Journal of Natural Products
Note
Table 2. Inhibitory Activity against HMT G9a and Cytotoxicity on P388 Cells of Compounds 1−7 (IC50)
1
2
3
4
5
6
7
HMT G9a (μM)
cytotoxicity (μM)
>100
3.4
55
0.058
>100
0.11
58
2.6
0.056
6.4
0.024
2.1
0.020
0.11
(1:1), followed by ODS-HPLC using gradient elution from 50% to
80% aqueous MeOH to yield five fractions (A−E). Fraction B was
then purified by semipreparative ODS-HPLC using a gradient of
MeCN in H2O (30−50%) to afford 3 (5.4 mg), 4 (2.6 mg), 5 (2.8
mg), and 6 (4.2 mg). Fractions C and D were combined and purified
by HPLC using gradient elution from 35% to 55% MeCN in H2O to
yield 1 (1.5 mg), 2 (2.6 mg), and 7 (3.0 mg).
Therefore, 2 was suggested to be the deoxy derivative of 5,
which was supported by the HMBC data (Table 1).13 Both
compound 1 and the co-isolated gliotoxin 614 have a 10aR
configuration, with the configuration of 6 being assigned by
comparison of specific rotation data. On the basis of biogenetic
considerations we also presume a 10aR configuration for 2.
The cytotoxic activities of compounds 1−7 were examined
against P388 murine leukemia cells. Gliotoxin (6) and gliotoxin
G (7) exhibited the most potent activity, whereas compounds
2−5 also showed significant activity (Table 2). However,
compound 1 had only marginal activity. We also examined the
inhibitory activity of 1−7 against HMT G9a and HMT Set7/9
(lysine-specific histone methyltransferase for lysine 4 in histone
H3). As expected from the previous report,4 compounds with a
disulfide or tetrasulfide bond (5, 6, and 7) exhibited potent
inhibitory activity. The weaker activity observed for 2, which
also has a disulfide bond, suggested that the C-6 hydroxy group
interfered with the G9a inhibitory activity. None of 1−7
inhibited HMT Set7/9 at 100 μM.
Bis(dethio)-10a-methylthio-3a-deoxy-3,3a-didehydrogliotoxin
(1): white solid; [α]25D −4.6 (c 0.03, MeOH); UV (MeOH) λmax (log
1
ε) 205 (4.20), 265 (4.07) nm; H NMR (CDCl3) and 13C NMR
(CDCl3), see Table 1; HRESIMS m/z 315.0759 [M + Na]+ (calcd for
C14H16N2O3S, 315.0779).
6-Deoxy-5a,6-didehydrogliotoxin (2): colorless oil; [α]25 −20 (c
D
0.05, MeOH); UV (MeOH) λmax (log ε) 206 (4.26), 265 (3.91) nm;
1H NMR (CDCl3) and 13C NMR (CDCl3), see Table 1; HRESIMS
m/z 331.0315 [M + Na]+ (calcd for C13H12N2O3S2, 331.0338).
Gliotoxin (6): [α]25 −440 (c 0.11, MeOH). The sign and
D
magnitude of the specific rotation value were comparable to those
of gliotoxin in the literature,14 which was [α]25D −290 (c 0.08, EtOH).
Preparation of MTPA Esters of 1a and 1b. Compound 1 (100 μg
for each) was reacted with either R-(−)- or S-(+)-MTPA Cl (5 μL) in
50 μL of CH2Cl2 and 50 μL of pyridine for 2 h. The reaction mixture
was diluted with H2O and extracted with CH2Cl2 three times. The
organic layers were combined and separated by ODS-HPLC (C18-
stationary phase, 10 × 50 mm; 40−60% MeCN in H2O) to afford the
S-(−)- or R-(+)-MTPA esters 1a and 1b.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a JASCO DIP-1000 digital polarimeter. UV spectra
were recorded on a Shimadzu Biospec 1600. NMR spectra were
recorded on a JEOL alpha 600 NMR spectrometer at 300 K. Chemical
shifts were referenced to solvent peaks: δH 7.27 and δC 77.2 for
CHCl3. ESI mass spectra were measured on a JEOL JMS-T 100LC.
HPLC was carried out on a Shimadzu LC 20AT with a SCL-10Avp
controller and a SPD-10Avp detector.
(S)-MTPA Ester of 1 (1a): 1H NMR (CDCl3) δ 6.32 (H-6), 5.99
(H-9), 5.94 (H-8), 5.67 (HZ-3a), 5.43 (H-7), 5.36 (H-5a), 4.94 (HE-
3a), 3.28 (N-CH3), 3.02 (H-10), 2.23 (S-CH3); ESIMS m/z 531 [M +
Na]+.
(R)-MTPA Ester of 1 (1b): 1H NMR (CDCl3) δ 6.41 (H-6), 6.04
(H-8), 6.03 (H-9), 5.69 (H-7), 5.50 (HZ-3a), 5.34 (H-5a), 4.91 (HE-
3a), 3.26 (N-CH3), 3.03 (H-10), 2.21 (S-CH3); ESIMS m/z 531 [M +
Na]+.
Fungal Material. Deep-sea sediments were collected by the
unmanned ROV KAIKO system from Fujikawa, Suruga-Bay, Japan, at
a depth of 1151 m, in July 1996. The sediment sample was stored in a
sterilized sampler and frozen with liquid nitrogen. Then the sample
was transported to the laboratory, where it was kept frozen until
processed. The Penicillium sp. JMF034 strain was isolated from this
sample. To investigate the taxonomic position of the strain, the 28S
rDNA-D1/D2 gene was amplified using the PCR method with primers
NL1 and NL4.15 The PCR product was sequenced with the
dideoxynucleotide chain-termination method, using a BigDye
Terminator v3.1 kit (Applied Biosystems) and ABI PRISM 3130xl
genetic analyzer system (Applied Biosystems). The 28S rDNA-D1/D2
gene sequence of the isolated fungus (DDBJ accession no. AB684325)
was compared with other sequences in the public database using the
BLAST program. Strain JMF034 showed the highest similarities to
strain Penicillium angulare NRRL28157T (sequence identity 99.1%), P.
adametzioides NRRL3405T (98.9%), and P. brocae NRRL31472T
(sequence identity 98.8%). Therefore JMF034 is included in the
Penicillium genus..
Fermentation, Extraction, and Isolation. The fungal strain was
cultured in 20 × 500 mL Erlenmeyer flasks each containing 100 mL of
production medium (0.3 g yeast extract, 0.3 g malt extract, 0.5 g
peptone, 1 g glucose, pH 7.2−7.4) at 27 °C. After 14 days of static
culture, the fermentation broth, including cells, was harvested and then
centrifuged to separate the mycelial mass from the aqueous layer.
The mycelial mass and the aqueous layer were exhaustively
extracted with acetone and EtOAc, respectively. Then each extract
was concentrated in vacuo. The EtOAc extract was subjected to C18
flash column chromatography (5 × 30 cm), eluting with a stepwise
gradient of 20%, 40%, 60%, 80%, and 100% (v/v) MeOH in H2O (2 L
each). The fraction that eluted with 60% MeOH was further
fractionated with a Sephadex LH-20 column using CHCl3/MeOH
Assay for Cytotoxicity against P388 Cells. P388 murine
leukemia cells were cultured in RPMI-1640 medium containing 10%
fetal bovine serum, 100 μg/mL kanamycin, and 10 μg/mL 2-
hydroxyethyl disulfide at 37 °C under an atmosphere of 5% CO2. To
each well of the 96-well microplate containing 100 μL of tumor cell
suspension (1 × 104 cells/mL) was added 100 μL of test solution
dissolved in RPMI-1640 medium, and the plate was incubated in a
CO2 incubator at 37 °C for 96 h. After the addition of 50 μL of 3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide saline sol-
ution (1 mg/mL) to each well, the plate was incubated for 3 h under
the same conditions to stain live cells. After the incubation, the plate
was centrifuged, the supernatants were removed, and the cells were
dissolved in 150 μL of DMSO to determine the IC50 values.
Histone Methyltransferase Inhibitory Activity Assay. A
mixture of 1 μL of GST (glutathione S-transferase)-G9a (200 ng) or
His (histone)-Set7/9 (931 ng), 1 μL of BSA (150 ng), 25 μL of 2×
HMT activity buffer (100 mM Tris-HCl (pH 8.5), 20 mM MgCl2, 40
mM KCl, 20 mM 2-mercaptoethanol, 500 mM sucrose), 1 μL of a
DMSO solution of the compound of interest, and 20 μL of H2O was
incubated at room temperature (rt) for 1 h in a total volume of 48 μL/
well. After the addition of 1 μL of S-adenosylmethionine (50 ng) and 1
μL of biotinylated H3 peptide (50 ng) into this reaction mixture, the
resulting mixture was further incubated at 37 °C for 2 h followed by
boiling at 96 °C for 30 min. The supernatant of the mixture was
transferred to a streptavidin-coated plate and incubated at rt for 1 h.
The supernatant was removed, and the remaining plate was washed
with 300 μL of PBS containing 0.5% Tween20 (PBST) three times.
Then, the plate was treated with an antimethylated-lysine antibody16
for G9a inhibitory activity assay or an antidimethyl-Histone H3 (Lys4)
113
dx.doi.org/10.1021/np200740e | J. Nat. Prod. 2012, 75, 111−114