JP2006213644A - Ap-1 activity inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a highly safe AP-1 (activator protein-1) activity inhibitor carrying out prophylaxis or amelioration of diseases (metastasis, etc., of cancer) caused by activation of MMP (matrix metalloprotease), to provide a gene expression inhibitor and to provide a pharmaceutical composition. <P>SOLUTION: The AP-1 activity inhibitor comprises a compound represented by ascochlorin, 4-O-methylascochlorin, 4-O-carboxymethylascochlorin, 4-O-methoxycarbonyloxyascochlorin, 4-O-(2-ethoxycarbonyloxy)ethylascochlorin, 4-O-nicotinoylascochlorin, ascofuranone, 4-O-methylascofuranone, 4-O-carboxymethylascofuranone, 4-O-methoxycarbonyloxyascofuranone, 4-O-(2-ethoxycarbonyloxy)ethylascofuranone or 4-O-nicotinoylascofuranone or a compound producing the compound in vivo or a pharmacologically acceptable salt thereof as an active ingredient. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to an activator protein 1 (hereinafter sometimes referred to as “AP-1”) inhibitor containing ascochlorin, ascofuranone and derivatives thereof, and more specifically, renal diseases (glomerulonephritis), cerebral infarction, etc. Prevention and suppression of various diseases caused by increased AP-1, such as ischemic psychiatric diseases, central excitatory diseases (such as epilepsy), Alzheimer's disease, bone metabolic diseases, cancer, ischemic heart diseases (such as myocardial infarction) Alternatively, the present invention relates to an AP-1 inhibitor that is effective in improving symptoms and is highly safe.
The present invention also relates to an expression inhibitor of a gene whose expression is regulated by AP-1 containing ascochlorin, ascofuranone and derivatives thereof, particularly a matrix metalloproteinase (hereinafter sometimes referred to as “MMP”). Effective in preventing, suppressing, or improving symptoms of various diseases caused by increased MMP, such as cancer metastasis, ulceration, rheumatoid arthritis, osteoporosis, periodontitis, skin aging, etc. The present invention relates to an expression inhibitor such as MMP.

  Cancer is one of the leading causes of death in most developed countries. In Japan, about 30% of people die from cancer. Recent developments in surgery and radiation therapy have made it possible to almost eliminate the primary cancer itself. However, the high mortality rate is still thought to be because cancer has the property of causing metastasis, and it is currently very difficult to treat cancer that has become metastatic. I must say there is. Therefore, in addition to conventional primary lesion treatment methods, establishing a method for suppressing metastasis is considered to be one of the major objectives of cancer treatment.

  Under such circumstances, the mechanism of metastasis is gradually elucidated at the molecular level, and recently, a degradation system of an extracellular matrix has attracted attention as one of the metastasis processes.

  The steps of tumor cell invasion are as follows: 1) cell migration and adhesion to normal tissue, 2) lysis of normal tissue via MMP, 3) migration into normal tissue and increase of cells in satellite lesions It is known that there is.

  Cancer metastasis means that cancer cells that have detached from the primary site are scattered throughout the body in the bloodstream, engraft other organs, and begin to grow again in that tissue. In general, tumor tissue is surrounded by a dense extracellular matrix, so that the extracellular degradation of the extracellular matrix is required for cancer cells to migrate from the primary site. The extracellular matrix is composed of various polymers such as collagen, elastin, fibronectin, laminin, and proteoglycan. A major group of enzymes involved in the degradation of these extracellular substrates is MMP. At least 20 members are known as MMPs, which are divided into four families (collagenase, gelatinase, stromelysin, and membrane-bound MMP) depending on structure and substrate specificity. For example, MMP-9, also called gelatinase-B, is one of the important enzymes that degrade type IV collagen.

For MMP, the expression level is increased and the activation of the enzyme occurs when a blood vessel is born in cancer tissue or when the cancer metastasizes. For example, “Retinoid-mediated tumor wetting and matrix metalloproteinase synthesis” “Inhibition” (Schoenermark MP et al., “Annals New York Academy of Sciences”, Vol. 878, pp. 466-486, 1999; Non-Patent Document 1). Therefore, it is considered that a decrease in the expression level or inhibition of activity of these enzymes suppresses the invasion of cancer cells and suppresses metastasis. In addition, it is known that there are several cis-acting regulatory factors including an AP-1 binding site in the promoter of MMP-9.
AP-1 is a transcriptional regulator composed of a heterodimer of Jun and Fos, and is involved in the regulation of the expression of many genes in addition to MMP.

  In the process of infiltration of macromolecules by cancer cells, collagen degradation is an important step. For example, in order for cancer cells to enter and escape from blood vessels, it is necessary to degrade the type IV collagen present in the basement membrane of the blood vessels. The fact that the action of the enzyme is an important factor that determines the metastatic potential of cancer, “correlation between metastatic potential and basement membrane collagen degradation” (Liotta LA et al., Nature, Vol. 284, 67-68. Page, 1980; non-patent document 2). Gelatinase belonging to MMP is an enzyme produced by fibroblasts, endothelial cells, cancer cells and the like, and degrades substrates such as type IV collagen, gelatin and elastin. Therefore, for example, a substance having an inhibitory activity on gelatinase is expected to have an effect of suppressing angiogenesis and cancer metastasis in cancer tissue, and is considered useful for the prevention and treatment of cancer diseases.

  Furthermore, MMP plays a central role in the degradation of extracellular matrix not only in cancer diseases but also in various pathologies such as ulceration, rheumatoid arthritis, osteoporosis, periodontitis and the like. Matrix metalloprotease ”(Mitsutoshi Nakata, Yasunori Okada“ Respiration ”, Vol. 18, No. 4, pp. 365-371, 1999; Non-Patent Document 3). In skin, MMP, whose activity is enhanced by external stimuli such as ultraviolet rays, decomposes components important for maintaining the structure of the skin, and thus has recently attracted particular attention as an aging promoting factor activated by ultraviolet rays.

  MMP inhibitors have been actively screened in various fields because they are thought to be useful in the treatment and improvement of the above-mentioned diseases caused by MMP enhancement. Such inhibitors are described in JP-A-9-40552 (Patent Document 1), JP-A-11-147833 (Patent Document 2), and JP-A 2000-226311 (Patent Document 3).

Ascochlorin and ascofuranone are antiviral and antitumor substances found by the present inventors from the product of the filamentous fungus Ascochyta visiae (Japanese Patent Publication No. 45-9832; Patent Document 4). Moreover, it is described in Japanese Patent Publication No. 1-41624 (patent document 5) and Japanese Patent Publication No. 3-6138 (patent document 6) that the derivative has a blood glucose lowering effect. However, the MMP expression inhibitory action or AP-1 inhibitory action of ascochlorin, ascofuranone or a derivative thereof has not been described so far.

JP-A-9-40552 JP-A-11-147833 JP 2000-22631 A Japanese Examined Patent Publication No. 45-9832 Japanese Patent Publication No. 1-41624 Japanese Patent Publication No. 3-6138 Schoenermark MP, etc., `` Annals New York Academy of Sciences '', Vol. 878, pp. 466-486, 1999 Liotta LA et al., “Nature”, Vol.284, pp. 67-68, 1980 Mitsutoshi Nakata, Yasunori Okada "Respiration", Vol.18, No.4, pp. 365-371, 1999

  The present invention inhibits the expression of MMP produced by a highly metastatic malignant tumor cell line, and enhances the expression of genes regulated by AP-1, such as cancer metastasis, particularly the enhancement of MMP. An object of the present invention is to provide a highly safe gene expression inhibitor that can prevent or ameliorate a disease that occurs as a cause.

  The present inventors screened compounds having MMP expression inhibitory activity. As a result, the present inventors have found that ascochlorin, ascofuranone and derivatives thereof have an MMP expression inhibitory effect, and further, when elucidating the mechanism of MMP expression inhibition by these compounds, ascochlorin, ascofuranone and It has also been found that the derivative exhibits activity as an AP-1 inhibitor, and the present invention has been completed.

または一般式（II）：

（式中、Ｒ^１は、−ＣＨＯまたは−ＣＯＯＨを表し；Ｒ^２は、水素原子、炭素数１〜５のアルキル基、−（Ｃ_ｎＨ_２ｎ）ＣＯＯＲ’（ここで、ｎは１〜５の整数、Ｒ’は水素原子または炭素数１〜３のアルキル基を表す）または−ＣＯＲ''（ここで、Ｒ''はピリジル基、炭素数１〜３のアルキル基で置換されたアミノ基、ベンゼン環上にハロゲン原子を有するフェノキシアルキル基、またはベンゼン環上に炭素数１〜３のアルコキシ基もしくは炭素数１〜３のアルコキシカルボニル基を有するフェニル基を表す）を表す）
で示される化合物もしくは生体内でこの化合物を生じうる化合物またはその薬理学的に許容できる塩を有効成分とする、アクチベータープロテイン１（ＡＰ−１）活性阻害剤；
〔２〕下記の一般式（Ｉ）：

または一般式（II）：

（式中、Ｒ^１は、−ＣＨＯまたは−ＣＯＯＨを表し；Ｒ^２は、水素原子、炭素数１〜５のアルキル基、−（Ｃ_ｎＨ_２ｎ）ＣＯＯＲ’（ここで、ｎは１〜５の整数、Ｒ’は水素原子または炭素数１〜３のアルキル基を表す）または−ＣＯＲ''（ここで、Ｒ''はピリジル基、炭素数１〜３のアルキル基で置換されたアミノ基、ベンゼン環上にハロゲン原子を有するフェノキシアルキル基、またはベンゼン環上に炭素数１〜３のアルコキシ基もしくは炭素数１〜３のアルコキシカルボニル基を有するフェニル基を表す）を表す）
で示される化合物もしくは生体内でこの化合物を生じうる化合物またはその薬理学的に許容できる塩を有効成分とする、ＡＰ−１によって発現調節される遺伝子の発現阻害剤；
〔３〕前記ＡＰ−１によって発現調節される遺伝子が、マトリックスメタロプロテアーゼ（ＭＭＰ）遺伝子である、前記〔２〕記載の阻害剤；
〔４〕前記〔１〕〜〔３〕のいずれか記載の阻害剤を含有する、ＭＭＰ亢進性疾患またはＡＰ−１亢進性疾患の予防または治療のための医薬組成物；
〔５〕前記ＭＭＰ亢進性疾患またはＡＰ−１亢進性疾患が、変形性関節症、関節リウマチ、角膜潰瘍、歯周炎、ウイルス感染症、閉塞性動脈硬化症、動脈硬化性動脈瘤、粥状動脈硬化症、再狭窄、敗血症、敗血症ショック、冠状血栓症、異常血管新生、強膜炎、多発性硬化症、開放角緑内障、網膜症、増殖性網膜症、血管新生緑内障、翼状皮膚、角膜炎、水泡性表皮剥離、乾癬、糖尿病、腎炎、神経性疾患、炎症、骨粗鬆症、骨吸収、歯肉炎、腫瘍増殖、腫瘍血管新生、眼腫瘍、血管線維腫、血管腫、熱病、出血、凝固、悪液質、食欲不振、急性感染症、ショック、自己免疫症、マラリア、クローン病、髄膜炎、心不全、喘息性気道炎、動脈硬化症、ガンの転移、潰瘍形成、慢性関節リウマチ、骨粗鬆症、歯周炎、皮膚の老化、胃腸潰瘍、腎疾患（糸球体腎炎）、脳梗塞、虚血性神経系疾患、中枢興奮性疾患、てんかん、アルツハイマー病、骨代謝疾患、ガン、虚血性心疾患、心筋梗塞等からなる群から選択される１以上の疾患である、前記〔４〕記載の医薬組成物、
を提供する。 Therefore, the present invention
[1] The following general formula (I):

Or general formula (II):

(In the formula, R ¹ represents —CHO or —COOH; R ² represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, — (C _n H _2n ) COOR ′ (where n is 1 to 5). , R ′ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms) or —COR ″ (where R ″ is a pyridyl group, an amino group substituted with an alkyl group having 1 to 3 carbon atoms). And a phenoxyalkyl group having a halogen atom on the benzene ring, or a phenyl group having an alkoxy group having 1 to 3 carbon atoms or an alkoxycarbonyl group having 1 to 3 carbon atoms on the benzene ring))
An activator protein 1 (AP-1) activity inhibitor, comprising as an active ingredient a compound represented by the formula: or a compound capable of producing this compound in vivo or a pharmacologically acceptable salt thereof;
[2] The following general formula (I):

Or general formula (II):

(In the formula, R ¹ represents —CHO or —COOH; R ² represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, — (C _n H _2n ) COOR ′ (where n is 1 to 5). , R ′ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms) or —COR ″ (where R ″ is a pyridyl group, an amino group substituted with an alkyl group having 1 to 3 carbon atoms). And a phenoxyalkyl group having a halogen atom on the benzene ring, or a phenyl group having an alkoxy group having 1 to 3 carbon atoms or an alkoxycarbonyl group having 1 to 3 carbon atoms on the benzene ring))
An inhibitor of the expression of a gene whose expression is regulated by AP-1, comprising as an active ingredient a compound represented by the formula (1) or a compound capable of producing this compound in vivo or a pharmacologically acceptable salt thereof;
[3] The inhibitor according to [2], wherein the gene whose expression is regulated by AP-1 is a matrix metalloprotease (MMP) gene;
[4] A pharmaceutical composition for preventing or treating an MMP-promoting disease or an AP-1 enhancing disease, comprising the inhibitor according to any one of [1] to [3] above;
[5] The above-mentioned MMP enhancing disease or AP-1 enhancing disease is osteoarthritis, rheumatoid arthritis, corneal ulcer, periodontitis, viral infection, obstructive arteriosclerosis, arteriosclerotic aneurysm, scab Arteriosclerosis, restenosis, sepsis, septic shock, coronary thrombosis, abnormal angiogenesis, scleritis, multiple sclerosis, open angle glaucoma, retinopathy, proliferative retinopathy, neovascular glaucoma, pterygium, keratitis , Blistering epidermis, psoriasis, diabetes, nephritis, neurological disease, inflammation, osteoporosis, bone resorption, gingivitis, tumor growth, tumor angiogenesis, eye tumor, angiofibroma, hemangioma, fever, bleeding, coagulation, evil Fluid quality, loss of appetite, acute infection, shock, autoimmunity, malaria, Crohn's disease, meningitis, heart failure, asthmatic airway inflammation, arteriosclerosis, cancer metastasis, ulceration, rheumatoid arthritis, osteoporosis, teeth Peritonitis, skin aging, gastrointestinal ulcer, kidney One or more selected from the group consisting of patients (glomerulonephritis), cerebral infarction, ischemic nervous system disease, central excitatory disease, epilepsy, Alzheimer's disease, bone metabolic disease, cancer, ischemic heart disease, myocardial infarction, etc. The pharmaceutical composition according to the above [4], which is a disease,
I will provide a.

  According to the present invention, cancer metastasis, ulceration, rheumatoid arthritis, osteoporosis, periodontitis, skin aging, and the like are effective in preventing, suppressing, or improving symptoms caused by increased MMP. A highly safe MMP expression inhibitor is provided. In addition, renal diseases (glomerulonephritis), ischemic nervous system diseases such as cerebral infarction, central excitatory diseases (such as epilepsy), Alzheimer's disease, bone metabolic diseases, cancer, ischemic heart diseases (such as myocardial infarction), AP, etc. A highly safe AP-1 inhibitor is provided that is effective in preventing, suppressing, or improving symptoms of various diseases caused by the increase in -1.

一般式（II）：

（式中、Ｒ^１は、−ＣＨＯまたは−ＣＯＯＨを表し；Ｒ^２は、水素原子、炭素数１〜５のアルキル基、−（Ｃ_ｎＨ_２ｎ）ＣＯＯＲ’（ここで、ｎは１〜５の整数、Ｒ’は水素原子または炭素数１〜３のアルキル基を表す）または−ＣＯＲ''（ここで、Ｒ''はピリジル基、炭素数１〜３のアルキル基で置換されたアミノ基、ベンゼン環上にハロゲン原子を有するフェノキシアルキル基、またはベンゼン環上に炭素数１〜３のアルコキシ基もしくは炭素数１〜３のアルコキシカルボニル基を有するフェニル基を表す）を表す） Ascochlorin, ascofuranone and derivatives thereof used as active ingredients in the present invention include compounds represented by the following general formula (I) or (II):
Formula (I):

General formula (II):

(In the formula, R ¹ represents —CHO or —COOH; R ² represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, — (C _n H _2n ) COOR ′ (where n is 1 to 5). , R ′ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms) or —COR ″ (where R ″ is a pyridyl group, an amino group substituted with an alkyl group having 1 to 3 carbon atoms). And a phenoxyalkyl group having a halogen atom on the benzene ring, or a phenyl group having an alkoxy group having 1 to 3 carbon atoms or an alkoxycarbonyl group having 1 to 3 carbon atoms on the benzene ring))

  Regarding R ″ in the above general formulas (I) and (II), an alkyl group on the amino group, a halogen atom on the benzene ring constituting the phenoxyalkyl group, an alkoxy group or alkoxy on the benzene ring constituting the phenyl group At least one carbonyl group may be present, and when two or more carbonyl groups are present, they may be the same or different from each other.

  Specific examples of the compound represented by the general formula (I) include the following compounds:

  Specific examples of the compound represented by the general formula (II) include the following compounds:

  Of the above, preferred are ascochlorin, 4-O-methylascochlorin, 4-O-carboxymethylascochlorin, ascofuranone, 4-O-methylascofuranone, and 4-O-carboxymethylascofuranone.

  In addition, ascochlorin, ascofuranone and derivatives thereof used in the present invention may be pharmacologically acceptable salts or hydrates thereof. Examples of such salts include salts with alkali metals (lithium, sodium, potassium, etc.), alkaline earth metals (magnesium, calcium, etc.), ammonium, organic bases or amino acids, or inorganic acids (hydrochloric acid, hydrogen bromide). And salts with organic acids (acetic acid, citric acid, maleic acid, fumaric acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.). These salts can be formed by a commonly performed method. Moreover, when these compounds form a hydrate, they may be coordinated with an arbitrary number of water molecules. The compound used in the present invention may be a compound (prodrug) capable of producing these compounds by metabolism in vivo.

  Furthermore, ascochlorin, ascofuranone and derivatives thereof used in the present invention are not limited to specific isomers, but include all possible isomers and racemates, which are also described below. As described in the Examples, it shows excellent expression inhibitory activity and AP-1 inhibitory activity of genes under the control of AP-1, such as MMP-9.

Ascochlorin, ascofuranone and derivatives thereof used in the present invention act mainly on the AP-1 (binding) site in the promoter region of the MMP gene, and as a result, can inhibit MMP expression. Similarly, the expression of other genes whose expression is regulated by AP-1 can be inhibited.
Examples of genes whose expression is regulated by AP-1 include CyclinD1, p53, p21, p19, and p16 genes.

  Therefore, these compounds can be used as research reagents as MMP gene expression inhibitors, other gene expression inhibitors regulated by AP-1, AP-1 activity inhibitors, etc. Thus, diseases caused by increased MMP such as cancer metastasis (sometimes referred to as “MMP-enhancing diseases”) can be prevented and improved. Furthermore, diseases caused by increased AP-1 (sometimes referred to as “AP-1 enhanced diseases”) can be prevented and ameliorated. In the context of the present invention, “prevention” means preventing illness against an unaffected disease, and “treatment” refers to any improvement of the disease (reduction of symptoms, suppression, complete cure). Etc.).

  Specific examples of MMP-promoting diseases include osteoarthritis, rheumatoid arthritis, corneal ulcer, periodontitis, viral infection (eg, HIV infection), obstructive arteriosclerosis, arteriosclerotic aneurysm, Atherosclerosis, restenosis, sepsis, septic shock, coronary thrombosis, abnormal angiogenesis, scleritis, multiple sclerosis, open angle glaucoma, retinopathy, proliferative retinopathy, neovascular glaucoma, pterygium, Keratitis, blistering epidermis, psoriasis, diabetes, nephritis, neurological disease, inflammation, osteoporosis, bone resorption, gingivitis, tumor growth, tumor angiogenesis, eye tumor, hemangiofibroma, hemangioma, fever, bleeding, coagulation , Cachexia, anorexia, acute infection, shock, autoimmune disease, malaria, Crohn's disease, meningitis, heart failure, asthmatic airway inflammation, arteriosclerosis, cancer metastasis, ulceration, rheumatoid arthritis, osteoporosis Periodontitis, skin aging and Intestinal ulcers, and the like.

  Specific examples of AP-1 hypersensitivity diseases include renal diseases (glomerulonephritis), ischemic nervous system diseases such as cerebral infarction, central excitatory diseases (epileptic disease, etc.), Alzheimer's disease, bone metabolic diseases, cancer, Examples include ischemic heart disease (myocardial infarction, etc.), rheumatoid arthritis, arteriosclerosis and the like.

  When the gene expression inhibitor of the present invention is administered to humans for the purpose of preventing or treating the above-mentioned diseases, it can be made into a pharmaceutical composition by methods known to those skilled in the pharmaceutical art.

  For example, to the effective amount of the gene expression inhibitor of the present invention, excipients, binders, wetting agents, disintegrating agents, lubricants and the like suitable for the desired dosage form may be added as necessary. Can be mixed and formulated. In the case of an injection, it is sterilized with an appropriate carrier to obtain a preparation. The dosage form and administration route may be any of those known in the art, for example, orally as powders, granules, tablets, capsules, pills, liquids, etc., or injections, suppositories, It can be administered parenterally as a transdermal absorption agent, an inhalation agent and the like.

The dose of the above-mentioned compound, which is an active ingredient contained in the pharmaceutical composition of the present invention, varies depending on the type of disease, the state of the disease, the route of administration, the age and weight of the patient, etc. In this case, it is usually 0.1 to 100 mg / kg / day, preferably 1 to 20 mg / kg / day.
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

In the following examples, the following materials were used unless otherwise indicated:
Caki-1 cells, Chang cells, MDA-MB-231 cells, U2OS cells and MX-1 cells were obtained from the American Type Culture Collection. These cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Grand Island) containing 10% fetal bovine serum (FBS) and 100 μg / ml gentamicin. Ascochlorin and Ascofuranone were obtained from NRL Pharma (Kawasaki City, Japan).
Lipofectamine reagent was obtained from Life Technologies, Inc. (Rockville, MD). Luciferase and β-galactosidase assay systems were obtained from Promega (Madison, Wis.). Phorbol 12-myristate 13-acetate (PMA) was obtained from SIGMA (St. Louis, USA).

Example 1: Inhibition of invasive activity of Caki-1 cells by ascochlorin Using Caki-1 cells, which are human kidney cancer cells, the effect of suppressing invasive activity by ascochlorin was examined.
Mix the upper part of Transwell (registered trademark; Corning Coastar, Cambridge, MA, USA) with Matrigel (trademark, Becton Dickinson, USA): Phosphate buffered saline (PBS), 1: 2. Coated with 30 μl of solution. Matrigel-coated transwells containing 5 × 10 ⁴ Caki-1 cells per chamber in various concentrations of ascochlorin (1, 10, 30, 50 μM) in the presence and absence of 50 nM PMA Plated below. These inserts were then cultured at 37 ° C. for 24 hours. Cells infiltrating the lower surface of the membrane were fixed with methanol, stained with hematoxylin and eosin and counted under a light microscope.

  The results are shown in FIG. PMA-treated Caki-1 cells (“B”) were promoted about 5-fold invasion compared to controls in the absence of PMA (“A”). In contrast, ascochlorin inhibited PMA-dependent invasion in a concentration-dependent manner and reached control levels in the absence of PMA at concentrations of 10-30 μM (“C”-“F”). .

Example 2: Inhibition of invasive activity of U2OS cells by ascofuranone In the same manner as in Example 1, the effect of suppressing invasive activity by ascofuranone was examined using human osteosarcoma cells U2OS cells. As a result, ascofuranone showed concentration-dependent suppression of infiltration in a concentration range of 1 to 50 μM.

Example 3: Inhibition of PMA-induced MMP-9 activity by ascochlorin Caki-1 cells were suspended in medium containing 10% FBS and 3 × 10 ⁵ cells were plated per 35 mm dish. . The dish was cultured until the cells reached 80% confluence. The medium was then replaced with fresh serum-free medium, and the supernatant was collected after further incubation for 24 hours in the presence and absence of 1, 10 or 30 μM ascochlorin. These media were subjected to SDS electrophoresis using a 10% polyacrylamide gel prepared in the presence of 1 mg / ml gelatin. After electrophoresis, the gel was washed several times for 1 hour at room temperature in 50 mM Tris-HCl, pH 7.6 buffer containing 2.5% Triton-X100. The gel was then shaken for 24 hours at 37 ° C. in the same buffer containing 5 mM CaCl ₂ and 1 μM ZnCl ₂ . Thereafter, the gel was stained with 0.25% Coomassei Brilliant Blue R250 (Bio-Rad, California, USA) for 1 hour and then decolorized. The presence of proteolytic activity is detected as a clear band against the blue background of stained gelatin.

  Similar experiments were performed using Chang (human hepatocytes), MDA-MB-231 (human breast cancer) and U2OS cells (human osteosarcoma) instead of Caki-1 cells.

  The results are shown in FIG. Caki-1 cells release precursor MMP-2 (proMMP-2) and precursor MMP-9 (proMMP-9) when cultured in serum-free medium. When Caki-1 cells are treated with PMA (50 nM) for 24 hours, there is no change in the expression level of progenitor MMP-2 (proMMP-2), but progenitor MMP-9 (proMMP-9) is induced. PMA-induced progenitor MMP-9 (proMMP-9) activity was reduced in a concentration-dependent manner by treatment with 1-50 μM ascochlorin.

  In order to confirm whether the suppression of progenitor MMP-9 (proMMP-9) activity by ascochlorin is a general phenomenon, using Chang, MDA-MB-231, and U2OS cells, the same ascochlorin sensitivity was observed. As a result of the examination, suppression of precursor MMP-9 (proMMP-9) activity by ascochlorin was observed in any cell. This indicates that this effect of ascochlorin has no cell type specificity.

Example 4: Inhibition of PMA-induced MMP-9 activity by ascofuranone The effect of ascofuranone on MMP-9 activity was examined using U2OS cells in the same manner as in Example 3. As a result, ascofuranone suppressed MMP-9 activity in a concentration-dependent manner at a concentration of 1 μM or more.

Example 5: Inhibition of PMA-induced MMP-9 enzyme expression by ascochlorin Is the inhibition of PMA-induced MMP-9 enzyme activity by ascochlorin due to inhibition of MMP-9 enzyme protein expression? I examined whether.
Using 3 × 10 ⁵ Caki-1 cells per 35 mm dish, in which MMP-9 is induced with 50 nM PMA, and cultured in the presence of various concentrations of ascochlorin (1, 10, 30, 50 nM) It was. Cells were treated with 30 μl lysis buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 1 mM DTT, 0.5% NP-40, 100 μM phenylmethylsulfonyl fluoride, 20 μM aprotinin and 20 μM leupeptin; pH Was adjusted to 8.0) and treated at 4 ° C. for 30 minutes to prepare a cell lysate. Proteins were electrophoretically transferred to Immobilin-P membrane (Millipore Corporation, Bredford, USA). Detection of the MMP-9 enzyme was performed using a rabbit anti-MMP-9 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, California, USA) using an enhanced chemiluminescence method (Amersham, UK). ).

  As a result, it was found that the expression level of MMP-9 protein was lowered depending on the concentration of ascochlorin. This indicates that suppression of precursor MMP-9 (proMMP-9) activity by ascochlorin is due to a decrease in MMP-9 protein.

Example 6: Involvement of AP-1 in suppression of MMP-9 gene expression by ascochlorin There are two AP-1 sites and one NF-κB site in the promoter region of the MMP-9 gene. Whether any of these were involved in the regulation of MMP-9 gene expression in Caki-1 cells was examined by luciferase reporter gene assay.

For this purpose, a wild-type MMP-9 promoter (pGL2-MMP-9WT), a mutant promoter in which one or two AP-1 promoters are mutated (pGL2-MMP-9mAP-1- 1 and pGL2-MMP-9mAP-1-2), or a plasmid having a reporter gene (luciferase) downstream of a promoter mutated in the NF-κB site (pGL2-MMP-9mNFκB), Clonetech (Palo Alto, From CA, USA). These were transiently transfected into Caki-1 cells. Cells were plated at a density of 3 × 10 ⁵ cells per 35 mm dish and grown overnight. Cells were co-transfected with 2 μg plasmid and 1 μg pCMV-β-galactosidase plasmid for 5 hours using Lipofectamine reagent according to the manufacturer's instructions.

  Next, the above Caki-1 cells were cultured in a medium containing various concentrations (1 to 50 μM) of ascochlorin for 24 hours, and then luciferase activity and β-galactosidase activity were measured according to the manufacturer's instructions. Luciferase activity was normalized for β-galactosidase activity in cell lysates and expressed as the average of three separate experiments.

  The result is shown in FIG. In the promoter in which the mutation was introduced into the AP-1 site, the induction by PMA was extremely reduced, and in the case of the mutation in the two promoters, the induction by PMA was completely lost. Even when the mutation was introduced into NF-κB, the induction by PMA was reduced, but it was weaker than the influence by the mutation of AP-1. When induction by PMA was examined in the presence of 10 μM ascochlorin, a decrease in luciferase activity was also observed under the AP-1 and NFκB promoters with mutations. A decrease in luciferase activity depending on the concentration of ascochlorin is observed.

Moreover, when the same experiment was performed also about the reporter gene which has a NF (kappa) B binding site, as shown in FIG. 4, the influence by a significant ascochlorin was not observed.
Thus, both AP-1 and NF-κB binding sites in the MMP-9 promoter contribute to promoter activity, and AP-1 sites against ascochlorin during PMA-dependent activation of the MMP-9 promoter. It was found to be a major site for regulatory responses.

Example 7: AP-1 involvement in suppression of MMP-9 gene expression by ascofuranone Using U2O2 cells, the effect of ascofuranone on AP-1 activity was examined in the same manner as in Example 6. As a result, ascofuranone attenuated the activity of the reporter gene (luciferase) controlled by the AP-1 promoter in a concentration-dependent manner in the concentration range of 1 to 50 μM. The results are shown in FIG.

Example 8: Suppression of gene expression under AP-1 control by ascochlorin Human breast cancer cells MX-1 (1 × 10 ⁵ / ml) deficient in estrogen receptor were cultured overnight, and then a reporter downstream of the AP-1 promoter. A plasmid (pTRE-luc) linked with a gene (luciferase), which was obtained from Y. Janssen-Heininger (University of Vermont, Canada). And the expression vector pCMV-β-gal and further cultured for 20 hours. After 1 μM of ascochlorin was added and treated for 30 minutes, PMA was added at 10 ng / ml, and further cultured for 20 hours to induce AP-1 activity. As a result, ascochlorin significantly suppressed the luciferase activity in the case where it was not induced with PMA, and was completely inhibited under the condition where induction with PMA was applied.
Therefore, it was found that ascochlorin can inhibit the expression of not only MMP-9 but also genes under AP-1.

  As described above, ascochlorin, ascofuranone, and derivatives thereof can inhibit the expression of genes controlled by AP-1 including MMP, and thus prevent or treat AP-1 or MMP-promoting diseases. It is effective for.

FIG. 1 is a diagram showing suppression of invasive activity of Caki-1 cells by ascochlorin. FIG. 2 shows inhibition of PMA-induced MMP-9 activity by ascochlorin. FIG. 3 is a figure which shows the result of the luciferase assay which shows that AP-1 is concerned in suppression of MMP-9 gene expression by an ascochlorin. As an MMP-9 promoter, (A) is a wild type, (B) is one having a mutation at one AP-1 site, (C) is one having a mutation at an NF-κB site, (D) is two places It is a result at the time of using what has a variation | mutation in AP-1 site | part. FIG. 4 is a diagram showing the results of conducting the same experiment as shown in FIG. 3 for a gene having an AP-1 binding site and a reporter gene having an NF-κB binding site. (A) shows the results for AP-1, and (B) shows the results for NF-κB. FIG. 5 is a diagram showing the results of experiments similar to those shown in FIGS. 3 and 4 using ascofuranone and other cells (U2O2 cells).

Claims (5)

The following general formula (I):

Or general formula (II):

(In the formula, R ¹ represents —CHO or —COOH; R ² represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, — (C _n H _2n ) COOR ′ (where n is 1 to 5). , R ′ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms) or —COR ″ (where R ″ is a pyridyl group, an amino group substituted with an alkyl group having 1 to 3 carbon atoms). And a phenoxyalkyl group having a halogen atom on the benzene ring, or a phenyl group having an alkoxy group having 1 to 3 carbon atoms or an alkoxycarbonyl group having 1 to 3 carbon atoms on the benzene ring))
An activator protein 1 (AP-1) activity inhibitor comprising the compound represented by the above or a compound capable of generating this compound in vivo or a pharmacologically acceptable salt thereof as an active ingredient. The following general formula (I):

Or general formula (II):

(In the formula, R ¹ represents —CHO or —COOH; R ² represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, — (C _n H _2n ) COOR ′ (where n is 1 to 5). , R ′ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms) or —COR ″ (where R ″ is a pyridyl group, an amino group substituted with an alkyl group having 1 to 3 carbon atoms). And a phenoxyalkyl group having a halogen atom on the benzene ring, or a phenyl group having an alkoxy group having 1 to 3 carbon atoms or an alkoxycarbonyl group having 1 to 3 carbon atoms on the benzene ring))
Or a compound capable of generating this compound in vivo or a pharmacologically acceptable salt thereof as an active ingredient, an inhibitor of gene expression regulated by AP-1.   The inhibitor according to claim 2, wherein the gene whose expression is regulated by AP-1 is a matrix metalloprotease (MMP) gene.   A pharmaceutical composition for preventing or treating an MMP-promoting disease or an AP-1-hypertensive disease, comprising the inhibitor according to any one of claims 1 to 3.   The above-mentioned MMP hypersensitivity disease or AP-1 hypersensitivity disease is osteoarthritis, rheumatoid arthritis, corneal ulcer, periodontitis, viral infection, obstructive arteriosclerosis, arteriosclerotic aneurysm, atherosclerosis Restenosis, sepsis, septic shock, coronary thrombosis, abnormal angiogenesis, scleritis, multiple sclerosis, open angle glaucoma, retinopathy, proliferative retinopathy, neovascular glaucoma, pterygium, keratitis, blister Epidermis peeling, psoriasis, diabetes, nephritis, neurological disease, inflammation, osteoporosis, bone resorption, gingivitis, tumor growth, tumor angiogenesis, eye tumor, hemangiofibroma, hemangioma, fever, bleeding, coagulation, cachexia, Anorexia, acute infection, shock, autoimmune disease, malaria, Crohn's disease, meningitis, heart failure, asthmatic airway inflammation, arteriosclerosis, cancer metastasis, ulceration, rheumatoid arthritis, osteoporosis, periodontitis, Skin aging, gastrointestinal ulcer, kidney disease ( Sphere nephritis), cerebral infarction, ischemic nervous system disease, central excitatory disease, epilepsy, Alzheimer's disease, bone metabolic disease, cancer, ischemic heart disease, one or more diseases selected from the group consisting of myocardial infarction, The pharmaceutical composition according to claim 4.

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