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NF- κ B-Inhibiting Naphthopyrones from the Fijian Echinoderm Comanthus parvicirrus

NF- κ B-Inhibiting Naphthopyrones from the Fijian Echinoderm Comanthus parvicirrus. Florence Folmer, † William T. A. Harrison, † Jioji N. Tabudravu, † Marcel Jaspars,*, † William Aalbersberg, ‡ Klaus Feussner, ‡ Anthony D. Wright, § Mario Dicato, ⊥ and Marc Diederich ⊥

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NF- κ B-Inhibiting Naphthopyrones from the Fijian Echinoderm Comanthus parvicirrus

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  1. NF-κB-Inhibiting Naphthopyrones from the Fijian Echinoderm Comanthus parvicirrus Florence Folmer,† William T. A. Harrison,† Jioji N. Tabudravu,† Marcel Jaspars,*,† William Aalbersberg,‡ Klaus Feussner,‡Anthony D. Wright,§ Mario Dicato,⊥ and Marc Diederich⊥ Department of Chemistry, UniVersity of Aberdeen, Old Aberdeen, AB24 3UE, U.K., Institute of Applied Sciences, Faculty of Science and Technology, UniVersity of the South Pacific, P.O. Box 1168, SuVa, Fiji Islands, Australian Institute of Marine Science, TownsVille 4810, Queensland, Australia, and Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Hôpital Kirchberg, 9, Rue Edward Steichen, L-2540 Luxembourg, Luxembourg Received June 18, 2007 學 生：賴建豪 簡嵐翔授課老師：詹于誼 老師日 期：97.06.10

  2. 結構解析

  3. naphthopyrones 6-methoxycomaparvin (1) brown needle-shaped crystals UV (MeOH) λmax (logε) 245 (4.57), 290 (4.37), 380 (3.77) nm IR (KBr) νmax 3341, 2950, 1667, 1615, 1571, 1537, 1448 cm-1 LRESIMS m/z 331 [M + H]+ (C18H18O6) HRESIMS m/z 331.1176 [M + H]+, (calc for C18H19O6) 1H NMR (400 MHz, CDCl3) δ: 1.8 (2H, m, H-12) Methylene (CH2) 1.01 (3H, t, H-13) 6.23 (2H, t J=7.7 Hz, H-11) Methyl (CH3) 3.94 (3H, s, H-16) 6.23 (1H, s, H-3) Methine (CH) 6.43 (1H, d, J=1.6 Hz, H-9) 12.90 (s, OH-5) 7.01 (1H, d, J=1.6 Hz, H-7) 3.95 (3H, s, H-15)

  4. 13C NMR : 20.0 (C-12) 13.7 (C-13) 36.5 (C-11) 56.2 (C-16) 170.1 (C-2) 109.5 (C-3) 152.4 (C-10b) 136.1 (C-10a) 183.4 (C-4) 160.2 (C-10) 96.7 (C-9) 147.1 (C-4a) 104.9 (C-8) 109.3 (C-5) 134.7 (C-6) 96.3 (C-7) 158.3 (C-6a) 60.4 (C-15)

  5. HMBC

  6. COSY

  7. single-crystal X-ray diffraction analysis

  8. 6-methoxycomaparvin 5-methyl ether (2) brown needle-shaped crystals UV (MeOH) λmax (logε) 240 (4.58), 280 (4.51), 365 (4.05) nm IR (KBr) νmax 3229, 2960, 1642, 1584, 1415 cm-1 LRESIMS m/z 345 [M + H]+ (C19H20O6) HRESIMS m/z 345.1337 [M + H]+, (calc for C19H21O6) 1H NMR (400 MHz, CDCl3) δ: 12.90 (s, OH-5) 3.93 (s, H-14) 6-methoxycomaparvin 5-methyl ether (2) naphthopyrones 6-methoxycomaparvin (1)

  9. 13C NMR : 62.0 (C-14) 6-methoxycomaparvin 5-methyl ether (2) naphthopyrones 6-methoxycomaparvin (1)

  10. HMBC

  11. 細胞活性試驗

  12. Reporter Gene AssaysLuciferase Reporter Gene Assay For rapid, sensitive determination of luciferase enzyme activity in transfected cells or in tissues isolated from transgenic animals Transient transfections of K562 cells- pNF-κBLuc K562 cells

  13. Figure 1. Luciferase activity of pNF-κBLuc K562 cells pretreated for 2 h with different concentrations (in μg/mL) of 6-methoxycomaparvin (1) (A) or 6-methoxycomaparvin 5-methyl ether (2) (B), and treated for 2 h with 20 ng/mL TNF-α.

  14. Electrophoretic mobility shift assay (EMSA) Oligonucleotide (contain NF-κB binding site ) The probe was hybridized and labeled with[γ-32P]ATP (Contain p50/p65)

  15. Figure 2. Inhibition of TNF-R-induced NF-κB-DNA binding activity by 6-methoxycomaparvin (1) (A) and 6-methoxycomaparvin 5-methyl ether (2) (B). For supershift/immunodepletion experiments, incubation with 2 μg of anti-p50, anti-p52, anti-p65, anti c-Rel, and anti-RelB antibodies (C).

  16. Figure 3. Western blot analysis showing that 6-methoxycomaparvin (1) and 6-methoxycomaparvin 5-methyl ether (2) inhibit TNF-α induced degradation of IκBα and the consequent translocation of p50 and p65 into the nucleus.

  17. Figure 4. Effects of 6-methoxycomaparvin (1) and 6-methoxycomaparvin 5-methyl ether (2) on the kinase activity of IKKβ. “No enzyme” refers to a control in the absence of IKKβ. The negative control was performed in the presence of IKKβ, but in the absence of any test compound. Calbio IV ,which is an IKKβinhibitor.

  18. Figure 5. Effects of 6-methoxycomaparvin (1) and 6-methoxycomaparvin 5-methyl ether (2) on the proteolytic activity of the 26S proteasome in K562 cells, at a concentration of 100 μg/mL (300μM). “Control” refers to a negative control without any test compound. Different concentrations of the known proteasome inhibitor MG132 (Z-Leu-Leu-Leu-CHO) were used as positive controls.

  19. 結論 • Both compounds inhibit the activation of the transcription factor NF-κB, which plays an important role in cancer development and inflammation, and the mechanism of action of the two compounds was investigated. • Both naphthopyrones 1 and 2 completely inhibit TNF-α-induced NF-κB activation by inhibiting the enzymatic activity of the kinase IKKβ.

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