KR101084729B1 - COMPOSITION FOR INHIBITING TGF-β COMPRISING ISOXAZOLE DERIVATIVES - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing 3-phenyl-3a,6a-dihydrothieno[2,3-d]isooxazole-4,4-dioxide is provided to suppress TFG-beta and to prevent or treat disease caused by TGF-beta overexpression. CONSTITUTION: A pharmaceutical composition for preventing or treating cancer, inflammation, fibrosis, or immune diseases contains isooxazole 4,4-dioxide of chemical formula A or pharmaceutically acceptable salt thereof. The isooxazole 4,4-dioxide compound is 3-phenyl-3a,6a-dihydrothieno[2,3-d]isooxazole 4,4-dioxide. A pharmaceutical composition for anticancer contains isooxazole 4,4-dioxide or pharmaceutically acceptable salt thereof. The anticancer agent is cisplatin, doxorubicin, fluorouracil, paclitaxel, or gemcitabine.

Description

Composition for inhibiting TGF-β activity including isoxazole derivatives {Composition for inhibiting TGF-β comprising isoxazole derivatives}

The present invention relates to 3-phenyl-3a, 6a-dihydrothieno [2,3-d] isoxazole-4,4-dioxide (3-phenyl-3a, 6a-dihydrothieno [2,3-d] isoxazole. It relates to a composition for inhibiting transforming growth factor-β (TGF-β) activity, including 4,4-dioxide) or a pharmaceutically acceptable salt thereof, and a use thereof.

The present invention is derived from research conducted as part of the nuclear research and development project of the Ministry of Education, Science and Technology.

[Job unique number: 2011-0002299, Title: Development of radiation combination therapy and efficacy evaluation technology]

TGF-β is expressed in almost all cells as a multifunctional cytokine that regulates various cell biological processes such as cell growth, death, differentiation, immunity, metastasis or extracellular matrix accumulation. Overexpression of TGF-β results in excessive accumulation of extracellular matrix (ECM), which promotes fibrosis / sclerosis in organs. Overexpression of TGF-β has been reported in most diseases related to fibrosis / curing, such as diabetic nephropathy, liver, lungs, interest, etc., which have a poor prognosis and a high mortality rate. 0998936A1; Pancreas, 2005, 30 (3), e71-9).

TGF-β also acts as a tumor suppressor in the early stages of the tumor, but later as a tumor-inducing factor. TGF-β is excessively produced from tumors, and directly inhibits natural killer (NK) cells or macrophages responsible for the initial immune response, and induces immaturation of dendritic cells, thereby reducing tumor antigen presentation and T Induction of regulatory lymphocytes is well known to inhibit immune activity and promote tumor growth, invasion and metastasis. In addition, TGF-β has been reported to be involved in angiogenesis indirectly by upregulating vascular endothelial growth factor (VEGF) production (Am. J. Respir Crit Care Med. 2002, 165, 88-94; Cancer Res. 2003 , 63, 1083-1092). Therefore, TGF-β can promote tumor growth, invasion, metastasis, etc. by angiogenesis and immunosuppressive activity. TGF-β is significantly increased and metastasis to lung is increased in anti-cancer drugs such as irradiation or doxorubicin in breast cancer transplant mouse model. Inhibiting activity has been shown to be effective (J Clin Invest. 1993, 92, 2569-2576; Curr. Cancer Drug targets 2006, 75, 565-578). In particular, recent studies have shown that TGF-β inhibition inhibits tumor cell susceptibility by inhibiting ATM (ataxia telangientasia-mutated gene), which repairs chromosomal duplexes caused by various stimuli such as radiation, anticancer agents, ultraviolet rays, and free radicals. It has been reported that it may be increased (Kirshner J. et al. Cancer Research, 2006, 66 (22), 10861-10869) .TGF-β activity inhibitors may be effective in combination with radiation or anticancer drugs. It is thought to be.

Inhibitors of TGF-β have been reported to be effective in the treatment of diseases such as fibrosis, cirrhosis, Crohn's disease, scleroderma, cardiomyopathy, rheumatoid arthritis, chronic graft versus host disease or cancer caused by TGF-β overexpression. For example, antibodies against TGF-β may include glomerulonephritis (Nature, 1990, 346, 371-374), scarring of nerves or skin (Eur. J. Neurosci. 1994, 6, 355-363; Lancet, 1992, 339 , 213-214), pulmonary fibrosis (Thorax 1993, 48, 959-966), radiation-induced fibrosis (US Pat. No. 5,616,561), myeloid fibrosis, burns, Duputurn's contracture, gastric ulcer, rheumatoid arthritis (J Exp. Medicine, 1993, 177, 225-230).

Connective tissue growth factor (CTGF) acts as a downstream signaler and a common mediator of the TGF-β signaling pathway. It is a glycoprotein that binds to multifunctional heparin and is expressed at low levels in normal conditions, but is a fibrotic disease. Increases significantly. CTGF regulates various processes related to fibrosis, including cell proliferation, migration, adhesion, survival, and secretion of extracellular matrix, and mesenchymal stem cells, hepatic stellate cells, podocytes, and mesangials involved in fibrosis. ), Cells, renal epithelial cells, type 2 alveolar epithelial cells, mesothelial cells, vascular smooth muscle cells, endothelial cells, cardiomyocytes, pericytes, fibroblasts, and the like. CTGF also induces the secretion of proinflammatory factors such as chemical principal components and activates NF-κB to infiltrate inflammatory cells in fibrotic lesions. NF-κB (nuclear factor-κB) is a factor that regulates the transcription of more than 200 target genes, and is involved in the regulation of innate, adaptive, and inflammatory responses, wound healing, and regulation of cell fate and function. do. Therefore, CTGF, which is involved in the subsignal transmission of TGF-β, may also be an excellent target for the treatment of fibrosis and immune diseases (Kidney Int. 2006, 69, 248-256).

Therefore, the development of drugs that can regulate the response of TGF-β plays an important role in the pathogenesis of various diseases is constantly required. Accordingly, the present inventors completed the present invention by searching for a substance exhibiting anti-inflammatory, anti-fibrosis, immunomodulatory effect, and anticancer auxiliary effect by inhibiting the activity of TGF-β.

It is an object of the present invention to provide isoxazole derivatives that inhibit TGF-β activity.

It is another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of cancer, inflammation, fibrosis or immune disease, including isoxazole derivatives that inhibit TGF-β activity.

Still another object of the present invention is to provide an anticancer adjuvant composition comprising an isoxazole derivative that inhibits TGF-β activity.

In order to achieve the above object, the present invention provides 3-phenyl-3a, 6a-dihydrothieno [2,3-d] isoxazole 4,4-dioxide of formula (A) or a pharmaceutically acceptable thereof. It provides a pharmaceutical composition for inhibiting TGF-β activity, including a possible salt,

In the above,

X and Y each independently represent hydrogen or halo,

R are each independently hydrogen, C ₁ -C ₄ alkyl, or, hydroxy, halo, C ₁ -C ₄ alkyl, and C ₁ -C ₄ halo-alkyl, optionally substituted by a moiety selected from the group consisting of C ₁ -C ₄ alkyl

Halo is fluoro, chloro, bromo, or iodo.

TGF-β is a multifunctional cytokine that is very important for regulating immune activity, mediates inflammation and fibrosis, and acts as a tumor-causing factor with increased secretion in the tumor environment. Thus, agents that inhibit TGF-β activity can be used for the prevention or treatment of various diseases through immunomodulatory, anticancer, anti-inflammatory and antifibrotic effects. Fibrosis is inhibited when the compound of formula A is treated to cells undergoing fibrosis by TGF-β, and NF-κB induced by phospholipid polysaccharide (LPS) when the compound of formula A is treated to hepatic stellate cells. It was confirmed that the activation of was suppressed. In addition, when the compound of formula A was treated to the cells causing inflammation, a significant decrease in inflammatory cytokines was observed.

In one embodiment of the present invention, the isoxazole 4,4-dioxide compound is 3-phenyl-3a, 6a-dihydrothieno [2,3-d] isoxazole 4,4- May be a dioxide.

 Formula I

The composition according to one embodiment of the present invention is a fibrosis or cirrhosis, multiple sclerosis, cardiomyopathy, Crohn's disease, sclerosis, glomerulonephritis, postoperative adhesion, keloid and hypertrophic scar formation, proliferative vitreous of various organs associated with overexpression of TGF-β. Use for the prevention and treatment of diseases associated with overexpression of TGF-β, including but not limited to retinopathy, corneal injury, chronic graft versus host disease, angiogenesis, tumors, burns, inflammation, rheumatoid arthritis and immune diseases Can be.

In one embodiment of the invention, the composition can be used for the prevention or treatment of cancer, inflammation, fibrosis or immune disease.

In one embodiment of the invention, the fibrosis may be fibrosis of the lung, liver, kidney or pancreas.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are conventionally used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, silicic acid Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. Do not. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and additives are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical compositions of the present invention are prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation of the pharmaceutical composition of the present invention is suitable for pharmaceutical preparations, including powders, granules, tablets, capsules, suspensions, oral formulations such as emulsions, syrups, aerosols, external preparations such as ointments, creams, suppositories, and sterile injectable solutions. It may be a formulation, and may further include a dispersant or stabilizer.

The present invention also provides an anticancer agent comprising 3-phenyl-3a, 6a-dihydrothieno [2,3-d] isoxazole 4,4-dioxide or a pharmaceutically acceptable salt thereof. Providing an auxiliary pharmaceutical composition,

In the above,

X and Y each independently represent hydrogen or halo,

R are each independently hydrogen, C ₁ -C ₄ alkyl, or, hydroxy, halo, C ₁ -C ₄ alkyl, and C ₁ -C ₄ halo-alkyl, optionally substituted by a moiety selected from the group consisting of C ₁ -C ₄ alkyl

Halo is fluoro, chloro, bromo, or iodo.

Inhibition of TGF-β activity has been shown to increase the sensitivity of cancer cells to anticancer agents, including radiation. Administration of the compound of Formula (A) prior to radiation therapy or administration of an anticancer agent such as doxorubicin enhanced the anticancer effect, that is, the killing effect of cancer cells.

The term "C ₁ -C _{4 as} used herein Alkyl "is straight and branched C ₁ -C ₄ By alkyl, it is to be understood to include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, sec-butyl, and isomers thereof.

The term "C ₁ -C _{4 as} used herein Haloalkyl "should be understood to mean that at least one hydrogen in the straight and branched alkyl as defined above is substituted in the same way or differently with halogen. For example, C ₁ -C ₄ Haloalkyl is, for example, chloromethyl, fluoropropyl, fluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, pentafluoroethyl, bromobutyl, trifluoromethyl, Iodoethyl, and isomers thereof.

As used herein, the term "pharmaceutical composition for inhibiting TGF-β activity" refers to a composition having an effect of inhibiting the expression of TGF-β or inhibiting its activity.

As used herein, the term "anticancer adjuvant pharmaceutical composition" means a composition that synergistically increases the effect of anticancer agents by increasing the sensitivity of cancer cells to anticancer agents when administered in combination with anticancer agents.

As used herein, the term “anticancer agent” means an agent that inhibits the proliferation of cancer cells or kills cancer cells, and includes radiation such as gamma rays, chemotherapeutic agents such as cisplatin, doxorubicin, vinblastine, and the like.

In one embodiment of the present invention, the anticancer agent may be selected from the group consisting of radiation, cisplatin, doxorubicin, vinblastine, fluorouracil, irinotecan, paclitaxel, vinorelbine, and gemcitabine, but is not limited thereto.

In one embodiment of the present invention, the isoxazole 4,4-dioxide compound is 3-phenyl-3a, 6a-dihydrothieno [2,3-d] isoxazole 4,4- Dioxide can be:

 Formula I

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are intended to illustrate the invention and should not be construed as limiting the invention.

The composition of the present invention can inhibit TGF-β activity, prevent or treat diseases caused by TGF-β overexpression, including cancer, inflammation, fibrosis or immune diseases, and increase the sensitivity of cancer cells to anticancer agents to increase anticancer activity. Can enhance the effect.

Figure 1 shows a system for screening TGF-β activity inhibitors by measuring luciferase activity constructed by stably transforming HEK 293 cells with a PAI promoter and SMAD binding site in response to TGF-β. It is schematic.
Figure 2 shows that luciferase activity is increased by TGF-β stimulation using the search system used in one embodiment of the present invention, confirming the effectiveness of the search system. In the figure, pGL2 refers to a cell line transformed with a vector containing only a gene encoding luciferase, 3TP-pGL2 refers to a cell line transformed with a vector containing a TGF-β response element (TRE), and TGF-b Denotes 3TP-pGL2 cell lines, each stimulated with the indicated concentrations of TGF-β.
Figure 3 shows that compounds according to one embodiment of the invention inhibit TGF-β signaling in a concentration dependent manner.
4 shows that the compounds according to one embodiment of the invention do not have their own toxicity to HEK 293 cells.
Figure 5 shows the effect of the compound according to one embodiment of the present invention on the proliferation of human hepatic stellate cells.
Figure 6 shows that the compound according to one embodiment of the present invention effectively inhibits fibrosis due to the increase of alpha-smooth muscle actin and extracellular matrix collagen and fibronectin in human hepatic stellate cells.
Figure 7 shows that the compound according to one embodiment of the present invention concentration-dependently inhibits NF-kB transcriptional activity stimulated by LPS in hepatic stellate cells.
Figure 8 shows that the effect of inhibiting NF-kB transcriptional activity by the compound according to an embodiment of the present invention in hepatic stellate cells is not due to apoptosis.
9 shows that the compounds according to one embodiment of the present invention inhibit fibrosis of human normal lung fibroblasts induced by TGF-β and reduce expression of CTGF and PAI-1.
10 shows that the compounds according to one embodiment of the invention effectively inhibit radiation induced pulmonary fibrosis.
Figure 11 shows that the compounds according to one embodiment of the present invention have an anti-inflammatory effect by inhibiting inflammatory cytokine activity in LPS-stimulated macrophages.
FIG. 12 shows that the compounds according to one embodiment of the present invention effectively inhibit fibrosis in a bleomycin-induced pulmonary fibrosis mouse model.
Figure 13 shows that fibrotic markers in bleomycin-induced pulmonary fibrosis tissue were significantly reduced by the compounds according to one embodiment of the invention.

Example  One. TGF Screening of -β-Activity Inhibitors

Cells stably transformed with luciferase expressing plasmids capable of measuring the effect on TGF-β activity were used to search for agents that inhibit TGF-β activity.

A plasmid with a SMAD binding site and a plasminogen activator inhibitor (PAI) promoter site, which plays an important role in TGF-β activity, was donated by Professor Carlos Arteaga of Vanderbilt University, USA to provide a pGL2-null reporter plasmid ( Promega, USA). Figure 1 shows the schematic structure of the 3TP-pGL2 plasmid containing the TGF-β Response Element (TRE), PAI-1 promoter and luciferase gene, which are SMAD binding sites. Using Lipofectamine 2000 (Invitrogen, Calsbad, Calif., USA), the plasmid was transformed into HEK (Human Embryonic Kidney) 293 cells according to the manufacturer's instructions to effect TGF-β through the activity of luciferase. We built a system that can search for. Luciferase activity was measured using a luciferase assay kit (Promega, Medison, WI, USA). HEK 293 cells were purchased from the American Type Culture Collection (ATCC), DMEM medium (Dulbecco's modified Eagle's) with 10% fetal bovine serum (FBS), 100 units / ml penicillin, and 100 μg / ml streptomycin. medium) at 5% CO ₂ and 37 ℃.

Transformed HEK 293 cells were inoculated in 96-well plates at 20,000 cells per well in multiples of 3, incubated for 24 hours under the above conditions, and then TGF-β was added at concentrations of 0.5, 1, and 5 ng / ml, respectively. After incubation for 15 hours, firefly luciferase activity was measured. Luciferase activity was increased 30-40 fold in TGF-β treatment and showed optimal efficacy in 0.5ng / ml TGF-β treatment. 2 shows that luciferase activity is increased by TGF-β treatment.

The search system constructed above was used to investigate the effect of inhibiting TGF-β activity of isoxazole derivatives (hereinafter referred to as compounds). Add to the 96-well plate inoculated with the transformed HEK 293 cells so that the final concentration of the compound is 0, 2.5, 5, 10 μM, 0.5 ng / ml of TGF-β after 2 hours incubation and Compounds were searched by measuring luciferase activity for each well by the described method. 3 shows that compounds inhibit TGF-β activity in a concentration dependent manner.

In addition, to confirm that inhibition of luciferase activity is not by cell death but by TGF-β activity inhibitors, the CCK-8 survival assay kit (Dojindo, Japan) for each well in which luciferase activity was measured. ) And the absorbance at 450 nm with a spectrometer (Lab Systems, USA) to determine the number of cells. Values shown are mean values obtained from three independent experiments. 4 is a cytotoxicity test results of the compounds measured by the CCK-8 assay, respectively, no significant decrease in cell number was observed, thereby confirming that the TGF-β activity is not reduced by apoptosis.

Example  2. Ixoxazole  By derivative In hepatic stellate cells  Inhibition of differentiation into myofibroblasts

It was tested whether the isoxoxazole derivative of formula I (hereinafter referred to as compound) which was found to effectively inhibit TGF-β activity in Example 1 can inhibit fibrosis of hepatic stellate cells. Hepatic stellate cells (HSCs), the major target cells of liver fibrosis, were used by SL Friedman of Mount Sinai, USA, to receive HSC-T6 cells from stable cell lines. HSC-T6 cells were cultured at 5% CO ₂ , 37 ° C. in DMEM medium supplemented with 10% fetal calf serum (FBS), 100 units / ml penicillin, and 100 μg / ml streptomycin. To determine whether the compound directly affects the proliferative capacity of hepatic stellate cells, the compound was incubated in serum-free medium for 24 hours and then treated with the compounds at concentrations of 0, 1, 10, and 20 μM 2 hours before serum addition. . Subsequently, after 48 hours of further incubation under the above conditions, the number of cells was measured by using a CCK-8 survival assay kit and measuring absorbance at 450 nm with a spectrometer (Lab Systems, USA). As shown in FIG. 5, in serum-free culture conditions, hepatic stellate cells are in a resting state, and then rapidly proliferate when serum is added, and the proliferation of hepatic stellate cells is suppressed in a concentration-dependent manner when the compound is treated. Recovered.

In addition, alpha-smooth muscle actin (α-SMA), which is commonly used as a molecular marker of fibrosis to confirm fibrosis of hepatic stellate cells, and type 1 collagen, an extracellular matrix substance. And fibronectin levels were confirmed using western blotting. Compounds were added to HSC-T6 cells cultured under the above conditions at different concentrations (0, 0.2, 1, 5 μM), and after 2 hours, TGF-β (5 ng / ml) was treated in a 5% CO ₂ , 37 ° C. incubator. Incubated. After 24 h incubation, RIPA buffer (50 mM Tris-Cl (pH 7.4), containing protease inhibitors (1 mM PMSF, 1 M / mL aprotinine, 1 g / mL leupetin, and 1 mM Na ₃ VO ₄ ), Cytoplasmic fractions were prepared by lysis with 1% NP-40, 150 mM NaCl, 1 mM EDTA), and proteins were isolated by performing a 4-10% gradient SDS-PAGE (Invitrogen). The protein separated on the SDS-PAGE gel was transferred to a nitrocellulose membrane (BioRad, Hercules, Calif., USA) and the membrane was blocked for 1 hour at room temperature with 5% fat free milk. Thereafter, anti-α-SMA antibody (Sigma, USA), anti-fibronectin antibody (Santa Cruz, USA), anti-type I collagen antibody (Santa Cruz, USA), and anti-GAPDH antibody (Ab Frontier, Korea) Was reacted to the membrane overnight at 4 ° C. Subsequently, the membrane was reacted with horseradish peroxidase (HRP) -conjugated secondary antibody at room temperature for 1 hour and then visualized using the ECL kit (Amersham, Inc.). FIG. 6 shows that compounds inhibit concentration dependently fibrosis with increasing alpha-smooth muscle actin and fibronectin in hepatic stellate cells.

Example  3. Ixoxazole  By derivative NF -κB activation inhibitory effect

Inflammatory cytokines are secreted due to excessive extracellular matrix production and NF-κB activation during liver damage, which induces fibrosis in which hepatic stellate cells differentiate into myofibroblasts. It was investigated whether the mechanism of inhibiting fibrosis by the compound of formula (I), which was found to effectively inhibit TGF-β activity in Example 1, was related to the inhibition of NF-κB activation.

Plasmids with NF-κB promoter sites were inserted into the pGL2-null reporter plasmid (Promega, USA). Using lipofectamine 2000 (Invitrogen, Calsbad, Calif., USA) as described in Example 1 above, the plasmid was transformed into HSC-T6 cells according to the manufacturer's instructions, thereby activating NF- through the activity of luciferase. We established a system that can detect κB activity. Transformed HSC-T6 cells contained 600 μg / ml G-418 (Invitrogen, USA) in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 units / ml penicillin, and 100 μg / ml streptomycin. And incubated at 5% CO ₂ and 37 ° C.

Transformed HSC-T6 cells were inoculated in 96-well plates at 10,000 cells per well in triplicate and incubated for 24 hours under these conditions. The method described above by incubating the compound of formula I in the cultured cells in different concentrations (0, 1.25, 2.5, 5, 10 μM), and incubated for 24 hours with the addition of 5 μg / ml of phospholipid polysaccharide (lipopolysaccharide, LPS) Luciferase activity was measured by. Under the same conditions, cell viability was confirmed using the CCK-8 Viability Assay Kit (Dojindo, Japan) as described in Example 1. As shown in FIG. 7, luciferase activity was increased by 2 to 2.5 times compared to before LPS treatment, and it was confirmed that the compound of Formula I inhibited LPS-induced NF-κB activation in hepatic stellate cells. 8 also shows that such inhibition of NF-κB activation is independent of decreased cell viability.

Example  4. Ixoxazole  Human by derivatives Pulmonary fibroblast  Fibrosis inhibition

It was tested whether the compound of formula (I), which was found to effectively inhibit TGF-β activity in Example 1, could inhibit fibrosis of human lung fibroblasts.

(1) inhibition of fibrosis induced by TGF-β

The CCD18-Lu cell line, a human normal lung fibroblast, was purchased from the American Type Culture Collection (ATCC), with 10% fetal bovine serum, 100 units / ml penicillin, and 100 μg / ml streptomycin, 1 mM sodium pyruvate. Incubated at 5% CO ₂ , 37 ° C for 24 hours in Modified Eagle's medium (MEM) medium containing 0.1 mM non-essential amino acids. Compounds of formula I were added to the cultured cells at different concentrations (0, 1, 5 and 10 μM) 2 hours before TGF-β (5ng / ml) treatment and incubated for 24 hours. Thereafter, Western blotting was performed as described in Example 2, and the degree of inhibition of differentiation of lung fibroblasts by the compound was confirmed by expression levels of α-SMA and fibronectin, which are fibrosis marker molecules. Figure 9 shows that as in hepatic stellate cells, the compound significantly reduces fibrosis marker expression in human lung fibroblasts in a concentration dependent manner.

Connective tissue growth factor (CTGF), a molecule involved in subcellular signal transduction of TGF-β, is also an important factor in inducing fibrosis, and reports on the interrelationship with TGF-β have been actively studied. Plasminogen activator inhibitor (PAI-1) is low in most cells and is involved in fibrinolysis and cell migration in steady state, but is significantly increased in fibrotic disease. It is widely known as a marker. Use of anti-CTGF antibodies (Santa Cruz, USA) and anti-PAI-1 antibodies (Santa Cruz, USA) to investigate the effect of compounds of formula (I) on increased expression of CTGF or PAI-1 by TGF-β Western blotting was performed. As shown in FIG. 9, the compounds significantly reduced CTGF and PAI-1 in a concentration-dependent manner.

(2) suppression of fibrosis caused by radiation

In addition to surgery, chemotherapy and radiation therapy are used as the main treatments for cancer patients, but few chemotherapy and radiation treatments have been reported to cause fibrosis in various organs as one of side effects. Rather than external stimulation of TGF-β, the radiation causing fibrosis was investigated internally by increasing TGF-β and free radicals, and it was confirmed whether the compound of formula (I) inhibited radiation-induced fibrosis. After culturing the lung fibroblasts as described in (1), the compound of formula (I) was treated at a concentration of 10 μM and incubated for 2 hours, followed by irradiation with 4 Gy dose and 48 hours later. Harvested. Thereafter, fibrotic markers as described in Example 2 were identified by western blotting from the cells. 10 shows that compounds of formula I significantly reduce α-SMA and collagen and fibronectin expression.

Example  5. Ixoxazole  By derivative LPS Inhibitory and anti-inflammatory effects of induced macrophage activation

Macrophages are the foremost immune cells responsible for the initial immune response to a variety of antigens. RAW 264.7 cells, a mouse macrophage line, were purchased from the American Type Culture Collection (ATCC) and added Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 units / ml penicillin, and 100 μg / ml streptomycin. Incubated in 5% CO ₂ and 37 ° C. incubator. After 2 hours of adding 10 μM of the compound of formula I to the cultured cells, 1 μg / ml of lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria, was further incubated for 7 hours, and then the cells were harvested to harvest TRIzol. Total RNA was isolated and quantified. Among them, 1 μg of RNA was taken to synthesize cDNA using reverse transcriptase, and a polymerase chain reaction (PCR) was performed to confirm the expression level of cytokines as a template. 30 at 94 ° C in the presence of a DNA polymerase to amplify the cDNA as a template and amplify the inflammatory cytokines IL-1β, IL-6, TNF-α, a nitric oxide synthase iNOS and a control β-actin, respectively. The polymerase chain reaction (PCR) was performed by repeating 30 cycles of seconds, 45 seconds at 55 ° C, and 45 seconds at 72 ° C. For amplification of IL-1β, IL-6, TNF-α, iNOS and β-actin, primers of SEQ ID NOS: 1 to 10 as shown in Table 1 below were used.

primer Sequence IL-1β Forward 5'-TGAAGGGCTGCTTCCAAACCTTTGACC-3 '(SEQ ID NO: 1) Reverse 5'-TGTCCATTGAGGTGGAGAGCTTTCAGC-3 '(SEQ ID NO: 2) IL-6 Forward 5'-TTCACAGAGGATACCACT-3 '(SEQ ID NO: 3) Reverse 5'-TAGCCACTCCTTCTGTGA-3 '(SEQ ID NO: 4) TNF-α Forward 5'-CAGGCAGGTTCTGTCCCTTTCA-3 '(SEQ ID NO: 5) Reverse 5'-CACTTGGTGGTTTGCTACGACG-3 '(SEQ ID NO: 6) iNOS Forward 5'-CCTTGTTCAGCTACGCCTTC-3 '(SEQ ID NO: 7) Reverse 5'-CTGAGGGCTCTGTTGAGGTC-3 '(SEQ ID NO: 8) β-actin Forward 5'-AGGCTGTGCTGTCCCTGTATGC-3 '(SEQ ID NO: 9) Reverse 5'-ACCCAAGAAGGAAGGCTGGAAA-3 '(SEQ ID NO: 10)

The amplification products obtained through the PCR reaction were electrophoresed on 1.5% agarose gel in the presence of ethidium bromide (dye) to measure the mRNA expression level of the cytokines. 11 shows the change in the expression level of inflammatory cytokines following treatment of the compound. LPS treatment significantly increased the inflammatory cytokines while IL-6, IL-1β and iNOS were significantly inhibited upon compound treatment.

Example  6. Ixoxazole  By derivative Bleomycin -Judo Pulmonary fibrosis  Inhibitory Effect of Fibrosis in a Mouse Model

Pulmonary fibrosis induced by bleomycin hydrochloride (Nippon Kayaku Co., Ltd., Japan) to confirm the antifibrotic effect of isoxazole derivatives in vitro and its effectiveness in vivo A mouse model was used. C57BL / 6 mice (Biolink, Korea), 6-8 weeks of bleomycin-sensitive strain, were used 6 per group. Induction of anesthesia by intraperitoneal injection of Zoletil 50 (Virbac, France) and xylazine, a combination of tiletamine and zolazepam, at 25 mg / kg and 5 mg / kg, respectively, followed by bleomycin suspended in saline (4 mg / kg) was injected intratracheally by surgical methods to build a pulmonary fibrosis model. Based on the day of bleomycin inoculation (day 0), the compound of formula I was diluted in physiological saline twice a day for two weeks, inoculated with 100 μl per mouse at a dose of 1 mg / kg. In the control group, the same amount of saline was administered by the same route. Lung tissues were collected at day 14 of bleomycin inoculation, and histological changes of lung tissues were observed through hematoxylin and eosin (H & E) staining, and collagen and α-SMA were examined to determine the expression level of fibrosis markers. Immunohistochemical staining and Western blotting were performed. Some of the recovered lung tissues were first fixed in 4% paraformaldehyde solution for 24 hours and then fixed in the formaldehyde solution by trimming the tissue and then fixed in the formaldehyde solution. Embedded in. Paraffin-embedded lung tissue was sectioned to 4 μm thickness using a microtome and then stained by tissue staining. As shown in FIG. 12, thickening of the alveolar wall was observed 14 days after the inoculation of bleomycin due to damage, edema, and inflammatory cell infiltration of alveolar epithelial cells. Lost. In addition, collagen accumulation was observed in the fibrotic lesion induced by bleomycin in Masson's Trichrome (Diagnostic Biosystems, USA) staining, and α- in immunostaining with anti-α-SMA antibody (Sigma, USA). SMA accumulation was confirmed. This confirmed that the compound of formula I inhibited pulmonary fibrosis by bleomycin. The remaining lung tissues were identified by Western blotting for expression of fibrosis markers and connective tissue growth factors in lung tissue proteins as described in Example 2 above. Figure 13 shows that the expression of collagen, α-SMA, connective tissue growth factor induced by bleomycin is reduced by the compound.

Attach an electronic file to a sequence list

Claims (6)

A pharmaceutical composition for the treatment or prophylaxis of cancer, inflammation, fibrosis or an immune disease, comprising isoxazole 4,4-dioxide of formula A or a pharmaceutically acceptable salt thereof:

Formula A
In the above,
X and Y each independently represent hydrogen or halo,
R are each independently hydrogen, C ₁ -C ₄ alkyl, or, hydroxy, halo, C ₁ -C ₄ alkyl, and C ₁ -C ₄ halo-alkyl, optionally substituted by a moiety selected from the group consisting of C ₁ -C ₄ alkyl, halo is fluoro, chloro, bromo, or iodo. The composition of claim 1, wherein the isoxazole 4,4-dioxide compound is 3-phenyl-3a, 6a-dihydrothieno [2,3-d] isoxazole 4,4-dioxide. . delete A pharmaceutical composition for adjuvant anticancer agents comprising isoxazole 4,4-dioxide of formula A or a pharmaceutically acceptable salt thereof:

Formula A
In the above,
X and Y each independently represent hydrogen or halo,
R are each independently hydrogen, C ₁ -C ₄ alkyl, or, hydroxy, halo, C ₁ -C ₄ alkyl, and C ₁ -C ₄ halo-alkyl, optionally substituted by a moiety selected from the group consisting of C ₁ -C ₄ alkyl, halo is fluoro, chloro, bromo, or iodo. The composition of claim 4, wherein the isoxazole 4,4-dioxide compound is 3-phenyl-3a, 6a-dihydrothieno [2,3-d] isoxazole 4,4-dioxide. . The composition of claim 4, wherein the anticancer agent is selected from the group consisting of radiation, cisplatin, doxorubicin, vinblastine, fluorouracil, irinotecan, paclitaxel, vinorelbine, and gemcitabine.

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