Pharmaceutical composition for preventing or treating renal fibrosis

Abstract

The present invention relates to a pharmaceutical composition comprising a compound of chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient for preventing or treating renal fibrosis, and a method for preventing or treating renal fibrosis, using same.

Description

Pharmaceutical composition for preventing or treating renal fibrosis

[0001] The present invention relates to a pharmaceutical composition for preventing or treating renal fibrosis, comprising a compound of formula 1 or a pharmaceutically acceptable salt thereof, and a method for preventing or treating renal fibrosis.

[0002]

[0003] Renal fibrosis refers to the fibrosis of kidney tissue due to excessive accumulation of extracellular matrix (ECM) accompanied by an intracellular inflammatory response caused by kidney damage. Renal fibrosis gradually reduces kidney function and increases the likelihood of developing chronic kidney disease (CKD) or renal failure.

[0004] Renal ischemia-reperfusion injury (IRI) can cause acute kidney injury (AKI) and may also contribute to the progression of AKI to chronic kidney disease (Non-patent Document 1). Acute kidney injury, such as renal ischemia-reperfusion injury, can induce ferroptosis (iron-dependent cell death) and inflammatory stimulation by increasing intracellular reactive oxygen species (ROS) and lipid peroxidation along with an inflammatory response (Non-patent Document 2). Reactive oxygen species (ROS) affect the epithelial-mesenchymal transition (EMT) induced by TGF-β and can modulate TGF-β-induced fibrosis through various pathways (Non-patent Document 3). Furthermore, 4-hydroxynonenal (4-HNE) can be generated by glutathione (GSH) deficiency and lipid peroxidation, and 4-HNE can induce iron homeostasis imbalance, which can induce ferroptosis (Non-patent literature 4, 5). Lipid ROS are mainly controlled by GPX4, which can reduce peroxidation of membrane phospholipids to lipid alcohols. Since GSH reacts with ROS and is oxidized by GPX4 while simultaneously reducing ROS, the GPX4 / GSH system can protect against ferroptosis (Non-patent literature 6).

[0005] The relationship between ferroptosis and renal fibrosis has not yet been elucidated, and the present inventors studied how inhibition of reactive oxygen species and lipid peroxidation and inhibition of ferroptosis affect renal fibrosis, and a method for inhibiting renal fibrosis through renal fibrosis initiating factors, and confirmed that the indole derivative compound according to the present invention is effective in preventing, improving, or treating renal fibrosis, thereby completing the present invention.

[0006] [Prior Art Literature]

[0007] [Patent Document]

[0008] (Patent Document 1) International Patent Publication No. WO2009 / 025478

[0009] [Spanish]

[0010] (Rev. 1) Guan, D. Nakano, Y. Zhang, L. Li, Y. Tian, ​​and A. Nishiyama, A mouse model of renal fibrosis. Sci Rep 9 (2019).

[0011] (Figure 2) Chen Y, ZM Fang, Yi X, Wei, and Jiang DS, The interaction between ferroptosis and inflammatory signaling pathways. Cell Death 14 (2023)

[0012] (Figure 3) J. Zhou, Y. Tan, R. Wang, and X. Li, Role of Ferroptosis in Fibrotic Diseases. J Inflamma Res 15(2022)3689–3708.

[0013] (Fig. 4) HF Yan, T. Zou, QZ Tuo, S. Xu, H. Li, AA Belaidi, and P. Lei, Ferroptosis: mechanisms and links with diseases. Signal Transduct Target Ther 6 (2021)

[0014] (Non-patent Document 5) X. Zheng, X. Jin, F. Ye, X. Liu, B. Yu, Z. Li, T. Zhao, W. Chen, Exp Hematol Oncol 12 (2023) 65.

[0015] (Non-patent Document 6) M. Oh, SY Jang, JY Lee, JW Kim, Y. Jung, J. Kim, J. Seo, TS Han, E. Jang, HY Son, D. Kim, MW Kim, JS Park, KH Song, KJ Oh, WK Kim, KH Bae, YM Huh, SH Kim, D. Kim, BS Han, SC Lee, GS Hwang, and EW Lee, The lipoprotein-associated phospholipase A2 inhibitor Darapladib sensitises cancer cells to ferroptosis by remodeling lipid metabolism. Nat Commun 14 (2023) 5728.

[0016]

[0017] The purpose of the present invention is to provide a use of a compound of chemical formula 1 or a pharmaceutically acceptable salt thereof for preventing, improving or treating renal fibrosis.

[0018]

[0019] The present invention provides a pharmaceutical composition for preventing or treating renal fibrosis, comprising a compound of chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0020] [Chemical Formula 1]

[0021]

[0022] In the above formula,

[0023] n is an integer from 1 to 3,

[0024] m is 0 or 1,

[0025] A represents phenyl,

[0026] R 1 is hydrogen, or C1-C6-alkyl,

[0027] R 2 represents hydrogen, halogen or C1-C6-alkoxy, or hydroxy-C 1- C6-alkyl, -(CH2) p CO2R 7 , -NHR 8 , -N(H)S(O)2R 7 or -NHC(O)R 7 , where p is an integer from 0 to 3, and R 7 represents hydrogen or C1-C3-alkyl, and R 8 Silver C 1- C3-alkylpiperidinyl, or C 1- It represents C3-alkylsulfonyl,

[0028] R 3 represents hydrogen, halogen, C1-C6-alkyl or phenyl, or the heterocycle contains 1 or 2 heteroatoms selected from S, N and O atoms and is a 5 to 6-membered ring -(CH2) p - represents a heterocycle, where p is an integer from 0 to 3, but when m is 0, R 3 is phenyl,

[0029] R 4 is halogen, C1-C6-alkyl, hydroxy-C 1- C6-alkyl, -O-phenyl, -(CH2) p CO2R 7 , a heterocycle containing 1 or 2 heteroatoms selected from S, N and O atoms and a 5 to 6 membered ring -(CH2) p-heterocycle, or proline-N-carbonyl, where p is an integer from 0 to 3, and R 7 is as defined above, and the group heterocycle may be substituted with one or more oxo (=O) substituents,

[0030] R 5 is hydrogen, or C1-C6-alkyl,

[0031] R 6 represents C1-C6-alkyl, C3-C6-cycloalkyl, heterocycle or heterocyclyl-C1-C6-alkyl, wherein the heterocycle is a 3 to 8-membered ring containing 1 to 3 heteroatoms selected from S, N and O atoms, and R 6 is C1-C6-alkylamine, hydroxy-C 1- It may be substituted with C6-alkyl or C1-C6-alkylsulfonyl.

[0032] The present invention also provides a method for preventing or treating renal fibrosis, comprising administering a compound of formula 1 or a pharmaceutically acceptable salt thereof to a subject in need thereof in a pharmaceutically acceptable amount.

[0033]

[0034] According to the composition or method of the present invention, the compound of formula 1 can regulate or improve the expression of factors associated with renal fibrosis in in vitro and in vivo models, suppress the occurrence of renal fibrosis and renal inflammation, and, in particular, suppress renal fibrosis occurring in kidneys damaged by renal ischemia-reperfusion. Therefore, the compound of formula 1 of the present invention can be usefully used for the prevention or treatment of diseases associated with renal fibrosis.

[0035]

[0036] Figure 1a shows a graph of the results of RT-PCR analysis for ROS-related genes in HK-2 cells according to Experimental Example 1.

[0037] Figure 1b shows the results of Western blot analysis on the expression of ferroptosis-related proteins in HK-2 cells according to Experimental Example 1.

[0038] Figure 1c shows a graph of the results of RT-PCR analysis for inflammation-related genes in HK-2 cells according to Experimental Example 1.

[0039] Figure 1d shows the results of Western blot analysis on the expression of renal fibrosis-related proteins in HK-2 cells according to Experimental Example 1.

[0040] Figure 2a shows a FACS analysis graph of the cellular lipid ROS and mitochondrial lipid ROS inhibition efficacy according to Experimental Example 2.

[0041] Figure 2b shows a GSH level graph according to Experimental Example 2.

[0042] Figure 2c shows the results of RT-PCR, Western blot, and GPX4 assay kit for GPX4 expression according to Experimental Example 2.

[0043] Figure 2d shows the Western blot results for GPX activity and Nrf2 and GPX4 according to Experimental Example 2.

[0044] Figure 3a shows a graph of blood urea nitrogen (Blood Urea Nitrogen) and serum creatinine (s-Cr) according to Experimental Example 3.

[0045] Figure 3b shows a microscopic image of hematoxylin and eosin (H&E) staining according to Experimental Example 3 (200 times the original magnification. Scale bar = 100 μm) and a tissue damage score graph.

[0046] Figure 4a shows a microscope image of dihydroethidium (DHE) staining according to Experimental Example 4 (200 times the original magnification. Scale bar = 50 μm).

[0047] Figure 4b shows a microscope image of TUNEL staining according to Experimental Example 4 (400 times the original magnification. Scale bar = 200 μm).

[0048] Figure 5a shows the results of Western blot analysis of each protein using the IR 3d experimental group according to Experimental Example 5.

[0049] Figure 5b shows the results of Western blot analysis of each protein using the IR 7d experimental group according to Experimental Example 5.

[0050] Figure 5c shows the results of immunohistochemical staining of 4-HNE in kidney sections of IR 3d and IR 7d mice according to Experimental Example 5.

[0051] Figure 6a shows the results of Western blot analysis of the proteins NF-κB, HMGB1, HO-1, and Nrf2 using the IR 3d experimental group according to Experimental Example 6.

[0052] Figure 6b shows the results of Western blot analysis of the proteins NF-κB, HMGB1, HO-1, and Nrf2 using the IR 7d experimental group according to Experimental Example 6.

[0053] Figure 6c shows the results of immunohistochemical staining for F4 / 80, a macrophage marker, according to Experimental Example 6.

[0054] Figure 7a shows the results of Western blot analysis for E-cadherin, collagen IV, α-SMA, Snail, and Twist proteins using the IR 3d experimental group according to Experimental Example 7.

[0055] Figure 7b shows the results of Western blot analysis for E-cadherin, collagen IV, α-SMA, Snail, and Twist proteins using the IR 7d experimental group according to Experimental Example 7.

[0056] Figure 8a shows the immunohistochemical staining results and MT (Masson's trichrome) staining results of E-cadherin, collagen IV α-SMA, and TGF-β using the IR 3d experimental group according to Experimental Example 8.

[0057] Figure 8b shows the immunohistochemical staining results and MT staining results of E-cadherin, collagen IV α-SMA, and TGF-β using the IR 7d experimental group according to Experimental Example 8.

[0058]

[0059] The present invention is described in detail below.

[0060] Meanwhile, each description and embodiment disclosed herein can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. Furthermore, the scope of the present invention is not limited by the specific descriptions described below.

[0061] When a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise stated.

[0062] The present invention provides a pharmaceutical composition for preventing or treating renal fibrosis, comprising a compound of chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0063] [Chemical Formula 1]

[0064]

[0065] In the above formula,

[0066] n is an integer from 1 to 3,

[0067] m is 0 or 1,

[0068] A represents phenyl,

[0069] R 1 is hydrogen, or C1-C6-alkyl,

[0070] R 2 represents hydrogen, halogen or C1-C6-alkoxy, or hydroxy-C 1- C6-alkyl, -(CH2) p CO2R 7 , -NHR 8 , -N(H)S(O)2R 7 or -NHC(O)R 7 , where p is an integer from 0 to 3, and R 7 represents hydrogen or C1-C3-alkyl, and R 8 Silver C 1- C3-alkylpiperidinyl, or C 1- It represents C3-alkylsulfonyl,

[0071] R 3 represents hydrogen, halogen, C1-C6-alkyl or phenyl, or the heterocycle contains 1 or 2 heteroatoms selected from S, N and O atoms and is a 5 to 6-membered ring -(CH2) p - represents a heterocycle, where p is an integer from 0 to 3, but when m is 0, R 3 is phenyl,

[0072] R 4 is halogen, C1-C6-alkyl, hydroxy-C 1- C6-alkyl, -O-phenyl, -(CH2) p CO2R 7 , a heterocycle containing 1 or 2 heteroatoms selected from S, N and O atoms and a 5 to 6 membered ring -(CH2) p -heterocycle, or proline-N-carbonyl, where p is an integer from 0 to 3, and R 7 is as defined above, and the group heterocycle may be substituted with one or more oxo (=O) substituents,

[0073] R 5 is hydrogen, or C1-C6-alkyl,

[0074] R 6represents C1-C6-alkyl, C3-C6-cycloalkyl, heterocycle or heterocyclyl-C1-C6-alkyl, wherein the heterocycle is a 3 to 8-membered ring containing 1 to 3 heteroatoms selected from S, N and O atoms, and R 6 is C1-C6-alkylamine, hydroxy-C 1- It may be substituted with C6-alkyl or C1-C6-alkylsulfonyl.

[0075] The compound of chemical formula 1 is a compound disclosed in International Patent Publication No. WO2009-025478, and is a substance known to exhibit a preventive or therapeutic and improvement effect on cell necrosis and related diseases.

[0076] The present invention has confirmed the effect of preventing, improving, or treating renal fibrosis by treating a cell model and an animal model of renal ischemia-reperfusion injury with the compound of the above chemical formula 1, inhibiting lipid peroxidation, increase in reactive oxygen species, and ferroptosis in the kidney caused by renal ischemia-reperfusion injury, reducing renal blood urea nitrogen and serum creatinine, and inhibiting renal inflammation and renal fibrosis. The present invention has identified a novel use of the compound of the above chemical formula 1.

[0077] The compound of the above chemical formula 1 of the present invention can be used in the form of a pharmaceutically acceptable salt thereof. In particular, the pharmaceutically acceptable salt may be an acid addition salt formed by a free acid. Here, the acid addition salt can be obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc.; non-toxic organic acids such as aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids, etc.; organic acids such as trifluoroacetic acid, acetate, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, etc. Such pharmaceutically acceptable salts may include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphate chlorides, bromides, iodides, fluorides, acetates, propionates, and the like.

[0078] The composition of the present invention may include the compound of formula 1, a pharmaceutically acceptable salt thereof, as well as all salts, isomers, hydrates and / or solvates that can be prepared by conventional methods.

[0079] As used herein, "isomer" may refer to a compound of the present invention or a salt thereof that has the same chemical formula or molecular formula but is structurally or sterically different. Such isomers include structural isomers such as tautomers, isomers such as R or S isomers having an asymmetric carbon center, geometric isomers (trans, cis), and optical isomers (enantiomers). All of these isomers and mixtures thereof are also included in the scope of the present invention.

[0080] As used herein, "hydrate" may mean a compound of the present invention or a salt thereof containing a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. The hydrate of the compound represented by the above formula 1 of the present invention may contain a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. The hydrate may contain at least 1 equivalent, preferably, 1 to 5 equivalents of water. Such a hydrate may be prepared by crystallizing the compound represented by the above formula 1 of the present invention, an isomer thereof, or a pharmaceutically acceptable salt thereof from water or a solvent containing water.

[0081] As used herein, the term "solvate" may refer to a compound of the present invention or a salt thereof comprising a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents include those that are volatile, non-toxic, and / or suitable for human administration.

[0082] As used herein, the term "alkyl" means an aliphatic hydrocarbon radical. An alkyl may be a "saturated alkyl" that does not contain an alkenyl or alkynyl moiety, or an "unsaturated alkyl" that contains at least one alkenyl or alkynyl moiety, and may have from 1 to 20 carbon atoms unless otherwise defined.

[0083] The term 'alkoxy', unless otherwise defined, means alkyl-oxy having 1 to 10 carbon atoms.

[0084] The term "cycloalkyl" refers to a saturated aliphatic 3- to 10-membered ring, unless otherwise defined. Typical cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0085] The term 'heterocycle', unless otherwise defined, means a 3-10 membered ring, preferably a 4-8 membered ring, more preferably a 5-6 membered ring, which contains 1 to 3 heteroatoms selected from the group consisting of N, O and S, which may be fused with benzo or C3-C8 cycloalkyl, and which is saturated or contains 1 or 2 double bonds. It may also be used interchangeably with the term 'heterocyclyl'. Examples of heterocycles include, but are not limited to, pyrroline, pyrrolidine, imidazoline, imidazolidine, pyrazoline, pyrazolidine, pyran, piperidine, morpholine, thiomorpholine, piperazine, hydrofuran, etc.

[0086] Unless otherwise defined, other terms and abbreviations used in this specification may be interpreted as having the meaning commonly understood by those skilled in the art to which the present invention pertains.

[0087] In one embodiment of the present invention, in the compound of formula 1,

[0088] R 3 represents hydrogen, halogen, or phenyl, or the heterocycle is morpholino, piperazinonyl -(CH2) p - represents a heterocycle, where p is an integer from 0 to 1, but when m is 0, R 3 may be phenyl.

[0089] In one embodiment of the present invention, in the compound of formula 1,

[0090] R 4 is halogen, C1-C3-alkyl, hydroxy-C 1- C3-alkyl, -O-phenyl, -(CH2)p CO2-ethyl, -(CH2) where the heterocycle is thiomorpholino, morpholino, piperazinonyl, or pyrrolidinyl p -heterocycle, or proline-N-carbonyl, where p can be an integer from 0 to 1. R 4 In the heterocycle, one or more oxo (=O) substituents may be substituted.

[0091] In one embodiment of the present invention, in the compound of formula 1,

[0092] R 5 is hydrogen, or C1-C3-alkyl,

[0093] R 6 represents C1-C3-alkyl, C3-C6-cycloalkyl, heterocycle or heterocyclyl-C1-C3-alkyl, wherein the heterocycle is tetrahydro-2H-pyran, or piperidinyl, and R 6 If this heterocycle or heterocyclyl-C1-C3-alkyl, C1-C6-alkylamine, hydroxy-C 1- It may be substituted with C6-alkyl or C1-C6-alkylsulfonyl.

[0094] In the present invention, examples of the compound of formula 1 include compounds 1 to 33 listed in Table 1 below or pharmaceutically acceptable salts thereof.

[0095] [Table 1]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101] In a preferred embodiment of the present invention, the compound of formula 1 may be a compound of formula 2 below.

[0102] [Chemical Formula 2]

[0103]

[0104] In the present invention, the compound of chemical formula 1 can inhibit, prevent or treat fibrosis of kidney damaged by ischemia-reperfusion injury.

[0105] In the present invention, the compound of chemical formula 1 can increase the expression of ROS-related genes such as Mn-SOD, GPX4, and UCP-1, and the protein expression of ferroptosis-related proteins such as xCT, SLC7A11, and GPX4, and can suppress the expression of 4-HNE protein and the gene expression of inflammation-related proteins such as OPN (Osteopontin) and MCP-1.

[0106] In the present invention, the compound of chemical formula 1 can increase the expression of E-cadherin protein, a protein associated with renal fibrosis, inhibit the protein expression of collagen IV, α-SMA, Snail, and Twist, inhibit the expression of HMGB1 and NF-κB protein, which are inflammation regulators, increase the expression of HO-1 and Nrf2 proteins, and inhibit the expression of F4 / 80.

[0107] In the present invention, the compound of chemical formula 1 can regulate the above genes, proteins, or regulatory factors abnormally expressed due to renal ischemia-reperfusion injury to a level close to the normal range.

[0108] In one embodiment of the present invention, it was confirmed that the compound of formula 1 inhibits ROS production in HK-2 cells induced with TGF-β, increases the mRNA expression levels of ROS-related genes Mn-SOD, GPX4, and UCP-1, increases the expression levels of ferroptosis-related proteins xCT, SLC7A11, and GPX4, and decreases the protein expression of 4-HNE. It was also confirmed that the gene expression levels of inflammation-related proteins OPN and MCP-1 are reduced, the protein expression level of E-cadherin is increased, and the protein expression levels of collagen IV and α-SMA (alpha smooth muscle actin) are decreased (Experimental Example 1).

[0109] In one embodiment of the present invention, it was confirmed that the compound of formula 1 can suppress cellular lipid ROS and mitochondrial lipid ROS in HK-2 cells treated with RSL3, increase the value of GSH, an antioxidant for ferroptosis, and increase the expression levels of GPX4 and Nrf2 (Experimental Example 2).

[0110] In one embodiment of the present invention, it was confirmed that the compound of formula 1 can reduce the amount of blood urea nitrogen (BUN) and serum creatinine (s-Cr) in the kidney after ischemia-reperfusion injury (IRI), and reduce tubulointerstitial damage (Experimental Example 3).

[0111] In one embodiment of the present invention, it was confirmed that the compound of formula 1 can suppress the production of reactive oxygen species (ROS) and inhibit cell death in the kidney after ischemia-reperfusion injury (IRI) (Experimental Example 4).

[0112] In one embodiment of the present invention, it was confirmed that the compound of formula 1 can inhibit ferroptosis by increasing the expression levels of xCT, SLC7A11, and GPX4 proteins and decreasing the expression level of 4-HNE protein in ischemia-reperfusion injured (IRI) kidneys (Experimental Example 5).

[0113] In one embodiment of the present invention, it was confirmed that the compound of formula 1 can suppress renal inflammation caused by ischemia-reperfusion injury (IRI) by reducing the expression levels of NF-κB and HMGB1 proteins, increasing the expression levels of HO-1 and Nrf2 proteins, and reducing the expression rate of F4 / 80 (Experimental Example 6).

[0114] In one embodiment of the present invention, the compound of formula 1 was confirmed to have a protective effect against fibrosis and an inhibitory effect against fibrosis in ischemia-reperfusion injured (IRI) kidneys by increasing the expression level of E-cadherin protein and decreasing the expression levels of collagen IV, α-SMA, Snail and Twist proteins and TGF-β (Experimental Examples 7 and 8).

[0115] In the present invention, renal fibrosis may correspond to one or more diseases selected from the group consisting of chronic kidney disease (CKD), glomerulosclerosis, diabetic renal fibrosis, tubulointerstitial fibrosis, hypertensive renal fibrosis, renal tubular fibrosis, renal failure, and end-stage renal disease (ESRD), but is not limited thereto as long as it corresponds to a disease causing fibrosis in renal tissue.

[0116] In the present specification, the compound of formula 1 or a pharmaceutically acceptable salt thereof in the composition may be characterized in that it is included at a concentration of 0.01 μM to 1000 μM. According to one specific example, the concentration may be, but is not limited to, 0.1 μM to 500 μM, 1 μM to 500 μM, 1 μM to 300 μM, 5 μM to 300 μM, 5 μM to 150 μM, 5 μM to 120 μM, 10 μM to 100 μM, or 20 μM to 40 μM.

[0117] In this specification, “treatment” means stopping or delaying the progression of a disease when used on a subject exhibiting symptoms of the disease, and “prevention” means stopping or delaying the signs of the disease when used on a subject not exhibiting symptoms of the disease but at high risk of such symptoms.

[0118] In the present invention, the “pharmaceutical composition” may include a pharmaceutically acceptable carrier as needed together with the compound of the present invention.

[0119] The compound of chemical formula 1 according to the present invention can be administered in various oral and parenteral dosage forms during clinical administration, and when formulated, it is manufactured using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0120] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc., and these solid preparations are manufactured by mixing one or more compounds of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, or syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included.

[0121] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, glycerol, and gelatin.

[0122] In addition, the present invention provides a method for preventing or treating renal fibrosis, comprising administering a compound of formula 1 or a pharmaceutically acceptable salt thereof to a subject in need thereof in a pharmaceutically acceptable amount.

[0123] The present invention also provides a use of a compound of formula 1 or a pharmaceutically acceptable salt thereof in the prevention or treatment of renal fibrosis. In the above preventive or therapeutic method and use, the descriptions of the compound of formula 1, the pharmaceutically acceptable salt, renal fibrosis, and the prevention or treatment can be applied in the same manner as described for the pharmaceutical composition.

[0124] In this specification, "administration" means introducing a given substance into a human or animal by any appropriate method, and the route of administration of the preventive or therapeutic composition according to the present invention may be oral or parenteral administration through any general route as long as it can reach the target tissue.

[0125] In the present invention, the "subject" requiring administration may include both mammals and non-mammals. Examples of mammals include, but are not limited to, non-human primates such as humans, chimpanzees, or monkeys, and livestock animals such as cows, horses, and sheep.

[0126] In addition, the effective dosage for the human body of the compound of chemical formula 1 of the present invention may vary depending on the patient's age, body weight, sex, dosage form, health condition, and disease severity, and is generally about 0.001-100 mg / kg / day, and preferably 0.01-35 mg / kg / day. Based on an adult patient weighing 70 kg, the dosage is generally 0.07-7000 mg / day, and preferably 0.7-2500 mg / day, and may be administered once or several times a day at regular intervals according to the judgment of a doctor or pharmacist.

[0127]

[0128] The numerical values ​​set forth in this specification are to be interpreted as including the equivalent range unless otherwise specified.

[0129]

[0130] Hereinafter, the present invention will be described in more detail through examples according to the present invention, but the scope of the present invention is not limited by the examples presented below.

[0131]

[0132]

[0133] Example

[0134] Experimental method

[0135] (1) Culturing of cells and treatment with Example Compound 1

[0136] In this example, as a representative example of the compound of chemical formula 1, (5-[(1,1-dioxido-4-thiomorpholinyl)methyl]-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine) (hereinafter referred to as 'Example Compound 1' or 'Compound 1') was used.

[0137] HK-2 cells were cultured in DMEM / F12 (Dulbecco's modified Eagle medium F12) containing 10% FBS (fetal bovine serum) and 1% antibiotic-antimycotic at 37°C and 5% CO2. HK-2 cells were plated and allowed to attach for 16–18 h, then exposed to 10 ng / mL TGF-β for 72 h. The exposed cells were then treated with the indicated concentrations of Example Compound 1 for 24 h.

[0138] For HK-2 cells induced with ferroptosis by RSL3, HK-2 cells were pretreated with the indicated concentrations of compound 1 for 20 min and then exposed to 0.3 or 0.75 μM RSL3 for 24 h.

[0139] (2) FACS (Fluorescence-activated cell sorting) analysis

[0140] 2.5 X 10 5 HK-2 cells were stained with fluorescent dyes for 20 min, washed with phosphate-buffered saline (PBS), resuspended in PBS, and then subjected to BODIPY for the measurement of cellular lipid ROS and mitochondrial lipid ROS, respectively. TM After fluorescent staining with 581 / 591 C11 (Invitrogen) and MitoPerOx (Abcam), BD FACSLyric TM FACS analysis was performed using a device (BD Bioscience) according to the manufacturer's protocol. Cellular lipid ROS or mitochondrial lipid ROS were expressed as relative values ​​to the control.

[0141] (3) Measurement of GSH (glutathione) levels

[0142] Cellular GSH levels were measured using a GSH assay kit (Abcam) according to the manufacturer's protocol. Specifically, 5 X 10 5HK-2 cells were homogenized in 0.1 mL of a 5% sulfosalicylic acid solution and centrifuged at 12,000 g for 20 min at 4°C. The supernatant was collected, diluted with assay buffer, and the diluted sample was mixed with enzyme buffer. The absorbance values ​​at a wavelength of 450 nm were measured using a SpectraMax iD3 multimode microplate reader. Relative GSH values ​​were expressed relative to the control group.

[0143] (4) GPX activity measurement

[0144] The activity value of glutathione peroxidase (GPX) was measured using a glutathione peroxidase activity assay kit (Glutathione Peroxidase Assay Kit (Colorimetric) (Abcam)) according to the manufacturer's protocol. Briefly, 5 X 10 5 HK-2 cells were homogenized in 0.2 mL of cold assay buffer to obtain 50 μL of enzyme extract, and the absorbance value at a wavelength of 340 nm was measured using a SpectraMax iD3 multi-mode microplate reader. Relative GPX activity values ​​were expressed relative to the control group.

[0145] (5) Preparation of animal model and treatment with Example Compound 1

[0146] Male C57BL / 6 mice (8 weeks old) were divided into six groups as shown in Table 2 below.

[0147] [Table 2]

[0148]

[0149] Mice subjected to renal ischemia-reperfusion injury (IRI) were anesthetized with intraperitoneal injection of ketamine (60 mg / kg) in wild-type mice. After abdominal incision, ischemia was induced by clamping both renal sacs, and the clamps were removed after 25 min. During this period, body temperature (33°C–34°C) was maintained with a heating pad, and blood and kidney tissues were collected from all mice at the end of each experimental protocol.

[0150] For IR 3d + M mice, Example Compound 1 was administered intraperitoneally once 24 hours after IRI (30 mg / kg), and for IR 7d + M mice, Example Compound 1 was administered intraperitoneally three times in total 24 hours, 3 days, and 5 days after IRI (30 mg / kg). For IR 3d and IR 7d mice, saline was administered instead at the same time as the administration of Example Compound 1 to IR 3d + M and IR 7d + M mice.

[0151] For WT + M, IR 3d + M and IR 7d + M mice, blood and kidney tissues were collected 24 hours after administration of the last example compound 1.

[0152] (6) Blood and tissue preparation

[0153] Blood was collected from the inferior vena cava of the mouse and centrifuged for 10 minutes in a microcentrifuge tube (4°C) to obtain serum, which was used to evaluate blood urea nitrogen (BUN) and serum creatinine (s-Cr).

[0154] The left kidney of each mouse was excised and cut into three pieces. Two kidney sections were flash frozen in liquid nitrogen and stored at -70°C for subsequent protein and RNA extraction analyses. The other kidney section was fixed in 4% paraformaldehyde at 4°C and embedded in Paraplast (Sherwood Medical, St. Louis, MO, USA). The paraffin-embedded kidney sections were cut at 4 μm and used for light microscopy.

[0155] (7) Tissue damage score

[0156] Paraffin-embedded kidney sections were deparaffinized with xylene, stained with hematoxylin and eosin (H&E) and Masson's trichrome (MT), and then observed under a light microscope (Olympus BX51).

[0157] Six consecutive fields were measured at 200x magnification, and the tissue damage score per slide was averaged. For H&E sections, renal cortical vacuolization, proximal tubular simplification, renal cortical vacuolization, and peritubular / proximal tubular leukocyte infiltration were assessed and scored as follows:

[0158] Normal: 0; <25% damage: 1 point; >25%~50% damage: 2 points; >50%~75% damage: 3 points; >75%~100% damage: 4 points.

[0159] MT staining was used to measure inflammatory cell accumulation and collagen deposition in renal tissue sections.

[0160] (8) Western blot analysis

[0161] Each protein was extracted with a buffer containing 1 M phosphate-buffered saline, 5 M EDTA, 0.5% Triton X-100, or NP-40 buffer (Invitrogen, Waltham, MA, USA) containing 1X phosphatase inhibitor cocktail and 1X protease inhibitor cocktail (Roche, Basel, Switzerland). Western blot analysis was performed on the supernatant obtained after centrifugation at 4 °C and 13,000 rpm for 10 min.

[0162] For Western blot analysis, proteins (20 μg / lane) were separated by electrophoresis on a 10%–15% SDS gel, transferred to a polyvinylidene fluoride membrane, and blocked with 5% skim milk powder for 1 h at room temperature.

[0163] For in vitro and in vivo experiments treated with TGF-β, membranes were incubated with α-tubulin, E-cadherin, or Snail (1:1000, 1:1000, and 1:1000, respectively: Cell Signaling Technology, Danvers, MA, USA); collagen IV, α-SMA, Twist, HMGB1, GPX4, or 4-HNE (1:1000, 1:1000, 1:1000, 1:1000, 1:1000, and 1:1000, respectively: Abcam, Cambridge, UK); GAPDH, NF-κBp65, or Nrf2 (1:1000, 1:1000, and 1:1000, respectively: Santa Cruz Biotechnology, Dallas, TX, USA); After overnight incubation at 4°C in HO-1 (1:1000, SPA-895: Enzo Life Sciences, Vienna, Austria) or SLC7A11 (1:1000, PA1-16893: Invitrogen, Waltham), the cells were incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G secondary antibody (1:2000: Abfrontier Co., Ltd.) and horseradish peroxidase-conjugated anti-mouse immunoglobulin G secondary antibody (1:2000: Abfrontier Co., Ltd.) for 2 h at room temperature. Analysis was then performed.

[0164] For in vitro experiments treated with RSL3, the membranes were incubated overnight at 4°C with primary antibodies against GPX4 (59735S, Cell signaling technology), Nrf2 (33649S, Cell signaling technology), or β-actin (A5441, Sigma), followed by incubation with anti-rabbit IgG, HRP-linked antibody (7074S, Cell signaling technology), or goat anti-mouse IgG secondary antibody [HRP] (HAF007, R&D Systems) for 2 h at room temperature. Analysis was then performed.

[0165] Protein bands were visualized using a chemiluminescence detection kit (Thermo Fisher Scientific, South Logan, UT, USA) and quantified using the Gel-Pro Analyzer software application (version 3.1: Media Cybernetics, Silver Spring, MD, USA).

[0166] (9) Immunohistochemistry and tissue staining

[0167] Paraffin-embedded kidney sections were deparaffinized with xylene and then hydrated with an alcohol solution. Peroxide was removed by treating with a mixture of 3% H2O2 and methanol for 10 minutes at room temperature.

[0168] The samples were incubated overnight at 4°C with collagen IV, α-SMA, 4-HNE, TGF-β (1:200, 1:200, 1:200, 1:500, respectively: Abcam), E-cadherin (1:200: Cell Signaling Technology), and F4 / 80 (1:200, MCA 497GA: Bio-Rad [AbD Serotec], Oxford, UK). After washing, secondary antibodies were added, and the samples were incubated for an additional 30 minutes at room temperature.

[0169] REAL TM EnVision TM The slides were immunostained with diaminobenzidine (DAB) using Detection System, Peroxidase / DAB+, Rabbit / Mouse Kit (Dako, Carpinteria, CA, USA), counterstained with hematoxylin, dehydrated, and covered with a coverslip.

[0170] (10) TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) staining method

[0171] Paraffin kidney sections (4 μm) were deparaffinized, cultured in 3% H2O2 at room temperature, and TUNEL staining was performed using the In Situ Cell Death Detection Kit, POD (Roche, Basel, Switzerland) according to the manufacturer's protocol.

[0172] TUNEL-positive cells were identified by their fluorescent signal using a fluorescence microscope, and apoptosis was semiquantitatively assessed under a microscope at 200× magnification. The apoptotic index (number of TUNEL-positive cells / DAPI-positive cells) was calculated using the Image Pro Plus 6.0 software application (Media Cybernetics).

[0173] (11) Dehydroethidium staining method

[0174] Paraffin kidney sections (4 μm) were deparaffinized, incubated with dihydroethidium (D11347: Invitrogen) solution at 37°C for 30 min, and covered with a coverslip containing DAPI (Roche), followed by observation under a fluorescence microscope.

[0175] (12) RT-PCR analysis

[0176] In in vitro and in vivo experiments treated with TGF-β, RNA was extracted using the Total RNA Miniprep Kit, and cDNA was amplified using PCR. The sequences of the PCR primers used for amplification of each gene are shown in Tables 3 and 4.

[0177] [Table 3]

[0178]

[0179] [Table 4]

[0180]

[0181] PCR was performed using 20 μL of amplification reaction solution consisting of 10 μL of iQ SYBR Green PCR Master Mix reagent (Qiagen Sciences Inc., Hilden, Germany), 2 μL of primers for each gene, 2 μL of cDNA, and 6 μL of H2O. After denaturation at 95 °C for 10 min, the amplification reaction was performed using a thermal cycler (Rotor-Gene TM Amplification was performed using a 6000: Corbett Research Pty Ltd., Mortlake, NSW, Australia) using 40 cycles of 95 °C for 10 s, annealing temperature (60 °C for GAPDH, UCP-1, MnSOD, GPX4, MCP-1, OPN, and 4-HNE) for 15 s, and 72 °C for 20 s. SYBR green fluorescence emission values ​​were measured at the end of each cycle using the comparative threshold cycle method, and the results were evaluated using the ΔΔCt method.

[0182]

[0183] Experimental results

[0184] Experimental Example 1. Evaluation of the effect of treatment with Example Compound 1 in a TGF-β-induced HK-2 cell fibrosis model.

[0185] HK-2 cells were exposed to TGF-β (10 ng / mL) for 72 h, and then treated with the indicated concentrations of Example Compound 1 (1, 5, or 20 μM) for 24 h. The inhibition of Example Compound 1 by TGF-β-induced ROS production was evaluated (*p < 0.01, **p < 0.001).

[0186] The mRNA expression levels of ROS-related genes, Mn-SOD, GPX4, and UCP-1, were analyzed by RT-PCR. The mRNA expression levels of each gene decreased in HK-2 cells exposed to TGF-β, but significantly increased in proportion to the concentration due to treatment with Example Compound 1 (Fig. 1a).

[0187] The expression levels of ferroptosis-related proteins, xCT, SLC7A11, GPX4, and 4-HNE, were analyzed by Western blot. In HK-2 cells exposed to TGF-β, the protein expression levels of xCT, SLC7A11, and GPX4 decreased, but were significantly increased in proportion to the concentration by treatment with Example Compound 1. In addition, the protein expression level of 4-HNE increased in HK-2 cells exposed to TGF-β, but was significantly decreased in proportion to the concentration by treatment with Example Compound 1 (Fig. 1b).

[0188] RT-PCR analysis of osteopontin (OPN) and MCP-1, which are inflammation-related proteins, was performed to evaluate whether Example Compound 1 improves inflammation induced by TGF-β. As a result, treatment with Example Compound 1 resulted in a gradual decrease in the mRNA expression levels of OPN and MCP-1 in proportion to the concentration (Fig. 1c).

[0189] When changes in the expression of fibrosis-related proteins in HK-2 cells were examined using Western blot, the expression level of E-cadherin decreased, and the expression levels of collagen IV and alpha smooth muscle actin (α-SMA) increased in HK-2 cells exposed to TGF-β. However, treatment with Example Compound 1 significantly increased the expression level of E-cadherin, and significantly decreased the expression levels of collagen IV and α-SMA (Fig. 1d).

[0190]

[0191] Experimental Example 2. Confirmation of the ferroptosis inhibitory effect of Example Compound 1 in HK-2 cells.

[0192] The ferroptosis inhibitory effect of Example Compound 1 was evaluated through RSL3, known as a GPX4 inhibitor.

[0193] The inhibitory efficacy of Example Compound 1 on cellular lipid ROS and mitochondrial lipid ROS was evaluated by FACS analysis. When treated with RSL3 (0.75 μM), cellular lipid ROS and mitochondrial lipid ROS increased, and were inhibited by treatment with Example Compound 1 (0.001, 0.01, and 0.1 μM) (Fig. 2a).

[0194] We examined the level of GSH, a major antioxidant for ferroptosis. GSH levels decreased in HK-2 cells treated with RSL3 (0.3 μM), and then increased when treated with Example Compound 1 (Fig. 2b).

[0195] RT-PCR, Western blot, and GPX4 assay kits were used to evaluate whether compound 1 enhances GPX4, which functions to repair oxidative damage of lipids. In HK-2 cells treated with RSL3 (0.75 μM), the mRNA expression level (using the primer set in Table 4), protein expression level, and GPX activity of GPX4 decreased, but were significantly increased by treatment with compound 1 (Figs. 2c, 2d). The protein expression of Nrf2 and GPX4, which are major antioxidant factors, decreased by treatment with RSL3 (0.75 μM), but were significantly increased by treatment with compound 1 (Fig. 2d).

[0196]

[0197] Experimental Example 3. Evaluation of renal function and histology after ischemia-reperfusion injury (IRI) in wild-type mice.

[0198] Blood urea nitrogen (BUN) and serum creatinine (s-Cr) were measured to evaluate renal function according to treatment with Example Compound 1. No significant difference was observed between wild-type mice (WT) and wild-type mice (WT+M) treated with Example Compound 1. BUN and s-Cr increased in ischemia-reperfusion injured mice (IR 3d, IR 7d mice), but in ischemia-reperfusion injured mice treated with Example Compound 1 (IR 3d + M, IR 7d + M mice), BUN and s-Cr were significantly decreased (Fig. 3a).

[0199] Tissue damage scores were assessed by hematoxylin and eosin (H&E) staining of kidney sections. WT and WT + M mice were observed to be similar, but IR 3d and IR 7d mice showed tubular interstitial injury and interstitial inflammatory cell infiltration, and the tubular interstitial damage scores were high, but the IR 3d + M and IR 7d + M mice treated with Example Compound 1 showed significantly lower scores (Fig. 3b).

[0200]

[0201] Experimental Example 4. Evaluation of the efficacy of Example Compound 1 on ROS and apoptosis generated in the kidney after IRI.

[0202] The effect of Example Compound 1 on renal tubular ROS in renal fibrosis caused by renal ischemia-reperfusion injury was evaluated using dehydroethidium (DHE) staining.

[0203] The fluorescence intensity of DHE staining in kidney sections from WT and WT + M mice was almost similar, but the fluorescence intensity of DHE staining in kidney sections from IR 3d + M and IR 7d + M mice was significantly reduced compared to IR 3d and IR 7d mice (Fig. 4a).

[0204] The extent of cell death in mice with renal ischemia-reperfusion injury was assessed using TUNEL staining analysis.

[0205] No difference in the number of TUNEL-positive cells was observed in kidney sections from WT and WT + M mice, but it was significantly reduced in kidney sections from IR 3d + M and IR 7d + M mice compared to kidneys from IR 3d and IR 7d mice (Fig. 4b).

[0206] Accordingly, it was confirmed that Example Compound 1, which has no particular effect on normal cells, inhibits the production of reactive oxygen species (ROS) and suppresses cell death in ischemia-reperfusion injured (IRI) kidneys.

[0207]

[0208] Experimental Example 5. Evaluation of the efficacy of compound 1 on ferroptosis in the kidneys of mice with ischemia-reperfusion injury (IRI).

[0209] The ability of compound 1 to inhibit ferroptosis induced in the kidney of a mouse model of ischemia-reperfusion injury (IRI) was evaluated.

[0210] The protein expressions of xCT, SLC7A11, and GPX4 did not show significant differences between WT and WT+M mice. The protein expressions of xCT, SLC7A11, and GPX4 were significantly reduced in the kidneys of IR 3d and IR 7d mice with renal ischemia-reperfusion injury, but were significantly increased in the kidneys of IR 3d + M and IR 7d + M mice treated with Example Compound 1. The expression levels of 4-HNE were significantly reduced in the kidneys of IR 3d + M and IR 7d + M mice (Figures 5a and 5b).

[0211] Immunohistochemical staining of 4-HNE showed no significant difference in the kidneys of WT and WT + M mice, but the positive area of ​​4-HNE was significantly increased in the kidney sections of IR 3d and IR 7d mice, whereas it was markedly decreased in the kidneys of IR 3d + M and IR 7d + M mice administered Example Compound 1 (Fig. 5c).

[0212] Accordingly, it can be confirmed that Example Compound 1, which has no particular effect on normal cells, inhibits ferroptosis in kidneys with ischemia-reperfusion injury (IRI).

[0213]

[0214] Experimental Example 6. Effect of Example Compound 1 on Renal Inflammation Caused by Ischemia-Reperfusion Injury (IRI)

[0215] HMGB1 and NF-κB are activated by ferroptosis expression, inducing an inflammatory response. The renal anti-inflammatory efficacy of Example Compound 1 was evaluated by Western blot and immunohistochemical staining on the kidneys of a mouse model of ischemia-reperfusion injury.

[0216] Protein expression of NF-κB, HMGB1, HO-1, and Nrf2 did not differ in the kidneys of WT and WT+M mice. NF-κB and HMGB1 expression increased in the kidneys of IR 3d and IR 7d mice, but significantly decreased in the kidneys of IR 3d + M and IR 7d + M mice administered Example Compound 1. HO-1 and Nrf2 expression decreased in the kidneys of IR 3d and IR 7d mice, but significantly increased in the kidneys of IR 3d + M and IR 7d + M mice administered Example Compound 1. (Figures 6a and 6b).

[0217] Regarding immunohistochemical staining for the macrophage marker F4 / 80, there was no significant difference in F4 / 80 expression in the kidneys of WT and WT+M mice, but the expression rate of F4 / 80 was reduced in the kidneys of IR 3d + M and IR 7d + M mice compared to IR 3d and IR 7d mice (Fig. 6c).

[0218] Accordingly, it can be confirmed that Example Compound 1, which has no particular effect on normal cells, suppresses renal inflammation occurring in kidneys subjected to ischemia-reperfusion injury (IRI).

[0219]

[0220] Experimental Example 7. Antifibrotic effect of compound 1 in the kidney of a mouse model of ischemia-reperfusion injury.

[0221] Western blot analysis was performed on the kidney of an ischemia-reperfusion injured mouse model to confirm the anti-fibrotic effect and its mechanism of Example Compound 1.

[0222] E-cadherin expression was downregulated in the kidneys of IR 3d and IR 7d mice compared to WT mice, but significantly increased in the kidneys of IR 3d + M and IR 7d + M mice. Collagen IV, α-SMA, Snail, and Twist protein expression was upregulated in the kidneys of IR 3d and IR 7d mice, but significantly decreased in the kidneys of IR 3d + M and IR 7d + M mice (Figures 7a and 7b).

[0223]

[0224] Experimental Example 8. Antifibrotic effect of Example Compound 1 in the kidney of an ischemia-reperfusion injured mouse model.

[0225] The antifibrotic effect of Example Compound 1 on renal fibrosis was confirmed in kidney sections of IRI mice. E-cadherin, collagen IV, and α-SMA, which are markers of epithelial-mesenchymal transition (EMT), and TGF-β, a key regulator of fibrosis, were identified by immunohistochemical staining, and MT staining was also performed to observe tissue fibrosis.

[0226] Although there was no significant difference in E-Cadherin staining intensity in the kidneys of WT and WT + M mice, it was increased in the kidneys of IR 3d + M and IR 7d + M mice compared to those of IR 3d and IR 7d mice. Even the staining intensities of collagen IV, α-SMA, and TGF-β did not differ significantly between WT and WT + M mice, but the staining intensities were significantly reduced in IR 3d + M and IR 7d + M mice compared to those of IR 3d and IR 7d mice. Image analysis of MT staining confirmed that fibrosis was reduced in the kidneys of IR 3d + M and IR 7d + M mice compared to those of IR 3d and IR 7d mice (Fig. 8a and 8b).

Claims

1. A pharmaceutical composition for preventing or treating renal fibrosis, comprising a compound of chemical formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: [Chemical Formula 1] In the above formula, n is an integer from 1 to 3, m is 0 or 1, A represents phenyl, R 1 is hydrogen, or C1-C6-alkyl, R 2 represents hydrogen, halogen or C1-C6-alkoxy, or hydroxy-C 1- C6-alkyl, -(CH2) p CO2R 7 , -NHR 8 , -N(H)S(O)2R 7 or -NHC(O)R 7 , where p is an integer from 0 to 3, and R 7 represents hydrogen or C1-C3-alkyl, and R 8 Silver C 1- C3-alkylpiperidinyl, or C 1- It represents C3-alkylsulfonyl, R 3 represents hydrogen, halogen, C1-C6-alkyl or phenyl, or the heterocycle contains 1 or 2 heteroatoms selected from S, N and O atoms and is a 5 to 6-membered ring -(CH2) p - represents a heterocycle, where p is an integer from 0 to 3, but when m is 0, R 3 is phenyl, R 4 is halogen, C1-C6-alkyl, hydroxy-C 1- C6-alkyl, -O-phenyl, -(CH2) p CO2R 7 , a heterocycle containing 1 or 2 heteroatoms selected from S, N and O atoms and a 5 to 6 membered ring -(CH2) p -heterocycle, or proline-N-carbonyl, where p is an integer from 0 to 3, and R 7 is as defined above, and the heterocycle may be substituted with one or more oxo (=O) substituents, R 5 is hydrogen, or C1-C6-alkyl, R 6 represents C1-C6-alkyl, C3-C6-cycloalkyl, heterocycle or heterocyclyl-C1-C6-alkyl, wherein the heterocycle is a 3 to 8-membered ring containing 1 to 3 heteroatoms selected from S, N and O atoms, and R 6 is C1-C6-alkylamine, hydroxy-C 1- It may be substituted with C6-alkyl or C1-C6-alkylsulfonyl.

2. In paragraph 1, R 3 represents hydrogen, halogen, or phenyl, or the heterocycle is morpholino, piperazinonyl -(CH2) p - represents a heterocycle, where p is an integer from 0 to 1, but when m is 0, R 3 is phenyl, R 4 is halogen, C1-C3-alkyl, hydroxy-C 1- C3-alkyl, -O-phenyl, -(CH2) p CO2-ethyl, -(CH2) where the heterocycle is thiomorpholino, morpholino, piperazinonyl, or pyrrolidinyl p -heterocycle, or proline-N-carbonyl, wherein p is an integer from 0 to 1, and the heterocycle may be substituted with one or more oxo (=O) substituents, R 5 is hydrogen, or C1-C3-alkyl, R 6 represents C1-C3-alkyl, C3-C6-cycloalkyl, heterocycle or heterocyclyl-C1-C3-alkyl, wherein the heterocycle is tetrahydro-2H-pyran, or piperidinyl, and R 6 If this heterocycle or heterocyclyl-C1-C3-alkyl, C1-C6-alkylamine, hydroxy-C 1- A pharmaceutical composition for preventing or treating renal fibrosis, which may be substituted with C6-alkyl or C1-C6-alkylsulfonyl.

3. In paragraph 1, A pharmaceutical composition for preventing or treating renal fibrosis, characterized in that the compound of the above chemical formula 1 is any one selected from the group of compounds below. <1> 5-[(1,1-dioxido-4-thiomorpholinyl)methyl]-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine; <2> Ethyl 7-(cyclopentylamino)-2-phenyl-1H-indole-5-carboxylate; <3> (7-(cyclopentylamino)-2-phenyl-1H-indol-5-yl)methanol; <4> 5-Chloro-N,1-dimethyl-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine; <5> 4-((7-(cyclopentylamino)-2-(3-fluorophenyl)-1H-indol-5-yl)methyl)piperazin-2-one; <6> 4-((2-phenyl-7-(((tetrahydro-2H-pyran-4-yl)methyl)amino)-1H-indol-5-yl)methyl)thiomorpholine 1,1-dioxide; <7> 5-Chloro-N-(1-methylpiperidin-4-yl)-2-phenyl-1H-indol-7-amine; <8> 5-Phenoxy-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine; <9> 5-chloro-3-(morpholinomethyl)-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine; <10> 2-(4-((5-fluoro-2-phenyl-1H-indol-7-yl)amino)piperidin-1-yl)ethan-1-ol; <11> N-(4-(5-chloro-7-(cyclopentylamino)-1H-indol-2-yl)phenyl)methanesulfonamide; <12> 5-Chloro-3-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine; <13> 4-((2-(3-fluorophenyl)-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-5-yl)methyl)thiomorpholine 1,1-dioxide; <14> 5-Chloro-N-cyclopentyl-2-(4-((1-methylpiperidin-4-yl)amino)phenyl)-1H-indol-7-amine; <15> 4-((7-(isopentylamino)-2-(4-methoxyphenyl)-1H-indol-5-yl)methyl)thiomorpholine 1,1-dioxide; <16> N-(4-(7-(cyclopentylamino)-5-((1,1-deoxidothiomorpholino)methyl)-1H-indol-2-yl)phenyl)acetamide; <17> 4-((3-bromo-2-phenyl-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-5-yl)methyl)thiomorpholine 1,1-dioxide;<18> 4-((5-chloro-2-phenyl-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-3-yl)methyl)piperazin-2-one; <19> 4-((7-(methyl(tetrahydro-2H-pyran-4-yl)amino)-2-phenyl-1H-indol-5-yl)methyl)thiomorpholine 1,1-dioxide; <20> 5-Methyl-N-(1-(methylsulfonyl)piperidin-4-yl)-2-phenyl-1H-indol-7-amine; <21> N-(4-(5-(1,1-deoxidothiomorpholino)-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-2-yl)phenyl)acetamide; <22> 4-((7-((1-(methylsulfonyl)piperidin-4-yl)amino)-2-phenyl-1H-indol-5-yl)methyl)thiomorpholine 1,1-dioxide; <23> N; 1 -(5-chloro-2-phenyl-1H-indol-7-yl)-N 4 -Methylcyclohexane-1,4-diamine; <24> Methyl 2-(3-(5-chloro-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-2-yl)phenyl)acetate; <25> (2-phenyl-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indole-5-carbonyl)-D-proline; <26> (3-(5-chloro-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-2-yl)phenyl)methanol; <27> N-Cyclopentyl-2-phenyl-5-(2-(pyrrolidin-1-yl)ethyl)-1H-indol-7-amine; <28> Methyl 2-(4-(5-chloro-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-2-yl)phenyl)acetate; <29> Methyl 4-(5-chloro-7-(cyclopentylamino)-1H-indol-2-yl)benzoate; <30> 2-(4-(5-chloro-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-2-yl)phenyl)ethan-1-ol; <31> 3-Bromo-5-(morpholinomethyl)-2-phenyl-N-(tetrahydro-2H-pyran-4-yl)-1H-indol-7-amine; <32> 4-((3-phenyl-7-((tetrahydro-2H-pyran-4-yl)amino)-1H-indol-5-yl)methyl)thiomorpholine 1,1-dioxide; and <33> N-Cyclopentyl-5-methyl-2-phenyl-1H-indol-7-amine.

4. In paragraph 1, A pharmaceutical composition for preventing or treating renal fibrosis, wherein the compound of chemical formula 1 is a compound of chemical formula 2 below. [Chemical Formula 2] 5. In paragraph 1, A pharmaceutical composition for preventing or treating renal fibrosis, wherein the renal fibrosis is at least one disease selected from the group consisting of chronic kidney disease (CKD), glomerulosclerosis, diabetic renal fibrosis, tubulointerstitial fibrosis, hypertensive renal fibrosis, renal tubular fibrosis, renal failure, and end-stage renal disease (ESRD).

6. In paragraph 1, A pharmaceutical composition for preventing or treating renal fibrosis, wherein the compound of the above chemical formula 1 or a pharmaceutically acceptable salt thereof inhibits fibrosis of a kidney subjected to renal ischemia-reperfusion injury (IRI).

7. In paragraph 1, A pharmaceutical composition for preventing or treating renal fibrosis, wherein the compound of the above chemical formula 1 or a pharmaceutically acceptable salt thereof increases the expression of Mn-SOD, GPX4 and UCP-1 genes; decreases the expression of OPN and MCP-1 genes; or increases the protein expression of xCT, SLC7A11 and GPX4 and decreases the protein expression of 4-HNE.

8. In paragraph 1, A pharmaceutical composition for preventing or treating renal fibrosis, which inhibits the expression of NF-κB, HMGB1, collagen IV, α-SMA, Snail and Twist proteins, increases the expression of E-cadherin HO-1 and Nrf2 proteins, and inhibits the expression of F4 / 80.

9. A method for preventing or treating renal fibrosis, comprising administering a compound of formula 1 or a pharmaceutically acceptable salt thereof to a subject in need thereof in a pharmaceutically acceptable amount: [Chemical Formula 1] In the above formula, R 1 Inland R 6 , A, n and m are each as defined in paragraph 1.

10. The present invention relates to the use of a compound of chemical formula 1 or a pharmaceutically acceptable salt thereof in the prevention or treatment of renal fibrosis: [Chemical Formula 1] In the above formula, R 1 Inland R 6 , A, n and m are each as defined in paragraph 1.

Images