WO2002048135A1 - Tak1 inhibitors - Google Patents

Tak1 inhibitors Download PDF

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Publication number
WO2002048135A1
WO2002048135A1 PCT/JP2001/010927 JP0110927W WO0248135A1 WO 2002048135 A1 WO2002048135 A1 WO 2002048135A1 JP 0110927 W JP0110927 W JP 0110927W WO 0248135 A1 WO0248135 A1 WO 0248135A1
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Prior art keywords
tak1
activation
inhibiting
inhibitor
kinase
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PCT/JP2001/010927
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French (fr)
Japanese (ja)
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Masayuki Tsuchiya
Toshihiko Ohtomo
Koichiro Ono
Masahiko Matsumoto
Tatsuya Ito
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Chugai Seiyaku Kabushiki Kaisha
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Priority to AU2002222627A priority Critical patent/AU2002222627A1/en
Publication of WO2002048135A1 publication Critical patent/WO2002048135A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D313/00—Heterocyclic compounds containing rings of more than six members having one oxygen atom as the only ring hetero atom

Definitions

  • the present invention provides a TAK1 inhibitor containing a specific zearalenone as an active ingredient, a TAK1 activation inhibitor containing a specific zearalenone as an active ingredient, and a TAK1 using a specific zearalenone as an active ingredient.
  • Inhibition method Use of specific zearalenones as an active ingredient
  • Inhibition of TAK1 activation Use of specific zearalenones to produce TAK1 inhibitors
  • the MAPK system is a conserved eukaryotic signaling system that converts receptor signals into various actions.
  • the MAPK system includes three types of protein kinases, namely, MAPKKK (Mitogen-Activated Protein Kinase Kinase), MAPKK (Mitogen-Activated Protein Kinase Kinase), and MAPK (Mitogen-Activated d. Protein Kinase).
  • MAPK is activated by phosphorylation by MAPKK.
  • MAPKK is activated by phosphorylation by MAPKKK (Trends Biochem. Sci. (1993) 18, 128; Trends Biochem. Sci. (1993) 19, 236; Trends Biochem. Sci. (1993) 19, 470; Cell (1995) 80, 179).
  • TAKl TGF-i8-Activated Kinase 1
  • MAPKKK MAPKKK family that functions in the intracellular signaling system
  • TAB1 TGF- / 3 signaling system that binds to and activates TAK1.
  • TAB1 binds to TAK1 to activate TAK1 kinase activity, and induces activation of c-Jun N-terminal kinase (JNK) and p38 MAPK through activation of MAPKK MKK4 and MKK3 / 6, and TGF- ; Signals such as 8 (J. Biol. Chem., (1996) 271, 13675-13679; J. Biol. Chem.,
  • TAK1 is also activated by various stresses such as stimulation of interleukin-1 (IL-1) and tumor necrosis factor (TNF) (J. Biol. Chem. (1997) 272, 8141-8144; J. Biol.
  • TAK1 is a MAPK cascade-mediated activation of JNK in IL-1 signaling, and nuclear factor- ⁇ It has been shown to be involved in the activation of NF- ⁇ through the activation of ⁇ ( ⁇ F— ⁇ ) -inducing kinase (NIK) and I ⁇ kinases (IKK) (Nature (1999) 398, 252) -256; Mol. Cell, (2000) 5, 649-658). Furthermore, LPS stimulation activated TAK1, induced NF- ⁇ F activation, and demonstrated that LPS is involved in signal transduction (FEBS Lett., (2000) 467, 160-164).
  • TGF-iS is a multifunctional factor that controls many cell functions, and one of them is TGF-J3, which is responsible for tissue repair and regeneration following various injuries (New Engl. J. Med., (1994) 331). , 1286-1292).
  • TGF-J3 is responsible for tissue repair and regeneration following various injuries.
  • TGF-jS may disrupt the balance of tissue repair and regeneration, resulting in pathological fibrosis.
  • TGF- ⁇ enhances the production of extracellular matrix proteins, inhibits the synthesis of extracellular matrix-degrading enzymes, and induces inhibitors of extracellular matrix-degrading enzymes. It has been shown to act as a major causative factor of fibrosis (New Engl. J. Med., (1994) 331, 1286-1292).
  • Inflammatory cytokines such as IL-1 and TNF play an important role as bioprotective factors such as inflammation due to invasion of foreign substances.However, it is known that excessive expression of this response causes various pathological conditions. Have been. IL-1 is thought to be an important mediator in immune and inflammatory responses, acting on various cells and enhancing their cellular functions.
  • TNF is released from cells such as macula phage activated by contact with foreign antigens that have invaded the living body, and not only enhances the chemotaxis and phagocytosis of macrophages, but also enhances the HLA of T lymphocytes. It enhances the expression of antigens, activates the function of T cells by increasing the expression of IL-2 receptor, and also enhances the chemotaxis and activation of neutrophils to remove foreign substances. It also promotes the proliferation of fibroblasts in inflamed areas and repairs their tissues. Furthermore, TNF Q! Promotes the expression of various cytokins in these cells, and is involved in the formation of cytokinetic networks, like IL-1.
  • TAK-] 3 IL-1, LPS, TNF, etc.
  • TAK-] 3 IL-1, LPS, TNF, etc.
  • TAK1 activation inhibitor no TAK1 kinase inhibitor or TAK1 activation inhibitor has been known to date.
  • An object of the present invention is to provide a TAK1 inhibitor or a TAK1 activation inhibitor.
  • the present inventors have conducted intensive studies to solve the above problems, and as a result, certain zearalenones have excellent TAK1 kinase inhibitory activity, and TGF-J3 and IL-1 mediated by activation of TAK1. The present inventors have found that such signal transduction is inhibited, and completed the present invention based on this finding.
  • the present invention provides a zearaleno having a hydroxyl group at each of the 8-position and the 9-position.
  • a TAK 1 inhibitor or a TAK 1 activation inhibitor comprising a pharmaceutically acceptable salt as an active ingredient.
  • the present invention also provides a method for inhibiting TAK1 or a method for inhibiting TAK1 activation, which comprises using an effective amount of a zearalenone having a hydroxyl group at each of the 8th and 9th positions.
  • the present invention provides the use of zearalenones having a hydroxyl group at both the 8-position and the 9-position for producing a TAK1 inhibitor or a TAK1 activation inhibitor.
  • the present invention provides a kit for inhibiting TAK1 or a kit for inhibiting TAK1 activation, which comprises a zearalenone having a hydroxyl group at each of positions 8 and 9 and an instruction manual.
  • FIG. 1 is a graph showing the inhibitory effect of compound (1) on signal transduction via TAK1.
  • zearalenones having a hydroxyl group at both the 8-position and 9-position include zearalenone (Zearal enone: the following formula (A)) or zearalenol (Ze arale nol: the following formula (B))
  • zearalenones having a hydroxyl group at both the 8-position and the 9-position include the compounds shown below:
  • Each of these compounds (1) to (13) is a compound known per se and described in the literature (for example, Int. J. Im Leak Pharmacol "21: 799-814, 1999; J. Org. Chem., 43: 2339). Chem. Pharm. Bull. 41: 373-375, 1993; J. Antibiotics, 52: 1077-1085, 1999; Biochem. Biophys. Res. Commun., 257: 19-23, 1999; Cytokine, 8: 751-761, 1996; Pharmacol. Commun., 7: 301-308, 1996; Japanese Patent Publication No. 8-40893; European Patent Publication No. 6 06444A; Biochemistry, 37: 9579-9585, 1998, etc.).
  • compounds (1) to (13) are preferable, compounds (1) to (3) are more preferable, and compound (1) is particularly preferable.
  • TAK1 inhibition means inhibiting the kinase activity of TAK1.
  • the TAK1 inhibitory activity can be measured, for example, by the method described in Example 1 below.
  • TAK1 activation inhibition refers to inhibiting TAK1-mediated intracellular signal transduction.
  • the TAK1 inhibitory activation inhibitory activity can be measured, for example, by the method described in Example 2 below.
  • the administration route and administration amount of the ⁇ kinase inhibitor or TAK1 activation inhibitor of the present invention are appropriately selected depending on the patient's body type, age, physical condition, type and degree of disease, elapsed time after onset, and the like. However, in the case of parenteral administration, a dose of 0.02 g to 2 mgZ body weight k days is generally expected, and in the case of oral administration, an effect of Sig SOOmgZ body weight k days is generally expected.
  • zearalenones having a hydroxyl group at both the 8-position and the 9-position may be used alone or in combination of two or more.
  • the TAK1 inhibitor or TAK1 activation inhibitor of the present invention may comprise one or more pharmaceutically acceptable diluents, wetting agents, emulsifiers, dispersants, adjuvants, preservatives, lubricants It can be administered as a pharmaceutical composition appropriately containing an impaction agent, a binder, a stabilizer and the like in an appropriate form depending on the intended administration route.
  • the kit of the present invention comprises a pharmaceutical composition
  • a pharmaceutical composition comprising a zearalenone having a hydroxyl group at each of the above-mentioned 8-position and 9-position, a diluent, and any one or more carriers including various carriers exemplified above. It contains the composition, as well as instructions for use.
  • TAK1 kinase activity was assessed by measuring TAK1 autophosphorylation and TAB1 phosphorylation. That is, a recombinant paculovirus vector containing the TAK1 and TAB1 genes (WO99 / 211010) was co-infected with insect cells Sf-9 and cultured for 3 days.
  • the cells are washed with PBS, and the extract solution is adjusted to 3.0 x 10 7 cells / mL (20 mM HEPES, pH 7.4, 150 mM NaCL 12.5 mM ⁇ -gycerophosphate, 1.5 mM MgCl 2 , 2 mM EGTA, 10 mM
  • the cells were suspended in mM NaF, 2 mM DTT, 1 mM Na 3 VO 4 1 mM PMSF, 20 ⁇ MAprotinin, 0.5% Triton X-100) to prepare a cell extract.
  • reaction was terminated by adding 50 L of a reaction stop solution (PBS containing 50% TCA) and further incubating at 4 for 10 minutes.
  • PBS containing 50% TCA a reaction stop solution
  • To remove free 33 P-ATP wash 5 times with PBS containing 0.05% Tween-20, add 50 ⁇ L of Supermix (Wallac), and incubate at room temperature for 30 minutes
  • TAK1 kinase activity was measured by measuring the amount of 33 P-ATP incorporated into TAK1 and TAB1 using micro-beta.
  • compounds (1) to (3) suppressed TAK1 kinase activity as shown in Table 1, and in particular, compound (1) exhibited IC 5 .
  • a TAK1 kinase inhibitory activity of 8.1 nM was shown.
  • Shimochi's Radicicol which is a compound that does not have a hydroxyl group at the position corresponding to the 8th and 9th positions of the compounds (1) to (3)
  • TAK1 activation of TAK1 induces activation of MAPK cascade and NF- ⁇ . Therefore, the effect of inhibition of TAK1 kinase activity on TAK1-mediated signal transduction was examined using the reporter Atsusei.
  • NFkB-Luc Genes Dev., 7, 1354-1363, 1993
  • NFkB-Luc incorporating a luciferase gene downstream of the response element was used to stimulate IL-1 or Activation of TAK1 was induced by forced expression of TAK1 / TAB1. That is, after seeding 1 x 10 6 293-IL1R cells (Mol. Cell, 5, 649-658, 2000), which forcibly expressed the IL-1 receptor in human embryonic kidney cell line 293 cells, and cultured overnight
  • a mixture of 2 g of NFkB-Luc and 10 FuGENE gene introduction reagents (Roche) was added to the cells, and the cells were further cultured for 8 hours to introduce the genes.
  • the cells into which the gene was introduced were detached with trypsin, the cells were seeded on a 96-well microplate (manufactured by Coaning Coaster) at 1 ⁇ 10 4 cells / well, and cultured overnight. After washing once with DMEM medium, compound (1) diluted with DMEM medium containing 0.2% fetal bovine serum was added in 50 portions. After incubating at 37 ° C for 1 hour, IL-1 (manufactured by PEPRO TECH) was added at 50 L each to a final concentration of 10 ng / mL, and the cells were further cultured for 24 hours. Luciferase activity is Steady Glo The measurement was performed using a Luciferase Assay Kit (promega).
  • FIG. 1 the vertical axis indicates the inhibitory activity ().
  • the horizontal axis represents the concentration of the compound (1) (M).
  • Closed squares indicate the inhibitory activity of p3TP-Lux Atsusei on TGF- / 3 signaling.
  • Closed triangles indicate the inhibitory activity of the IL-1 signaling system on NFkB-LucAssy.
  • the open triangle indicates the inhibitory activity of the signal transduction system by forced expression of TAK1 / TAB1 in NFkB-Luc assay.
  • compound (1) suppressed the expression of luciferase due to forced expression of TGF-3, IL-la, and TAK1 / TAB1 in a dose-dependent manner. This result suggests that inhibition of TAK1 kinase activity can inhibit downstream signal transduction.
  • the compound of the present invention has a TAK1 inhibitory activity, it inhibits various signal transduction systems through activation of TAK1 and various diseases involving activation of TAK1 as a cause of the disease, for example, TGF-j3, It is expected to be useful as an agent for treating and preventing various diseases known to be caused by IL-1, LPS, TNF- ⁇ and the like.

Abstract

TAK1 inhibitors or TAK1 activation inhibitors containing as the active ingredient zearalenones having hydroxyl groups at both of the 8- and 9-positions; a method of inhibiting TAK1 or a method of inhibiting the activation of TAK1 by using as the active ingredient zearalenones having hydroxyl groups at both of the 8- and 9-positions; utilization of zearalenones having hydroxyl groups at both of the 8- and 9-positions for producing TAK1 inhibitors or TAK1 activation inhibitors; and kits for inhibiting TAK1 or kits for inhibiting the activation of TAK1 containing zearalenones having hydroxyl groups at both of the 8- and 9-position and a manual.

Description

明 細 書  Specification
T A K l阻害剤 技術分野  TAKl inhibitor Technical field
本発明は、 特定のゼァラレノン類を有効成分とする T AK 1阻害剤、 特定のゼ ァラレノン類を有効成分とする T A K 1活性化阻害剤、 特定のゼァラレノン類を 有効成分として使用する T AK 1を阻害する方法、 特定のゼァラレノン類を有効 成分として使用する T A K 1活性化を阻害する方法、 T AK 1阻害剤を製造する ための特定のゼァラレノン類の使用、 T A K 1活性ィヒ阻害剤を製造するための特 定のゼァラレノン類の使用、 特定のゼァラレノン類および使用説明書を含む T A K 1を阻害するためのキット、 並びに特定のゼァラレノン類および使用説明書を 含む T AK 1の活性化を阻害するためのキットに関する。 背景技術  The present invention provides a TAK1 inhibitor containing a specific zearalenone as an active ingredient, a TAK1 activation inhibitor containing a specific zearalenone as an active ingredient, and a TAK1 using a specific zearalenone as an active ingredient. Inhibition method, Use of specific zearalenones as an active ingredient Inhibition of TAK1 activation, Use of specific zearalenones to produce TAK1 inhibitors, Production of TAK1 activity inhibitors Kits for inhibiting TAK1 containing specific zearalenones and instructions for use, and for inhibiting activation of TAK1 containing specific zearalenones and instructions for use About the kit. Background art
細胞内のシグナル伝達に関与する一連の系として、 マイトジェン-活性化プロ ティンキナーゼ （Mitogen-Activated Protein Kinase; MAPK) 系が知られてい る。 MAPK系は受容体のシグナルを種々の作用に転換する保存された真核細胞 性シグナル伝達系である。 MAPK系は 3種類のプロテインキナーゼ、 すなわち MAPKKK (Mitogen-Activated Protein Kinase Kinase Kinase ) 、 MAPKK (Mitogen-Activated Protein Kinase Kinase) 、 MAPK (Mito gen- Activate d. Protein Kinase ) を含む。 MAPKは MAPKKによるリン酸化で活性化される。 MAPKKは MAPKKKによるリン酸ィ匕で活性化される （Trends Biochem. Sci. (1993) 18, 128； Trends Biochem. Sci. (1993) 19, 236； Trends Biochem. Sci. (1993) 19， 470； Cell (1995) 80， 179) 。  As a series of systems involved in intracellular signal transduction, the mitogen-activated protein kinase (MAPK) system is known. The MAPK system is a conserved eukaryotic signaling system that converts receptor signals into various actions. The MAPK system includes three types of protein kinases, namely, MAPKKK (Mitogen-Activated Protein Kinase Kinase), MAPKK (Mitogen-Activated Protein Kinase Kinase), and MAPK (Mitogen-Activated d. Protein Kinase). MAPK is activated by phosphorylation by MAPKK. MAPKK is activated by phosphorylation by MAPKKK (Trends Biochem. Sci. (1993) 18, 128; Trends Biochem. Sci. (1993) 19, 236; Trends Biochem. Sci. (1993) 19, 470; Cell (1995) 80, 179).
細胞内のシグナル伝達系において機能する MAPKKKファミリ一の一つであ る TAKl (TGF- i8 -Activated Kinase 1) は Yamaguchi, K.らにより同定された 蛋白質であり （Science (1995)270, 2008) 、 TGF- /3のシグナル伝達に関与し TGF- )3により活性ィ匕されることが明らかになつている。 また、 TAK1に結合し、 TAK1 を活性化する TGF- /3のシグナル伝達系に関与する蛋白質である TAB1 (TAK1 Binding Protein 1) が Shibuya, H.らにより同定された （ScienceTAKl (TGF-i8-Activated Kinase 1), a member of the MAPKKK family that functions in the intracellular signaling system, is a protein identified by Yamaguchi, K. et al. (Science (1995) 270, 2008) It has been clarified that it is involved in TGF- / 3 signal transduction and is activated by TGF-) 3. TAB1 is a protein involved in the TGF- / 3 signaling system that binds to and activates TAK1. (TAK1 Binding Protein 1) was identified by Shibuya, H. et al. (Science
(1996) 272, 1179-1182) 。 TAB1は TAK1に結合して TAK1のキナーゼ活性を 活性化し、 MAPKKである MKK4や MKK3/6 の活性化を通じ、 c-Jun N- terminal kinase (JNK) や p38 MAPKの活性化を誘導し、 TGF- ;8等のシグナ ルを伝達する （J. Biol. Chem., (1996) 271, 13675-13679； J. Biol. Chem.,(1996) 272, 1179-1182). TAB1 binds to TAK1 to activate TAK1 kinase activity, and induces activation of c-Jun N-terminal kinase (JNK) and p38 MAPK through activation of MAPKK MKK4 and MKK3 / 6, and TGF- ; Signals such as 8 (J. Biol. Chem., (1996) 271, 13675-13679; J. Biol. Chem.,
(1997) 272, 8141-8144) 。 さらに、 活性化 TAK1の発現により心肥大、 さらに は心不全を誘導することが明らかとなつた （Nature Med., (2000) 6, 556-563) 。 また最近になり、 TAK1 がインターロイキン- 1 (IL-1 ) と腫瘍壊死因子 (TNF) の刺激等の各種ストレスによっても活性化されることが明らかになり (J. Biol. Chem. (1997) 272， 8141-8144； J. Biol. Chem., (1999) 274, 10641- 10648) 、 TAK1は IL-1のシグナル伝達系において MAPKカスケ一ドを介した JNK の活性化、 ならびに nuclear factor- κ Β (Ν F— κ Β ) -inducing kinase (NIK), I κ kinases (IKK)の活性化を通じた N F— κ Βの活性化に関与する ことが明らかになった (Nature (1999) 398， 252-256； Mol. Cell, (2000) 5, 649- 658) 。 さらに、 LPS刺激により TAK1 が活性化され、 N F— κ Βの活性化を 誘導し、 LPS のシグナル伝達への関与が明らかになった （FEBS Lett., (2000) 467, 160-164) 。 (1997) 272, 8141-8144). Furthermore, it has been revealed that the expression of activated TAK1 induces cardiac hypertrophy and further heart failure (Nature Med., (2000) 6, 556-563). Recently, it has been revealed that TAK1 is also activated by various stresses such as stimulation of interleukin-1 (IL-1) and tumor necrosis factor (TNF) (J. Biol. Chem. (1997) 272, 8141-8144; J. Biol. Chem., (1999) 274, 10641-10648), TAK1 is a MAPK cascade-mediated activation of JNK in IL-1 signaling, and nuclear factor-κ It has been shown to be involved in the activation of NF-κΒ through the activation of Β (ΝF—κΒ) -inducing kinase (NIK) and Iκ kinases (IKK) (Nature (1999) 398, 252) -256; Mol. Cell, (2000) 5, 649-658). Furthermore, LPS stimulation activated TAK1, induced NF-κ F activation, and demonstrated that LPS is involved in signal transduction (FEBS Lett., (2000) 467, 160-164).
TGF- iSは多くの細胞機能を制御する多機能因子であり、 その一つとして TGF- J3は様々な傷害に伴う組織の修復および再生を司る （ New Engl. J. Med., (1994) 331, 1286-1292) 。 しかしながら、 慢性化した傷害においては TGF- jSの 異常産生により、 組織の修復、 再生のパランスが崩れ病的な線維化を生ずること がある。 TGF- βは細胞外マトリックス蛋白質の産生を亢進させ、 細胞外マトリ ックス分解酵素の合成の阻害、 ならびに細胞外マトリックス分解酵素の阻害因子 を誘導する事により、 種々の臓器、 例えば肝臓、 腎臓等の線維症の主要な原因因 子として作用することが明らかになつている （New Engl. J. Med., (1994) 331, 1286-1292) 。 また、 その他の作用としては、 細胞増殖阻害活性 （Cell (1990) 63, 245-247) 、 単球遊走活性 （Proc. Natl. Acad. Sci. USA, (1987) 84, 5788- 5792) 、 生理活性物質誘導活性 （Proc. Natl. Acad. Sci. USA, (1987) 84, 5788- 5792) 、 アミロイド蛋白質沈着作用 (Nature, (1997) 389， 603-606) が知ら れている。 肝線維症、 肺線維症、 糸球体腎炎、 糖尿病性腎症、 腎硬化症、 血管再 狭窄、 ケロイド症、 強皮症、 眼科手術後の瘢痕化、 増殖性網膜症、 自己免疫疾患 およびアルツハイマー症などの疾病と TGF- )3の異常産生の関連が報告されてい る。 TGF-iS is a multifunctional factor that controls many cell functions, and one of them is TGF-J3, which is responsible for tissue repair and regeneration following various injuries (New Engl. J. Med., (1994) 331). , 1286-1292). However, in chronic injuries, abnormal production of TGF-jS may disrupt the balance of tissue repair and regeneration, resulting in pathological fibrosis. TGF-β enhances the production of extracellular matrix proteins, inhibits the synthesis of extracellular matrix-degrading enzymes, and induces inhibitors of extracellular matrix-degrading enzymes. It has been shown to act as a major causative factor of fibrosis (New Engl. J. Med., (1994) 331, 1286-1292). Other effects include cell growth inhibitory activity (Cell (1990) 63, 245-247), monocyte migration activity (Proc. Natl. Acad. Sci. USA, (1987) 84, 5788-5792), Active substance-inducing activity (Proc. Natl. Acad. Sci. USA, (1987) 84, 5788-5792) and amyloid protein deposition (Nature, (1997) 389, 603-606) are known. Have been. Liver fibrosis, pulmonary fibrosis, glomerulonephritis, diabetic nephropathy, renal sclerosis, vascular restenosis, keloidosis, scleroderma, scarring after ophthalmic surgery, proliferative retinopathy, autoimmune disease and Alzheimer's disease It has been reported that such diseases are associated with abnormal production of TGF-) 3.
IL-1や TNF等の炎症性サイトカインは、 異物の侵入による炎症などの生体防 御因子として重要な役割を果たしているが、 この反応が過剰に発現すると、 逆に 様々な病態を引き起こす事が知られている。 IL-1 は免疫及び炎症反応において 重要なメデイエ一夕一であると考えられ、 様々な細胞に働いて、 それぞれの細胞 機能を亢進する。 その結果、 T、 Β リンパ球の分化及び増殖、 IL-2やコロニー 刺激因子、 IL-6、 IL-8、 TN 等の炎症性サイトカインの産生、 発熱 ·睡眠 ·食 欲低下 ·低血圧の誘導、 脳下垂体からのホルモン分泌亢進、 コラゲナーゼの産生 亢進による関節軟骨の破壌、 プロスタグランジン産生亢進による痛覚閾値の低下 をそれぞれ誘導し、 さらにランゲルハンス島の 細胞の破壊、 骨髄性白血病細胞 の増殖、 関節炎、 腸炎等の炎症への関与、 動脈硬化プラークの形成など多彩な生 理反応への関与が明らかになつている。 また、 IL-1 の過剰産生が原因と考えら れている疾病として慢性関節リウマチ、 骨粗鬆症、 敗血症、 炎症性腸疾患、 イン スリン依存性糖尿病、 動脈硬化症、 乾癬、 喘息、 アルコール性肝炎等が知られて いる （ TIPS,  Inflammatory cytokines such as IL-1 and TNF play an important role as bioprotective factors such as inflammation due to invasion of foreign substances.However, it is known that excessive expression of this response causes various pathological conditions. Have been. IL-1 is thought to be an important mediator in immune and inflammatory responses, acting on various cells and enhancing their cellular functions. As a result, differentiation and proliferation of T and Β lymphocytes, production of inflammatory cytokines such as IL-2 and colony stimulating factors, IL-6, IL-8 and TN, fever, sleep, decreased appetite, and induction of hypotension Increases hormone secretion from the pituitary gland, induces articular cartilage rupture due to increased collagenase production, lowers pain threshold due to increased prostaglandin production, further destroys cells in the islets of Langerhans, and proliferates myeloid leukemia cells Involvement in various inflammations such as arthritis and enteritis, and various physiological responses such as formation of atherosclerotic plaque have been clarified. Diseases that are thought to be caused by overproduction of IL-1 include rheumatoid arthritis, osteoporosis, sepsis, inflammatory bowel disease, insulin-dependent diabetes mellitus, arteriosclerosis, psoriasis, asthma, and alcoholic hepatitis. Known (TIPS,
(1988) 9, 171-177； New Engl. J. Med., (1993) 328， 106-113； Blood, (1996) 87, 2095-2147) 。  (1988) 9, 171-177; New Engl. J. Med., (1993) 328, 106-113; Blood, (1996) 87, 2095-2147).
また、 TNF は生体内に侵入した異物抗原との接触により活性化されたマク 口ファージ等の細胞より放出され、 マクロファージの走化性、 貪食能を亢進させ るばかりでなく、 T リンパ球の HLA抗原の発現を増強し、 また IL-2受容体の 発現の増加を通じて T細胞の機能を賦活化、 さらに好中球の走化性の亢進、 活 性化を誘導し、 異物の除去に働く。 また、 炎症局所の線維芽細胞に対しては増殖 促進に働き、 その組織修復を行っている。 さらに、 TNF Q!はこれらの細胞より 種々のサイト力インの発現を宂進させ、 IL-1 と同様にサイト力インネットヮ一 クの形成に関与している。 しかし、 これらの反応が過剰に発現すると増悪因子と して働くことがあり、 TNF Q!の過剰産生が知られている疾患として、 敗血症、 慢性関節性リウマチ、 炎症性腸疾患、 潰瘍性大腸炎、 クローン病、 SLE、 川崎 病等が報舎されている （日本臨床、 （1999) 57、 Suppl:794-797) 。 In addition, TNF is released from cells such as macula phage activated by contact with foreign antigens that have invaded the living body, and not only enhances the chemotaxis and phagocytosis of macrophages, but also enhances the HLA of T lymphocytes. It enhances the expression of antigens, activates the function of T cells by increasing the expression of IL-2 receptor, and also enhances the chemotaxis and activation of neutrophils to remove foreign substances. It also promotes the proliferation of fibroblasts in inflamed areas and repairs their tissues. Furthermore, TNF Q! Promotes the expression of various cytokins in these cells, and is involved in the formation of cytokinetic networks, like IL-1. However, when these reactions are overexpressed, they may act as exacerbating factors, and as diseases for which overproduction of TNF Q! Is known, sepsis, Rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, SLE, Kawasaki disease, etc. have been reported (Japanese clinical practice, (1999) 57, Suppl: 794-797).
従って、 TAK1キナーゼ活性もしくは TAK1 の活性ィ匕を抑制する物質が見出 されれば、 TAK1がシグナル伝達に関与する事が知られている TGF- ]3、 IL-1、 LPS, TNF等が原因因子として知られている各種疾病、 並びに心肥大、 心不全 の治療および予防薬の開発につながると考えられる。 しかしながら、 現在までに TAK1キナーゼ阻害剤、 もしくは TAK1活性化阻害剤は知られていない。  Therefore, if a substance that suppresses TAK1 kinase activity or TAK1 activity is found, TAK-] 3, IL-1, LPS, TNF, etc., which are known to be involved in signal transduction, may be the cause. It will lead to the development of drugs for treating and preventing various diseases known as factors, as well as for cardiac hypertrophy and heart failure. However, no TAK1 kinase inhibitor or TAK1 activation inhibitor has been known to date.
ところで、 ゼァラレノン類は、 これまでに種々の化合物が報告されており、 ま た、 種々の作用、 例えば、 サイト力イン、 特に I L一 1の産生抑制 （特開平 8— 4 0 8 9 3号公報、 欧州第 6 0 6 0 4 4 A号公報など） 、 チロシンキナーゼ阻害 (WO 9 6 / 1 3 2 5 9号公報） 、 ME K 1キナーゼ阻害に基づく T細胞の活性 化及び活性化に伴う増殖阻害 （Biochemistry, 37; 9579-9585， 1998) 、 J NK/ p 3 8活性化の阻害 （Biochem. Biophys. Res. Commun., 257;19-23, 1999) 、 ME K阻害 （J. Antibiotics, 52;1086-1094, 1999) 、 L P S刺激後のサイトカイ ン （ I L— 1、 I L一 6、 T N F - α ) 産生阻害 （Int. J. Imunop armacol., 21;799-814， 1999) 、 LPS、 IFN- T刺激後のサイト力イン （IL-l j3、 TNF- α) 産生抑制 （Cytokine, 8;751-761， 1996) など、 についても報告されている。  By the way, various compounds of zearalenones have been reported so far, and various actions, for example, inhibition of production of cytokins, particularly IL-11 (Japanese Patent Application Laid-Open No. 8-48093) , European Patent No. 664044A), Tyrosine kinase inhibition (WO96 / 13259), T cell activation based on MEK1 kinase inhibition and proliferation accompanying activation Inhibition (Biochemistry, 37; 9579-9585, 1998), inhibition of JNK / p38 activation (Biochem. Biophys. Res. Commun., 257; 19-23, 1999), inhibition of MEK (J. Antibiotics, 52; 1086-1094, 1999), inhibition of cytokine production (IL-1, IL-16, TNF-α) after LPS stimulation (Int. J. Imunoparmacol., 21; 799-814, 1999), LPS And the suppression of cytokin (IL-lj3, TNF-α) production after IFN-T stimulation (Cytokine, 8; 751-761, 1996).
しかし、 これまで、 ゼァラレノン類が TAK1あるいは TAK1活性化を阻害す る作用を有することは知られていない。 また、 8位および 9位のいずれにも水酸 基を有するゼァラレノン類が特に優れた該作用を有することも全く知られていな い。 発明の開示  However, it has not been known that zearalenones have an effect of inhibiting TAK1 or TAK1 activation. Further, it is not known at all that zearalenones having a hydroxyl group at both the 8-position and the 9-position have the particularly excellent action. Disclosure of the invention
本発明の目的は、 TAK 1阻害剤、 あるいは、 TAK 1活性化阻害剤を提供す ることである。 本発明者らは、 上記課題を解決するため鋭意研究を重ねた結果、 ある種のゼァラレノン類が優れた TAK1キナーゼ阻害活性を持ち、 また、 TAK1 の活性化を介した TGF- J3や IL-1等のシグナル伝達を阻害することを見出し、 この知見に基づき本発明を完成した。  An object of the present invention is to provide a TAK1 inhibitor or a TAK1 activation inhibitor. The present inventors have conducted intensive studies to solve the above problems, and as a result, certain zearalenones have excellent TAK1 kinase inhibitory activity, and TGF-J3 and IL-1 mediated by activation of TAK1. The present inventors have found that such signal transduction is inhibited, and completed the present invention based on this finding.
すなわち、 本発明は、 8位および 9位のいずれにも水酸基を有するゼァラレノ ン類を有効成分とする、 TAK 1阻害剤または TAK 1活性化阻害剤を提供する。 また、 本発明は、 8位及び 9位のいずれにも水酸基を有するゼァラレノン類を 有効量用いる、 T AK 1を阻害する方法または TAK 1の活性化を阻害する方法 を提供する。 That is, the present invention provides a zearaleno having a hydroxyl group at each of the 8-position and the 9-position. To provide a TAK 1 inhibitor or a TAK 1 activation inhibitor comprising a pharmaceutically acceptable salt as an active ingredient. The present invention also provides a method for inhibiting TAK1 or a method for inhibiting TAK1 activation, which comprises using an effective amount of a zearalenone having a hydroxyl group at each of the 8th and 9th positions.
さらに、 本発明は、 TAK1阻害剤または TAK1活性化阻害剤を製造するた めの 8位及び 9位のいずれにも水酸基を有するゼァラレノン類の使用を提供する。 加えて、 本発明は、 8位及び 9位のいずれにも水酸基を有するゼァラレノン類 および使用説明書を含む T AK 1を阻害するためのキットまたは T AK 1の活性 化を阻害するためのキットを提供する。 図面の簡単な説明  Furthermore, the present invention provides the use of zearalenones having a hydroxyl group at both the 8-position and the 9-position for producing a TAK1 inhibitor or a TAK1 activation inhibitor. In addition, the present invention provides a kit for inhibiting TAK1 or a kit for inhibiting TAK1 activation, which comprises a zearalenone having a hydroxyl group at each of positions 8 and 9 and an instruction manual. provide. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 化合物 （1) の TAK1を介したシグナル伝達阻害作用を示すグラフ である。 発明を実施するための最良の形態  FIG. 1 is a graph showing the inhibitory effect of compound (1) on signal transduction via TAK1. BEST MODE FOR CARRYING OUT THE INVENTION
本発明において、 8位および 9位のいずれにも水酸基を有するゼァラレノン類 には、 ゼァラレノン （Z e a r a l enone ：下記式 （A) ) またはゼァラレ ノール （Ze a r a l e no l ：下記式 （B) ) In the present invention, zearalenones having a hydroxyl group at both the 8-position and 9-position include zearalenone (Zearal enone: the following formula (A)) or zearalenol (Ze arale nol: the following formula (B))
Figure imgf000008_0001
Figure imgf000008_0001
Figure imgf000008_0002
Figure imgf000008_0002
から誘導される化合物であって、 その 8位および 9位のいずれにも水酸基 （―〇 H) を有する化合物が含まれる。 これら化合物には、 光学異性体、 幾何異性体が 存在し得るがそれらのいずれも、 およびそれら任意の割合の混合物も本発明に含 まれる。 And a compound having a hydroxyl group (-〇H) at both the 8-position and the 9-position. These compounds may exist in the form of optical isomers and geometric isomers, and any of these, and mixtures thereof in any ratio are also included in the present invention.
8位および 9位のいずれにも水酸基を有するゼァラレノン類としては、 具体的 には、 例えば、 以下に示されるような化合物が含まれる： Specific examples of zearalenones having a hydroxyl group at both the 8-position and the 9-position include the compounds shown below:
Figure imgf000009_0001
これら化合物 （1) 〜 （13) は、 各々それ自体公知の化合物であり、 文献 (例えば、 Int. J. Im漏 Pharmacol" 21:799-814, 1999； J. Org. Chem., 43: 2339-2343, 1978； Chem. Pharm. Bull. 41:373-375, 1993 ； J. Antibiotics, 52:1077-1085, 1999； Biochem. Biophys. Res. Commun., 257:19-23, 1999； Cytokine, 8:751-761, 1996； Pharmacol. Commun., 7:301-308, 1996； 日本特許 公開平 8— 40 8 9 3号公報； 欧州特許公開第 6 0 6 044 A号公報 ； Biochemistry, 37: 9579-9585, 1998など） に記載の方法により調製することが できる。
Figure imgf000009_0001
Each of these compounds (1) to (13) is a compound known per se and described in the literature (for example, Int. J. Im Leak Pharmacol "21: 799-814, 1999; J. Org. Chem., 43: 2339). Chem. Pharm. Bull. 41: 373-375, 1993; J. Antibiotics, 52: 1077-1085, 1999; Biochem. Biophys. Res. Commun., 257: 19-23, 1999; Cytokine, 8: 751-761, 1996; Pharmacol. Commun., 7: 301-308, 1996; Japanese Patent Publication No. 8-40893; European Patent Publication No. 6 06444A; Biochemistry, 37: 9579-9585, 1998, etc.).
8位および 9位のいずれにも水酸基を有するゼァラレノン類としては、 化合物 (1) 〜 (13) が好ましく、 化合物 (1) 〜 (3) がさらに好ましく、 化合物 (1) が特に好ましい。  As the zearalenones having a hydroxyl group at both the 8-position and the 9-position, compounds (1) to (13) are preferable, compounds (1) to (3) are more preferable, and compound (1) is particularly preferable.
本発明において、 TAK1阻害とは、 TAK1のキナーゼ活性を阻害すること を意味する。 TAK1の阻害活性は、 例えば、 後述の実施例 1に記載された方法 により測定することができる。  In the present invention, TAK1 inhibition means inhibiting the kinase activity of TAK1. The TAK1 inhibitory activity can be measured, for example, by the method described in Example 1 below.
TAK 1活性化阻害とは、 TAK 1を介した細胞内シグナル伝達を阻害するこ とを意味する。 TAK1阻害活性化阻害作用は、 例えば、 後述の実施例 2に記載 された方法により測定することができる。  TAK1 activation inhibition refers to inhibiting TAK1-mediated intracellular signal transduction. The TAK1 inhibitory activation inhibitory activity can be measured, for example, by the method described in Example 2 below.
疾病の原因として TAK 1の活性化が関与する疾病には、 TAK 1の活性化を 介した細胞内のシグナル伝達系に関与する TGF— |8、 I L— 1、 LPS, TN F等を原因因子とする各種疾病、 並びに、 心肥大、 心不全などが含まれる。  Diseases in which TAK 1 activation is involved as the cause of the disease include TGF- | 8, IL-1, LPS, TNF, etc., which are involved in intracellular signal transduction through TAK 1 activation. And various diseases, as well as cardiac hypertrophy and heart failure.
本発明の Τ ΑΚΊキナーゼ阻害剤または TAK 1活性化阻害剤の投与経路、 投 与量は、 患者の体型、 年齢、 体調、 疾患の種類や度合い、 発症後の経過時間等に より、 適宜選択することができるが、 非経口投与の場合には、 一般に 0.02 g〜 2mgZ体重 k 日の用量で、 経口投与の場合には、 一般に S ig SOOmgZ 体重 k 日の用量で効果が期待できる。  The administration route and administration amount of the Τ kinase inhibitor or TAK1 activation inhibitor of the present invention are appropriately selected depending on the patient's body type, age, physical condition, type and degree of disease, elapsed time after onset, and the like. However, in the case of parenteral administration, a dose of 0.02 g to 2 mgZ body weight k days is generally expected, and in the case of oral administration, an effect of Sig SOOmgZ body weight k days is generally expected.
本発明において 8位および 9位のいずれにも水酸基を有するゼァラレノン類は 1種用いてもよいし、 あるいは 2種以上を組み合わせて用いてもよい。  In the present invention, zearalenones having a hydroxyl group at both the 8-position and the 9-position may be used alone or in combination of two or more.
本発明の T AK 1阻害剤または T AK 1活性化阻害剤は、 1種もしくはそれ以 上の薬学的に許容し得る希釈剤、 湿潤剤、 乳化剤、 分散剤、 補助剤、 防腐剤、 緩 衝剤、 結合剤、 安定剤などを適宜含む薬学的組成物として、 目的とする投与経路 に応じ、 適当な任意の形態にして投与することができる。 The TAK1 inhibitor or TAK1 activation inhibitor of the present invention may comprise one or more pharmaceutically acceptable diluents, wetting agents, emulsifiers, dispersants, adjuvants, preservatives, lubricants It can be administered as a pharmaceutical composition appropriately containing an impaction agent, a binder, a stabilizer and the like in an appropriate form depending on the intended administration route.
また、 本発明のキットは、 前記の 8位及び 9位のいずれにも水酸基を有するゼ ァラレノン類、 希釈剤および上で例示した種々の担体を含む任意の 1種以上の担 体を含む薬学的組成物、 並びに使用説明書等を含む。 実施例  Further, the kit of the present invention comprises a pharmaceutical composition comprising a zearalenone having a hydroxyl group at each of the above-mentioned 8-position and 9-position, a diluent, and any one or more carriers including various carriers exemplified above. It contains the composition, as well as instructions for use. Example
以下、 実施例により本発明をさらに詳細に説明するが、 本発明はこれらの実施 例によりなんら限定されるものではない。  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
TAK1キナーゼ阻害活性の評価 Evaluation of TAK1 kinase inhibitory activity
(実施例 1 ) 化合物 （1 ) 〜 （3 ) の TAK1キナーゼ阻害活性の検討  (Example 1) Examination of TAK1 kinase inhibitory activity of compounds (1) to (3)
TAK1キナーゼ活性は TAK1の自己リン酸化、 ならびに TAB1のリン酸化を 測定することで評価した。 すなわち、 TAK1及び TAB1遺伝子を含有した組み 換えパキュロウィルスベクター （WO 9 9 / 2 1 0 1 0号公報） をそれぞれ昆虫 細胞 Sf-9に共感染させ、 3日間培養した。 培養後、 細胞を PBSで洗浄し、 3.0 X 107細胞/ mLとなるように抽出液 （20 mM HEPES, pH 7.4、 150 mM NaCL 12.5 mM β -gycerophosphate, 1.5 mM MgCl2、 2 mM EGTA、 10 mM NaF、 2 mM DTT、 1 mM Na3VO4 1 mM PMSF、 20 ^ MAprotinin、 0.5% Triton X- 100) に懸濁し細胞抽出液を調製した。 細胞抽出液 10 Lに抗 TAK1抗体 Μ-Γ7 、 SantaCruz 社 ) 100 ng、 及び ProtemG- Sepharose ( Amersham Pharmacia社製） 1 Lを加え、 4°Cでインキュベートした後、 免疫沈降物を洗 浄液 （20 mM HEPES, pH 7.4、 500 mM NaCL 10 mM MgCl2) で 3回洗浄、 続いてキナーゼ溶液 (10 mM HEPES, pH 7.4、 1 mM DTT、 5 mM MgCl2) で 2回洗浄し、 TAK1/TAB1精製物を調製した。 TAK1/TAB1精製物をキナーゼ溶 液に再懸濁し、 Multiscreenプレート （Millipore社製） の各穴に 25 t Lずつ加 えた後、 DMSOに溶解した試験化合物を 1 Lずつ加え、 室温にて 30分間イン キュペートした。 続いて、 1ゥエル当たり O.l C iとなるように [ τ -33Ρ] - ATP (Amersham Pharmacia社製） を加えた溶液 （100 mM MOPS, pH 7.2、 10 mM MgCl2 2 M ATP) を 25 i Lずつ加え、 37でにて 30分間インキュべ 一卜しキナーゼ反応を行った。 反応停止液 （50% TCA含有 PBS) を 50 Lず つ加え、 さらに 4 にて 10分間インキュベートし、 キナーゼ反応を停止した。 フリーの 33P-ATPを除去するために、 0.05% Tween-20を含有する PBSで 5回 洗浄し、 50 ^ Lずつの Supermix (Wallac社製） を加え、 さらに室温にて 30分 間清置した後 TAK1及び TAB1に取り込まれた 33P-ATP量を micro-betaにより 測定することで TAK1 キナーゼ活性を測定した。 その結果、 化合物 （1 ) 〜 ( 3 ) は表 1 に示すように TAK1キナーゼ活性を抑制し、 特に化合物.（1 ) は IC5。値として 8.1 nMの TAK1キナーゼ阻害活性を示した。 しかしながら、 化合 物 （1 ) 〜 （3 ) の 8位および 9位に対応する位置に水酸基を有しない化合物で める下己の Radicicol TAK1 kinase activity was assessed by measuring TAK1 autophosphorylation and TAB1 phosphorylation. That is, a recombinant paculovirus vector containing the TAK1 and TAB1 genes (WO99 / 211010) was co-infected with insect cells Sf-9 and cultured for 3 days. After culturing, the cells are washed with PBS, and the extract solution is adjusted to 3.0 x 10 7 cells / mL (20 mM HEPES, pH 7.4, 150 mM NaCL 12.5 mM β-gycerophosphate, 1.5 mM MgCl 2 , 2 mM EGTA, 10 mM The cells were suspended in mM NaF, 2 mM DTT, 1 mM Na 3 VO 4 1 mM PMSF, 20 ^ MAprotinin, 0.5% Triton X-100) to prepare a cell extract. To 10 L of cell extract, add 100 ng of anti-TAK1 antibody Μ-Γ7, SantaCruz) and 1 L of ProtemG-Sepharose (Amersham Pharmacia), incubate at 4 ° C, and wash the immunoprecipitate. Wash 3 times with 20 mM HEPES, pH 7.4, 500 mM NaCL 10 mM MgCl 2 ), then wash twice with kinase solution (10 mM HEPES, pH 7.4, 1 mM DTT, 5 mM MgCl 2 ), and TAK1 / TAB1 A purified product was prepared. Resuspend the purified TAK1 / TAB1 in kinase solution, add 25 tL to each well of Multiscreen plate (Millipore), add 1 L of test compound dissolved in DMSO, and allow to stand at room temperature for 30 minutes. Incubated. Subsequently, 1 so that Ueru per Ol C i [τ - 33 Ρ ] - ATP (Amersham Pharmacia Co. Ltd.) was added a solution (100 mM MOPS, pH 7.2, 10 mM MgCl2 2 M ATP) was added in 25 iL portions, and the mixture was incubated at 37 for 30 minutes to perform a kinase reaction. The reaction was terminated by adding 50 L of a reaction stop solution (PBS containing 50% TCA) and further incubating at 4 for 10 minutes. To remove free 33 P-ATP, wash 5 times with PBS containing 0.05% Tween-20, add 50 ^ L of Supermix (Wallac), and incubate at room temperature for 30 minutes After that, TAK1 kinase activity was measured by measuring the amount of 33 P-ATP incorporated into TAK1 and TAB1 using micro-beta. As a result, compounds (1) to (3) suppressed TAK1 kinase activity as shown in Table 1, and in particular, compound (1) exhibited IC 5 . A TAK1 kinase inhibitory activity of 8.1 nM was shown. However, Shimochi's Radicicol, which is a compound that does not have a hydroxyl group at the position corresponding to the 8th and 9th positions of the compounds (1) to (3),
radictcoi radictcoi
Figure imgf000012_0001
Figure imgf000012_0001
, ゼァラレノン （式 (Α) ) 、 ゼァラレノール （式 （Β) )
Figure imgf000013_0001
, Zearalenone (formula (Α)), zarelenol (formula (Β))
Figure imgf000013_0001
Figure imgf000013_0002
は TAKlキナーゼ阻害活性はほぼ認められなかつた。
Figure imgf000013_0002
Showed almost no TAKl kinase inhibitory activity.
表 1 table 1
I C5。 （M) 化合物 (1) 8. 1 X 10 -9 化合物 (2) 1. 2 X 1 0-7 化合物 (3) 6. 9 X 1 0-6 IC 5. (M) Compound (1) 8. 1 X 10 - 9 Compound (2) 1. 2 X 1 0-7 Compound (3) 6. 9 X 1 0- 6
Radicicol > 1 X 1 0 -5 ゼァラレノン > 1 X 1 0- 4 ゼァラレノール 〉 1 X 1 0-4 (実施例 2 ) TAK1を介したシグナル伝達阻害作用の検討 Radicicol> 1 X 1 0 - 5 Zeararenon> 1 X 1 0- 4 Zeararenoru> 1 X 1 0- 4 (Example 2) Examination of TAK1-mediated signal transduction inhibitory action
TAK1の活性化により MAPKカスケ一ドならびに NF- κ Βの活性化が誘導さ れることが知られている。 そこで、 TAK1 キナーゼ活性の阻害により TAK1 を 介したシグナル伝達への影響をレポーターアツセィを用い検討した。  It is known that activation of TAK1 induces activation of MAPK cascade and NF-κΒ. Therefore, the effect of inhibition of TAK1 kinase activity on TAK1-mediated signal transduction was examined using the reporter Atsusei.
初めに、 TGF- )3応答性エレメントの下流にルシフェラーゼの遺伝子を組み込 んだ p3TP-Lux (Cell, 71, 1003-1014, 1992) を用い、 化合物 ( 1 ) の影響を検 討した。 ヒト fibrosarcoma細胞株 HT-1080細胞 1 x 106個を播種し終夜培養し た後、 2 gの p3TP-Luxと 10 Lの FuGENE遺伝子導入試薬 （Roche社製） の混合物を細胞に添加し、 さらに 8時間培養し遺伝子を導入した。 遺伝子を導 入した細胞をトリプシンにより剥がした後、 1ゥエル当たり 1 X 104個となるよ うに 96穴マイクロプレート （Falcon社製） に播種し、 さらに終夜培養した。 First, the effect of compound (1) was examined using p3TP-Lux (Cell, 71, 1003-1014, 1992) in which a luciferase gene was incorporated downstream of the TGF-) 3 responsive element. After inoculating 1 x 10 6 human fibrosarcoma cell line HT-1080 cells and culturing overnight, a mixture of 2 g of p3TP-Lux and 10 L of FuGENE gene transfer reagent (Roche) was added to the cells. After culturing for 8 hours, the gene was introduced. The cells into which the gene was introduced were detached with trypsin, and then seeded on a 96-well microplate (Falcon) at 1 × 10 4 cells / well, and cultured overnight.
DMEM培地で 1回洗浄した後、 0.2%ゥシ胎児血清を含む DMEM培地で希釈し た化合物 （1 ) を 50 Lずつ添加した。 37°Cで 1時間インキュベートした後、 終濃度で 10 ng/mLとなるように TGF- β (Promega社製） を 50 Lずつ加え、 さらに 24時間培養した。 ルシフェラーゼ活性は Steady Glo Luciferase Assay Kit (Promega社製） を用いて測定した。 After washing once with DMEM medium, 50 L of compound (1) diluted with DMEM medium containing 0.2% fetal bovine serum was added. After incubating at 37 ° C for 1 hour, TGF-β (Promega) was added at 50 L each to a final concentration of 10 ng / mL, and the cells were further cultured for 24 hours. Luciferase activity was measured using the Steady Glo Luciferase Assay Kit (Promega).
また、 NF- κ Bの活性化の測定には応答性ェレメントの下流にルシフェラーゼ の遺伝子を組み込んだ NFkB-Luc (Genes Dev., 7, 1354-1363, 1993) を用い、 IL-1刺激、 又は TAK1/TAB1強制発現により TAK1の活性化を誘導し行った。 すなわち、 ヒト胎児腎細胞株 293 細胞に IL-1 受容体を強制発現させた 293- IL1R細胞 （ Mol. Cell, 5, 649-658, 2000) を 1 x 106個を播種し終夜培養した後、 2 gの NFkB-Lucと 10 の FuGENE遺伝子導入試薬 （Roche社製） の混 合物を細胞に添加し、 さらに 8時間培養し遺伝子を導入した。 遺伝子を導入し た細胞をトリプシンにより剥がした後、 1ゥエル当たり 1 X 104個となるように 96穴マイクロプレート （Coaning Coaster社製） に播種し、 さらに終夜培養し た。 DMEM培地で 1回洗浄した後、 0.2%ゥシ胎児血清を含む DMEM培地で希 釈した化合物 （1 ) を 50 しずつ添加した。 37°Cで 1時間インキュベートした 後、 終濃度で 10 ng/mLとなるように IL-1 (PEPRO TECH社製） を 50 L ずつ加え、 さらに 24 時間培養した。 ルシフェラーゼ活性は Steady Glo Luciferase Assay Kit (Promega社製） を用いて測定した。 TAK1/TAB1強制発 現による NFkBの活性化に関しては、 NFkB-Lucの遺伝子導入と共に、 TAK1、 TAB1発現ベクター pTAKl-HAならびに pCOS-FTABlをそれぞれ 250 ngずつ を同時に加え、 上記と同様に遺伝子を導入した。 遺伝子を導入した細胞は上記と 同様に 96穴マイクロプレート （Coaning Coaster社製） に播種し、 終夜培養し た後、 DMEM培地で 1回洗浄し、 0.2%ゥシ胎児血清を含む DMEM培地で希 釈した化合物 （1 ) を 50 Lずつ添加した。 37°Cで 24時間培養した後、 ルシ フェラーゼ活性を測定した。 To measure NF-κB activation, NFkB-Luc (Genes Dev., 7, 1354-1363, 1993) incorporating a luciferase gene downstream of the response element was used to stimulate IL-1 or Activation of TAK1 was induced by forced expression of TAK1 / TAB1. That is, after seeding 1 x 10 6 293-IL1R cells (Mol. Cell, 5, 649-658, 2000), which forcibly expressed the IL-1 receptor in human embryonic kidney cell line 293 cells, and cultured overnight A mixture of 2 g of NFkB-Luc and 10 FuGENE gene introduction reagents (Roche) was added to the cells, and the cells were further cultured for 8 hours to introduce the genes. After the cells into which the gene was introduced were detached with trypsin, the cells were seeded on a 96-well microplate (manufactured by Coaning Coaster) at 1 × 10 4 cells / well, and cultured overnight. After washing once with DMEM medium, compound (1) diluted with DMEM medium containing 0.2% fetal bovine serum was added in 50 portions. After incubating at 37 ° C for 1 hour, IL-1 (manufactured by PEPRO TECH) was added at 50 L each to a final concentration of 10 ng / mL, and the cells were further cultured for 24 hours. Luciferase activity is Steady Glo The measurement was performed using a Luciferase Assay Kit (promega). Regarding the activation of NFkB by forced expression of TAK1 / TAB1, together with the gene transfer of NFkB-Luc, simultaneously add 250 ng each of TAK1, TAB1 expression vector pTAKl-HA and pCOS-FTABl, and transfer the gene as above. did. Cells into which the gene has been introduced are seeded in a 96-well microplate (manufactured by Coaning Coaster) in the same manner as described above, cultured overnight, washed once with DMEM medium, and then diluted with DMEM medium containing 0.2% fetal serum. The diluted compound (1) was added in 50 L portions. After culturing at 37 ° C for 24 hours, luciferase activity was measured.
結果を図 1に示す。 図 1において、 縦軸は、 阻害活性 ( ) を示す。 横軸は、 化合物 （1 ) 濃度 （M) を示す。 黒塗り四角は、 p3TP-Lux アツセィにおける TGF- /3シグナル伝達経の阻害活性を示す。 黒塗り三角は NFkB-Lucアツセィに おける IL-1シグナル伝達系の阻害活性を示す。 白抜き三角は NFkB-Lucアツセ ィにおける TAK1/TAB1強制発現によるシグナル伝達系の阻害活性を示す。 図 1に示すように、 化合物 （1 ) は容量依存的に TGF- 3、 IL-l a、 ならびに TAK1/TAB1 強制発現によるルシフェラーゼの発現を抑制した。 この結果は TAK1キナーゼ活性の阻害により下流へのシグナル伝達を阻害できることを示唆 する。 産業上の利用の可能性  The results are shown in Figure 1. In FIG. 1, the vertical axis indicates the inhibitory activity (). The horizontal axis represents the concentration of the compound (1) (M). Closed squares indicate the inhibitory activity of p3TP-Lux Atsusei on TGF- / 3 signaling. Closed triangles indicate the inhibitory activity of the IL-1 signaling system on NFkB-LucAssy. The open triangle indicates the inhibitory activity of the signal transduction system by forced expression of TAK1 / TAB1 in NFkB-Luc assay. As shown in FIG. 1, compound (1) suppressed the expression of luciferase due to forced expression of TGF-3, IL-la, and TAK1 / TAB1 in a dose-dependent manner. This result suggests that inhibition of TAK1 kinase activity can inhibit downstream signal transduction. Industrial applicability
本発明の化合物は、 は TAK1 阻害作用を有するので、 TAK1 の活性化を介し た各種シグナル伝達系を阻害し、 疾病の原因として TAK 1の活性化が関与する 各種疾病、 例えば、 TGF- j3、 IL-1, LPS, TNF- α等が原因因子として知られる 各種疾病の治療および予防する薬剤として有用であることが期待される。  Since the compound of the present invention has a TAK1 inhibitory activity, it inhibits various signal transduction systems through activation of TAK1 and various diseases involving activation of TAK1 as a cause of the disease, for example, TGF-j3, It is expected to be useful as an agent for treating and preventing various diseases known to be caused by IL-1, LPS, TNF-α and the like.

Claims

請 求 の 範 囲 The scope of the claims
1. 8位及び 9位のいずれにも水酸基を有するゼァラレノン類を有効成分とす る、 T AK 1阻害剤または T AK 1活性化阻害剤。 1. A TAK1 inhibitor or TAK1 activation inhibitor comprising, as an active ingredient, a zearalenone having a hydroxyl group at each of the 8th and 9th positions.
2. 8位および 9位のいずれにも水酸基を有するゼァラレノン類が、 下記の化 合物 （1) 〜 （13)  2. Zearalenones having a hydroxyl group at each of the 8- and 9-positions are represented by the following compounds (1) to (13)
Figure imgf000016_0001
Figure imgf000017_0001
から選択される、 請求項 1に記載の阻害剤。
Figure imgf000016_0001
Figure imgf000017_0001
The inhibitor according to claim 1, which is selected from:
3. 化合物が、 化合物 （1) 、 （2) または （3) である、 請求項 2に記載の 阻害剤。  3. The inhibitor according to claim 2, wherein the compound is the compound (1), (2) or (3).
4. 8位及び 9位のいずれにも水酸基を有するゼァラレノン類を有効量用いる， TAK 1を阻害する方法または TAK 1の活性化を阻害する方法。  4. A method of inhibiting TAK 1 or inhibiting the activation of TAK 1, using an effective amount of a zearalenone having a hydroxyl group at each of positions 8 and 9.
5. TAK1阻害剤または TAK 1活性化阻害剤を製造するための 8位及び 9 位のいずれにも水酸基を有するゼァラレノン類の使用。  5. Use of a zearalenone having a hydroxyl group at each of the 8th and 9th positions for producing a TAK1 inhibitor or a TAK1 activation inhibitor.
6. 8位及び 9位のいずれにも水酸基を有するゼァラレノン類および使用説明 書を含む TAK1を阻害するためのキットまたは TAK1の活性化を阻害するた めのキッ卜。  6. A kit for inhibiting TAK1 or a kit for inhibiting TAK1 activation, comprising a zearalenone having a hydroxyl group at each of the 8th and 9th positions and instructions for use.
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