemical synthetics, natural additives, and mixed preparations according to their ingredients, and into sweeteners, flavorings, preservatives, emulsifiers, acidifiers, and thickeners according to their functions.

[0044] Sweeteners are used to impart appropriate sweetness to food. Natural and synthetic sweeteners can be used in the food compositions of the present invention. Natural sweeteners are preferably used. Examples of natural sweeteners include carbohydrate sweeteners, such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

[0045] Flavorings are used to enhance taste and aroma. Both natural and synthetic flavorings can be used. Natural flavorings are recommended.

[0046] In addition to enhancing flavor, natural flavorings can also enhance nutritional value. These include flavors derived from apples, lemons, oranges, grapes, strawberries, and peaches, as well as green tea leaves, polygonum multiflorum, bamboo leaves, cinnamon, chrysanthemum leaves, and jasmine flowers. Ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo biloba can also be used. Natural flavorings can be liquid concentrates or solid extracts. In some cases, synthetic flavorings, including esters, alcohols, aldehydes, and terpenes, can be used.

[0047] Available preservatives include calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, and ethylenediaminetetraacetic acid (EDTA). Available emulsifiers include gum arabic, carboxymethyl cellulose, xanthan gum, and pectin. Available acidulants include citric acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, and phosphoric acid.

[0048] Acidulants can be added to food compositions to maintain appropriate acidity while enhancing flavor and inhibiting microbial growth. Thickeners include suspending agents, precipitants, gelling agents, and fillers.

[0049] In addition to the above-mentioned food additives, the food composition of the present invention may further include physiologically active substances or minerals known in the art and guaranteed to be safe as food additives, for supplementing and enhancing functions and nutrition.

[0050] Examples of such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, and dibenzoylthiamine. Examples of minerals include calcium preparations such as calcium citrate, magnesium preparations such as magnesium stearate, iron preparations such as ferrous citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, and zinc.

[0051] The food composition of the present invention can be added with the above-mentioned food additives in an appropriate amount according to the product type to achieve the desired purpose. Information about other food additives that can be added in the food composition of the present invention can be obtained from applicable Food Codex or Food Additives Codex according to the laws of various countries.

[0052] In other embodiments, the compositions of the present invention may be considered as pharmaceutical compositions.

[0053] The pharmaceutical compositions of the present invention can be prepared as oral or parenteral formulations by conventional methods known in the art, depending on the route of administration. In addition to the active ingredient, they also contain a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" herein means that the composition does not inhibit the activity of the active ingredient and does not exhibit toxicity exceeding the level tolerable to the intended subject.

[0054] When the pharmaceutical composition of the present invention is prepared into an oral dosage form, it can be prepared into powders, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, suspensions, wafers and the like using appropriate carriers and methods known in the art.

[0055] Examples of suitable pharmaceutically acceptable carriers include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol and xylitol; starches such as corn starch, potato starch and wheat starch; celluloses such as cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose and hydroxypropyl methylcellulose; polyvinylpyrrolidone, water, methylparaben, propylparaben, magnesium stearate, mineral oil, malt, gelatin, talc, polyols and vegetable oils.

[0056] In the case of a formulation, the formulation may include a diluent and / or an excipient, such as a filler, a swelling agent, a binder, a wetting agent, a disintegrant, and a surfactant, as needed.

[0057] When the pharmaceutical composition of the present invention is prepared into a parenteral preparation, it can be formulated into the form of eye drops, injections, transdermal administration agents, inhalants (medicaments that use a nebulizer to deliver the drug directly to the nasal cavity, oral cavity, respiratory tract, bronchi, etc.), suppositories, etc. using suitable carriers and methods known in the art.

[0058] When formulated as eye drops, suitable carriers include sterile water, saline solutions and isotonic solutions, such as 5% glucose, and if necessary, benzalkonium chloride, methylparaben and ethylparaben may be added for preservative purposes.

[0059] When formulated for injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, preferably Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, or isotonic solutions such as 5% glucose.

[0060] When formulated as a transdermal agent, it can be formulated in the form of an ointment, cream, lotion, gel, external solution, paste, liniment or aerosol.

[0061] For inhalation, it can be formulated into an aerosol form using a suitable propellant (e.g., dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide); for suppositories, bases that can be used include witepsol, Tween 61, polyethylene glycol, cocoa butter, glyceryl laurate, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate and sorbitan fatty acid esters.

[0062] Specific formulations of pharmaceutical compositions are known in the art and can be found in, for example, Remington's Pharmaceutical Sciences (19th ed. 1995), which is incorporated herein by reference.

[0063] The preferred dosage range of the pharmaceutical composition of the present invention is 0.001 mg / kg to 10 g / kg per day, preferably 0.001 mg / kg to 1 g / kg, depending on the patient's condition, weight, sex, age, severity of the condition, and route of administration. Administration can be once daily or in divided doses. This dosage should not be construed in any way as limiting the scope of the present invention.

[0064] As described above, according to the present invention, a composition for improving pulmonary fibrosis using a novel flavonoid derivative can be provided, wherein the flavonoid derivative exerts an EMT-inhibiting effect by inhibiting the TGF-β1 signal transduction pathway, and does not show particular cytotoxicity to A549 cells derived from lung cancer, Hulec-5a cells derived from pulmonary blood vessels, and HLF cells derived from human lung fibroblasts, and is capable of regulating the expression of pulmonary fibrosis-related factors in A549 cells and Hulec-5a cells that cause pulmonary fibrosis and are treated with EGF or bleomycin.

[0065] The composition of the present invention can be commercialized as a food such as a health functional food or a medicine such as a drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 and Figure 2 These are the 1H-NMR and 13C-NMR results of the new flavonoid derivative I, respectively.

[0067] Figure 3This is the LC-MS spectrum of the new flavonoid derivative I.

[0068] Figures 4 to 6 These are the results of cytotoxicity evaluation of the novel flavonoid derivative I.

[0069] Figure 7 and Figure 8 The effect of the novel flavonoid derivative I on A549 cells treated with TGF-β1.

[0070] Figure 9 Shown are the effects of the novel flavonoid derivative I on Hulec-5a cells treated with TGF-β1.

[0071] Figure 10 Shown are the effects of the novel flavonoid derivative I on HLF cells treated with TGF-β1.

[0072] Figure 11 and Figure 12 Shown are the effects of the novel flavonoid derivative I on EGF-treated A549 cells.

[0073] Figure 13 Shown are the effects of the novel flavonoid derivative I on bleomycin-treated A549 cells.

[0074] Figure 14 Shown are the effects of the novel flavonoid derivative I on Hulec-5a cells treated with bleomycin.

[0075] Figure 15 and 16 These are the 1H-NMR and 13C-NMR results of the new flavonoid derivative II, respectively.

[0076] Figures 17 to 19 These are the results of cytotoxicity evaluation of the novel flavonoid derivative II.

[0077] Figure 20 and 21 This is the effect of the novel flavonoid derivative II on A549 cells treated with TGF-β1.

[0078] Figure 22 This is the effect of the novel flavonoid derivative II on Hulec-5a cells treated with TGF-β1.

[0079] Figure 23 This is the effect of the novel flavonoid derivative II on HLF cells treated with TGF-β1.

[0080] Figure 24 This is the effect of the novel flavonoid derivative II on A549 cells treated with EGF.

[0081] Figure 25 and 26 These are the 1H-NMR and 13C-NMR results of the new flavonoid derivative III, respectively.

[0082] Figures 27 to 29 These are the results of cytotoxicity evaluation of the novel flavonoid derivative III.

[0083] Figure 30 This is the effect of the novel flavonoid derivative III on A549 cells treated with TGF-β1.

[0084] Figure 31 This is the effect of the novel flavonoid derivative III on Hulec-5a cells treated with TGF-β1.

[0085] Figure 32 This is the effect of the novel flavonoid derivative III on HLF cells treated with TGF-β1. DETAILED DESCRIPTION

[0086] The present invention will be described below with reference to Examples and Experimental Examples, but the scope of the present invention is not limited to these Examples and Experimental Examples.

[0087] <Example> Preparation of Novel Flavonoid Derivatives I to III and Testing of Their Pulmonary Fibrosis-Ameliorating Activity

[0088] <Example 1> Preparation of Novel Flavonoid Derivative I and Testing of Its Pulmonary Fibrosis Improvement Activity

[0089] 1. Preparation of Novel Flavonoid Derivative I

[0090] Step 1

[0091]

[0092] Potassium carbonate (K2CO3, 32.86 g, 238.10 mmol) and benzyl bromide (BnBr, 22.39 g, 130.95 mmol) were added to a solution (25°C) of methyl 3,4-dihydroxybenzoate (1) (10 g, 59.52 mmol) in acetonitrile (100 mL), and the mixture was stirred at room temperature for 5 hours.

[0093] The solvent was removed, and the residue was diluted with 100 mL of water, extracted with ethyl acetate (100 mL x 2), dried over anhydrous sodium sulfate, and concentrated.

[0094] The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1) to obtain methyl 3,4-bis(benzyloxy)benzoate (2) (19 g, yield: 91.73%) as a yellow solid.

[0095] LC-MS (ESI) m / z 349.2 [M+1] +

[0096] Step 2

[0097]

[0098] 1 M NaOH (aq) (60 mL, 60.0 mmol) was added to a solution of methyl 3,4-bis(benzyloxy)benzoate (2) (19 g, 54.60 mmol) in THF (tetrahydrofuran) / MeOH (50 mL / 50 mL), and the mixture was stirred at 20°C for 12 hours.

[0099] The mixture was concentrated, diluted with water, adjusted to pH 5 with 1 M HCl (aq), and extracted with DCM (dichloromethane). The organic solvent was dried over Na2SO4, filtered, and concentrated to give 3,4-bis(benzyloxy)benzoic acid (3) (13 g, crude) as a white solid.

[0100] LC-MS (ESI) m / z 335.2[M+1] +

[0101] Step 3

[0102]

[0103] To a solution of 3,4-bis(benzyloxy)benzoic acid (3) (7 g, 20.96 mmol) in DCM (50 mL) were added thionyl chloride (4.99 g, 41.92 mmol) and DMF (15.33 mg, 0.21 mmol) at 0°C, and the mixture was stirred at 0°C for 4 hours. The mixture was concentrated to give 3,4-bis(benzyloxy)benzoyl chloride (4) (8 g, crude) as a yellow solid, which was used immediately in the next step 4.

[0104] Step 4

[0105]

[0106] To a solution of 3,4-bis(benzyloxy)benzoyl chloride (4) (8 g, 20.96 mmol) in DCM (50 mL) (0°C) were added TEA (triethylamine, 2.12 g, 20.96 mmol) and 1H-benzo[d][1,2,3]triazole (5) (2.49 g, 20.96 mmol), and the mixture was stirred at 0°C for 4 hours.

[0107] Saturated aqueous ammonium chloride solution was added to the resulting product, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with a 3 M aqueous sodium hydroxide solution, dried over Na2SO4, concentrated under reduced pressure, and recrystallized from n-hexane-DCM to obtain (1H-benzo[d][1,2,3]triazol-1-yl)(3,4-bis(benzyloxy)phenyl)methanone (6) (8 g, yield 87.74%) as a white solid.

[0108] LC-MS (ESI) m / z 436.2[M+1] +

[0109] Step 5

[0110]

[0111] To a solution of 1-(2-((tert-butyldiphenylsilyl)oxy)-4,5-dimethoxyphenyl)ethan-1-one (8 g, 18.43 mmol) (14, prepared in Step 11 below) in THF (50 mL) (-70°C) was added LiHMDS (lithium bis(trimethylsilyl)amide, CAS No. 4939-32-1) (55 mL, 55 mmol, 1 M THF solution) and the mixture was stirred at -70°C for 1 hour.

[0112] To the obtained product was added a THF (30 mL) solution (-70°C) of (1H-benzo[d][1,2,3]triazol-1-yl)(3,4-bis(benzyloxy)phenyl)methanone (6) (8 g, 18.43 mmol), and the mixture was stirred at 0°C for 2 hours.

[0113] Saturated aqueous ammonium chloride solution was added to the reaction product, extracted with ethyl acetate, and the organic solvent was dried over Na2SO4 and concentrated under reduced pressure to obtain 1-(3,4-bis(benzyloxy)phenyl)-3-(2-((tert-butyldiphenylsilyl)oxy)-4,5-dimethoxyphenyl)propane-1,3-dione (7) (19 g, crude product) as a yellow oil, which was immediately transferred to the next step 6.

[0114] Step 6

[0115]

[0116] TBAF (tetrabutylammonium fluoride, 5.23 g, 20.00 mmol) was added to a solution of 1-(3,4-bis(benzyloxy)phenyl)-3-(2-((tert-butyldiphenylsilyl)oxy)-4,5-dimethoxyphenyl)propane-1,3-dione (7) (19 g, crude) in THF (50 mL) and stirred at 20°C for 3 h.

[0117] Saturated aqueous ammonium chloride solution was added to the reaction product, and the product was extracted with ethyl acetate three times. The organic solvent was dried over Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1) to obtain 1-(3,4-bis(benzyloxy)phenyl)-3-(2-hydroxy-4,5-dimethoxyphenyl)propane-1,3-dione (8) (5.3 g, yield 56.17%) as a yellow solid.

[0118] LC-MS (ESI) m / z 513.3[M+1] +

[0119] Step 7

[0120]

[0121] TFA (7.92 mL, 103.50 mmol) was added to a solution of 1-(3,4-bis(benzyloxy)phenyl)-3-(2-hydroxy-4,5-dimethoxyphenyl)propane-1,3-dione (8) (5.3 g, 10.35 mmol) in MeOH / THF=1 / 5 (30 mL) at 0°C, and the mixture was stirred at 60°C for 5 hours.

[0122] The solvent was removed from the obtained product, and recrystallized with n-hexane-DCM to give 2-(3,4-bis(benzyloxy)phenyl)-6,7-dimethoxy-4H-chroman-4-one (9) (3 g, yield: 58.68%) as a white solid.

[0123] LC-MS (ESI) m / z 495.2[M+1] +

[0124] Step 8

[0125]

[0126] TBAB (tetrabutylammonium bromide, 2.93 g, 9.11 mmol) was added to a solution of PhI(OAc)2 (2.93 g, 9.11 mmol) in DCM (30 mL), and the mixture was stirred at 20°C for 1 h under a hydrogen atmosphere.

[0127] To the resulting product was added a solution of 2-(3,4-bis(benzyloxy)phenyl)-6,7-dimethoxy-4H-chroman-4-one (9) (3 g, 6.07 mmol) in anhydrous DCM (20 ml), and the mixture was stirred at 20°C for 8 hours. Saturated aqueous ammonia was added to the resulting product, and the mixture was extracted three times with DCM. The organic solvent was removed with NaSO, and the mixture was concentrated under reduced pressure.

[0128] The obtained product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain 2-(3,4-bis(benzyloxy)phenyl)-3-bromo-6,7-dimethoxy-4H-chroman-4-one (10) (3.2 g, yield: 92.16%) as a yellow solid.

[0129] LC-MS (ESI) m / z 575.2[M+1] +

[0130] Step 9

[0131]

[0132] To a solution of 2-(3,4-bis(benzyloxy)phenyl)-3-bromo-6,7-dimethoxy-4H-chroman-4-one (10) (3.2 g, 5.59 mmol) in THF / EtOH = 1 / 1 (30 mL) were added TBAB (1.80 g, 5.59 mmol), K2CO3 (1.54 g, 11.18 mmol) and Pd(PPh3)4 (347 mg, 0.30 mmol), and the mixture was stirred under H2 at 80°C for 5 h.

[0133] The solvent was removed, and the resulting product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to give 2-(3,4-bis(benzyloxy)phenyl)-6,7-dimethoxy-3-(4-methoxyphenyl)-4H-chroman-4-one (11) (2.3 g, yield: 68.57%) as a yellow solid.

[0134] LC-MS (ESI) m / z 601.3[M+1] +

[0135] Step 10

[0136]

[0137] To a solution of 2-(3,4-bis(benzyloxy)phenyl)-6,7-dimethoxy-3-(4-methoxyphenyl)-4H-chroman-4-one (11) (1.6 g, 2.67 mmol) in THF / EtOH = 1 / 1 (30 mL) was added Pd / C (500 mg), and the mixture was stirred at room temperature under H2 overnight.

[0138] The obtained product was filtered through celite, the filtrate was concentrated, and the concentrate was recrystallized from diethyl ether to obtain the final product, a novel flavonoid derivative Ⅰ (Achem Ⅰ), which was a green solid 2-(3,4-dihydroxyphenyl)-6,7-dimethoxy-3-(4-methoxyphenyl)-4H-chroman-4-one (UC-224425) (750 mg, yield: 66.88%).

[0139] The results of 1H NMR (DMSO-d6) and 13C NMR (DMSO-d6) were as follows: Figure 1 and Figure 2 As shown, LC-MS data are Figure 3 As shown (LC-MS (ESI) m / z 421.1 [M+1] +).

[0140] Step 11

[0141]

[0142] To a DMF (5 mL) solution (0°C) containing 1-(2-hydroxy-4,5-dimethoxyphenyl)ethan-1-one (12) (1 g, 5.1 mmol) were added imidazole (624 mg, 9.18 mmol) and tert-butylchlorodiphenylsilane (13) (3.0 g, 11.2 mmol), and the mixture was stirred at 100°C for 10 hours.

[0143] Saturated aqueous ammonium chloride solution was added to the reaction product, and the product was extracted three times with DCM. The organic layer was dried over sodium sulfate (NaSO), concentrated under reduced pressure, and then recrystallized from n-hexane-EA to obtain 1-(2-((tert-butyldiphenylsilyl)oxy)-4,5-dimethoxyphenyl)ethan-1-one (14) (800 mg, yield: 87.74%) as a white solid. The resulting product was used as the reactant in the fifth step.

[0144] 2. Experimental study on the anti-pulmonary fibrosis activity of a novel flavonoid derivative I

[0145] 2.1 Cytotoxicity evaluation

[0146] 2.1.1 A549 cytotoxicity evaluation

[0147] To determine whether the novel flavonoid derivative I affects cell viability, the cell viability of lung cancer A549 cells was measured using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide].

[0148] A549 cells were suspended in culture medium and seeded into 96-well plates for 24 hours. After 24 hours, 200 μL of culture medium containing 0-20 μM BGC was added to each well and cultured for an additional 24 or 48 hours. Following incubation, 20 μL of MTT (0.5 mg / mL) diluted in phosphate-buffered saline (PBS) was added to each well and the cells were incubated in a CO2 incubator for 4 hours.

[0149] After 4 hours, the MTT solution was removed, and 200 μL of dimethyl sulfoxide (DMSO) was added to each well for 5 minutes to dissolve the formazan precipitate, and the absorbance was measured at 595 nm.

[0150] Figure 4 shows cell viability after 24, 48, and 72 hours as a percentage of the control (untreated) group. No cytotoxicity was observed after 24 hours of incubation at a treatment concentration of 5 μM, and approximately 80% cell viability was observed after 48 hours of incubation at a treatment concentration of 5 μM.

[0151] 2.1.2 Cytotoxicity Evaluation on HULEC-5a Cells

[0152] To determine whether the novel flavonoid derivative I affects cell viability, cell viability of HULEC-5a cells (human pulmonary microvascular endothelial cells) was assessed using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide].

[0153] HULEC-5a cells were suspended in culture medium and seeded into 96-well plates for 24 hours. After 24 hours, 200 μL of culture medium containing 0-10 μM novel flavonoid derivative I was added to each well of the plate and cultured for 24, 48, and 72 hours, respectively.

[0154] After incubation, all culture medium was discarded, and 200 μL of MTT (0.5 mg / mL) diluted in phosphate-buffered saline (PBS) was added to each well. The cells were incubated in a CO2 incubator for 4 hours. After 4 hours, the MTT solution was discarded, and 160 μL of dimethyl sulfoxide (DMSO) was added to each well. The cells were incubated for 30 minutes to dissolve the formazan precipitate, and the absorbance was measured at 570 nm.

[0155] Figure 5 shows the percentage of cell viability after 24, 48, and 72 hours compared to the control (untreated) group. No cytotoxicity was observed during the 24, 48, and 72 hours of incubation up to a treatment concentration of 5 μM.

[0156] 2.1.3 HLF cytotoxicity assessment

[0157] To determine whether the novel flavonoid derivative I affects cell viability, we used human lung fibroblasts (HLF) and assessed cell viability using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. HLF cells were suspended in culture medium, seeded in 96-well plates, and cultured for 24 hours.

[0158] After 24 hours, 200 μL of culture medium containing 0–10 μM novel flavonoid derivative I was added to each well and incubated for 24, 48, and 72 hours, respectively. After the incubation period, all culture medium was discarded, and 200 μL of MTT (0.5 mg / mL) diluted in phosphate-buffered saline (PBS) was added to each well. The cells were incubated in a CO2 incubator for 4 hours. After 4 hours, the MTT solution was discarded, and 160 μL of dimethyl sulfoxide (DMSO) was added to each well for 30 minutes to dissolve the formazan precipitate. The absorbance was measured at 570 nm.

[0159] Cell viability after 24, 48, and 72 hours is expressed as a percentage of the untreated control (Figure 5). After 24, 48, and 72 hours of incubation, cell viability exceeded 80% for all treatment concentrations.

[0160] The incubation time and sample concentration of subsequent experiments were determined according to the results of the above cytotoxicity test.

[0161] 2.2 Effects of TGF-β1 treatment on A549 cells

[0162] (1) Experimental methods

[0163] A549 cells derived from lung cancer were suspended in culture medium at a density of 3.5 x 105 cells, seeded in a 60 mm culture plate, and cultured for 48 hours.

[0164] After 48 hours of culture, TGF-β1 (10 ng / ml) alone or various or fixed concentrations of novel flavonoid derivative I and / or NIDB (nintedanib), a drug for the treatment of idiopathic pulmonary fibrosis and interstitial lung disease, were added to each plate as a positive control. After 48 hours of culture, cells were lysed using RIPA lysis buffer (RIPA lysis buffer [50 mM Tris]-Cl (pH 7.4), 1% NP40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF (phenylmethylsulfonyl fluoride)) containing MG-132 (10 μM, a proteasome inhibitor that protects HIF-1α subunits from proteasomal degradation).

[0165] Next, the supernatant was collected after centrifugation at 4°C (14,000 rpm for 10 minutes), and proteins were separated by SDS-PAGE (SDS-polyacrylamide gel electrophoresis). The separated proteins were transferred to a PVDF membrane (polyvinylidene fluoride membrane), reacted with 5% skim milk powder for 3 hours, and then reacted with the primary antibody at 4°C for 12 hours. After 12 hours, the samples were further reacted with the secondary antibody for each primary antibody, and protein bands were confirmed using an ECL kit according to the manufacturer's instructions.

[0166] (2) Experimental results

[0167] The results are as follows Figure 7 and Figure 8 shown.

[0168] like Figure 7 As shown, when A549 cells were treated with 10 ng / mL TGF-β1 and 5 μM novel flavonoid derivative I for 24 h, the protein levels of fibronectin, HIF-1α, NOX2, NOX4, vimentin, p-EGFR, EGFR, and p-AKT, which were increased after TGF-β1 treatment, tended to be reduced after novel flavonoid derivative I treatment.

[0169] In addition, reference Figure 8 When A549 cells were treated with 10 ng / mL TGF-β1 and 5 μM novel flavonoid derivative I for 48 hours, the results showed that the fibronectin, HIF-1α and pc-Src proteins increased by TGF-β1 treatment tended to be reduced by the novel flavonoid derivative I.

[0170] TGF-β1 is a typical profibrotic cytokine in type II alveolar epithelial cells in mice and humans. Numerous studies in this field have shown that in the presence of TGF-β1, epithelial cells first transform into mesenchymal cells, then into fibroblasts, and finally into myofibroblasts (TGF-β-Induced Endothelial-Mesenchymal Transition in Fibrotic Diseases, Int J Mol Sci., 2017, 18(10):2157-2179).

[0171] 2.3 Effects of TGF-β1 treatment on Hulec-5a cells

[0172] (1) Experimental methods

[0173] Hulec-5a cells derived from pulmonary vessels were suspended in culture medium at a cell density of 1.2×106, seeded in 100 mm culture plates, and cultured for 24 hours.

[0174] After 24 hours of culture, each plate was treated with TGF-β1 (10 ng / ml) alone or in combination with varying or fixed concentrations of novel flavonoid derivative I and / or NIDB (nintedanib), a drug for the treatment of idiopathic pulmonary fibrosis and interstitial lung disease. After 3, 24, and 48 hours of culture, cells were lysed using RIPA lysis buffer (RIPA lysis buffer [50 mM Tris]-Cl (pH 7.4), 1% NP40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF (phenylmethylsulfonyl fluoride)) containing MG-132 (10 μM, a proteasome inhibitor that protects HIF-1α subunits from proteasomal degradation).

[0175] Next, the supernatant was collected by centrifugation at 4°C (13,000 rpm, 7 min), and the proteins were separated by SDS-PAGE (SDS-polyacrylamide gel electrophoresis).

[0176] The separated proteins were transferred to a PVDF membrane and incubated in 5% nonfat dry milk for 2 hours. The membrane was then incubated with primary antibodies overnight at 4°C. Following this overnight incubation, the membrane was further incubated with secondary antibodies specific for each primary antibody. Protein bands were identified using an ECL kit according to the manufacturer's instructions.

[0177] (2) Experimental results

[0178] The results are as follows Figure 9 As shown. Figure 9It can be seen that when HULEC-5a cells were treated with 10 ng / mL TGF-β1 and / or 5 μM novel flavonoid derivative I for 3, 24 and 48 hours, the fibrosis-related factors (fibronectin, α-SMA, NOX1, NOX2, NOX4) increased by TGF-β1 treatment tended to decrease after treatment with novel flavonoid derivative I.

[0179] 2.4 Effects of TGF-β1 treatment on HLF cells

[0180] (1) Experimental methods

[0181] 5×106 human lung fibroblasts (HLF) were suspended in culture medium, seeded in 100 mm culture plates, and cultured for 24 hours.

[0182] After 24 hours of culture, each plate was treated with TGF-β1 (10 ng / ml) alone or in combination with novel flavonoid derivative I and / or nintedanib (nintedanib), a drug used to treat idiopathic pulmonary fibrosis and interstitial lung disease, at varying concentrations or a constant concentration. After 24 and 48 hours of culture, cells were lysed using RIPA lysis buffer (RIPA lysis buffer [50 mM Tris]-Cl (pH 7.4), 1% NP40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF (phenylmethylsulfonyl fluoride)) containing MG-132 (10 μM, a proteasome inhibitor that protects HIF-1α subunits from proteasomal degradation).

[0183] Next, the supernatant was collected after centrifugation at 4°C (13,000 rpm for 7 minutes), and proteins were separated by SDS-PAGE (SDS-polyacrylamide gel electrophoresis). The separated proteins were transferred to a PVDF membrane (polyvinylidene fluoride membrane), reacted with 5% nonfat dry milk for 2 hours, and then reacted with the primary antibody overnight at 4°C. After the overnight reaction, the proteins were further reacted with the secondary antibody for each primary antibody, and protein bands were confirmed using an ECL kit according to the manufacturer's instructions.

[0184] (2) Experimental results

[0185] The results are as follows Figure 10 As shown. Figure 10 It can be seen that when TGF-β1 (5 ng / mL) and the new flavonoid derivative I were treated at concentrations of 1 μM and 3 μM, respectively, for 24 hours, the fibrosis-related factors (fibronectin, COL4, α-SMA, HIF-1α) increased by TGF-β1 treatment tended to be reduced by the new flavonoid derivative I.

[0186] 2.5 Effects of EGF treatment on A549 cells

[0187] (1) Experimental methods

[0188] A549 lung cancer cells were cultured at varying cell numbers and adjusted for treatment duration. 4.5 × 105 cells (30 minutes to 12 hours), 4.0 × 105 cells (24 hours), and 3.5 × 105 cells (48 hours) were suspended in culture medium, seeded in 60 mm culture plates, and cultured for 24 hours.

[0189] After 24 hours, each plate was treated with EGF (2 ng / ml) alone or in combination with different or constant concentrations of the novel flavonoid derivative I. After incubation for a certain period of time (30 minutes to 48 hours), the cells were lysed with RIPA lysis buffer (RIPA lysis buffer [50 mM Tris-Cl (pH, 7.4), 1% NP40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF (phenylmethylsulfonyl fluoride)]) containing MG-132 (10 μM).

[0190] Next, the supernatant was collected after centrifugation at 4°C (14,000 rpm for 10 minutes), and proteins were separated by SDS-PAGE (SDS-polyacrylamide gel electrophoresis). The separated proteins were transferred to a PVDF membrane (polyvinylidene fluoride membrane), reacted with 5% skim milk powder for 3 hours, and then reacted with the primary antibody at 4°C for 12 hours. After 12 hours, the samples were further reacted with the secondary antibody for each primary antibody, and protein bands were confirmed using an ECL kit according to the manufacturer's instructions.

[0191] (2) Experimental results

[0192] The results are as follows Figure 11 and Figure 12 As shown. Epidermal growth factor (EGFR ligand) has been reported to induce pulmonary fibrosis (rapamycin can prevent transforming growth factor α-induced pulmonary fibrosis. Am J Respir Cell Mol Biol. 2009, 41(5):562-572). Therefore, we evaluated the inhibitory effect of the novel flavonoid derivative I on the expression of pulmonary fibrosis-related factors that are increased after EGF treatment in A549 cells.

[0193] When A549 cells were treated with 2 ng / mL EGF and 1, 3, and 5 / 2 μM of the novel flavonoid derivative I for 1 hour, the results showed that the fibrosis-related factors (p-EGFR, EGFR, p-AKT, HIF-1α, and NOX2) increased by EGF treatment tended to be reduced by the novel flavonoid derivative I.

[0194] 2.6 Effects of bleomycin treatment on A549 cells

[0195] (1) Experimental methods

[0196] A549 lung cancer cell culture: The cell number varied depending on the treatment time. 4.5×105 cells (30 minutes to 12 hours), 4.0×105 cells (24 hours), and 3.5×105 cells (48 hours) were suspended in culture medium, seeded into 60 mm culture plates, and cultured for 24 hours.

[0197] After 24 h, each plate was treated with bleomycin (BLM) alone or in combination with a certain concentration of novel flavonoid derivative I. After incubation for a certain period of time, the cells were lysed with RIPA lysis buffer (RIPA lysis buffer [50 mM Tris-Cl (pH 7.4), 1% NP40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF (phenylmethylsulfonyl fluoride)]) containing MG-132 (10 μM).

[0198] Next, the supernatant was collected after centrifugation at 4°C (14,000 rpm for 10 minutes), and proteins were separated by SDS-PAGE (SDS-polyacrylamide gel electrophoresis). The separated proteins were transferred to a PVDF membrane (polyvinylidene fluoride membrane), reacted with 5% skim milk powder for 3 hours, and then reacted with the primary antibody at 4°C for 12 hours. After 12 hours, the samples were further reacted with the secondary antibody for each primary antibody, and protein bands were confirmed using an ECL kit according to the manufacturer's instructions.

[0199] (2) Experimental results

[0200] Bleomycin (BLM) is a substance used to establish animal models of pulmonary fibrosis. It is known to induce abnormal extracellular matrix deposition and fibroblast aggregation in lesioned tissues. Currently, bleomycin is being used to elucidate the molecular mechanisms of pulmonary fibrosis and screen for treatments (Role of cellular senescence and EMT crosstalk in the pathogenesis of bleomycin-induced pulmonary fibrosis - an in vitro analysis, Cell Biol Int. 2020, 44(2):477-487).

[0201] Depend on Figure 13 It can be seen that when A549 cells were treated with BLM and / or novel flavonoid derivative I for 3, 24 and 48 hours, the pulmonary fibrosis-related factors (fibronectin, α-SMA, HIF-1α, NOX1, NOX2, NOX4) increased by BLM treatment showed a downward trend due to novel flavonoid derivative I treatment.

[0202] 2.7 Effects of bleomycin treatment on Hulec-5a cells

[0203] (1) Experimental methods

[0204] Hulec-5a cells derived from pulmonary vessels were suspended in culture medium at a cell density of 1.2 × 10⁶ and seeded onto 100 mm culture plates for 24 hours. After 24 hours, each plate was treated with bleomycin (BLM) alone or in combination with various or fixed concentrations of the novel flavonoid derivative I. After incubation for the specified time, the cells were lysed in RIPA lysis buffer (50 mM Tris-Cl (pH 7.4), 1% NP40, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF)) containing MG-132 (10 μM).

[0205] Next, the supernatant was collected after centrifugation at 4°C (14,000 rpm for 10 minutes), and proteins were separated by SDS-PAGE (SDS-polyacrylamide gel electrophoresis). The separated proteins were transferred to a PVDF membrane (polyvinylidene fluoride membrane), reacted with 5% skim milk powder for 3 hours, and then reacted with the primary antibody at 4°C for 12 hours. After 12 hours, the samples were further reacted with the secondary antibody for each primary antibody, and protein bands were confirmed using an ECL kit according to the manufacturer's instructions.

[0206] (2) Experimental results

[0207] The results are as follows Figure 14 Similar to the results of A549 cells treated with bleomycin, when Hulec-5a cells, derived from pulmonary vascular tissue, were treated with bleomycin, the levels of pulmonary fibrosis-related factors (fibronectin, α-SMA, HIF-1α, NOX2, and NOX4) showed a decreasing trend with treatment with the novel flavonoid derivative I.

[0208] <Example 2> Preparation of novel flavonoid derivative II and experimental study of its anti-pulmonary fibrosis activity

[0209] 1. Preparation of Novel Flavonoid Derivative II

[0210] Step 1

[0211]

[0212] Under nitrogen protection at room temperature, 2-(4-methoxyphenyl)acetic acid (CAS No: 104-01-8) (25.6 g, 154 mmol) was added to a solution of 3,4-dimethoxyphenol (1) (25 g, 162 mmol) in boron trifluoride and diethyl ether (40 mL). The reaction mixture was heated to 120°C and stirred for 15 minutes.

[0213] The mixture was cooled to room temperature, poured into 100 mL of water, and extracted with ethyl acetate (EA). The organic phase was collected, washed with brine, and dried over sodium sulfate. After filtration, the solution was concentrated in vacuo to obtain 50 g of crude oil, which was purified on a silica gel column (PE / EA = 10%–50%) to obtain 26 g of 1-(2-hydroxy-4,5-dimethoxyphenyl)-2-(4-methoxyphenyl) ketene (2) as a white solid (yield: 55%).

[0214] 1 H NMR (400 MHz, CDCl3) δ: 12.62 (s, 1H), 7.02 - 7.16 (m, 3H), 6.90 (d, J = 8.4 Hz, 2H), 6.45 (s, 1H), 4.15 (s, 1H), 3.90 (s, 3H), 3.82 (s, 3H),3.79 (s, 3H).

[0215] Step 2

[0216]

[0217] To a solution of 1-(2-hydroxy-4,5-dimethoxyphenyl)-2-(4-methoxyphenyl) ketene (2) (15.5 g, 51.3 mmol) and 4-(benzyloxy)-3-methoxybenzoic acid (15.9 g, 61.5 mmol) (CAS No.: 1486-53-9) in DCM (dichloromethane) (1000 mL) were added DCC (N,N'-dicyclohexylcarbodiimide) (21.2 g, 102.6 mmol) and DMAP (dimethylaminopyridine) (1.3 g, 10.6 mmol), and the mixture was stirred at room temperature for 3 hours.

[0218] The reaction mixture was then concentrated in vacuo to obtain a white solid. Purification by silica gel column gave 11 g of the target compound, 4,5-dimethoxy-2-(2-(4-methoxyphenyl)acetyl)phenyl 4-(benzyloxy)-3-methoxybenzoate (3), as a white solid (yield 38%).

[0219] 1 H NMR (400 MHz, DMSO-d6) δ: 7.50 - 7.24 (m, 7H), 7.15 - 7.05 (m, 4H), 6.97 - 6.92 (m, 2H), 6.90 (s, 1H), 5.12 (s, 2H), 3.96 (s, 3H), 3.90 (s, 3H), 3.78 (s, 3H), 3.51 (s, 3H).

[0220] Step 3

[0221]

[0222] A mixture of 4,5-dimethoxy-2-(2-(4-methoxyphenyl)acetyl)phenyl 4-(benzyloxy)-3-methoxybenzoate (3) (3.2 g, 5.9 mmol) and glycerol (30 mL) was degassed three times with nitrogen and heated at 200°C for 3 h.

[0223] The reaction product was cooled to room temperature, added to 100 mL of water, and extracted with ethyl acetate (EA). The organic phases were combined, washed with brine, and dried over sodium sulfate. The crude product was concentrated and purified on a silica gel column to obtain 800 mg of 2-(4-benzyloxy)-3-methoxyphenyl)-6,7-dimethoxy-3-(4-methoxyphenyl)-4H-chroman-4-one (4) as a white solid (yield 22%).

[0224] 1 H NMR (400 MHz, CDCl3) δ: 7.44 - 7.34 (m, 7H), 7.15 - 7.05 (m, 4H), 6.97 - 6.92 (m, 2H), 6.85 (s, 1H), 5.12 (s, 2H), 3.96 (s, 3H), 3.90 (s, 3H), 3.78 (s, 3H), 3.50 (s, 3H).

[0225] Step 4

[0226]

[0227] To a solution of 2-(4-(benzyloxy)-3-methoxyphenyl)-6,7-dimethoxy-3-(4-methoxyphenyl)-4H-chroman-4-one (800 mg, 1.3 mmol) in ethyl acetate (50 mL) was added Pd / C (palladium on carbon) (80 mg), and the mixture was stirred under hydrogen pressure at 60 psi for 2 hours.

[0228] The resulting mixture was filtered, concentrated, and then triturated with methanol to give 525 mg of 2-(3-methoxy-4-hydroxyphenyl)-6,7-dimethoxy-3-(4-methoxyphenyl)-4H-chroman-4-one

[0229] 2-(4-Hydroxy-3-methoxyphenyl)-6,7-dimethoxy-3-(4-methoxyphenyl)-4H-chroman-4-one (UC-230171), a new flavonoid derivative II (Achem II), is a white solid.

[0230] The results of nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry analysis of the final substance, the novel flavonoid derivative II, obtained in this manner, are as follows.

[0231] LC-MS (ESI): m / z 435.0 [M+H] +

[0232] 1 H NMR(DMSO-d6) 및 13 C NMR(DMSO-d6) 결과는각각도15 및도16에나타내었다.

[0233] The results of 1H NMR (DMSO-d6) and 13C NMR (DMSO-d6) were as follows: Figure 15 and 16 shown.

[0234] 2. Experimental study on the anti-pulmonary fibrosis activity of a novel flavonoid derivative II

[0235] 2.1 Cytotoxicity evaluation

[0236] The cytotoxicity of the novel flavonoid derivative II (Achem II) to A549 cells, HULEC-5a cells and HLF cells was detected by MTT assay using the same method as in Example 1. Figures 17 to 19 The results are shown as cell viability (%) compared with the untreated control group after 24, 48, and 72 hours.

[0237] As shown in Figure 17, A549 cell viability exceeded 80% after 24 and 48 hours of culture at all treatment concentrations. However, at concentrations of 3 μM or higher, cell viability dropped below 80% after 72 hours of culture.

[0238] As shown in Figures 18 and 19 , the cell viability of HULEC-5a and HLF cells exceeded 80% at all treatment concentrations up to 10 μM after 24, 48, and 72 h of culture.

[0239] The incubation time and sample treatment concentrations in subsequent experiments were determined based on the results of the above cytotoxicity tests.

[0240] 2. Effects of TGF-β1 on A549 cells

[0241] The same method as in Example 1 was used to evaluate the effect of the novel flavonoid derivative II on TGF-β1-treated A549 cells. Figure 20 and Figure 21 shown.

[0242] like Figure 20 and Figure 21 As shown, when A549 cells were treated with 10 ng / mL TGF-β1 and novel flavonoid derivative II for 24 hours, TGF-β1 treatment led to an increase in the levels of Col3, Col4, fibronectin and NOX4 proteins, while novel flavonoid derivative II tended to reduce their levels.

[0243] 3. Effects of TGF-β1 on Hulec-5a cells

[0244] The same method as in Example 1 was used to evaluate the effect of the novel flavonoid derivative II on Hulec-5a cells treated with TGF-β1. Figure 22 shown.

[0245] refer to Figure 22 When treated with the novel flavonoid derivative II, the fibronectin, COL4, α-SMA, and HIF-1α that were increased by TGF-β1 treatment were all reduced.

[0246] 4. Effects of TGF-β1 treatment on HLF cells

[0247] The same method as in Example 1 was used to evaluate the effect of the novel flavonoid derivative II on HLF cells treated with TGF-β1. Figure 23 shown.

[0248] refer to Figure 23When treated with the novel flavonoid derivative II, the fibronectin, α-SMA, HIF-1α and COL1 that were increased by TGF-β1 treatment were all reduced.

[0249] 5. Effects of EGF-treated A549 cells

[0250] The same method as in Example 1 was used to evaluate the effect of the novel flavonoid derivative II on EGF-treated A549 cells. Figure 24 shown.

[0251] When A549 cells were treated with 2 ng / mL EGF and novel flavonoid derivative II for 1 hour, the results showed that the fibrosis-related factor NOX2 increased after EGF treatment, while the novel flavonoid derivative II tended to reduce NOX2.

[0252] <Example 3> Preparation, Identification and Activity Testing of Novel Flavonoid Derivative III

[0253] 1. Preparation of Novel Flavonoid Derivative III

[0254] Step 1

[0255]

[0256] 2-(4-Methoxyphenyl)acetic acid (CAS No. 104-01-8) (5.4 g, 32.47 mmol) was added to a solution of 3,4-dimethoxyphenol (1) (5.0 g, 32.47 mmol) in BF3.Et2O (10 mL) at room temperature under nitrogen.

[0257] The reaction mixture was heated to 120° C., stirred for 15 minutes, cooled to room temperature, added to 30 mL of water, and extracted three times with 30 mL of ethyl acetate (EA).

[0258] The organic phases were combined, washed with brine, and dried over sodium sulfate. After filtration, the solution was concentrated in vacuo to obtain 13.2 g of crude oil, which was purified by silica gel column (PE / EA = 10% to 50%) to obtain 4.2 g of 1-(2-hydroxy-4,5-dimethoxyphenyl)-2-(4-methoxyphenyl) ketene (2) as a white solid (yield: 42.9%).

[0259] LC-MS (M+H + ) m / z 303.3.

[0260] Step 2

[0261]

[0262] 1-(2-Hydroxy-4,5-dimethoxyphenyl)-2-(4-methoxyphenyl) ketene (2) (4.0 g, 13.24 mmol) and 3-(benzyloxy)-4-methoxybenzoic acid (4.1 g, 15.84 mmol) (CAS No.: 58452-00-9) were dissolved in dichloromethane (100 mL). DCC (N,N'-dicyclohexylcarbodiimide) (5.5 g, 26.4 mmol) and DMAP (dimethylaminopyridine) (0.32 g, 2.64 mmol) were added to the mixture. The mixture was stirred at room temperature for 3 hours and concentrated under reduced pressure to obtain a white solid.

[0263] The substance was purified using a silica gel column (PE / EA = 10% to 30%) to obtain 4.8 g of the target compound 4,5-dimethoxy-2-(2-(4-methoxyphenyl)acetyl)phenyl 3-(benzyloxy)-4-methoxybenzoate (3) as a white solid (yield: 66.8%).

[0264] LC-MS (M+H + ) m / z 543.4

[0265] Step 3

[0266]

[0267] A mixture of 4,5-dimethoxy-2-(2-(4-methoxyphenyl)acetyl)phenyl 3-(benzyloxy)-4-methoxybenzoate (3) (4.8 g, 8.86 mmol) and glycerol (50 mL) was degassed three times with nitrogen and heated at 200 °C for 3 h.

[0268] The reaction product was cooled to room temperature, added to 100 mL of water, and extracted three times with 50 mL of ethyl acetate (EA). The organic phases were combined, washed with brine, and dried over sodium sulfate. The crude product was concentrated and purified on a silica gel column (PE / EA = 10% to 50%) to obtain 1.3 g of 2-(3-benzyloxy)-4-methoxyphenyl)-6,7-dimethoxy-3-(4-methoxyphenyl)-4H-chroman-4-one (4) as a white solid (yield 28%).

[0269] LC-MS (M+H + ) m / z 525.4

[0270] Step 4

[0271]

[0272] To a solution of 2-(3-benzyloxy)-4-methoxyphenyl)-6,7-dimethoxy-3-(4-methoxyphenyl)-4H-chroman-4-one (4) (1.3 g, 2.48 mmol) in ethyl acetate (50 mL) was added Pd / C (palladium on carbon) (150 mg), and the mixture was stirred under a hydrogen atmosphere at 60 psi for 2 hours. The mixture was then filtered and the filtrate was concentrated.

[0273] Chromatographic separation was performed using the following silica gel column (PE / EA = 10% to 70%) to obtain 510 mg of 2-(3-hydroxy-4-methoxyphenyl)-6,7-dimethoxy-3-(4-methoxyphenyl)-4H-chroman-4-one (UC-230172), a new flavonoid derivative III (Achem III), as a white solid.

[0274] LC-MS (M+H + ) m / z 435.1

[0275] The 1H NMR (DMSO-d6) and 13C NMR (DMSO-d6) results of the new flavonoid derivatives III are as follows: Figure 25 and 26 shown.

[0276] 2. Experimental study on the anti-pulmonary fibrosis activity of a novel flavonoid derivative III

[0277] 1. Cytotoxicity Assessment

[0278] The cytotoxicity of the novel flavonoid derivative III (Achem III) to A549 cells, HULEC-5a cells and HLF cells was detected by MTT assay using the same method as in Example 1. Figures 27 to 29 The results are shown as cell viability (%) compared with the untreated control group after 24, 48, and 72 hours.

[0279] As shown in Figure 27, A549 cells maintained cell viability exceeding 80% after 24, 48, and 72 hours of culture at concentrations up to 20 μM. As shown in Figure 28, HULEC-5a cells maintained cell viability exceeding 80% after 24, 48, and 72 hours of culture at concentrations up to 15 μM, but at 20 μM, cell viability fell below 80% after 24 hours of culture.

[0280] like Figure 29As shown, HLF cells maintained cell viability exceeding 80% after 24, 48, and 72 hours of culture at all treatment conditions up to 10 μM. At concentrations of 15 and 20 μM, cell viability fell below 80% after 24 hours of culture. The incubation time and sample treatment concentrations for subsequent experiments will be determined based on the results of the cytotoxicity assay.

[0281] 2. Effects of TGF-β1 on A549 cells

[0282] The same method as in Example 1 was used to evaluate the effect of the novel flavonoid derivative III on TGF-β1 treated A549 cells. Figure 30 shown.

[0283] like Figure 30 As shown, when A549 cells were treated with 5 ng / mL of TGF-β1 and the novel flavonoid derivative III for 24 hours, the increasing trend of fibronectin, COL4 and COL3 levels induced by TGF-β1 was reduced by the novel flavonoid derivative III.

[0284] 3. Effects of TGF-β1 Treatment on Hulec-5a Cells

[0285] The same method as in Example 1 was used to evaluate the effect of the novel flavonoid derivative III on Hulec-5a cells treated with TGF-β1. Figure 31 shown.

[0286] like Figure 31 As shown, when Hulec-5a cells were treated with 5 ng / mL of TGF-β1 and the novel flavonoid derivative III for 24 hours, fibronectin, COL4, and COL3, which were elevated after TGF-β1 treatment, were decreased.

[0287] 4. Effects of TGF-β1 on HLF cells

[0288] The same method as in Example 1 was used to evaluate the effect of the novel flavonoid derivative III on TGF-β1 treated HLF cells. Figure 32 shown.

[0289] like Figure 32 As shown, when HLF cells were treated with 5 ng / mL TGF-β1 and the novel flavonoid derivative III for 24 h, the levels of fibronectin, COL4, COL1, α-SMA, HIF-1α, and NOX2, which were elevated after TGF-β1 treatment, were decreased.

[0290] [Industrial Applicability]

[0291] The composition of the present invention can be commercialized as a food such as a health functional food or a medicine such as a drug.

Claims

1. A compound, characterized in that It is a flavonoid derivative of the following <Chemical Formula 4>, a hydrate thereof, or a solvate thereof: <Chemical Formula 4> ； wherein R1, R2, R3 and R4 are each independently hydrogen (H), hydroxyl (-OH), methyl (-CH3) or methoxy (-OCH3).

2. The compound according to claim 1, characterized in that R1 is hydrogen or hydroxy, R2 is hydroxy or methoxy, R3 is hydrogen, methoxy or hydroxy, and R4 is methoxy.

3. The compound according to claim 1, characterized in that The flavonoid derivative is a compound represented by the following <Chemical Formula 1>: <Chemical Formula 1> 。 4. The compound according to claim 1, characterized in that The flavonoid derivative is a compound represented by the following <Chemical Formula 2>: <Chemical Formula 2> 。 5. The compound according to claim 1, characterized in that The flavonoid derivative is a compound shown in the following <Chemical Formula 3>: <Chemical Formula 3> 。 6. A composition for improving pulmonary fibrosis, characterized in that: It contains the following <Chemical Formula 4> flavonoid derivatives, hydrates or solvates thereof as active ingredients: <Chemical Formula 4> 。 7. The composition according to claim 6, characterized in that wherein R1 is hydrogen or hydroxy, R2 is hydroxy or methoxy, R3 is hydrogen, methoxy or hydroxy, and R4 is methoxy.

8. The composition according to claim 6, characterized in that The flavonoid derivative is a composition having the following <Chemical Formula 1>: <Chemical Formula 1> 。 9. The composition according to claim 6, characterized in that The flavonoid derivative is a compound represented by the following <Chemical Formula 2>: <Chemical Formula 2> 。 10. The composition according to claim 6, characterized in that The flavonoid derivative is a composition having the following <Chemical Formula 3>: <Chemical Formula 3> 。 11. The composition according to claim 6, characterized in that The pulmonary fibrosis is idiopathic pulmonary fibrosis.

12. The composition according to claim 6, characterized in that The composition is a pharmaceutical composition.

13. The composition according to claim 6, characterized in that The composition is a food composition.

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