Cytotoxic 5-alkylresorcinol metabolites from the leaves of Grevillea robusta

Article abstract of DOI:10.1021/np0605687

Bioassay-guided fractionation of the MeOH extract of the leaves of Grevillea robusta led to the isolation of six new 5-alkylresorcinols, gravicycle (1), dehydrogravicycle (2), bisgravillol (3), dehydrobisgravillol (4), dehydrograviphane (5), and methyldeh

Full text of DOI:10.1021/np0605687

J. Nat. Prod. 2007, 70, 319-323
319
Cytotoxic 5-Alkylresorcinol Metabolites from the Leaves of GreWillea robusta
Ta-Hsien Chuang† and Pei-Lin Wu*,‡
Department of Chemistry, National Cheng Kung UniVersity, Tainan, 701, Taiwan, Republic of China, and Department of Cosmetic Science,
Chung Hwa College of Medical Technology, Tainan, 717, Taiwan, Republic of China
ReceiVed NoVember 12, 2006
Bioassay-guided fractionation of the MeOH extract of the leaves of GreVillea robusta led to the isolation of six new
5-alkylresorcinols, gravicycle (1), dehydrogravicycle (2), bisgravillol (3), dehydrobisgravillol (4), dehydrograviphane
(5), and methyldehydrograviphane (6), as well as eight known compounds. The structures of these compounds were
determined by spectroscopic and chemical methods. Graviphane (7) and methylgraviphane (8) were isolated in the pure
form for the first time from a natural source. The compounds all showed marginal cytotoxicity against MCF-7, NCI-
H460, and SF-268 cell lines.
GreVillea robusta A. CUNN (Proteaceae), known commonly as
“silky oak”, is a tropical ornamental tree native to Australia.1,2 In
Taiwan, it is cultivated as a shade tree. In our continuing search
for bioactive molecules from the plants of Taiwan, a MeOH extract
of the leaves of G. robusta was selected for bioassay-guided
fractionation, as it showed significant cytotoxicity against human
breast carcinoma (MCF-7), lung carcinoma (NCI-H460), and central
nervous system carcinoma (SF-268) cell lines.
made to estimate the amount of each compound in the subfractions.
Herein, we describe the isolation, structural elucidation, and
cytotoxic properties of these resorcinols.
The methanol extract of the leaves of G. robusta was suspended
in H2O and partitioned into hexane-, CHCl3-, and EtOAc-soluble
fractions. The hexane fraction was chromatographed to afford seven
active subfractions, A-G, containing alkylresorcinols with different
skeletons and found to be extremely difficult to separate into pure
1
components.2 In the H NMR spectrum of each subfraction, one
set of signals between δ 5.8 and 6.5 for aromatic protons and signals
between δ 1.0 and 1.7 typical for long chain methylenes were
noticed. The spectrum also exhibited olefinic protons near δ 5.3,
allylic protons near δ 2.0, and methylene protons adjacent to two
double bonds at about δ 2.8, which represent varying degrees of
unsaturation in the carbon chain. The integration among the
aromatic methines, olefinic methines, and allylic methylenes was
not consistent, indicating that each active subfraction was a mixture
of resorcinols having the same ring system but with both saturated
and unsaturated alkyl substituents. Moreover, mass spectral analysis
inferred that the major component in each subfraction was a
resorcinol containing a 14-carbon chain having a double bond
(C14,1). Except the molecular ion peak for C14,1, the minor peaks at
M + 2, M + 26, and M + 28 were attributable to resorcinols
bearing saturated (C14,0) and unsaturated (C16,2, C16,1) chains,
respectively. After hydrogenation with Pd/C, each sample exhibited
only M + 2 and M + 30 peaks in the mass spectra, corresponding
to saturated long chain C14,0 and its double homologue C16,0
.
Eventually, by prolonged chromatographic separations on silica gel,
small amounts of 14 pure 5-alkylresorcinol metabolites were
isolated. Of these, gravicycle (1), dehydrogravicycle (2), bisgravillol
(3), dehydrobisgravillol (4), dehydrograviphane (5), and methyl-
dehydrograviphane (6) were new compounds; graviphane (7) and
methylgraviphane (8) were isolated as pure compounds for the first
time; and robustol (9),3 dehydrorobustol A (10),3 bis-norstriatol
(11),4 5-[14′-(3′′,5′′-dihydroxyphenyl)-cis-tetradec-6′-en-1-yl]resor-
cinol (12),3 cis-5-n-pentadecylresorcinol (13),5 and cis-5-n-pentadec-
8′-enylresorcinol (14)6 were known compounds. The hydrogenated
products of 2, 4, 5, 6, 10, 12, and 14 were identical to the saturated
analogues of 1, 3, 7, 8, 9, 11, and 13, respectively. No attempt was
Gravicycle (1) was isolated as a white, amorphous powder. The
molecular formula was determined to be C26H36O5 from the
1
molecular ion at m/z 428.2565. From the H NMR and COSY
spectra, the upfield shifted aromatic signals were assigned as two
symmetrical protons [δ 6.46 (s, H-4 and -6)] of a 2,5-disubstituted
resorcinol and three m-coupled protons [δ 5.83 (t, J ) 2.1 Hz, H-6′),
6.26 (t, J ) 2.1 Hz, H-4′), and 6.48 (t, J ) 2.1 Hz, H-2′)] of a
5′-substituted resorcinol. A 14-carbon long chain containing typical
methylenes at δ 1.27 (20H, m, H-3′′-12′′), 1.45 (2H, m, H-13′′),
and 1.72 (2H, m, H-2′′), benzylic protons at δ 2.34 (2H, t, J ) 7.8
Hz, H-14′′), and a proton on a benzylic carbon bearing an OH
functionality at δ 4.46 (1H, t, J ) 6.6 Hz, H-1′′) were also observed.
The HMBC correlations between H-1′′ and C-4, -6 (δ 106.7) and
C-5 (δ 143.9) as well as between H-14′′ and C-4′ (δ 109.9), C-5′
(δ 146.0), and C-6′ (δ 105.3) indicated that the resorcinols were
* To whom correspondence should be addressed. Fax: 886-6-2740552.
E-mail: wupl@mail.ncku.edu.tw.
† National Cheng Kung University.
‡ Chung Hwa College of Medical Technology.
10.1021/np0605687 CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/23/2007

320 Journal of Natural Products, 2007, Vol. 70, No. 2
Notes
Scheme 1
Scheme 2
the molecular formula C27H40O4. The 1H NMR and COSY spectra
exhibited two slightly different sets of 5-alkylresorcinol signals,
one set at δ 6.25 (1H, s, H-2), 6.26 (1H, s, H-4), and 6.33 (1H, s,
H-6) and the other at δ 6.17 (1H, s, H-2′) and 6.24 (2H, s, H-4′
and -6′). A methoxyl group at δ 3.76 showed a NOE with H-2 and
H-6, indicating its attachment to C-1. Ten methylenes at δ 1.25
for H-3′′ to H-12′′, two homobenzylic methylenes at δ 1.56 for
H-2′′ and H-13′′, and two benzylic methylenes at δ 2.49 for H-1′′
and H-14′′ suggested that 3 should be a bis-5-alkylresorcinol
derivative with a chain length of 14 carbon atoms. The HMBC
correlations of aromatic methines H-4, -6, -4′, and -6′ with the
benzylic carbons C-1′′ and -14′′ (δ 35.8 and 36.0) further confirmed
the above structure. Thus, bisgravillol (3) was defined as 1-O-
methyl-5-[14′′-(1′,3′-dihydroxyphenyl)tetradec-1′′-yl]resorcinol.
attached to both ends of the 1′′-hydroxytetradecyl chain. On the
basis of the chemical shift of C-2 (δ 130.4), an ether linkage via
C-1′ and C-2 between two aromatic rings to form a macrocyclic
molecule, as in robustol (9), was deduced. This could also be
supported by an unusual upfield shift of H-6′ (δ 5.83) compared
with H-4′ (δ 6.26) due to the shielding effect by the other non-
coplanar aromatic ring. The stereochemistry at C-1′′ was determined
using a modified Mosher ester method to elucidate the absolute
1
configuration of the secondary alcohol by the application of H
NMR spectroscopy.7,8 On the basis of the drawing of a Mosher
plane, the positive ∆δ for H-2′′ (+0.07) as well as the negative
∆δ for H-4 (-0.10) and H-6 (-0.01) indicated the R configuration
at C-1′′. Consequently, the structure (R)-1′′-hydroxyrobustol was
assigned for gravicycle (1).
Dehydrobisgravillol (4) was isolated as a colorless syrup. The
HREIMS showed the molecular ion at m/z 426.2773 for C27H38O4,
two hydrogen atoms less than that of 3. The NMR data of these
two compounds showed that they were closely related, but indicated
the presence of a C14,1 alkenyl chain in 4. The existence of a vinylic
proton signal at δ 5.35 (2H, m) and allylic proton signals at δ 2.02
Dehydrogravicycle (2) was determined to have the molecular
formula C26H34O5, two atomic mass units less than that of 1, by
HREIMS. Compounds 2 and 1 exhibited almost identical 1H NMR
spectra, except for the appearance of two vinylic protons at δ 5.33
and four allylic protons at δ 2.00 in 2, indicating the presence of
an unsaturation in the long chain. The configuration of the double
bond appeared to be Z, as evidenced by the allylic carbon resonances
at δ 27.6 (C-7′′ and -10′′) and olefinic carbon resonances at δ 129.8
(C-8′′ and -9′′).9 The long chain with a double bond has been
proposed as -(CH2)9CHdCH(CH2)3- and/or -(CH2)7CHdCH-
(CH2)5- by chemical degradation and biosynthetic consider-
ation.10,11 The presence of HMBC correlations between benzylic
H-1′′ (δ 4.46), -14′′ (δ 2.35) and methylene carbons at δ 27.9-
30.2 instead of the allylic carbon at δ 27.6 ruled out the former
possibility. Owing to the small amount of 2, the position of the
double bond in the cyclic structure was determined by chemical
transformation using dehydrorobustol A (10) as a model (Scheme
1). Methylation of 10 with (trimethylsilyl)diazomethane12 followed
by opening the ring with sodium in liquid ammonia13 gave 10a. Its
structure was confirmed by the presence of one methoxyl singlet
at δ 3.76 (3-OCH3), two equivalent methoxyl singlets at δ 3.78
(1′- and 3′-OCH3), and two sets of 1,3,5-trisubstituted benzene ring
signals, one set at δ 6.30 (H-2′) and 6.35 (H-4′ and -6′) and the
other set at δ 6.23 (H-2), 6.25 (H-6), and 6.32 (H-4). Acylation of
10a with acetyl chloride and addition of diethyl disulfide afforded
10b.14 The EIMS of 10b (C35H54O5S2) showed a base peak at m/z
309, corresponding to either C18H29O2S or C17H25O3S, indicating
the location of a double bond between carbons 8′′ and 9′′.
Comparison of the specific rotation with 1 and the double-bond
position with 10 led us to deduce that compound 2 possessed the
structure (R)-1′′-hydroxy-(Z)-dehydrorobustol A, and it was named
dehydrogravicycle.
1
(4H, m) in the H NMR spectrum indicated that 4 was simply an
unsaturated analogue of 3. The Z geometry of the double bond was
evidenced by the signals of allylic carbons at δ 27.1 and olefinic
carbons at δ 129.8. Finally, the position of the double bond was
established by the treatment of 4 with acetyl chloride followed by
the addition of diethyl disulfide to give 4a (Scheme 2). The EIMS
of 4a (C37H54O7S2) showed a base peak at m/z 337, corresponding
to either C19H29O3S or C18H25O4S. This clearly inferred that the
double bond was between carbons 8′′ and 9′′ in the 14-carbon chain.
Therefore, the structure of dehydrobisgravillol (4) was (Z)-1-O-
methyl-5-[14′′-(1′,3′-dihydroxyphenyl)tetradec-8′′-en-1′′-yl]resor-
cinol.
Dehydrograviphane (5) showed its molecular ion at m/z 410.2455
(C26H34O4). Signals assignable to a 14-carbon chain with a double
bond were observed at δ 1.25 (12H, m, 6 × CH2), 1.36 (2H, m,
H-13′′), 1.58 (2H, m, H-2′′), 1.95 (4H, m, 2 × allylic CH2), 2.26
(2H, t, J ) 7.8 Hz, H-14′′), 2.59 (2H, t, J ) 6.3 Hz, H-1′′), and
5.32 (2H, m, 2 vinyl protons). In the aromatic region, a two-proton
singlet at δ 6.47 for H-4 and H-6 and two singlets at δ 6.45 and
6.48 for H-2′ and -4′, respectively, confirmed the 2,5- and 5′,6′-
disubstituted resorcinol moieties. HMBC connectivities of H-4 and
H-6 with C-1′′ (δ 36.0) and only H-4′ with C-14′′ (δ 34.2) indicated
that the terminals of the long chain were attached to C-5 and C-5′
of two resorcinol units. The upfield chemical shifts of quaternary
carbons C-2 (δ 103.4) and C-6′ (δ 105.7) suggested a carbon-
carbon linkage between two resorcinol rings through C-2 and C-6′
to form a non-coplanar biphenyl skeleton. No optical activity ([R]D
) 0) for 5 proved the presence of a conformation with a plane of
symmetry. Four hydroxyl signals at δ 4.73 (2H, s, 1- and 3-OH),
5.07 (1H, br s, 1′-OH), and 5.18 (1H, br s, 3′-OH) confirmed the
structure of 5 as a turriane derivative, a kind of (14-p,0-o)-
cyclophane.11 The Z geometry of the double bond was supported
by the allylic carbons at δ 26.5 and vinylic carbons at δ 129.8 as
Bisgravillol (3) was isolated as a white amorphous powder. The
HREIMS showed a molecular ion at m/z 428.2924, consistent with

Notes
Journal of Natural Products, 2007, Vol. 70, No. 2 321
Table 1. 1H NMR Data of Alkylresorcinols 1-8 (δ, multiplicity, J, Hz in parentheses)a
1
2
3
4
5
6
7
8
H-2
H-4
H-6
H-2′
H-4′
H-6′
H-1′′
H-2′′
H-3′′-12′′
H-3′′-6′′, 11′′, 12′′
H-7′′, 10′′
H-8′′, 9′′
H-13′′
H-14′′
1-OH
6.25 s
6.26 s
6.33 s
6.17 s
6.24 s
6.24 s
2.49 m
1.56 m
1.25 brs
6.23 s
6.27 s
6.31s
6.16 s
6.23 s
6.23 s
2.47 m
1.53 m
6.46 s
6.46 s
6.46 s
6.46 s
6.47 s
6.47 s
6.45 s
6.48 s
6.42 s
6.42 s
6.41 d (2.1)
6.48 d (2.1)
6.41 s
6.41 s
6.21 s
6.32 s
6.43 s
6.43 s
6.43 s
6.48 s
6.48 t (2.1)
6.26 t (2.1)
5.83 t (2.1)
4.46 t (6.6)
1.72 m
6.48 t (2.1)
6.26 t (2.1)
5.82 t (2.1)
4.46 t (6.4)
1.72 m
2.59 t (6.3)
1.58 m
2.57 t (6.6)
1.59 m
2.50 t (6.6)
1.60 m
2.58 t (6.4)
1.60 m
1.27 m
1.21 m
1.20 m
1.27 br s
2.00 m
5.33 m
1.45 m
2.35 t (7.8)
1.26 br s
2.02 m
5.35 m
1.53 m
2.47 m
1.25 br s
1.95 m
5.32 m
1.36 m
2.26 t (7.8)
4.73 s
1.23 m
1.94 m
5.30 m
1.38 m
2.29 t (7.8)
4.59 s
1.45 m
2.34 t (7.8)
1.56 m
2.49 m
1.30 m
1.27 m
2.18 t (7.8)
5.28 br s
5.28 br s
5.78 br s
6.38 brs
2.29 t (7.8)
4.59 br s
4.59 br s
3-OH
4.95 s
4.95 s
4.95 s
3.76 s
5.97 br s
5.97 br s
5.97 br s
3.73 s
4.73 s
5.07 brs
5.18 br s
4.59 s
1′-OH
3′-OH
1-OCH3
1′-OCH3
5.16 br s
3.72 s
5.25 br s
3.71 s
a 1 and 2 were measured in CD3OD; 3-8 were measured in CDCl3.
Department of Life Sciences, National Cheng Kung University. A
voucher specimen (No: PLW-0303) was deposited in the Herbarium
of the same university.
in 2 and 4. According to the structure of tetramethoxyturriene and
the possible biogenetic route to cyclophane,11 the alkenyl chain
pattern would be the same as that in 4 and 10. Hence, (Z)-1,1′,3,3′-
tetrahydroxyturri-8′′-ene was deduced for the structure of dehy-
drograviphane (5).
Methyldehydrograviphane (6) was determined to have the
molecular formula C27H36O4 by HREIMS. The 1H NMR data of 6
were almost the same as that of 5. The difference was that 6
contained three hydroxyls at δ 4.59 (s, 1- and 3-OH) and 5.16 (br
s, 3′-OH) and a methoxyl at δ 3.72 (3H, s, 1′-OCH3) on the phenyl
rings. The location of the methoxyl group was determined from its
NOE cross-peak with H-2′ (δ 6.41) but not with H-4′ (δ 6.48).
Thus, the structure of 6 was assigned as (Z)-1′-methoxy-1,3,3′-
trihydroxyturri-8′′-ene, and it was named methyldehydrograviphane.
Compound 6 has been synthesized by Furstner et al.15
The molecular formulas of graviphane (7) and methylgraviphane
(8) were established as C26H36O4 and C27H38O4 from the molecular
ion peaks at m/z 412.2612 and 426.2773, respectively. By com-
parison of their spectroscopic data with those of 5 and 6, they were
found to be the saturated analogues of 5 and 6 with the same turriane
ring system. Consequently, 1,1′,3,3′-tetrahydroxyturriane and
1′-methoxy-1,3,3′-trihydroxyturriane were assigned for the struc-
tures of graviphane (7) and methylgraviphane (8), respectively.
Although the structures of 7 and 8 were proposed from a mixture
obtained from an extract of G. robusta and synthesized by Ridley
et al.,11 this represents their first isolation as pure compounds from
a natural source.
The alkylresorcinols 1-14 were all subjected to cytotoxic
evaluation against MCF-7, NCI-H460, and SF-268 cell lines. They
all showed marginal cytotoxicity (Table 3). The similar IC50 values
of these compounds led us to conclude that there was no impact of
alkyl chain, either cyclic or straight chain, on the cytotoxic activity
of these alkylresorcinols.
Extraction and Isolation. The dried leaves of G. robusta (7.7 kg)
were extracted with MeOH under reflux. The extract was concentrated
under reduced pressure to give a dark green syrup. The syrup was
suspended in H2O and then partitioned with hexane. The concentrated
hexane layer (150 g) was chromatographed on a silica gel column by
eluting with a gradient of hexane-(CH3)2CO (4:1 to pure (CH3)2CO)
to yield eight fractions. Cytotoxic assay indicated that fractions 4-7
were active. Therefore, further chromatography of fraction 4 on a silica
gel column eluting with a gradient of CHCl3-MeOH (50:1 to pure
MeOH) gave three active subfractions, A (105 mg), B (2.15 g), and C
(825 mg). Repeated chromatography of A-C afforded small amounts
of 6 and 8, 13 and 14, and 9 and 10, respectively. Fraction 5 was
chromatographed on silica gel eluting with a gradient of CHCl3-MeOH
(30:1 to pure MeOH) to give two active subfractions, D (35 mg) and
E (180 mg). Repeated chromatography of D and E yielded 3 and 4
and 5 and 7, respectively. Fraction 6 was chromatographed on silica
gel eluting with a gradient of CHCl3-MeOH (20:1 to pure MeOH)
and gave active subfraction F (545 mg), which was subjected to
chromatographic separation to give pure compounds 1 and 2. Using
the same separation procedure, fraction 7 was separated to produce
one active subfraction, G (52 mg). Chromatographic purification of G
gave 11 and 12.
Gravicycle (1): white, amorphous powder; [R]D +13.1 (c 0.45,
CHCl3); UV (MeOH) λmax (log ꢀ) 228 (4.1), 274 (3.5) nm; IR (KBr)
1
νmax 3346, 1597, 1502, 1459 cm-1; H NMR data, Table 1; 13C NMR
data, Table 2; EIMS m/z (rel int) 428 (100, M+), 410 (18), 369 (8),
260 (26), 232 (12), 124 (18); HREIMS m/z 428.2565 [M]+ (calcd for
C26H36O5 428.2563).
Acylation of 1 with MTPACl to 1a. (R)- or (S)-MTPACl (10 mg)
was added to a pyridine solution (0.2 mL) containing 1 (1.0 mg). The
reaction mixture was stirred at room temperature for 4 h, then
evaporated to dryness. The residue was washed with water, extracted
with EtOAc, and dried with anhydrous MgSO4. The filtered organic
solution was concentrated and subjected to chromatography on silica
gel using hexane-EtOAc (10:1) as eluent. Pure 1a-(S)-MTPA (2.7 mg)
or 1a-(R)-MTPA (2.5 mg) was thus obtained. 1H NMR data for
1a-(S)-MTPA: δ 1.26 (20H, m), 1.35 (2H, m), 1.95 (2H, m, H-2′′),
2.38 (2H, t, J ) 7.8 Hz), 3.31 (3H, s), 3.42 (3H, s), 3.53 (3H, s), 3.67
(3H, s), 5.94 (1H, t, J ) 6.6 Hz, H-1′′), 6.08 (1H, s), 6.52 (1H, s), 6.60
(1H, s), 7.00 (1H, s, H-4), 7.09 (1H, s, H-6), 7.2-7.7 (20H, m); for
1a-(R)-MTPA: δ 1.25 (20H, m), 1.38 (2H, m), 1.88 (2H, m, H-2′′),
2.39 (2H, t, J ) 8.1 Hz), 3.36 (6H, S), 3.50 (3H, s), 3.68 (3H, s), 6.02
(1H, t, J ) 6.6 Hz, H-1′′), 6.12 (1H, s), 6.56 (1H, s), 6.60 (1H, s), 7.10
(2H, s, H-4 and -6), 7.2-7.7 (20H, m).
Experimental Section
General Experimental Procedures. Optical rotations were mea-
sured on a Jasco DIP-370 digital polarimeter. UV spectra were recorded
on an Agilent 8453 spectrophotometer. IR spectra were recorded on a
Nicolet Magna FT-IR spectrophotometer. NMR spectra were recorded
on Bruker Avance 300 and AMX 400 FT-NMR spectrometers; all
chemical shifts are given in ppm from tetramethylsilane as an internal
standard. Mass spectra were obtained on a VG 70-250S spectrometer
using a direct inlet system.
Plant Material. The leaves of GreVillea robusta were collected on
the campus of National Cheng Kung University, Tainan, Taiwan, in
September 2003. The collection was identified by Professor C. S. Kuoh,
Dehydrogravicycle (2): white, amorphous powder; [R]D +13.3 (c
0.20, CHCl3); UV (MeOH) λmax (log ꢀ) 217 (4.1), 275 (3.3) nm; IR
(KBr) νmax 3396, 1598, 1505, 1458 cm-1; 1H NMR data, Table 1; 13
C

322 Journal of Natural Products, 2007, Vol. 70, No. 2
Table 2. 13C NMR Data of Alkylresorcinols 1-8a
Notes
1
2
3
4
5
6
7
8
C-1
151.8
151.8
160.7
160.6
154.5
153.5
154.3
153.5
C-2
C-3
C-4
C-5
C-6
C-1′
C-2′
C-3′
C-4′
C-5′
C-6′
C-1′′
130.4
151.8
106.7
143.9
106.7
160.8
102.7
159.3
109.9
146.0
105.3
75.3
130.4
151.8
106.7
143.9
106.7
160.8
102.7
159.3
109.9
146.0
105.2
75.4
98.7
156.4
107.9
145.8
106.8
156.6
100.1
156.6
108.0
146.2
108.0
35.8
98.8
156.4
108.1
145.8
106.8
156.5
100.3
156.5
108.1
145.8
108.1
36.0
103.4
154.5
108.3
146.8
108.3
156.4
100.9
158.2
109.8
147.5
105.7
36.0
107.8
153.5
107.6
144.7
107.6
159.9
97.5
157.9
109.3
148.1
108.7
35.1
104.4
154.3
108.2
146.4
108.2
155.7
100.9
157.7
109.8
147.5
106.6
35.5
107.3
153.5
107.6
144.8
107.6
160.0
97.5
158.0
109.0
148.1
108.5
35.6
C-2′′
40.0
40.0
31.1
31.1
30.8
29.9
30.3
30.4
C-3′′
26.4
26.4
27.9-30.2
27.6
129.8
27.9-30.2
32.2
29.2-29.6
29.2-29.6
29.2-29.6
29.2-29.6
29.2-29.6
31.1
29.1-29.8
29.1-29.8
27.1
129.8
29.1-29.8
31.1
27.3-29.9
27.3-29.9
26.5
129.8
27.3-29.9
31.3
27.0-29.4
27.0-29.4
26.7
129.9
27.0-29.4
31.4
26.8-29.4
26.8-29.4
26.8-29.4
26.8-29.4
26.8-29.4
31.2
26.8-29.7
26.8-29.7
26.8-29.7
26.8-29.7
26.8-29.7
31.4
C-4′′-6′′
C-7′′, 10′′
C-8′′, 9′′
C-11′′, 12′′
C-13′′
C-14′′
1-OCH3
1′-OCH3
27.9-30.2
27.9-30.2
27.9-30.2
27.9-30.2
32.2
37.0
37.0
36.0
55.2
36.0
55.3
34.2
33.4
33.6
33.6
55.9
55.9
a 1 and 2 were measured in CD3OD; 3-8 were measured in CDCl3.
Table 3. Cytotoxicity of 1-14 toward Three Cancer Linesa
IC50 (µM)
EtOAc (2:1) to obtain the diethyldifulfide adduct 4a: EIMS m/z (rel
int) 674 (1, M+), 337 (100), 320 (38).
Dehydrograviphane (5): colorless syrup; UV (CHCl3) λmax (log ꢀ)
243 (3.8), 281 (3.6), 324 (2.8) nm; IR (KBr) νmax 3408, 1621, 1569,
1455 cm-1; 1H NMR data, Table 1; 13C NMR data, Table 2; EIMS m/z
(rel int) 410 (100, M+), 246 (17), 229 (15), 161 (5), 149 (3), 123 (8);
HREIMS m/z 410.2455 [M]+ (calcd for C26H34O4 410.2457).
Methyldehydrograviphane (6): white, amorphous powder; UV
(MeOH) λmax (log ꢀ) 244 (4.0), 280 (3.9), 324 (2.9) nm; IR (KBr) νmax
3405, 1604, 1585, 1458 cm-1; 1H NMR data, Table 1; 13C NMR data,
Table 2; EIMS m/z (rel int) 424 (100, M+), 260 (14), 243 (11), 166
(6), 149 (9), 137 (6); HREIMS m/z 424.2610 [M]+ (calcd for C27H36O4
424.2613).
compound
MCF-7
NCI-H460
SF-268
1
2
3
4
5
6
7
8
30.6 ( 0.3
30.0 ( 0.1
29.4 ( 1.2
29.8 ( 0.3
30.7 ( 1.5
29.3 ( 0.3
28.6 ( 3.2
30.8 ( 1.9
32.4 ( 0.1
31.8 ( 0.8
29.2 ( 0.8
28.6 ( 1.5
37.0 ( 1.1
37.1 ( 1.9
27.0 ( 0.6
27.9 ( 1.8
28.7 ( 1.4
27.0 ( 2.1
25.4 ( 2.9
27.9 ( 3.9
22.8 ( 1.3
28.9 ( 1.5
24.0 ( 5.0
28.0 ( 3.2
28.8 ( 0.3
27.7 ( 1.2
34.2 ( 1.2
35.4 ( 1.7
32.1 ( 0.8
31.3 ( 0.4
29.4 ( 1.0
30.2 ( 1.3
33.4 ( 1.1
29.1 ( 1.1
27.7 ( 1.5
31.1 ( 0.1
32.7 ( 1.2
33.1 ( 0.4
29.8 ( 0.2
30.6 ( 0.8
39.8 ( 0.3
39.2 ( 0.7
9
10
11
12
13
14
Graviphane (7): colorless syrup; UV (CHCl3) λmax (log ꢀ) 244 (3.8),
1
281 (3.6) nm; IR (KBr) νmax 3408, 1621, 1573, 1455 cm-1; H NMR
data, Table 1; 13C NMR data, Table 2; EIMS m/z (rel int) 412 (100,
M+), 246 (16), 229 (14), 211 (11), 197 (5), 149 (13), 124 (9), 123 (6);
HREIMS m/z 412.2612 [M]+ (calcd for C26H36O4 412.2614).
a Values are mean ( SD (n ) 3-8). MCF-7 ) human breast tumor
cell line; NCI-H460 ) human lung tumor cell line; SF-268 ) human
central nervous system tumor cell line.
Methylgraviphane (8): white, amorphous powder; UV (MeOH) λmax
(log ꢀ) 248 (3.9), 280 (3.8), 323 (2.5) nm; IR (KBr) νmax 3413, 1584,
1459 cm-1; 1H NMR data, Table 1; 13C NMR data, Table 2; EIMS m/z
(rel int) 426 (100, M+), 260 (10), 243 (9), 137 (5); HREIMS m/z
426.2773 [M]+ (calcd for C27H38O4 426.2770).
NMR data, Table 2; EIMS m/z (rel int) 426 (100, M+), 369 (12), 260
(40), 246 (14), 229 (15), 163 (10), 149 (8), 137 (12), 123 (34); HREIMS
m/z 426.2403 [M]+ (calcd for C26H34O5 426.2406).
Diethyldisulfide Adduct 10a from 10. A mixture of 9 and 10 (15
mg), (trimethylsilyl)diazomethane (2.0 M solution in hexane, 0.2 mL),
and MeOH (0.8 mL) in benzene (3 mL) was placed in a sealed tube
and heated at 65 °C for 6 h. After the excess (trimethylsilyl)-
diazomethane was decomposed with acetic acid, the reaction mixture
was evaporated to dryness. The residue was purified by column
chromatography on silica gel eluting with hexane-EtOAc (10:1) to
give pure methylated dehydrorobustol A (7.5 mg). The methylated
dehydrorobustol A was added to dry liquid NH3 (5 mL). Sodium (500
mg) was then added in small pieces, and the solution was continuously
stirred until it retained an intense blue color for 2 h. The coolant was
withdrawn, and the mixture was stirred for another 2 h in a Dewar
flask without additional cooling. A surplus of NH4Cl was added, and
the NH3 was removed in a stream of N2. The residue was purified by
column chromatography on silica gel eluting with hexane-EtOAc
(1:1) to give 10a (5.1 mg): 1H NMR (CDCl3, 300 MHz) δ 1.27 (12H,
m, H-3′′-6′′, -11′′, and -12′′), 1.58 (4H, m, H-2′′ and -13′′), 2.00 (4H,
m, H-7′′ and -10′′), 2.52 (4H, m, H-1′′ and -14′′), 3.76 (3H, s, 3-OCH3),
3.78 (6H, s, 1′- and 3′-OCH3), 4.74 (1H, s, 1-OH), 5.32 (2H, m, H-8′′
and -9′′), 6.23 (1H, s, H-2), 6.25 (1H, s, H-6), 6.30 (1H, s, H-2′), 6.32
(1H, s, H-4), 6.35 (2H, s, H-4′ and -6′); 13C NMR (CDCl3, 75 MHz)
δ 27.1 (C-7′′ and -10′′), 29.1-29.7 (C-3′′-6′′, -11′′, and -12′′), 31.1
and 31.3 (C-2′′ and -13′′), 36.0 and 36.3 (C-1′′ and -14′′), 55.2 (1′, 3,
and 3′-OCH3), 97.5 (C-2′), 98.6 (C-2), 106.5 (C-4′ and -6′), 106.8
(C-4), 107.8 (C-6), 129.9 (C-8′′ and -9′′), 145.4 (C-5), 145.8 (C-5′),
Bisgravillol (3): white, amorphous powder; UV (CHCl3) λmax (log
ꢀ) 242 (3.6), 280 (3.5) nm; IR (KBr) νmax 3362, 1597, 1464 cm-1; H
1
NMR data, Table 1; 13C NMR data, Table 2; EIMS m/z (rel int) 428
(49, M+), 180 (4), 163 (4), 151 (15), 138 (100), 124 (42); HREIMS
m/z 428.2924 [M]+ (calcd for C27H40O4 428.2927).
Dehydrobisgravillol (4): colorless syrup; UV (CHCl3) λmax (log ꢀ)
242 (3.6), 281 (3.5) nm; IR (KBr) νmax 3372, 1598, 1463 cm-1; H
1
NMR data, Table 1; 13C NMR data, Table 2; EIMS m/z (rel int) 426
(52, M+), 246 (6), 217 (19), 205 (8), 189 (14), 177 (24), 163 (32), 138
(100), 124 (56); HREIMS m/z 426.2773 [M]+ (calcd for C27H38O4
426.2770).
Diethyldisulfide Adduct 4a from 4. Acetyl chloride (12 mg) was
added to a pyridine solution (0.5 mL) containing dehydrobisgravillol
(4) (3.4 mg). The solution was stirred at room temperature for 4 h.
The reaction mixture was quenched with water and extracted with
EtOAc. The organic extract was dried with MgSO4 and chromato-
graphed on silica gel eluting with hexane-EtOAc (3:1) to give pure
acetylated dehydrobisgravillol. The acetylated dehydrobisgravillol was
added to a hexane (0.5 mL) solution that contained a catalytic amount
of iodine. Diethyldisulfide (0.1 mL) was then added, and the solution
was heated at 50 °C for 12 h. After evaporating the solvent, the residue
was chromatographed on a silica gel column eluting with hexane-

Notes
Journal of Natural Products, 2007, Vol. 70, No. 2 323
(2) Cannon, J. R.; Chow, P. W.; Fuller, M. W.; Hamilton, B. H.; Metcalf,
B. W.; Power, A. J. Aust. J. Chem. 1973, 26, 2257-2275.
(3) Ahmed, A. S.; Nakamura, N.; Meselhy, M. R.; Makhboul, M. A.;
El-Emary, N.; Hattori, M. Phytochemistry 2000, 53, 149-154.
(4) Varma, R. S.; Manju, M.; Parthasarathy, M. R. Phytochemistry 1976,
15, 1418-1419.
156.4 (C-1), 160.6 (C-1′ and -3′), 160.8 (C-3); EIMS m/z (rel int) 454
(25, M+), 152 (100), 138 (43). Using the same procedure for the
preparation of 8a from 8, acylation with acetyl chloride and addition
with diethyldisulfide of 10a (5.1 mg) gave 10b (4.3 mg): 1H NMR
(CDCl3, 300 MHz) δ 1.25 (6H, t, J ) 7.3 Hz, 2 × SCH2CH3), 1.32
(16H, br s, H-3′′-7′′ and 10′′-12′′), 1.60 (4H, m, H-2′′ and -13′′),
2.28 (3H, s, 1-OCOCH3), 2.52 (4H, q, J ) 7.3 Hz, 2 × SCH2CH3),
2.56 (4H, m, H-1′′ and -14′′), 2.74 (2H, m, H-8′′ and -9′′), 3.78 (9H,
s, 1′-, 3-, and 3′-OCH3), 6.30 (1H, s, H-2′), 6.34 (2H, s, H-4′ and -6′),
6.46 (1H, s, H-2), 6.51 (1H, s, H-6), 6.60 (1H, s, H-4); 13C NMR
(CDCl3, 75 MHz) δ 15.1 (2 × SCH2CH3), 21.2 (1-OCOCH3), 26.3 (2
× SCH2CH3), 29.1-29.7 (C-3′′-7′′ and 10′′-12′′), 31.4 (C-2′′ and
-13′′), 35.9 (C-1′′), 36.3 (C-14′′), 51.1 (C-8′′ and -9′′), 55.2 (1′- and
3′-OCH3), 55.3 (3-OCH3), 97.5 (C-2′), 104.6 (C-2), 106.5 (C-4′ and
-6′), 111.9 (C-4), 113.8 (C-6), 145.4 (C-5 and -5′), 151.4 (C-1), 160.2
(C-3), 160.7 (C-1′ and -3′), 169.5 (1-OCOCH3); EIMS m/z (rel int)
618 (10, M+), 369 (21), 309 (100), 295 (41), 152 (46), 151 (37), 137
(28).
(5) Sumino, M.; Sekine, T.; Ruangrungsi, N.; Igarashi, K.; Ikegami, F.
Chem. Pharm. Bull. 2002, 50, 1484-1487.
(6) Lytollis, W.; Scannell, R. T.; An, H.; Murty, V. S.; Reddy, K. S.;
Barr, J. R.; Hecht, S. M. J. Am. Chem. Soc. 1995, 117, 12683-
12690.
(7) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092-4096.
(8) Liu, L.; Li, W.; Koike, K.; Nikaido, T. Heterocycles 2004, 63, 1429-
1436.
(9) Rossi, R.; Carpita, A.; Quirici, M. G.; Veracini, C. A. Tetrahedron
1982, 38, 639-644.
(10) Ridley, D. D.; Ritchie, E.; Taylor, W. C. Aust. J. Chem. 1968, 21,
2979-2988.
(11) Ridley, D. D.; Ritchie, E.; Taylor, W. C. Aust. J. Chem. 1970, 23,
147-183.
Cytotoxicity Assay. The cytotoxicity assay was carried out according
to the procedure described in the literature.16
(12) Veluri, R.; Weir, T. L.; Bais, H. P.; Stermitz, F. R.; Vivanco, J. M.
J. Agric. Food Chem. 2004, 52, 1077-1082.
Acknowledgment. The authors thank the National Science Council
of the Republic of China for the financial support (NSC 94-2113-M-
273-001) and the Division of Biotechnology and Pharmaceutical
Research in the National Health Research Institutes for the cytotoxicity
assay.
(13) Glombitza, K. W.; Lentz, G. Tetrahedron 1981, 37, 3861-3866.
(14) Buser, H. R.; Arn, H.; Guerin, P.; Rauscher, S. Anal. Chem. 1983,
55, 818-822.
(15) Furstner, A.; Stelzer, F.; Rumbo, A.; Krause, H. Chem.-Eur. J. 2002,
8, 1856-1871.
(16) Chuang, T. H.; Lee, S. J.; Yang, C. W.; Wu, P. L. Org. Biomol.
Chem. 2006, 4, 860-867.
References and Notes
(1) Ritchie, E.; Taylor, W. C.; Vautin, S. T. K. Aust. J. Chem. 1965, 18,
2105-2020.
NP0605687