Compositions for treating KCa3.1 channel-mediated diseases, comprising phenylalkyl carbamate compounds

Abstract

The present invention relates to a composition for treating K Ca 3.1 channel-mediated diseases. More specifically, the composition contains, as an active ingredient, solriamfetol which is currently used as a therapeutic agent for narcolepsy, that is, a phenylalkylcarbamate compound represented by 2-amino-3-phenylpropyl carbamate, and can inhibit the activity of K Ca 3.1 channels in cell membranes, and thus can be used for treating pharmaceutical compositions for K Ca 3.1 channel-mediated diseases such as fibrotic diseases, autoimmune diseases, and cancers.

Description

Technical Field

[0001] The present invention relates to a composition for treating K Ca 3.1 channel-mediated diseases, specifically, a composition containing, as an active ingredient, a phenylalkyl carbamate compound such as solriamfetol, which is currently used as a drug for treating narcolepsy, i.e., tert-butyl 2-amino-3-phenylethylcarbamate, and inhibits the expression of K Ca 3.1 channels in cell membranes, and thus can be used as a pharmaceutical composition for treating K Ca 3.1-mediated diseases such as fibrotic diseases, autoimmune diseases, and cancer diseases. Background Art

[0002] K Ca 3.1 channels are distributed in the human body in non-excitable cells such as fibroblasts, hepatic stellate cells, vascular endothelial cells, nerve cells, cancer cells, and immune cells (T cells, B cells). Generally, K + channels increase the intracellular influx of Ca 2+ through membrane potential hyperpolarization, thereby playing a role in activating cells. However, Ca 2+ acts as a second messenger or a co-factor for various intracellular enzymes and plays a very important role in the intracellular signal transduction process. Therefore, when K Ca 3.1 channels are activated, the intracellular Ca 2+ increases, promoting cell proliferation, epithelial-mesenchymal transition, cell migration, the production and secretion of substances such as extracellular matrix or nitric oxide.

[0003] The formation of myofibroblasts (or activated hepatic stellate cells) is the most important stage in the fibrosis process and occurs through epithelial-mesenchymal transition. After the formation of myofibroblasts and the activation of hepatic stellate cells, the proliferation of these cells becomes active, and the formation of extracellular matrix occurs in these cells, making fibrosis active. It is known that the epithelial-mesenchymal transition from vascular endothelial cells or epithelial cells to form myofibroblasts is a Ca 2+ -dependent process. Moreover, the epithelial-mesenchymal transition induced by the fibrosis inducer TGF β is particularly sensitive to Ca 2+ . K Ca3.1 channels can regulate Ca within cells such as myofibroblasts and hepatic stellate cells 2+ , thereby regulating epithelial-mesenchymal transition.

[0004] K Ca After the 3.1 channels are activated, other reactions occur in the cells expressing these K + channels. Through immune cells, inflammation and immune responses are further increased through immune cell proliferation and cytokine secretion, etc. Angiogenesis is promoted through vascular endothelial cells, and vasodilation is induced through the secretion of nitric oxide. Fibrotic diseases are induced through fibroblasts and hepatic stellate cells. Further, myofibroblasts are generated or hepatic stellate cells are activated, inducing the generation of extracellular matrix such as collagen, etc., thereby promoting the formation of connective tissue. In addition, it also plays an important role in the proliferation and metastasis of some cancer cells. Therefore, it can be inferred that K Ca 3.1 channels play a very important role in the development of inflammation and autoimmune diseases, fibrotic diseases, and cancer.

[0005] Pro-inflammatory agents such as cytokines and H2O2 can exacerbate immune and inflammatory responses such as immune cell proliferation, and pro-fibrotic agents including growth factors such as TGF β and PDGF can exacerbate fibrotic responses. However, these pro-inflammatory agents and pro-fibrotic agents will increase the expression of K Ca 3.1 channels. Further, during the exacerbation of inflammation, immune responses, and fibrotic responses induced by pro-inflammatory agents and pro-fibrotic agents, the increase in the expression of K Ca 3.1 channels plays an important role.

[0006] As mentioned above, when the expression of K Ca 3.1 channels increases, it will accelerate the development of proliferative diseases such as fibrotic diseases and cancer, and inflammatory diseases such as autoimmune diseases. Therefore, these diseases are classified as K Ca 3.1 channel-mediated diseases. Recently, efforts have been continuously made to develop drugs for the treatment of inflammation and autoimmune diseases, fibrotic diseases, and cancer by using K Ca 3.1 channel inhibitors. Among them, the relatively representative K Ca 3.1 inhibitor, senicapoc, is under development as a treatment for sickle cell anemia retinopathy, various inflammatory and autoimmune diseases, and fibrotic diseases.

[0007] The present inventor has proposed in the U.S. Patent No. 9,259,412 B2 of the Industry-Academic Cooperation Group of Ewha Womans University and the Korean registered patent No. 10-1414831 as its family patent that modafinil and its derivatives currently used as therapeutic agents for narcolepsy contain K Ca 3.1 Compositions for the treatment or prevention of channel-mediated diseases. Ca 3.1 Channel-Mediated Diseases Sickle cell anemia retinopathy, immune diseases including acute immune reactions or autoimmune diseases, cancers including prostate cancer or pancreatic cancer, traumatic brain injury, neurodegenerative diseases, secretory diarrhea, etc. are mentioned as examples.

[0008] In addition, solriamfetol, i.e., 2-amino-3-phenylpropyl carbamate, is a therapeutic agent for narcolepsy and is developed and sold by SK Corporation of Korea under the product name of "Sunosi". In this regard, SK Corporation's U.S. Patents US 5,955,499 B2 and US 6,140,532 B2 and their family patents, i.e., Korean registered patents No. 10-197892 and No. 10-173863, describe that solriamfetol and its derivatives are useful as central nervous system therapeutic agents, especially antidepressants and antianxiety drugs.

[0009] U.S. Patent No. 9,464,041 B2 and its equivalent patent Korean Patent No. 10-2019-105675 disclose methods for treating or preventing fatigue associated with diseases such as depression, cancer, multiple sclerosis, Parkinson's disease, Alzheimer's disease, chronic fatigue, fibromyalgia, chronic pain, traumatic brain injury, AIDS and osteoarthritis using thiamethoxazole and its derivatives. U.S. Patent No. 10,351,517 B2 and its equivalent patent Korean Registered Patent No. 10-1335941 disclose methods for treating sleep-wake disorders including excessive daytime sleepiness and pathological somnolence.

[0010] [Prior art literature]

[0011] [Patent Document]

[0012] U.S. Patent US9,259,412B2 (Korean registered patent No. 10-1414831);

[0013] U.S. Patent No. US5,955,499B2 (Korean Registered Patent No. 10-197892);

[0014] U.S. Patent No. US6,140,532B2 (Korean Registered Patent No. 10-173863);

[0015] U.S. Patent No. US9,464,041B2 (Korean Published Patent No. 10-2019-105675);

[0016] U.S. Patent No. US10,351,517B2 (Korean Registered Patent No. 10-1335941). Summary of the Invention

[0017] Technical Problem

[0018] The present inventors were studying a method for treating K + channel-mediated diseases using a substance that inhibits the K Ca channel, and surprisingly found that phenylalkylcarbamate compounds currently containing solriamfetol, which is used as a therapeutic agent for narcolepsy, can inhibit the expression of the K Ca channel, and proved these possibilities through various experiments, thus completing the present invention.

[0019] Therefore, an object of the present invention is to provide a novel composition for treating K Ca channel-mediated diseases containing the phenylalkylcarbamate compound as an active ingredient.

[0020] Technical Solution

[0021] The composition for treating K Ca channel-mediated diseases according to the present invention preferably contains a phenylalkylcarbamate compound represented by the following [Structural Formula 1] or a pharmaceutically acceptable salt thereof as an active ingredient.

[0022] [Structural Formula 1]

[0023]

[0024] In the [Structural Formula 1], R1 is one or two functional groups selected from hydrogen, halogen, hydroxyl, amine, nitro, hydrogen sulfide, methyl, methyl halide, ethyl, propyl, methoxy, ethoxy, vinyl, and aryl, R2 and R3 are each a functional group selected from hydrogen, methyl, ethyl, and amide, and the * symbol represents a chiral center.

[0025] The composition for treating K Ca3.1 The composition for treating channel-mediated diseases is, preferably, in the [Structural Formula 1], a compound in which R2 and R3 are hydrogen.

[0026] The K treatable according to the present invention Ca 3.1 The composition for treating channel-mediated diseases is, preferably, in the [Structural Formula 1], a compound in which R1 is one or two functional groups selected from hydrogen, F, Cl, Br, and I.

[0027] The K treatable according to the present invention Ca 3.1 The composition for treating channel-mediated diseases is, preferably, in the [Structural Formula 1], a compound in which R1 is one or two Fs, and R2 and R3 are each one functional group selected from hydrogen, methyl, and amide.

[0028] Preferably, the compound represented by the [Structural Formula 1] is a chiral compound in which the content of the R-enantiomer or S-enantiomer is 90% or more.

[0029] Preferably, the compound represented by the [Structural Formula 1] is selected from 2-amino-3-phenylpropyl carbamate, 2-amino-3-(3-fluorophenyl)propyl carbamate, 2-amino-3-(3,4-dichlorophenyl)propyl carbamate, 2-amino-3-phenylpropyl methyl carbamate, 2-amino-3-phenylpropyl (aminocarbonyl)carbamate, 2-amino-3-(4-hydroxyphenyl)propyl carbamate, 2-amino-3-[3-(trifluoromethyl)phenyl]propyl carbamate.

[0030] Preferably, the K Ca 3.1 Channel-mediated diseases include fibrotic diseases such as liver fibrosis and pulmonary fibrosis, autoimmune diseases, and cancers.

[0031] Beneficial effects

[0032] The compound of Structural Formula 1 of the present invention was proven to have the effect of inhibiting the expression of K Ca 3.1 channels in in vitro experiments using fibroblasts, as well as the effects of inhibiting inflammation and fibrosis. Further, it was proven to have the effects of inhibiting inflammation and fibrosis in in vivo experiments on a mouse model induced with liver disease.

[0033] Therefore, it is expected that the compound of Structural Formula 1 can be effectively used as a new pharmaceutical composition for treating K Ca 3.1 channel-mediated diseases, namely various inflammations, autoimmune diseases, fibrotic diseases, and cancers in the human body. According to needs, it can also be developed into a veterinary drug. Description of the drawings

[0034] Figure 1 Shows the effect of the compound of Structural Formula 2 of the present invention on K Ca 3.1 current generation;

[0035] Figure 2 Shows the effect of the compounds of Structural Formulas 3 to 8 of the present invention on K Ca 3.1 current generation;

[0036] Figure 3 Shows the effect of the compounds of Structural Formulas 2 to 8 of the present invention on the expression of inflammatory markers in fibroblasts exposed to LPS (lipopolysaccharides) that induces inflammation for 24 hours;

[0037] Figure 4 Shows the effect of the compounds of Structural Formulas 2 to 8 of the present invention on the expression of fibrosis markers in fibroblasts exposed to TGF β for 24 hours to induce fibrosis;

[0038] Figure 5 Shows the results of confirming the fibrosis inhibitory effect of the compound of Structural Formula 2 of the present invention in a mouse model of fibrosis induced by bleomycin by Masson's trichrome staining of collagen;

[0039] Figure 6 Shows the effect of the compound of Structural Formula 2 of the present invention on the expression of fibrosis markers, i.e., mRNA, in a mouse model of pulmonary fibrosis induced by bleomycin;

[0040] Figure 7 Shows the results of confirming the fibrosis inhibitory effect of the compound of Structural Formula 2 of the present invention in a mouse model of liver fibrosis induced by CDAHFD diet by Masson's trichrome staining of collagen;

[0041] Figure 8 Shows the effect of the compound of Structural Formula 2 of the present invention on the expression of fibrosis marker mRNA in a mouse model of liver fibrosis induced by CDAHFD diet. Detailed implementation method

[0042] The phenylalkyl carbamate compound of Structural Formula 1 of the present invention specifically includes the compounds of the following Structural Formulas 2 to 8.

[0043] [Structural Formula 2]

[0044]

[0045] [Structural Formula 3]

[0046]

[0047] [Structural Formula 4]

[0048]

[0049] [Structural Formula 5]

[0050]

[0051] [Structural Formula 6]

[0052]

[0053] [Structural Formula 7]

[0054]

[0055] [Structural Formula 8]

[0056]

[0057] The chemical names of the compounds of Structural Formula 2 to Structural Formula 8 and the representative names written by the inventors are as follows.

[0058] [Table 1]

[0059]

[0060]

[0061] The compound of formula 2 is generally known as solriamfetol and is currently used as a therapeutic agent for narcolepsy. The method for preparing the compound of formula 3 is disclosed in U.S. Pat. No. 6,140,532 B2 and Korean registered patent No. 10-173863 as a family patent. The compound of formula 4 can be prepared according to the method described in U.S. patent publication US 2005 / 0080268 A1.

[0062] The preparation method of the compound of structural formula 5 has been disclosed in U.S. Patent No. 5,705,640 B2, the preparation method of the compound of structural formula 6 has been disclosed in U.S. Patent No. 9,403,761 B2 or its equivalent patent Korean Patent No. 10-2016-0126988, and the compound of structural formula 7 can be prepared according to the method described in [Example 9] of U.S. Patent No. 6,140,532 B2 and [Example 9] of International Patent Publication WO 98 / 15526.

[0063] The compound of structural formula 8 can be prepared according to the method described in US Pat. No. 9,180,120 B2 applied by Bayer or in [Example 48A] of Korean Patent Publication No. 10-2013-0138216, which is a patent of the same family.

[0064] For reference, the Bayer patent describes that the compound of structural formula 8 can be used for diseases such as liver cirrhosis. However, in the Bayer patent, the compound of structural formula 8 is used as a V1a and V2 receptor antagonist to inhibit the action of antidiuretic hormone, i.e., vasopressin, and its purpose is to treat heart failure and cardiovascular disorders and liver cirrhosis through kidney and hemodynamic effects.

[0065] On the contrary, in the present invention, the purpose is that the compound of formula 8 inhibits K Ca The expression of 3.1 channel is used to solve the direct cause of fibrosis, so the present invention is significantly different from the liver cirrhosis treatment produced by the Bayer patent.

[0066] The pharmaceutical composition of the present invention comprises a pharmaceutically acceptable salt of the compound of formula 1. Here, "pharmaceutically acceptable salt" generally refers to metal salts, organic base salts, inorganic acid salts, organic acid salts, basic or acidic amino acid salts, etc.

[0067] The pharmaceutical composition of the present invention includes all solvates and water compounds of the compound of structural formula 1, including racemic salts and all possible stereoisomers, and may further include the crystalline form or non-crystalline form of each compound.

[0068] The pharmaceutical composition of the present invention can be prepared into dosage forms such as tablets, pills, acid preparations, granules, capsules, suspensions, liquid preparations, emulsions, syrups, aerosols, and sterile injections by conventional methods. It can be administered orally or parenterally according to the purpose of use. When administered parenterally, it can be applied externally to the skin or injected intraperitoneally, rectally, subcutaneously, intravenously, intramuscularly, or by chest injection.

[0069] The dosage of the pharmaceutical composition of the present invention can be changed according to the patient's weight, age, gender, health status, diet, administration time, administration method, excretion rate and severity of the disease. The preferred daily dosage is 0.2 to 20 mg / kg based on the active ingredient, and more preferably 0.5 to 10 mg / kg, once or twice a day, but is not limited thereto.

[0070] Next, the pharmacological effects of the phenylalkylcarbamate compounds of the present invention will be described.

[0071] 1) Experimental methods

[0072] 1-1) Culturing fibroblasts

[0073] Fibroblasts (CRL-2795; American Type Culture Collection, VA) were cultured in Dulbecco's Modified Eagle Medium (Hyclone, Logan, UT). All cells were maintained at 37 °C under 5% carbon dioxide in a humidified condition. The fibroblasts cultured as such were exposed to an inflammatory inducer, i.e., LPS (lipopolysaccharides), and TGF, a fibrotic inducer, β , and the compounds of Structural Formulas 2 to 8 of the present invention (hereinafter referred to as SF-2 to SF-8 compounds) for 24 hours, and then the anti-inflammatory and anti-fibrotic effects were tested.

[0074] 1-2) Establishing a mouse model of pulmonary fibrosis

[0075] To verify the fibrotic inhibitory effect of the SF-2 compound of the present invention, the following experiment was carried out. First, C57BL / 6 wild-type mice (purchased from Orient Bio) were divided into three groups of 6 - 8 mice each, and were used as a disease induction group, a drug administration group, and a normal control group, respectively. The test mice in each group were treated as follows.

[0076] (1) Disease induction group: 1.5 units of bleomycin was injected into the airways of the test mice to induce pulmonary fibrosis. In addition, the same amount of distilled water as the SF-2 compound injected into the following drug administration group was intraperitoneally injected five times a week (100 mg / kg). In the figures, BLM (bleomycin) represents this disease induction group.

[0077] (2) Drug administration group: 1.5 units of bleomycin was injected into the airways of the test mice, and then the SF-2 compound of the present invention was intraperitoneally injected five times a week (100 mg / kg).

[0078] In the figures, BLM+SF-2 represents these drug administration groups.

[0079] (3) Normal control group: The same amount of distilled water as the bleomycin injected into the disease induction group was injected into the airways. In addition, the same amount of distilled water as the SF-2 compound injected into the drug administration group was intraperitoneally injected five times a week. In the figures, C or Control represents this normal control group.

[0080] The mouse models of each group were treated with drugs for four weeks according to the described method, then injected with an overdose of anesthetic and immediately sacrificed, and then the lungs were removed for the following tests.

[0081] 1-3) Establish a mouse model of fatty liver disease

[0082] In order to verify the therapeutic effect of the SF-2 compound of the present invention on hepatitis and fibrosis, the following experiment was carried out. First, C57BL / 6 natural mice (purchased from Orient Bio) were divided into three groups of 6-8 mice each, and were used as the disease induction group, the drug administration group and the normal control group respectively. The following treatments were carried out on the test mice of each group.

[0083] (1) Disease induction group: The test mice were fed with CDAHFD diet (choline deficient, L-amino-acid-defined, high-fat diet with 0.1% methionine. A06071302, Research Diets, New Brunswick, NJ) to induce fatty liver disease and fibrosis. In addition, the same amount of distilled water as the SF-2 compound injected into the following drug administration group was administered to the mice through an oral tube five times a week. CDAHFD in the attached figure indicates this disease induction group.

[0084] (2) Drug administration group: The SF-2 compound of the present invention was administered to the test mice together with the CDAHFD diet five times a week (100 mg / kg / day). CDAHFD+SF-2 in the attached figure indicates these drug administration groups.

[0085] (3) Normal control group: The test mice were fed with a normal diet. In addition, the same amount of distilled water as the SF-2 compound administered to the drug administration group was administered to the mice through an oral tube five times a week. C or Control in the attached figure indicates these normal control groups.

[0086] The mouse models of each group were treated with drugs for 16 weeks according to the described method, then administered an overdose of anesthetic and immediately sacrificed, and then the livers were removed for the following tests.

[0087] 1-4) Preparation of paraffin tissue specimens of lung and liver tissues and observation of morphological changes

[0088] After fixing the lung and liver tissues removed from the mouse models with paraformaldehyde solution, they were cut into a thickness of 1 to 2 mm. The cut tissues were embedded in paraffin, cut into a thickness of 4 μm, then the paraffin was removed with xylene, and then the xylene was removed with ethanol, and then washed with tap water to prepare paraffin tissue specimens.

[0089] The immunohistochemistry of the fibrosis marker, i.e., collagen, in the lung and liver tissues was performed using Masson's trichrome staining method.

[0090] 1 - 5) Real time PCR analysis

[0091] In the lung and liver tissues excised from the mouse model, the mRNA expression levels of inflammatory or fibrosis factors were measured by real time polymerase chain reaction. The RNA of the tissues was extracted using Trizol reagent (Molecular Research Center, Cincinnati, OH), and after synthesizing single-stranded cDNA using BcaBEST polymerase (Takara Shuzo), polymerase chain reaction was performed.

[0092] The primer sequences (sequence numbers 1 to 30) of the inflammatory cytokine and fibrosis marker used at this time are shown in Table 1 and Table 3 below.

[0093] [Table 2] Primer sequences of cytokines inducing inflammation

[0094]

[0095] [Table 3] Primer sequences of fibrosis markers

[0096]

[0097] 1 - 6) Electrophysiological analysis

[0098] The whole cell current through the cell membrane in isolated and cultured single fibroblasts was measured by the patch-clamp technique. In whole-cell voltage clamp cells, a voltage ramp from -100 mV to +100 mV was applied through a microglass electrode, and the generated current was amplified by an amplifier (EPC-10, HEKA, Lambrecht, Germany) and then recorded at a sample extraction rate of 1 to 4 kHz.

[0099] The standard external solution contains 150 mM of NaCl, 6 mM of KCl, 1.5 mM of CaCl2, 1 mM of MgCl2, 10 mM of HEPES, 10 mM of glucose, pH 7.4 (titrated with NaOH), and the microelectrode (pipette) solution contains 40 mM of KCl, 100 mM of K-aspartate, 2 mM of MgCl2, 0.1 mM of EGTA, 4 mM of Na2ATP, 10 mM of HEPES, pH 7.2 (titrated with KOH) solution. The free Ca in the pipette solution 2+ concentration is, in the presence of 5 mM of EGTA, by adding an appropriate amount of Ca 2+ , adjusted to 1 μM (calculated as CaBuf, G. Droogmans, Leuven and Belgium).

[0100] K Ca 3.1 current was isolated as follows. In whole-cell voltage-clamped cells, 1 μM of Ca was injected through a glass electrode 2+ , and 1-ethyl-2-benzimidazolinone (1-EBIO, 100 μM) used to activate the K Ca 3.1 current was applied, and in the recorded current, the current inhibited by the K Ca 3.1 channel inhibitor, TRAM-34 (10 μM), was determined as the K Ca 3.1 current, and the recorded current was normalized by dividing by the cell capacitance.

[0101] 1 - 7) Statistical analysis

[0102] The experimental results were expressed as mean ± standard error (S.E.M). Statistical analysis was performed by Student's t-test, and a significant difference was determined at a significance level of less than 0.05.

[0103] 2) Experimental results using cultured cells

[0104] 2 - 1) Effect of the SF-2 compound on the K Ca 3.1 current

[0105] Attached Figure 1 shows the effect of the SF-2 compound on the K Ca 3.1 channel current in the fibroblasts. The K 2+ activated by intracellular Ca Ca and 1-EBIO, the 3.1 current was inhibited by SF-2 in a concentration-dependent manner.

[0106] K Ca3.1 The magnitude of the current was 37.49 ± 1.51 pA / pF in cells not exposed to the SF-2 compound, and was 26.65 ± 1.89 mV / pF, 11.69 ± 1.66 mV / pF, 7.12 ± 1.33 mV / pF, 1.87 ± 0.24 mV / pF, 1.95 ± 0.44 mV / pF in cells exposed to 10, 30, 100, 300, 1000 nM of the SF-2 compound respectively, showing that the K Ca 3.1 current decreased significantly.

[0107] 2-2) Effects of SF-3 to SF-8 compounds on K Ca 3.1 current

[0108] Appendix Figure 2 shows the effects of SF-3 to SF-8 compounds on the K Ca 3.1 channel current in the fibroblasts. The K 2+ 3.1 current activated by intracellular Ca Ca and 1-EBIO was inhibited by SF-3 to SF-8 compounds in a concentration-dependent manner.

[0109] 2-3) Inhibitory effects of SF-3 to SF-8 compounds on inflammation

[0110] Appendix Figure 3 shows the inhibitory effects of SF-2 to SF-8 compounds on LPS-induced inflammation in the fibroblasts. The inflammatory induction effect by LPS was determined by the mRNA expression level of inflammatory markers. After exposing the fibroblasts to LPS (10 ug / ml) for 24 hours, the mRNA levels of IL6 and others as inflammatory markers increased.

[0111] In addition, after exposing the fibroblasts to one of the SF-2 compound to SF-8 compounds together with LPS for 24 hours, the mRNA expression level of the inflammatory markers decreased. Appendix Figure 3 The test results in the figure are marked as mean ± SE, indicating n = 6 - 8, ## < 0.01 (normal control group: disease induction group), ** < 0.01 (disease induction group: drug administration group).

[0112] 2-4) Inhibitory effects of SF-3 to SF-8 compounds on fibrosis

[0113] Appendix Figure 4 shows the fibrosis inhibitory effects of SF-2 to SF-8 on TGF β induced fibrosis in fibroblasts. Here, the fibrosis induction effect by TGF β was determined by the mRNA expression level of fibrosis markers. After exposing the fibroblasts to TGFβ After exposure for 24 hours in (10 ng / ml), the mRNA levels of α-smooth muscle actin (α-SMA), collagen 1α (Col1α), and collagen 3α (Col3α), which are fibrosis markers, increased.

[0114] In addition, after fibroblasts were exposed to one of the SF-2 to SF-8 compounds for 24 hours together with TGF β the mRNA expression levels of inflammatory markers decreased. In the attached Figure 4 the test results are marked as mean ± SE, indicating n = 6 - 8 ## < 0.01 (normal control group: disease-induced group), ** < 0.01 (disease-induced group: drug-administered group).

[0115] 3) Effects of the SF-2 compound on a mouse model of lung disease

[0116] 3-1) Results of histological or immunohistochemical analysis

[0117] Paraffin tissue specimens were prepared from the lung tissues excised from the mouse model, and immunohistochemical staining of collagen, a fibrosis marker, was performed. In the attached Figure 5 is the result of Masson's trichrome staining of the lung tissue, showing an increased degree of fibrosis in the disease-induced group (BLM) compared with the normal control group (Sham control), while a decreased degree of fibrosis in the drug-administered group (BLM + SF-2).

[0118] Therefore, it can be confirmed that the SF-2 compound of the present invention has an inhibitory effect on pulmonary fibrosis. In the attached Figure 5 "BLM" in it represents the disease-induced group treated with 1.5 units of bleomycin, and "BLM + SF-2" represents the drug-administered group in which the SF-2 compound at 10 mg / kg / day or 100 mg / kg / day is administered together with 1.5 units of bleomycin.

[0119] 3-2) Results of analysis of mRNA expression of fibrosis markers

[0120] To confirm the effect of the SF-2 compound on the mRNA expression of fibrosis markers, i.e., Col1α (collagen 1α), Col3α (collagen 3α), and α-SMA (α-smooth muscle actin), in the mouse model, RT-PCR (reverse transcription-polymerase chain reaction) detection of these fibrosis markers was performed.

[0121] As Figure 6 shown, compared with the normal control group (C), the mRNA expression levels of Col1α, Col3α, and α-SMA in the lung tissues of the disease-induced group (BLM) increased significantly, indicating the development of fibrosis. However, in the drug-administered group (BLM+SF-2), the mRNA expression levels of Col1α, Col3α, and α-SMA decreased significantly. Therefore, it can be confirmed that fibrosis was inhibited. These results suggest that the SF-2 compound of the present invention has an inhibitory effect on pulmonary fibrosis. Figure 6 The test results in

[0122] 4) Experimental results on a mouse model of liver disease

[0123] 4-1) Histological or immunohistochemical analysis

[0124] Attached Figure 7 is the result of Masson's trichrome staining showing the collagen fibers in the liver tissue. The liver tissue of the normal control group (Sham control) is in a healthy state without fibrosis. In contrast, the liver tissue of the disease-induced group (CDAHFD) is stained blue, indicating that fibrosis is in progress. However, in the liver tissue of the drug-administered group (CDAHFD+SF-2) where the SF-2 compound was administered to the disease-induced group, the degree of blue staining decreased significantly, and thus a significant inhibition of fibrosis was observed.

[0125] 4-2) Analysis results of the mRNA expression of fibrosis markers

[0126] To confirm the effect of the SF-2 compound on the mRNA expression of fibrosis markers in the mouse model, RT-PCR (reverse transcription-polymerase chain reaction) detection of fibrosis markers, namely Col1α (collagen 1α, type I collagen), Col3α (collagen 3α, type III collagen), and α-SMA (α-smooth muscle actin), was performed.

[0127] As Figure 8 shown, compared with the normal control group (C), the mRNA expression levels of Col1α, Col3α, and α-SMA in the lung tissues of the disease-induced group (CDAHFD) increased significantly, indicating that fibrosis has occurred. However, in the drug-administered group (CDAHFD+SF-2), the mRNA expression levels of Col1α, Col3α, and α-SMA decreased significantly. Therefore, it can be confirmed that fibrosis was inhibited.

[0128] These results can indicate that the SF-2 compound of the present invention has an inhibitory effect on pulmonary fibrosis. Figure 8 The test results in are marked as mean ± SE, indicating n = 6, * < 0.05, ** < 0.01.

[0129] 5) Evaluation of experimental results

[0130] According to the experimental results described above, it can be confirmed that the compound of Structural Formula 2 of the present invention, namely Soriamphetol, has an inhibitory effect on the mouse model of bleomycin-induced pulmonary fibrosis and the mouse model of CDAHFD diet-induced liver fibrosis, respectively.

[0131] The compounds of Structural Formulas 2 to 8 of the present invention have been confirmed to have inflammatory and fibrosis inhibitory effects on fibroblasts induced by LPS and fibroblasts induced by TGF β induced fibrosis, respectively. These results indicate that the compounds of Structural Formulas 2 to 8 of the present invention have an inhibitory effect on the expression of K Ca 3.1 channels.

[0132] In addition, as described in the background art above, K Ca 3.1 channels play a very important role in the development of autoimmune diseases and cancers in addition to inflammatory and fibrotic diseases. Therefore, various research groups and pharmaceutical companies around the world have used K Ca 3.1 channel inhibitors such as TRAM-34 and senicapoc in the development of drugs for the treatment of autoimmune diseases, inflammatory and fibrotic diseases, and cancers.

[0133] As described above, inhibiting K Ca 3.1 channels can not only inhibit inflammation and fibrosis, but also inhibit the development of autoimmune diseases and cancers. Therefore, it can be fully shown that the compounds of Structural Formulas 2 to 8 of the present invention also have effects on the treatment of autoimmune diseases and cancers.

[0134] In the present invention, due to practical limitations, the above-mentioned experiments could not be carried out on all compounds belonging to Structural Formula 1. However, in view of the chemical activity and metabolic mechanism in vivo, it can be speculated that the compounds of Structural Formula 1 all have pharmacological effects similar to those of the compounds of Structural Formulas 2 to 8.

Claims

1. Use of a composition in the preparation of a medicament for treating KCa3.1 channel-mediated diseases, characterized in that, the composition is (R)-2-amino-3-phenylpropyl carbamate hydrochloride; Said K Ca 3.1 Channel-mediated diseases are fibrotic diseases including liver fibrosis or pulmonary fibrosis.

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