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

結構解析

naphthopyrones 6-methoxycomaparvin (1)brown needle-shaped crystalsUV (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 :

OOCH3

HO

OCH3

OH

O

11

4

4a

66a

10a 10b

6.23 (1H, s, H-3)

7.01 (1H, d, J=1.6 Hz, H-7)

6.43 (1H, d, J=1.6 Hz, H-9)

6.23 (2H, t J=7.7 Hz, H-11)

1.8 (2H, m, H-12)

Methine (CH)

Methylene (CH2)1.01 (3H, t, H-13)

3.94 (3H, s, H-16)

3.95 (3H, s, H-15)

Methyl (CH3)

12.90 (s, OH-5)

13C NMR :

OOCH3

HO

OCH3

OH

O

11

4

4a

66a

10a 10b

170.1 (C-2)

183.4 (C-4)

147.1 (C-4a)

109.3 (C-5)

134.7 (C-6)

158.3 (C-6a)

104.9 (C-8)

160.2 (C-10)136.1 (C-10a) 152.4 (C-10b)

109.5 (C-3)

96.3 (C-7)

96.7 (C-9)

36.5 (C-11)

20.0 (C-12)

13.7 (C-13)

60.4 (C-15)

56.2 (C-16)

HMBC

OOCH3

O

OCH3

O

O

H

H

H

H

H

H

HH

11

4

4a

66a

10a 10b

COSY

OOCH3

O

OCH3

O

O

H

H

H

H

H

H

HH

11

4

4a

66a

10a 10b

single-crystal X-ray diffraction analysis

6-methoxycomaparvin 5-methyl ether (2)brown needle-shaped crystalsUV (MeOH) λmax (logε) 240 (4.58), 280 (4.51), 365 (4.05) nmIR (KBr) νmax 3229, 2960, 1642, 1584, 1415 cm-1LRESIMS m/z 345 [M + H]+ (C19H20O6) HRESIMS m/z 345.1337 [M + H]+, (calc for C19H21O6)1H NMR (400 MHz, CDCl3) δ :

OOCH3

HO

OCH3

OH

O

11

4

4a

66a

10a 10b

OOCH3

HO

OCH3

OCH3

O10b10a

6a6

4a

4

11

naphthopyrones 6-methoxycomaparvin (1)6-methoxycomaparvin 5-methyl ether (2)

12.90 (s, OH-5)3.93 (s, H-14)

OOCH3

HO

OCH3

OH

O

11

4

4a

66a

10a 10b

OOCH3

HO

OCH3

OCH3

O10b10a

6a6

4a

4

11

naphthopyrones 6-methoxycomaparvin (1)6-methoxycomaparvin 5-methyl ether (2)

13C NMR :

62.0 (C-14)

HMBC

OOCH3

HO

OCH3

OCH3

O10b10a

6a6

4a

4

11

細胞活性試驗

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

Figure 1. Luciferase activity of pNF-κBLuc K562 cells pretreatedfor 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.

Electrophoretic mobility shift assay (EMSA)Oligonucleotide (contain NF-κB binding site )

The probe was hybridized and labeled with[γ-32P]ATP

(Contain p50/p65)

Figure 2. Inhibition of TNF-R-induced NF-κB-DNA bindingactivity by 6-methoxycomaparvin (1) (A) and 6-methoxycomaparvin5-methyl ether (2) (B). For supershift/immunodepletion experiments, incubationwith 2 μg of anti-p50, anti-p52, anti-p65, anti c-Rel, and anti-RelBantibodies (C).

Figure 3. Western blot analysis showing that 6-methoxycomaparvin(1) and 6-methoxycomaparvin 5-methyl ether (2) inhibit TNF-α induceddegradation of IκBα and the consequent translocation ofp50 and p65 into the nucleus.

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 IKKβ inhibitor.

Figure 5. Effects of 6-methoxycomaparvin (1) and 6-methoxycomaparvin5-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.

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.