α-Tocopherols from the formosan soft coral lobophytum crassum

Article abstract of DOI:10.1246/bcsj.20110051

In our screening of marine organisms for bioactive metabolites we have obtained two new α-tocopherols, designated as crassumtocopherols A (1) and B (2), from the Formosan soft coral Lobophytum crassum. The structures of 1 and 2 were determined on the basis of comprehensive NMR and HR-ESI-MS analyses. The cytotoxicity of 1, 2, natural vitamin E, and synthetic vitamin E against cancer cell lines and antiviral activity against human cytomegalovirus were evaluated in vitro. Compounds 1 and 2 exhibited moderate cytotoxicity against P-388 (mouse lymphocytic leukemia) cell lines with IC₅₀ of 3.2 and 2.7 μgmL^-1, respectively. In addition, compound 2 also displayed moderate cytotoxicity against HT-29 (human colon adenocarcinoma) with an IC ₅₀ of 3.9 μgmL^-1. However, natural vitamin E and synthetic vitamin E were not cytotoxic to the selected cancer cell lines and the results for inhibition of antiviral activity against human cytomegalovirus are all negative at a concentration of 1 μgmL^-1.

Full text of DOI:10.1246/bcsj.20110051

© 2011 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 84, No. 7, 783-787 (2011)
783
¡-Tocopherols from the Formosan Soft Coral Lobophytum crassum
Shi-Yie Cheng,1 Shih-Tseng Lin,1 Shang-Kwei Wang,*2 and Chang-Yih Duh*1,3
1Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan
2Department of Microbiology, Kaohsiung Medical University, Kaohsiung 807, Taiwan
3Asia-Pacific Ocean Research and Biopharmaceutical Translational Center, National Sun Yat-sen University,
Kaohsiung 804, Taiwan
Received February 17, 2011; E-mail: yihduh@mail.nsysu.edu.tw
In our screening of marine organisms for bioactive metabolites we have obtained two new ¡-tocopherols, designated
as crassumtocopherols A (1) and B (2), from the Formosan soft coral Lobophytum crassum. The structures of 1 and 2 were
determined on the basis of comprehensive NMR and HR-ESI-MS analyses. The cytotoxicity of 1, 2, natural vitamin E,
and synthetic vitamin E against cancer cell lines and antiviral activity against human cytomegalovirus were evaluated in
vitro. Compounds 1 and 2 exhibited moderate cytotoxicity against P-388 (mouse lymphocytic leukemia) cell lines with
IC50 of 3.2 and 2.7 ¯g mL¹1, respectively. In addition, compound 2 also displayed moderate cytotoxicity against HT-29
(human colon adenocarcinoma) with an IC50 of 3.9 ¯g mL¹1. However, natural vitamin E and synthetic vitamin E were
not cytotoxic to the selected cancer cell lines and the results for inhibition of antiviral activity against human
¹1
cytomegalovirus are all negative at a concentration of 1 ¯g mL
.
Soft corals belonging to the genus Lobophytum (Alcyoni-
idae) have been well recognized as a rich source of secondary
metabolites1-20 that have attracted much interest for synthetic
and natural products chemists due to their structural complexity
and remarkable bioactivities such as cytotoxicity,2-9 antimicro-
bial activities,10 and anti-inflammatory properties.10-12 Our
previous research of the Formosan soft coral L. crassum (von
Marenzeller, 1886) resulted in the purification of two cytotoxic
cembranoids, lobocrassolide,4 and lobocrasol.9 As part of our
continuing investigation of this organism collected at Dongsha
Atoll led to the discovery of two new ¡-tocopherols,
designated as crassumtocopherols A (1) and B (2) (Figure 1).
Tocols, both tocopherols and tocotrienols, comprising
naturally occurring ¡-, ¢-, £-, and ¤-homologs, are present in
various oils in different proportions.21 The main function of
¡-tocopherol (¡-T) is to protect cell membranes against UV
irradiation or oxidative damage.22 It is believed to play a major
role in the prevention of light-induced pathologies of human
skin and eyes and degenerative disorders such as atheroscle-
rosis, cardiovascular diseases, and cancer.23 Freshwater micro-
algae Euglena gracilis yields a large amount of ¡-T.24 The
marine species Dunaliella tertiolecta widely used in aqua-
culture as feed for fish and mollusk larvae produces relatively
high concentrations of ¡-T.25 A marine-derived tocopherol
(MDT) was first obtained from the lipophilic fraction of chum
salmon eggs, and from the tissues of a variety of fish.26 To the
best of our knowledge, this is the first report on the occurrence
of ¡-T derived from marine soft corals.
Compounds 1 and 2 were isolated by cytotoxicity-guided
fractionations and characterized by interpretation of detailed
1D and 2D NMR experiments, employing correlation spec-
troscopy (COSY), distortionless enhancement by polarization
transfer (DEPT), heteronuclear multiple bond correlation
(HMBC), heteronuclear single quantum correlation (HSQC),
and nuclear Overhauser effect spectroscopy (NOESY) experi-
ments. Moreover, the cytotoxicity against A-549 (human lung
epithelial carcinoma), HT-29 (human colon adenocarcinoma),
and P-388 (mouse lymphocytic leukemia) cancer cell lines,
along with antiviral activity against human cytomegalovirus for
1, 2, natural vitamin E, and synthetic vitamin E were evaluated.
Results and Discussion
Conventional extraction procedures were used, and the
acetone extract of the Formosan soft coral L. crassum was
exhaustively partitioned between EtOAc and H2O to afford the
EtOAc-soluble fraction, which was evaporated under vacuum
to yield a brown gum (20 g). The residue was successively
subjected to fractionation with column chromatography and
high-performance liquid chromatography, leading to the puri-
fication of crassumtocopherols A (1) and B (2) (see Exper-
imental Section).
4
5
RO
6
7
4a
8a
3
2
OH
OH
2'
1
11'
4'
8'
12'
O
13'
8
1'
3'
The positive high-resolution electrospray ionization mass
spectrometry (HR-ESI-MS) of 1 exhibited a pseudomolecular
ion peak at m/z 501.3553 [M + Na]+, consistent with a
molecular formula of C29H50O5, requiring five degrees of
unsaturation. Its NMR experiments acquired in C5D5N pro-
OH
1
R= H
2 R= Ac
Figure 1. Structures of 1 and 2.
Published on the web June 25, 2011; doi:10.1246/bcsj.20110051

784
Bull. Chem. Soc. Jpn. Vol. 84, No. 7 (2011)
¡-Tocopherols from Lobophytum crassum
hydroxy group by an acetoxy group. The presence of the
acetoxy moiety was identified by the 1H NMR signal at ¤H 2.29
(3H, s) and 13C NMR signals at ¤C 169.6 (qC) and 20.4 (CH3).
Although there were no direct HMBC correlations available,
the acetoxy group attached to C-6 was supported by the
13C NMR data of the aromatic ring and IR absorption of the
carbonyl at higher frequency (1758 cm¹1) due to the conjuga-
tion between single-bonded oxygen and the aromatic ring. The
absolute configuration of 2 was elucidated as 2R,4¤R,8¤R,11¤S
after determination of the stereochemistry of 1 as they had the
same sign of specific rotations and similar CD spectra.
Moreover, compounds 1 and 2 were respectively acetylated
with Ac2O in pyridine at room temperature to afford the same
product, further confirming the same stereochemistry between
1 and 2. From the aforementioned observations, the structure of
crassumtocopherol B (2) was proposed unambiguously.
HO
OH
OH
O
OH
Figure 2. Selected 1H-1H COSY (®) and HMBC (¼)
correlations of 1.
−0.03
−0.02
−0.02
−0.05
+0.02
HO
OH
OH
−0.01
O
+0.01
−0.02
−0.01
+0.05
OR
+0.01
1a R= S-MTPA
As noted in the introduction, a large number of ¡-
tocopherols and their analogs exhibited remarkable biological
activities.23 Crassumtocopherols A (1) and B (2) in the present
study were evaluated in vitro for cytotoxicity against P-388,
A-549, and HT-29 cancer cell lines using the MTT assay, as
well as antiviral activity against human cytomegalovirus.
Preliminary cytotoxic screening revealed that 1 and 2 displayed
modest cytotoxicity against P-388 cell line with IC50 values of
3.2 and 2.7 ¯g mL¹1, respectively. Compound 2 exhibited
modest cytotoxicity against HT-29 cell line with an IC50 value
of 3.9 ¯g mL¹1. The anticancer agent mithramycin was used as
the positive control and exhibited IC50 values of 0.06, 0.07, and
1b
R= R-MTPA
Figure 3. 1H NMR chemical shift differences (¦¤ = ¤S ¹
¤R) of the MTPA esters in CDCl3.
vided better resolution than in CDCl3, avoiding overlapped
signals. An analysis of the NMR data coupled with 1H-1H
COSY, HSQC, and HMBC correlations (Figure 2) were
diagnostic in determining that the gross framework of
crassumtocopherol A, possessing a tocopherol skeleton, was
assigned as 1. The above findings suggest that 1 has the
identical benzopyranol (chromanol) structure of ¡-T and differs
only by having three hydroxy groups at C-8¤, C-11¤, and C-12¤
in the isoprenoid side chain.
¹1
0.08 ¯g mL against P-388, A-549, and HT-29 cells, respec-
tively. With the exception of the above finding, the obtained
¹1
The circular dichroism (CD) spectrum of 1 exhibited a
negative Cotton effect at -max (¦¾) 289 nm (¹0.98) due to the
chromenol. The absolute configuration at C-2 was deduced to
be R on the basis of its CD data comparable to that of the
standard (2R)-D-¡-T acetate.27 The above assignment is also in
good agreement with that of naturally occurring ¡-T, all of
which have a (2R)-benzopyranol ring. The 4¤R configuration in
1 could be deduced by comparison of the 13C NMR spectro-
scopic data (measured in acetone-d6, 100 MHz) of 4¤-Me
(¤C 20.1) with those of (2R,4¤R,8¤R)-¡-T (natural vitamin E)
(¤C 20.1) and (2R,4¤S,8¤R)-¡-T (synthetic vitamin E) (¤C
20.0).28 Because of biogenetic considerations, the absolute
configurations of C-2 and C-4¤ of 1, having a similar
substitution pattern at each of the above carbons, are suggested
to be the same as those of naturally occurring ¡-T. Further-
more, we determined the absolute configuration at C-11¤ using
a modified Mosher’s esterification.29 Analysis of ¦¤S-R values
(Figure 3) for the protons neighboring C-11¤ led to the
assignment of the S configuration. The absolute configurations
of C-8¤ and C-11¤ were also proposed on the basis of
comparison of the NMR data with those of (8¤R,11¤S)-
(+)-heptaol30 and (8¤R,11¤R)-aurilol,31 suggesting the 8¤R and
11¤S configurations. Accordingly, the structure of crassum-
tocopherol A (1) was established unambiguously.
negative result showed that compounds 1 and 2 (1 ¯g mL )
exhibited no discernible antiviral activity against human
cytomegalovirus. Morever, natural vitamin E and synthetic
vitamin E were not cytotoxic to P-388, A-549, and HT-29
cancer cell lines and the results for inhibition of antiviral
activity against human cytomegalovirus are all negative at a
¹1
concentration of 1 ¯g mL
.
Experimental
General Experimental Procedures. Optical rotations were
determined with a JASCO P1020 digital polarimeter. CD, IR,
and UV spectra were recorded on JASCO J-815, JASCO FT/
IR-4100, and JASCO V-650 spectrophotometers, respectively.
The NMR spectra were recorded on Bruker Avance 300 NMR
and Varian MR 400 NMR spectrometers (300 and 400 MHz for
1H, and 75 and 100 MHz for 13C, respectively), using TMS as
internal standard. Chemical shifts are given in ¤ (ppm) and
coupling constants in Hz. ESI-MS were recorded by ESI
FT-MS on a Bruker APEX II mass spectrometer. Silica gel 60
(Merck, 230-400 mesh) and Sephadex LH-20 (Pharmacia)
were used for column chromatography. Precoated silica gel
plates (Merck, Kieselgel 60 F254, 0.25 mm) were used for thin-
layer chromatography (TLC) analysis. High-performance liq-
uid chromatography (HPLC) was carried out using a Hitachi
L-7100 pump equipped with a Hitachi L-7400 UV detector at
220 nm and a preparative reversed-phase column (Merck, Hibar
Licrospher RP-18e, 5 ¯m, 250 © 25 mm). S-(+)- and R-(¹)-¡-
methoxy-¡-trifluoromethylphenylacetyl chloride were obtained
from ACROS Organics (Geel, Belgium).
The positive HR-ESI-MS of 2 showed a pseudomolecular
ion peak at m/z 543.3659 [M + Na]+, corresponding to a
molecular formula of C31H52O6 and six degrees of unsatura-
tion. The NMR spectroscopic data of 2 were highly compatible
with those obtained for 1, except for the replacement of the

S.-Y. Cheng et al.
Bull. Chem. Soc. Jpn. Vol. 84, No. 7 (2011)
785
Animal Material. The Formosan soft coral L. crassum was
collected at Dongsha Atoll in April 2007, at a depth of 8 m, and
was immediately frozen at ¹20 °C until further processed for
extraction in the laboratory. Identification was kindly verified
by Professor Chang-Feng Dai, Institute of Oceanography,
National Taiwan University, Taiwan. A voucher specimen (TS-
11) has been deposited at the Department of Marine Bio-
technology and Resources, National Sun Yat-sen University.
Extraction and Isolation. The frozen soft coral L. crassum
was chopped into small pieces and extracted with acetone for
24 h at room temperature. The quantity of solvent used for each
extraction (2.0 L) was at least three times the amount of the soft
coral material used (1.5 kg). The combined extracts were
concentrated in vacuo (under 35 °C) to obtain 25 g of dry
extract, which was suspended in water and extracted with
EtOAc. The EtOAc phase was evaporated to dryness in vacuo
to give a dark brown residue (20 g). The resulting EtOAc
residue was subjected to silica gel chromatography using a
stepwise gradient mixture of n-hexane-EtOAc-MeOH for
elution and separated into 40 fractions based on TLC and
1H NMR analysis. Fraction 20 (223 mg) eluted with n-hexane/
EtOAc (1:10) was submitted to repeated chromatography over
silica gel using n-hexane-EtOAc mixtures of increasing polar-
ity as eluent. Altogether, three subfractions were obtained, of
which subfraction 20-3 (142 mg) was subjected column
chromatography on RP-18 gel column eluting with 53%
MeOH in H2O to yield a mixture (72 mg). In turn, the mixture
was further purified by RP-18 HPLC using an isocratic solvent
system of 90% MeOH in H2O to allow the isolation of 1
(26 mg) and 2 (4 mg), respectively.
(1H, m, H-10¤), 3.34 (1H, dd, J = 9.6, 6.8 Hz, H-11¤), 1.17 (3H,
s, H-13¤), 1.24 (3H, s, 2-CH3), 2.11 (3H, s, 5-CH3), 2.16 (3H, s,
7-CH3), 2.11 (3H, s, 8-CH3), 0.85 (3H, d, J = 6.4 Hz, 4¤-CH3),
1.15 (3H, s, 8¤-CH3), 1.21 (3H, s, 12¤-CH3); 13C NMR (CDCl3,
100 MHz): ¤C 74.4 (qC, C-2), 31.7 (CH2, C-3), 20.7 (CH2,
C-4), 117.3 (qC, C-4a), 118.9 (qC, C-5), 145.5 (qC, C-6), 122.5
(qC, C-7), 121.4 (qC, C-8), 144.5 (qC, C-8a), 39.0 (CH2, C-1¤),
20.7 (CH2, C-2¤), 37.1 (CH2, C-3¤), 32.3 (CH, C-4¤), 37.2 (CH2,
C-5¤), 21.3 (CH2, C-6¤), 43.3 (CH2, C-7¤), 72.6 (qC, C-8¤), 38.7
(CH2, C-9¤), 25.7 (CH2, C-10¤), 79.1 (CH, C-11¤), 73.1 (qC,
C-12¤), 23.3 (CH3, C-13¤), 24.0 (CH3, 2-Me), 11.8 (CH3,
5-Me), 12.3 (CH3, 7-Me), 11.3 (CH3, 8-Me), 19.6 (CH3,
4¤-Me), 26.3 (CH3, 8¤-Me), 26.5 (CH3, 12¤-Me); 1H NMR
(acetone-d6, 400 MHz): ¤H 1.78 (2H, m, H-3), 2.58 (2H, t,
J = 6.4 Hz, H-4), 1.51 (2H, m, H-1¤), 1.48 (2H, m, H-2¤), 1.08
(2H, m, H-3¤), 1.43 (1H, m, H-4¤), 1.29 (2H, m, H-5¤), 1.32
(2H, m, H-6¤), 1.39 (2H, m, H-7¤), 1.76 (1H, m, H-9¤), 1.44
(1H, m, H-9¤), 1.65 (1H, m, H-10¤), 1.36 (1H, m, H-10¤), 3.25
(1H, br d, J = 10.0 Hz, H-11¤), 1.10 (3H, s, H-13¤), 1.21 (3H, s,
2-CH3), 2.04 (3H, s, 5-CH3), 2.12 (3H, s, 7-CH3), 2.08 (3H, s,
8-CH3), 0.86 (3H, d, J = 6.4 Hz, 4¤-CH3), 1.10 (3H, s, 8¤-CH3),
1.10 (3H, s, 12¤-CH3); 13C NMR (acetone-d6, 100 MHz): ¤C
75.0 (qC, C-2), 32.6 (CH2, C-3), 21.5 (CH2, C-4), 117.9 (qC,
C-4a), 120.7 (qC, C-5), 146.4 (qC, C-6), 123.1 (qC, C-7), 122.5
(qC, C-8), 146.1 (qC, C-8a), 40.1 (CH2, C-1¤), 21.7 (CH2,
C-2¤), 38.3 (CH2, C-3¤), 33.4 (CH, C-4¤), 38.5 (CH2, C-5¤), 22.1
(CH2, C-6¤), 43.5 (CH2, C-7¤), 72.2 (qC, C-8¤), 40.2 (CH2,
C-9¤), 26.8 (CH2, C-10¤), 80.1 (CH, C-11¤), 73.0 (qC, C-12¤),
25.1 (CH3, C-13¤), 24.3 (CH3, 2-Me), 12.1 (CH3, 5-Me), 12.9
(CH3, 7-Me), 11.9 (CH3, 8-Me), 20.1 (CH3, 4¤-Me), 27.5 (CH3,
8¤-Me), 26.1 (CH3, 12¤-Me); ESI-MS m/z 501 [M + Na]+;
HR-ESI-MS m/z 501.3553 [M + Na]+ (Calcd for C29H50O5Na,
501.3556).
25
Crassumtocopherol A (1): Colorless oil; ½¡ꢀD = ¹66 (c 0.1,
CHCl3); CD (1.33 © 10¹4 M, MeOH): -max (¦¾) 245 (+1.00),
289 nm (¹0.98); UV (MeOH): -max (log ¾) 245 (3.72), 289 nm
25
(3.28); IR (KBr): ¯max 3394, 2973, 2933, 1640, 1558, 1459,
Crassumtocopherol B (2): Colorless oil; ½¡ꢀD = ¹50 (c 0.1,
¹1
1377, 1258, 1216, 1163, 1081, 919, 755 cm
;
1H NMR
CHCl3); CD (1.23 © 10¹4 M, MeOH): -max (¦¾) 237 (+2.66),
285 nm (¹1.51); UV (MeOH): -max (log ¾) 237 (3.80), 285 nm
(3.16); IR (KBr): vmax 3408, 2970, 2933, 1758, 1648, 1575,
(C5D5N, 400 MHz): ¤H 1.75 (2H, m, H-3), 2.58 (2H, dd,
J = 9.6, 6.4 Hz, H-4), 1.44 (1H, m, H-1¤), 1.59 (1H, m, H-1¤),
1.53 (1H, m, H-2¤), 1.43 (1H, m, H-2¤), 1.27 (1H, m, H-3¤),
1.03 (1H, m, H-3¤), 1.40 (1H, m, H-4¤), 1.31 (1H, m, H-5¤),
1.11 (1H, m, H-5¤), 1.61 (2H, m, H-6¤), 1.68 (2H, m, H-7¤),
2.33 (1H, m, H-9¤), 1.83 (1H, m, H-9¤), 2.19 (1H, m, H-10¤),
1.92 (1H, m, H-10¤), 3.76 (1H, dd, J = 9.6, 6.8 Hz, H-11¤), 1.49
(3H, s, H-13¤), 1.25 (3H, s, 2-CH3), 2.36 (3H, s, 5-CH3), 2.42
(3H, s, 7-CH3), 2.29 (3H, s, 8-CH3), 0.84 (3H, d, J = 6.4 Hz,
4¤-CH3), 1.40 (3H, s, 8¤-CH3), 1.46 (3H, s, 12¤-CH3); 13C NMR
(C5D5N, 100 MHz): ¤C 74.6 (qC, C-2), 32.1 (CH2, C-3), 21.2
(CH2, C-4), 117.6 (qC, C-4a), 121.4 (qC, C-5), 146.9 (qC, C-6),
123.9 (qC, C-7), 122.3 (qC, C-8), 145.8 (qC, C-8a), 40.1 (CH2,
C-1¤), 21.5 (CH2, C-2¤), 37.9 (CH2, C-3¤), 33.0 (CH, C-4¤), 38.3
(CH2, C-5¤), 22.0 (CH2, C-6¤), 43.5 (CH2, C-7¤), 71.7 (qC,
C-8¤), 40.7 (CH2, C-9¤), 27.0 (CH2, C-10¤), 80.0 (CH, C-11¤),
72.8 (qC, C-12¤), 26.1 (CH3, C-13¤), 24.1 (CH3, 2-Me), 12.6
(CH3, 5-Me), 13.5 (CH3, 7-Me), 12.4 (CH3, 8-Me), 19.8 (CH3,
4¤-Me), 27.7 (CH3, 8¤-Me), 26.1 (CH3, 12¤-Me); 1H NMR
(CDCl3, 400 MHz): ¤H 1.79 (2H, m, H-3), 2.60 (2H, t,
J = 6.4 Hz, H-4), 1.45 (2H, m, H-1¤), 1.43 (2H, m, H-2¤), 1.08
(2H, m, H-3¤), 1.39 (1H, m, H-4¤), 1.24 (2H, m, H-5¤), 1.35
(1H, m, H-6¤), 1.25 (1H, m, H-6¤), 1.37 (2H, m, H-7¤), 1.71
(1H, m, H-9¤), 1.55 (1H, m, H-9¤), 1.57 (1H, m, H-10¤), 1.44
1
1456, 1372, 1212, 1162, 1110, 1077, 923, 755 cm¹1; H NMR
(C5D5N, 400 MHz): ¤H 1.63 (2H, m, H-3), 2.47 (2H, dd,
J = 9.6, 6.4 Hz, H-4), 1.43 (2H, m, H-1¤), 1.48 (1H, m, H-2¤),
1.38 (1H, m, H-2¤), 1.24 (1H, m, H-3¤), 1.02 (1H, m, H-3¤),
1.40 (1H, m, H-4¤), 1.31 (1H, m, H-5¤), 1.11 (1H, m, H-5¤),
1.59 (2H, m, H-6¤), 1.68 (2H, m, H-7¤), 2.36 (1H, m, H-9¤),
1.84 (1H, m, H-9¤), 2.20 (1H, m, H-10¤), 1.92 (1H, m, H-10¤),
3.76 (1H, dd, J = 9.6, 6.8 Hz, H-11¤), 1.49 (3H, s, H-13¤), 1.18
(3H, s, 2-CH3), 2.01 (3H, s, 5-CH3), 2.05 (3H, s, 7-CH3), 2.15
(3H, s, 8-CH3), 0.84 (3H, d, J = 6.4 Hz, 4¤-CH3), 1.40 (3H, s,
8¤-CH3), 1.46 (3H, s, 12¤-CH3), 2.29 (3H, s, 6-OAc); 13C NMR
(C5D5N, 100 MHz): ¤C 75.4 (qC, C-2), 31.2 (CH2, C-3), 20.8
(CH2, C-4), 118.0 (qC, C-4a), 125.7 (qC, C-5), 141.5 (qC, C-6),
127.3 (qC, C-7), 123.0 (qC, C-8), 140.2 (qC, C-8a), 40.2 (CH2,
C-1¤), 21.4 (CH2, C-2¤), 37.8 (CH2, C-3¤), 33.0 (CH, C-4¤), 38.3
(CH2, C-5¤), 22.0 (CH2, C-6¤), 43.6 (CH2, C-7¤), 71.7 (qC,
C-8¤), 40.8 (CH2, C-9¤), 27.0 (CH2, C-10¤), 80.0 (CH, C-11¤),
72.8 (qC, C-12¤), 26.1 (CH3, C-13¤), 24.0 (CH3, 2-Me), 12.4
(CH3, 5-Me), 13.2 (CH3, 7-Me), 12.1 (CH3, 8-Me), 19.8 (CH3,
4¤-Me), 27.7 (CH3, 8¤-Me), 26.1 (CH3, 12¤-Me), 169.6 (qC,
1
6-OAc), 20.4 (CH3, 6-OAc); H NMR (CDCl3, 400 MHz): ¤H
1.78 (2H, m, H-3), 2.59 (2H, t, J = 6.4 Hz, H-4), 1.55 (2H, m,

786
Bull. Chem. Soc. Jpn. Vol. 84, No. 7 (2011)
¡-Tocopherols from Lobophytum crassum
H-1¤), 1.42 (2H, m, H-2¤), 1.12 (2H, m, H-3¤), 1.41 (1H, m,
H-4¤), 1.29 (2H, m, H-5¤), 1.30 (2H, m, H-6¤), 1.44 (2H, m,
H-7¤), 1.74 (1H, m, H-9¤), 1.58 (1H, m, H-9¤), 1.58 (1H, m,
H-10¤), 1.45 (1H, m, H-10¤), 3.35 (1H, dd, J = 9.6, 6.8 Hz,
H-11¤), 1.17 (3H, s, H-13¤), 1.22 (3H, s, 2-CH3), 1.98 (3H, s,
5-CH3), 2.02 (3H, s, 7-CH3), 2.09 (3H, s, 8-CH3), 0.87 (3H,
d, J = 7.2 Hz, 4¤-CH3), 1.18 (3H, s, 8¤-CH3), 1.22 (3H, s,
12¤-CH3), 2.33 (3H, s, 6-OAc); 13C NMR (CDCl3, 100 MHz):
6.8 Hz, H-11¤), 1.39 (3H, s, H-13¤), 1.18 (3H, s, 2-CH3), 2.01
(3H, s, 5-CH3), 2.04 (3H, s, 7-CH3), 2.15 (3H, s, 8-CH3), 0.84
(3H, d, J = 6.8 Hz, 4¤-CH3), 1.34 (3H, s, 8¤-CH3), 1.39 (3H,
s, 12¤-CH3), 2.28 (3H, s, 6-OAc), 2.01 (3H, s, 11¤-OAc);
13C NMR (C5D5N, 100 MHz): ¤C 75.4 (qC, C-2), 31.4 (CH2,
C-3), 20.9 (CH2, C-4), 118.1 (qC, C-4a), 125.7 (qC, C-5),
141.6 (qC, C-6), 127.4 (qC, C-7), 123.1 (qC, C-8), 150.3 (qC,
C-8a), 40.4 (CH2, C-1¤), 21.4 (CH2, C-2¤), 37.9 (CH2, C-3¤),
33.0 (CH, C-4¤), 38.3 (CH2, C-5¤), 22.0 (CH2, C-6¤), 43.1 (CH2,
C-7¤), 71.4 (qC, C-8¤), 39.8 (CH2, C-9¤), 24.9 (CH2, C-10¤),
81.3 (CH, C-11¤), 71.6 (qC, C-12¤), 27.0 (CH3, C-13¤), 24.2
(CH3, 2-Me), 12.2 (CH3, 5-Me), 13.2 (CH3, 7-Me), 12.4 (CH3,
8-Me), 19.8 (CH3, 4¤-Me), 27.6 (CH3, 8¤-Me), 26.1 (CH3,
12¤-Me), 169.7 (qC, 6-OAc), 20.5 (CH3, 6-OAc), 171.1 (qC,
11¤-OAc), 21.2 (CH3, 11¤-OAc).
¤
75.0 (qC, C-2), 31.0 (CH2, C-3), 20.6 (CH2, C-4), 117.3
C
(qC, C-4a), 124.9 (qC, C-5), 140.5 (qC, C-6), 126.6 (qC, C-7),
123.0 (qC, C-8), 149.4 (qC, C-8a), 39.6 (CH2, C-1¤), 20.9
(CH2, C-2¤), 37.3 (CH2, C-3¤), 32.5 (CH, C-4¤), 37.5 (CH2,
C-5¤), 21.4 (CH2, C-6¤), 43.2 (CH2, C-7¤), 72.7 (qC, C-8¤), 38.7
(CH2, C-9¤), 25.8 (CH2, C-10¤), 79.1 (CH, C-11¤), 73.1 (qC,
C-12¤), 23.3 (CH3, C-13¤), 24.5 (CH3, 2-Me), 12.1 (CH3,
5-Me), 12.9 (CH3, 7-Me), 11.8 (CH3, 8-Me), 19.6 (CH3,
4¤-Me), 26.6 (CH3, 8¤-Me), 26.5 (CH3, 12¤-Me), 169.8 (qC,
6-OAc), 20.6 (CH3, 6-OAc); ESI-MS m/z 543 [M + Na]+;
HR-ESI-MS m/z 543.3659 [M + Na]+ (Calcd for C31H52O6Na,
543.3661).
Cytotoxicity Assay. To measure the cytotoxicity of natural
product against P-388 (mouse lymphocytic leukemia), HT-29
(human colon adenocarcinoma), and A-549 (human lung
epithelial carcinoma), each cell line was initiated at 1500,
750, 750 cells/well, respectively, in 96-well microplates. Three
to eight concentrations encompassing an 8- to 128-fold range
were on each cell line. P-388, A-549, and HT-29 cells were
enumerated using MTT after the exposure to test samples for 3,
Preparation of Mosher’s Esters of 1. In separate vials,
duplicate (1.0 mg) samples of 1 were dissolved in 0.5 mL of dry
pyridine and allowed to react overnight at room temperature
with (R)- and (S)-MTPA chloride (one drop), respectively. The
reaction was quenched by the addition of 1.0 mL of H2O,
followed by extraction with EtOAc (3 © 1.0 mL). The EtOAc-
soluble extracts were combined and evaporated. The resulting
residue was subjected to a short silica gel column eluting with
n-hexane-EtOAc (2:1) to yield (S)-MTPA ester 1a (0.6 mg). In
the same manner, the (R)-MTPA ester 1b (0.5 mg) was prepared
with (S)-MTPA chloride according to the same procedure as
¹1
6, and 6 days, respectively. Fifty ¯L of 1 mg mL MTT were
added to each well, and plates were incubated at 37 °C for 5 h.
Supernatant was aspirated. Formazan crystals were redissolved
in DMSO for 10 min with shaking, and the plate was read
immediately on a microplate reader at a wavelength of
540 nm.32
Anticytomegalovirus Assay. To determine the effects of
natural product upon human cytomegalovirus (HCMV) cyto-
pathic effect (CPE), confluent human embryonic lung (HEL)
cells grown in 24-well plates were incubated for 1 h in the
presence or absence of various concentrations of tested natural
product. Then, cells were infected with HCMV at an input of
1000 pfu (plaque forming units) per well of 24-well dish.
Antiviral activity is expressed as IC50 (50% inhibitory
concentration), or compound concentration required to reduce
virus induced CPE by 50% after 7 days as compared with the
untreated control. To monitor the cell growth upon treating
with natural products, an MTT-colorimetric assay was em-
ployed.32
1
described above. Selected H NMR (CDCl3, 300 MHz) of 1a:
¤
7.41-7.61 (5H, m, Ph), 4.98 (1H, br d, J = 8.8 Hz, H-11¤),
H
3.58 (3H, s, OCH3), 2.59 (2H, t, J = 6.2 Hz, H-4), 1.79 (1H, m,
H-9¤), 1.24 (3H, s, H-13¤), 1.24 (3H, s, 2-CH3), 2.08 (3H, s,
5-CH3), 2.09 (3H, s, 7-CH3), 2.17 (3H, s, 8-CH3), 0.85 (3H,
d, J = 6.2 Hz, 4¤-CH3), 1.07 (3H, s, 8¤-CH3), 1.18 (3H, s,
12¤-CH3); Selected 1H NMR (CDCl3, 300 MHz) of 1b: ¤H
7.40-7.61 (10H, m, Ph), 4.99 (1H, br d, J = 8.8 Hz, H-11¤),
3.56 (3H, s, OCH3), 2.61 (2H, t, J = 6.2 Hz, H-4), 1.78 (1H, m,
H-9¤), 1.19 (3H, s, H-13¤), 1.23 (3H, s, 2-CH3), 2.11 (3H, s,
5-CH3), 2.11 (3H, s, 7-CH3), 2.16 (3H, s, 8-CH3), 0.87 (3H, d,
J = 6.2 Hz, 4¤-CH3), 1.12 (3H, s, 8¤-CH3), 1.16 (3H, s,
12¤-CH3).
Acetylation of 1 and 2. A mixture of 1 (2.0 mg), Ac2O
(a drop), and pyridine (1.0 mL) stood at room temperature
overnight and then was diluted with 1.0 mL H2O. The crude
residue was suspended in H2O and extracted with EtOAc. The
EtOAc extract was purified by a short silica gel column using
n-hexane-EtOAc (5:1) to give an acetylated product (1.9 mg).
The 1H NMR data of the product were in good accordance with
those of the product acetylated from 2 according to the same
procedure as described above. 1H NMR (C5D5N, 400 MHz): ¤H
1.63 (2H, m, H-3), 2.47 (2H, dd, J = 9.6, 6.4 Hz, H-4), 1.42
(2H, m, H-1¤), 1.45 (1H, m, H-2¤), 1.34 (1H, m, H-2¤), 1.22
(1H, m, H-3¤), 1.04 (1H, m, H-3¤), 1.37 (1H, m, H-4¤), 1.32
(1H, m, H-5¤), 1.12 (1H, m, H-5¤), 1.55 (2H, m, H-6¤), 1.67
(2H, m, H-7¤), 2.33 (1H, m, H-9¤), 1.82 (1H, m, H-9¤), 2.10
(1H, m, H-10¤), 1.92 (1H, m, H-10¤), 5.32 (1H, dd, J = 9.6,
Financial support was provided by the National Science
Council (No. NSC99-2628-B-110-002-MY3) and Ministry of
Education of the Republic of China (Taiwan) awarded to
C.-Y.D.
Supporting Information
1H NMR and 13C NMR spectra of compounds 1 and 2. This
material is available free of charge on the web at http://
www.csj.jp/journals/bcsj/.
References
1
J. W. Blunt, B. R. Copp, W.-P. Hu, M. H. G. Munro, P. T.
Northcote, M. R. Prinsep, Nat. Prod. Rep. 2009, 26, 170, and
literature cited in previous reviews.
2
R. Higuchi, T. Miyamoto, K. Yamada, T. Komori, Toxicon
1998, 36, 1703.

S.-Y. Cheng et al.
Bull. Chem. Soc. Jpn. Vol. 84, No. 7 (2011)
787
3
G. F. Matthée, G. M. König, A. D. Wright, J. Nat. Prod.
1159.
1998, 61, 237.
17 Y. Kashman, S. Carmely, A. Groweiss, J. Org. Chem. 1981,
46, 3592.
4
S.-K. Wang, C.-Y. Duh, Y.-C. Wu, Y. Wang, M.-C. Cheng,
K. Soong, L.-S. Fang, J. Nat. Prod. 1992, 55, 1430.
18 Z. Kinamoni, A. Groweiss, S. Carmely, Y. Kashman, Y.
Loya, Tetrahedron 1983, 39, 1643.
5
S. J. Coval, R. W. Patton, J. M. Petrin, L. James, M. L.
Rothofsky, S. L. Lin, M. Patel, J. K. Reed, A. T. McPhail, W. R.
Bishop, Bioorg. Med. Chem. Lett. 1996, 6, 909.
19 B. F. Bowden, J. C. Coll, D. M. Tapiolas, Aust. J. Chem.
1983, 36, 2289.
6
C.-Y. Duh, S.-K. Wang, B.-T. Huang, C.-F. Dai, J. Nat.
20 B. F. Bowden, J. C. Coll, M. S. L. de Costa, M. F. Mackay,
M. Mahendran, R. H. Willis, E. D. de Silva, Aust. J. Chem. 1984,
37, 545.
Prod. 2000, 63, 884.
7
C.-H. Chao, H.-C. Huang, Y.-C. Wu, C.-K. Lu, C.-F. Dai,
J.-H. Sheu, Chem. Pharm. Bull. 2007, 55, 1720.
21 N. Gotoh, H. Watanabe, T. Oka, D. Mashimo, N. Noguchi,
K. Hata, S. Wada, Lipids 2009, 44, 133.
8
W. Zhang, K. Krohn, J. Ding, Z.-H. Miao, X.-H. Zhou,
S.-H. Chen, G. Pescitelli, P. Salvadori, T. Kurtan, Y.-W. Guo,
J. Nat. Prod. 2008, 71, 961.
22 M. J. Fryer, Plant Cell Environ. 1992, 15, 381.
23 R. Brigelius-Flohé, M. G. Traber, FASEB J. 1999, 13, 1145.
24 M. R. Brown, M. Mular, I. Miller, C. Farmer, C. Trenerry,
J. Appl. Phycol. 1999, 11, 247.
9
S.-T. Lin, S.-K. Wang, S.-Y. Cheng, C.-Y. Duh, Org. Lett.
2009, 11, 3012.
10 S.-Y. Cheng, C.-T. Chuang, Z.-H. Wen, S.-K. Wang, S.-F.
Chiou, C.-H. Hsu, C.-F. Dai, C.-Y. Duh, Bioorg. Med. Chem. 2010,
18, 3379.
11 S.-Y. Cheng, Z.-H. Wen, S.-K. Wang, S.-F. Chiou, C.-H.
Hsu, C.-F. Dai, C.-Y. Duh, Bioorg. Med. Chem. 2009, 17, 3763.
12 S.-Y. Cheng, Z.-H. Wen, S.-K. Wang, S.-F. Chiou, C.-H.
Hsu, C.-F. Dai, M. Y. Chiang, C.-Y. Duh, J. Nat. Prod. 2009, 72,
152.
13 K. Yamada, K. Ryu, T. Miyamoto, R. Higuchi, J. Nat. Prod.
1997, 60, 798.
14 C. Subrahmanyam, C. V. Rao, V. Anjaneyulu, P.
Satyanarayana, P. V. S. Rao, R. S. Ward, A. Pelter, Tetrahedron
1992, 48, 3111.
25 J. Abalde, J. Fabregas, C. Herrero, Bioresour. Technol.
1991, 38, 121.
26 Y. Yamamoto, N. Maita, A. Fujisawa, J. Takashima, Y.
Ishii, W. C. Dunlap, J. Nat. Prod. 1999, 62, 1685.
27 R. H. Cichewicz, V. A. Kenyon, S. Whitman, N. M.
Morales, J. F. Arguello, T. R. Holman, P. Crews, J. Am. Chem. Soc.
2004, 126, 14910.
28 S. Brownstein, G. W. Burton, L. Hughes, K. U. Ingold,
J. Org. Chem. 1989, 54, 560.
29 I. Ohtani, T. Kusumi, Y. Kashman, H. Kakisawa, J. Am.
Chem. Soc. 1991, 113, 4092.
30 C. M. Adams, I. Ghosh, Y. Kishi, Org. Lett. 2004, 6, 4723.
31 K. Suenaga, T. Shibata, N. Takada, H. Kigoshi, K. Yamada,
J. Nat. Prod. 1998, 61, 515.
32 M. Stevens, J. Balzarini, O. Tabarrini, G. Andrei, R.
Snoeck, V. Cecchetti, A. Fravolini, E. De Clercq, C. Pannecouque,
J. Antimicrob. Chemother. 2005, 56, 847.
15 B. F. Bowden, J. C. Coll, A. Heaton, G. König, M. A.
Bruck, R. E. Cramer, D. M. Klein, P. J. Scheuer, J. Nat. Prod.
1987, 50, 650.
16 Y. Kashman, A. Groweiss, Tetrahedron Lett. 1977, 18,