Journal of Natural Products
Article
quenched with H2O and extracted with EtOAc (50 mL). The organic
layer was sequentially washed with HCl (0.2 N), H2O, saturated
NaHCO3, H2O, and saturated NaCl (50 mL each) and dried over
Na2SO4. The organic filtrate was concentrated under reduced pressure,
and the resulting residue was purified over an HPLC column
(CapcellPak UG120, ⦶ 20 mm × 250 mm, 80% aqueous acetonitrile)
to give 8a (5.4 mg) and 8b (1.4 mg).
108.5 (C-4), 108.1 (C-6, C-9a), 74.7 (C-1′), 56.2 (OCH3-1′), 32.5 (C-
2′), 31.2 (C-4′), 25.4 (C-3′), 22.0 (C-5′), 13.9 (C-6); HRESIMS m/z
385.1295 (calcd for C21H21O7, 385.1282).
Methanolysis of 5. A solution of 5 (3.6 mg) in 5% HCl−MeOH
(1.0 mL) was stirred for 42 h at room temperature. The reaction
mixture was extracted with EtOAc (80 mL) and H2O (80 mL). The
organic layer was washed with saturated NaCl solution (80 mL) and
dried over Na2SO4. The organic filtrate was concentrated under
reduced pressure, and the resulting residue was purified by silica gel
column chromatography (eluent CHCl3) to give 10 (1.8 mg). The
aqueous layer was concentrated in vacuo to give 7 (2.5 mg). Methyl D-
galactopyranoside (7) was p-bromobenzoylated and purified in a
Methyl 2,3,4,6-tetra-O-p-bromobenzoyl-α-D-galactopyranoside
(8a): colorless, amorphous solid; [α]25 +177.5 (c 0.29, CHCl3)
D
{lit.17 [α]25D +178.1 (c 1.16, CHCl3)}; 1H NMR (CDCl3, 400 MHz) δ
8.0−7.4 (16H, p-bromobenzoyl × 4), 5.96 (1H, d, J = 3.2 Hz, H-4),
5.93 (1H, dd, J = 3.2, 10.8 Hz, H-3), 5.62 (1H, dd, J = 3.6, 10.8 Hz, H-
2), 5.28 (1H, d, J = 3.6 Hz, H-1), 4.58 (2H, m, H-5, H-6a), 4.38 (1H,
m, H-6b), 3.48 (3H, s, OCH3-1); 13C NMR (CDCl3, 100 MHz) δ
165.3 (ester), 165.3 (ester), 164.9 (ester), 164.7 (ester), 132.1−127.8
(aromatic carbons), 97.5 (C-1), 69.3 (C-4), 69.2 (C-2), 68.5 (C-3),
66.5 (C-5), 62.5 (C-6), 55.8 (OCH3); HRESIMS m/z 944.8142 (calcd
for C35H26O10Br4Na, 944.8152).
manner similar to that described above to afford 8a (0.8 mg; [α]24
D
+182.9 (c 0.04, CHCl3) and 8b (<0.1 mg).
Versicolorin B (10): orange-yellow, amorphous solid; [α]26 −230
D
(c 0.07, dioxane); 1H NMR (CDCl3/CH3OD = 1:1, 400 MHz) δ 7.53
(1H, d, J = 2.6 Hz, H-4), 7.22 (1H, s, H-6), 6.59 (1H, d, J = 2.6 Hz, H-
2), 6.49 (1H, d, J = 5.8 Hz, H-11), 4.17 (2H, m, H-12, H-14a), 3.86
(1H, m, H-14b), 2.37 (1H, m, H-13a), 2.30 (1H, m, H-13b); 13C
NMR (CDCl3/CH3OD = 1:1, 100 MHz) δ 190.4 (C-10), 182.6 (C-
5), 166.2 (C-7), 166.0 (C-9), 165.4 (C-1), 160.2 (C-3), 136.5 (C-5a),
135.6 (C-4a), 120.3 (C-8), 113.6 (C-11), 111.9 (C-9a), 110.2 (C-4),
109.4 (C-10a), 108.9 (C-2), 102.8 (C-6), 48.2 (C-14), 44.7 (C-12),
31.0 (C-13); HRESIMS m/z 341.0655 (calcd for C18H13O7,
341.0656).
Methyl 2,3,4,6-tetra-O-p-bromobenzoyl-β-D-galactopyranoside
(8b): colorless, amorphous solid; [α]25 +149.3 (c 0.06, CHCl3)
D
{lit.17 [α]25D +132.6 (c 1.16, CHCl3)}; 1H NMR (CDCl3, 400 MHz) δ
8.0−7.4 (16H, p-bromobenzoyl × 4), 5.93 (1H, dd, J = 1.0, 3.6 Hz, H-
4), 5.70 (1H, dd, J = 7.8, 10.8 Hz, H-2), 5.55 (1H, dd, J = 3.6, 10.8 Hz,
H-3), 4.73 (1H, d, J = 7.8 Hz, H-1), 4.67 (1H, dd, J = 6.8, 11.0 Hz, H-
6a), 4.40 (1H, d, J = 7.0, 11.0 Hz, H-6b), 4.30 (1H, br dd, J = 6.8, 7.0
Hz, H-5), 3.60 (3H, s, OCH3-1); 13C NMR (CDCl3, 100 MHz) δ
165.3 (ester), 164.9 (ester), 164.8 (ester), 164.6 (ester), 132.1−127.4
(aromatic carbons), 102.2 (C-1), 71.8 (C-3), 71.0 (C-5), 69.8 (C-2),
68.2 (C-4), 61.9 (C-6), 57.4 (OCH3); HRESIMS m/z 944.8143 (calcd
for C35H26O10Br4Na, 944.8152).
Cell Culture. LNZTA3 cells, stably transfected human glioblastoma
LN-Z308 cells that express wild-type p53 by a tetracycline-regulated
system, were obtained from the American Type Culture Collection
(Rockville, MD, USA).9 LNZTA3 cells were grown at 37 °C with 5%
CO2 in Opti-MEM (Life Technologies, Carlsbad, CA, USA)
supplemented with 1% heat-inactivated fetal bovine serum (Nichirei
Biosciences, Tokyo, Japan), penicillin G (100 units/mL), and
tetracycline (2.3 μM).
p53-Dependent Growth Suppression Assay. LNZTA3 cells in
the presence or absence of tetracycline (2.3 μM) were plated into 96-
well plates (8 × 103 cells/well) and precultured for 12 h. Various
amounts of quinofuracins were added to the wells, and the cells were
subsequently cultured for 3 days. 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT; 10 μL; 12 mM) was added to the
treated cells, and the incubation at 37 °C continued for 6 h. After
incubation, cells were supplemented with sodium dodecyl sulfate and
incubated at 37 °C for 12 h. Absorbance was measured at 570 nm
using a spectrophotometer.
Preparation of Authentic Methyl 2,3,4,6-Tetra-O-p-bromo-
benzoyl-α-D- galactopyranoside (8a). To a solution of methyl α-D-
galactopyranoside (10 mg) in pyridine (1 mL) were added p-
bromobenzoyl chloride (200 mg) and DMAP (5 mg), and the mixture
was stirred for 12 h at 80 °C. The reaction mixture was quenched with
H2O and extracted with EtOAc (50 mL). The organic layer was
sequentially washed with HCl (0.2 N), H2O, saturated NaHCO3, H2O,
and saturated NaCl (50 mL, each) and dried over Na2SO4. The filtrate
was concentrated under reduced pressure, and the resulting residue
was purified by silica gel chromatography (n-hexane−EtOAc = 10:1)
to give an authentic sample of 8a (37.8 mg): colorless, amorphous
solid; [α]26D +189.0 (c 0.88, CHCl3); HRESIMS m/z 944.8164 (calcd
for C35H26O10Br4Na, 944.8152).
Preparation of Authentic Methyl 2,3,4,6-Tetra-O-p-bromo-
benzoyl-β-D- galactopyranoside (8b). In a procedure similar to
that used in the synthesis of 8a, methyl β-D-galactopyranoside was
used to generate an authentic sample of 8b (44.9 mg): colorless,
ASSOCIATED CONTENT
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* Supporting Information
1
CD, H NMR, and 13C NMR spectra for quinofuracins are
amorphous solid; [α]26 +139.7 (c 1.05, CHCl3); HRESIMS m/z
D
944.8169 (calcd for C35H26O10Br4Na, 944.8152).
Methanolysis of 1. A solution of 1 (16.7 mg) in 5% HCl−MeOH
(1.67 mL) was stirred for 18 h at room temperature. The reaction
mixture was extracted with EtOAc (84 mL) and H2O (84 mL). The
organic layer was washed with H2O (80 mL) and saturated NaCl
solution (80 mL) and dried over Na2SO4. The organic filtrate was
concentrated under reduced pressure, and the resulting residue was
purified by silica gel column chromatography (eluent CHCl3) to give 9
(10.7 mg). The aqueous layer was concentrated in vacuo to give 7 (6.0
mg). Methyl D-galactopyranoside (7) was p-bromobenzoylated and
purified in a manner similar to that described above to afford 8a {7.1
mg; [α]27D +173.6 (c 0.34, CHCl3)} and 8b {1.1 mg; [α]27D +147.5 (c
0.06, CHCl3)}.
AUTHOR INFORMATION
Corresponding Author
*Phone: +81-55-924-0601. Fax: +81-55-922-6888. E-mail:
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by JST and Grant-in-Aid
for Young Scientists (B) Number 21790217. We thank Mr. S.
Ohba for the spectroscopic measurements, Ms. S. Kakuda for
technical assistance, and Dr. M. Hatano and Dr. M. Igarashi for
helpful advice.
1′-O-Methylaverufin (9): red, amorphous solid; [α]27 0 (c 0.1,
D
CHCl3/MeOH = 5:1); 1H NMR (DMSO-d6, 400 MHz) δ 12.82
(OH), 12.14 (OH), 11.20 (OH), 7.20 (1H, s, H-6), 7.08 (1H, d, J =
2.6 Hz, H-4), 6.56 (1H, d, J = 2.6 Hz, H-2), 4.81 (1H, dd, J = 6.4, 7.2
Hz, H-1′), 3.20 (3H, s, OCH3-1′), 2.00 (1H, m, H-2′a), 1.80 (1H, m,
H-2′b), 1.30 (1H, m, H-3′a), 1.24 (4H, m, H2-4′, H2-5′), 1.20 (1H, m,
H-3′b), 0.84 (3H, s, H-6′); 13C NMR (DMSO-d6, 100 MHz) δ 188.9
(C-10), 181.3 (C-5), 165.2 (C-1), 164.2 (C-3), 163.7 (C-7), 163.1 (C-
9), 134.8 (C-4a), 133.3 (C-5a), 119.3 (C-8), 108.7 (C-2, C- 10a),
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