Lehualides A-D, metabolites from a Hawaiian sponge of the genus Plakortis

Article abstract of DOI:10.1021/np0500528

A collection of an undescribed marine sponge of the genus Plakortis yielded four new "polyketide-derived" metabolites, lehualides A-D (1-4). The structures of compounds 1-4 were elucidated by interpretation of spectral data. Compound 2 demonstrated cytotoxicity against an ovarian cancer cell line, while compound 4 was active against both ovarian cancer and leukemia cell lines.

Full text of DOI:10.1021/np0500528

1400
J. Nat. Prod. 2005, 68, 1400-1403
Lehualides A-D, Metabolites from a Hawaiian Sponge of the Genus Plakortis
Noriko Sata,† Hazel Abinsay,† Wesley Y. Yoshida,† F. David Horgen,‡ Namthip Sitachitta,*,† Michelle Kelly,§ and
Paul J. Scheuer†
Department of Chemistry, University of Hawaii at Manoa, Honolulu, Hawaii 96822, College of Natural Sciences, Hawaii
Pacific University, Kaneohe, Hawaii 96744, and National Institute of Water and Atmospheric Research (NIWA), Private Bag,
109-695 Newmarket, Auckland, New Zealand
Received February 17, 2005
A collection of an undescribed marine sponge of the genus Plakortis yielded four new “polyketide-derived”
metabolites, lehualides A-D (1-4). The structures of compounds 1-4 were elucidated by interpretation
of spectral data. Compound 2 demonstrated cytotoxicity against an ovarian cancer cell line, while
compound 4 was active against both ovarian cancer and leukemia cell lines.
Marine sponges in the family Plakinadae are known to
be rich sources of structurally unique and biologically active
metabolites. Plakortin1 and structurally related cycloper-
oxide ring-containing polyketides1b-d are commonly found
in the extracts of Plakortis. These compounds have a broad
range of biological activities such as antibacterial,1b
antileishmanical,1c and cytotoxic.1d,e In addition, other
recent examples include potent immunosuppressive agents,
the plakosides,2 and the activators of cardiac SR-Ca2+
-
pumping ATPase, the plakortones.3 In our continuing
program to search for new bioactive compounds from
marine organisms, it was found that the crude extract of
an undescribed Hawaiian Plakortis sp. exhibited toxicity
against brine shrimp. Bioassay-guided fractionation of the
extract led to the isolation of four new metabolites, lehua-
lides A-D (1-4). Herein, we report the details of the
isolation, structure elucidation, and cytotoxicity of these
new metabolites.
The sample of sponge was collected by hand using scuba
from waters between Lehua Rock and Niihau Island,
Hawaii, in July 2003. The freeze-dried sponge was repeat-
edly extracted with dichloromethane and 2-propanol (1:1).
The crude extracts were combined, concentrated in vacuo,
and partitioned between hexane and 80% aqueous MeOH.
The aqueous MeOH layer was further extracted with
dichloromethane. The dichloromethane-soluble materials
were first chromatographed over silica gel vacuum liquid
chromatography and followed by silica gel column chro-
matography. The brine shrimp toxic fractions were further
purified by normal-phase HPLC to provide lehualides A
(1, 26.4 mg), B (2, 28.7 mg), C (3, 10.6 mg), and D (4, 10.8
mg).
The HREIMS of lehualide A (1) revealed a molecular ion
at m/z 422.2461, which was consistent with a molecular
formula of C27H34O4 and required 11 degrees of unsatura-
tion. The 1H NMR signals of 1 resonating in the aromatic
region (δ 7.15 (3H) and 7.26 (2H)) and the presence of three
aromatic signals [δ 141.7 (1 C), 128.3 (4 CH), and 125.7
(1CH)] in the 13C NMR spectrum indicated the presence
of a phenyl ring. Further analysis of the 13C NMR spectrum
of 1 showed that 10 out of the remaining 20 carbons
resonated in the olefinic region and one was a carbonyl
carbon (δ 160.2). Thus, 10 out of 11 degrees of unsaturation
are now accounted for and lehualide A (1) must contain
one additional ring.
Inspection of the 1H-1H COSY NMR spectrum of 1
allowed us to establish three spin systems: C-7 to C-8, C-10
to C-12, and C-16 to C-17 (Figure 1). The three-bond HMBC
correlations from C-9 (δ 138.4) to H2-7 (δ 3.19) and H2-11
(δ 2.11) readily placed C-9 between C-8 (δ 117.4) and C-10
(δ 39.7). The C-9 olefinic quaternary carbon was shown to
possess a methyl group subtituent based on the observed
HMBC correlations between C-9 and a vinyl methyl group
at δ 1.68 (H3-25). Similarly, C-13, a quaternary olefinic
carbon resonating at δ 133.5, was located between C-12 (δ
126.0) and an isolated methylene carbon, C-14 (δ 41.8), due
to HMBC correlations from it to H2-11 (δ 2.11) and H2-14
(δ 2.73). The placement of the vinyl methyl group C-26 (δ
23.3) on C-13 was possible through HMBC correlations
from C-12 and C-13 to H3-26 (δ 1.59). The HMBC cross-
peaks shown from C-15 (δ 134.0) to H2-14 and H2-17 (δ
3.36) clearly indicated that C-15 is adjacent to C-14 and
C-16 (δ124.0). In addition, a vinyl methyl group (C-27; δ
15.9) was also found to be attached to C-15, due to the
HMBC correlation shown from C-15 to H3-27 (δ 1.63).
Dedicated to the late Dr. Noriko Sata for her major contribution to
this work.
* To whom correspondence should be addressed. Tel: (808) 956-9878.
Fax: (808) 956-5908. E-mail: namthip@gold.chem.hawaii.edu.
† University of Hawaii at Manoa.
‡ Hawaii Pacific University.
§ NIWA, Auckland.
10.1021/np0500528 CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/19/2005

Notes
Journal of Natural Products, 2005, Vol. 68, No. 9 1401
Lehualide C (3) was assigned the molecular formula
C23H36O5S by HREIMS, requiring six degrees of unsatura-
tion. Analysis of the 1H NMR spectrum of compound 3
(Table 2) revealed, in contrast to lehualides A and B, the
presence of only one vinyl proton at δ 5.17 (H-8) and the
absence of phenyl proton signals. The observed resonances
at δ 159.8 (C-2), 127.4 (C-3), 176.7 (C-4), 119.8 (C-5), and
156.8 (C-6) in the 13C NMR spectrum (Table 2) clearly
indicated that compound 3 possesses a γ-pyrone moiety,
which accounted for four out of six degrees of unsaturation.
The substitution pattern on the γ-pyrone ring of 3 was
shown to be identical to that of 2 through interpretation
of the HMBC spectral data. Of eight olefinic carbons shown
in the 13C NMR spectrum of 3, five were assigned to the
γ-pyrone system. Thus, compound 3 must contain a double
bond [δ 116.4 (C-8) and 139.6 (C-9)] and a carbonyl carbon
(δ 196.1), which also accounted for the last two degrees of
unsaturation. As it was for compounds 1 and 2, the HMBC
correlations shown from both C-8 and C-9 to H2-7 (δ 3.27)
linked the double bond to the isolated methylene carbon
(C-7). The cross-peaks shown from C-5 and C-6 to H2-7 in
the HMBC spectrum led to the attachment of the γ-pyrone
ring to C-7. The C-9 olefinic quaternary carbon was shown
to possess a methyl group substituent through an observed
HMBC correlation shown between it and a vinyl methyl
group at δ 1.70 (H3-20). The carbonyl carbon was assigned
to an acetyl group due to its HMBC correlation with a
methyl proton at δ 2.32 in the HMBC spectrum.
Figure 1. Spin systems deduced by the COSY spectrum (bold
linkages) and key HMBC correlations (CfH arrows) of lehualide A
(1).
Comparison of the molecular formula of lehualide A (1)
with the assembled carbon framework required the eluci-
dation of a C7H7O4 subunit. The strong absorption at 1720
cm-1 in the IR spectrum and absorbance in the UV
spectrum (λmax ) 242 and 292 nm) of compound 1 are in
accordance with an extended R-pyrone chromophore.4 The
carbon signals observed at δ 160.2 (C-2), 124.9 (C-3), 157.8
(C-4), 105.6 (C-5), and 157.1 (C-6) in the 13C NMR spectrum
of 1 further confirmed the presence of an R-pyrone ring.
Since all of the carbons of the R-pyrone ring are nonpro-
tonated, as indicated by the HSQC and DEPT spectra, the
R-pyrone ring must be fully substituted. A vinyl methyl
group at C-24 (δ 9.28) was placed on C-5 due to the
observed HMBC correlations from the polarized olefinic
carbons (C-4 and C-6) to H3-24 (δ 1.95). Oxygen atoms were
placed on C-3 and C-4 of 1 since both carbons resonated at
relatively high field. One of these oxygen atoms belonged
to a methoxy group, which was placed at C-3 on the basis
of a HMBC correlation shown from C-3 to the methoxy
protons (δ 3.90). Thus, the remaining hydroxyl group is
located on C-4. This placement was assured by an observed
Comparison of the above assigned partial structure with
the molecular formula, the final assembling of the carbon
framework of compound 3, requires the assignment of a
saturated hydrocarbon chain with an attached sulfur atom
(C9H18S). The assignment of each nonoverlapped carbon
and proton signal was accomplished by analysis of the 1D
and 2D NMR data. The HMBC correlation shown from
C-10 (δ 39.5) to H3-20 allowed us to join the C-10 end of
the hydrocarbon chain to the double bond. Therefore, the
other end of the hydrocarbon chain must bear a thioester
group. This conclusion was supported by the diagnostic
chemical shift of C-18 (δ 29.1)6 and the HMBC correlation
shown from the carbonyl carbon to the methylene protons
at δ 2.85 (H2-18), completing the structure of lehualide C
(3).
upfield shift of the C-4 carbon signal by 7.1 ppm in the 13
C
NMR spectrum of lehualide A acetate (5). The HMBC cross-
peaks shown from C-5 and C-6 to H2-7 linked the 4-hy-
droxyl-3-methoxy-5-methyl-R-pyrone to the C-7 methylene
group. Finally, the HMBC correlation shown from C-17 to
H-19/H-23 attached the phenyl moiety to C-17, the meth-
ylene carbon of the olefinic chain, concluding the structure
of lehualide A (1).
The geometry of the double bonds in the structure of
lehualide A was primarily accomplished by interpretation
of NOESY spectral data. The ∆8 double bond was assigned
as E stereochemistry on the basis of H2-7/H3-25 and H-8/
H2-10 NOESY correlations. The observed H2-11/H2-14 and
H-12/H3-26 NOESY cross-peaks supported the Z geometry
of the ∆12 double bond. Finally, the H2-17/H3-27 NOESY
correlation established the E stereochemistry of the ∆15
double bond.
1
The H and 13C NMR spectra (Table 2) of lehualide D
(4) were found to be strikingly similar to those of lehualide
C (3) except for the absence of the acetyl group. This was
supported by the HREIMS, which revealed the molecular
formula of 4 to be C21H34O4S. Detailed analyses of the 1D
and 2D NMR spectra further confirmed the structure of
lehualide D (4) to be a thiol analogue of lehualide C (3).
Previous chemical studies of sponges in the family
Plakinadae have resulted in simple polyketide-derived
compounds1d and numerous structurally modified poly-
ketides with cyclic peroxides,1 peroxylactones,7 and perox-
yketals.8 Lehualides A (1) and B (2) are structurally similar
to other phenyl-containing compounds isolated from Pla-
kortis sp.1a and P. halichondrioides.9 However, the lehua-
lides appear to be the first set of polyketide metabolites
from Plakortis sp. to possess R- and γ-pyrone moieties.
Interestingly, lehualides C (3) and D (4) represent the only
metabolites from the sponge of this genus to contain the
thioester and thiol functionalities.
HREIMS of lehualide B (2) gave an [M]+ peak at
436.2640, yielding a molecular formula of C28H36O4, with
1
11 degrees of unsaturation. The H NMR of compound 2
was strikingly similar to that of 1 except for an additional
methoxy group. The observed carbon signals at δ 159.7 (C-
2), 128.2 (C-3), 176.6 (C-4), 119.7 (C-5), and 156.6 (C-6) in
the 13C NMR spectrum of 2 are consistent with the
presence of a γ-pyrone moiety.5 This was further supported
by a strong absorption band at 1655 cm-1 in the IR
spectrum of 2.5 The two OMe groups were assigned to C-2
and C-3 of the γ-pyrone ring due to the observed HMBC
cross-peaks shown from C-2 to δ 3.97 (OMe-1) and from
C-3 to δ 3.78 (OMe-2). The placement of the vinyl methyl
group (C-24) on C-5 was based on HMBC correlations from
both conjugated carbonyl carbon C-4 and the polarized
olefinic carbon C-6 to H3-24 (δ 1.97). Further analysis of
1D and 2D NMR spectra of 2 confirmed that the remainder
of its chemical structure as well as its double-bond geom-
etries were identical to those of lehualide A (1).
Lehualide A (1) showed mild brine shrimp toxicity (LD50
50 µg/mL), while lehualides B (2), C (3), and D (4) were
moderately toxic to brine shrimp (LD50 15 µg/mL). Lehua-
lide B (2) showed moderate cytotoxicity in vitro against an
ovarian cancer cell line (IGROV-ET) with a GI50 value of

1402 Journal of Natural Products, 2005, Vol. 68, No. 9
Notes
Table 1. NMR Spectral Data of Lehualides A (1) and B (2) Recorded in CDCl3
lehualide A (1)
lehualide B (2)
δH (J in Hz)
C/H no.
δC
δH (J in Hz)
HMBCa
δC
2
160.2 qC
124.9 qC
157.8 qC
105.6 qC
157.1 qC
30.2 CH2
117.4 CH
138.4 qC
39.7 CH2
26.4 CH2
126.0 CH
133.5 qC
41.8 CH2
134.0 qC
124.0 CH
34.2 CH2
141.7 qC
128.3 CH
128.3 CH
125.7 CH
128.3 CH
128.3 CH
9.3 CH3
159.7 qC
128.2 qC
176.6 qC
119.7 qC
156.6 qC
3
4
5
6
7
3.19 d (6.9)
C: 5, 6, 8, 9
29.9 CH2
116.7 CH
139.0 qC
39.5 CH2
26.2 CH2
125.6 CH
133.4 qC
41.7 CH2
133.7 qC
124.0 CH
34.1 CH2
141.5 qC
128.2 CH
128.2 CH
125.6 CH
128.2 CH
128.2 CH
9.6 CH3
3.25 d (7.2)
5.18 br t (7.2)
8
5.18 br t (6.9)
C: 6, 7, 10, 25
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
OMe-1
OMe-2
OH
2.05 t (6.9)
2.11 t (6.9)
5.19 m
C: 8, 9, 11, 12, 25
C: 9, 10, 12, 13
C: 11, 14, 26
2.01 t (7.2)
2.11 t (7.2)
5.18 m
2.73 br s
C: 12, 13,15, 16, 26, 27
2.72 br s
5.36 tq (7.5, 1.0)
3.36 d (7.5)
C: 14, 17, 18, 27
C: 15, 16, 18, 19, 23
5.34 br t (7.5)
3.35 d (7.5)
7.15 m
C: 17, 21, 23
C: 18, 22
C: 19, 23
C: 18, 20
C: 17, 21, 19
C: 4, 5, 6
C: 8, 9, 10
C: 12, 13, 14
C: 14, 15, 16
C: 3
7.13 m
7.25 m
7.13 m
7.25 m
7.13 m
1.97 s
7.26 m
7.15 m
7.26 m
7.15 m
1.95 br s
1.68 br s
1.59 br s
1.63 d (1.0)
3.90 s
16.4 CH3
23.3 CH3
15.9 CH3
59.8 CH3
16.2 CH3
23.2 CH3
15.8 CH3
56.1 CH3
60.2 CH3
1.69 br s
1.58 br s
1.62 br s
3.97 s
3.78 s
6.70 br s
n
a Correlations were observed after optimization for JCH ) 7 Hz. Protons showing long-range correlation with indicated carbon.
300 and 500 MHz NMR spectrometers. 1H chemical shifts are
referenced to the residual CDCl3 signal (δ 7.26), and 13C
chemical shifts are referenced to the CDCl3 solvent peak (δ
77.0). Low- and high-resolution EIMS were recorded on a VG-
70SE mass spectrometer. Silica gel plates were used for
analytical thin-layer chromatography. Chromatographic silica
gel (200-425 mesh) was used for normal flash and column
chromatography. All solvents were distilled from glass prior
to use.
Animal Material. The sponge was collected from the
vertical walls of large caves at depths of 10-20 m between
Lehua Rock and the north shore of Niihau Island, Hawaii. The
sponge forms small oval encrustations about 1 cm thick; the
surface is covered in short thick nodules about 2-4 mm high
and wide. The texture is corky and leathery. In life the sponge
is dark chocolate brown, some specimens being somewhat
lighter in color. The sponge has diod spicules in one to two
size categories, about 180-250 µm in length and occasional
triods 110 µm in total length. The sponge is an undescribed
species of Plakortis (order Homosclerophorida, family Pla-
kinidae) closest to P. ceylonica (Dendy, 1905) in the large size
of the diods. A voucher specimen has been deposited in the
Natural History Museum, London (BMNH 2003.9.25.2).
Extraction of Plakortis and Isolation of Lehualides
A-D (1-4). A freeze-dried sample of sponges (58.0 g dry
weight) was immersed in dichloromethane/2-propanol (1:1) at
room temperature. The combined extracts were concentrated
in vacuo. The crude extract was partitioned between hexane
and MeOH/H2O (80:20). The aqueous MeOH layer was sub-
sequently extracted with dichloromethane. The dichloromethane
layer (4.11 g) was subjected to silica gel vacuum liquid
chromatography using stepwise gradient elution (hexane/
EtOAc/CH2Cl2/MeOH). The fraction eluted with 100% hexane
was further fractionated by silica gel gravity column chroma-
tography [hexane/EtOAc (2:1)] to provide a total of 11 fractions.
Normal-phase HPLC [Phenomenex Sphereclone silica, 10 µm,
250 × 21.2 mm, gradient elution from hexane/2-propanol (95:
5) to 100% 2-propanol in 50 min] of the brine shrimp toxic
fractions (fractions 8-11) provided four new compounds,
Table 2. NMR Spectral Data of Lehualides C (3) and D (4)
Recorded in CDCl3
lehualide C (3)
δC δH (J in Hz)
lehualide D (4)
δC δH (J in Hz)
C/H no.
2
159.8 qC
127.4 qC
176.7 qC
119.8 qC
156.8 qC
159.8 qC
127.4 qC
176.8 qC
119.8 qC
156.9 qC
3
4
5
6
7
30.0 CH2 3.27 d (7.2)
30.0 CH2 3.27 d (6.9)
8
116.4 CH 5.17 t (7.2, 1.2) 116.4 CH 5.17 t (6.9, 1.0)
9
139.6 qC
39.5 CH2 2.00 t (7.2)
139.6 qC
39.5 CH2 2.05 t (7.1)
10
11
12
13
14
15
16
17
18
19
20
OMe-1
OMe-2
SAc
27.8 CH2 1.33 quint (7.2) 27.8 CH2 1.34 quint (7.1)
29.4 CH2 1.25 m
29.4 CH2 1.25 m
29.4 CH2 1.25 m
29.4 CH2 1.25 m
29.4 CH2 1.25 m
29.5 CH2 1.59 m
29.1 CH2 2.85 t (7.2)
9.7 CH3 1.95 s
16.2 CH3 1.70 d (1.2)
56.3 CH3 3.90 s
60.3 CH3 3.79 s
196.1 CH3 2.32 s
30.6 CH3
29.4 CH2 1.24 m
29.4 CH2 1.24 m
29.4 CH2 1.24 m
29.4 CH2 1.24 m
29.4 CH2 1.24 m
29.2 CH2 1.66 m
39.0 CH2 2.66 t 7.5
9.7 CH3 1.95 s
16.2 CH2 1.69 d 1.0
56.3 CH3 3.99 s
60.3 CH3 3.78 s
0.83 µM. Lehualide D (4) exhibited moderate cytotoxicity
to ovarian (IGROV-ET) and leukemia (K562) cell lines with
GI50 values of 0.73 and 0.23 µM, respectively. Interestingly,
lehualides A (1) and C (3) did not show significant levels
of cytotoxicity against any of the cancer cell lines.
Experimental Section
General Experimental Procedures. Ultraviolet spectra
were recorded on a Hewlett-Packard 8452A diode array
spectrometer. IR spectra were recorded on a Perkin-Elmer
1600 FTIR. The 1H and 13C NMR spectra were recorded on

Notes
Journal of Natural Products, 2005, Vol. 68, No. 9 1403
lehualides A (1, 26.4 mg), B (2, 28.7 mg), C (3, 10.6 mg), and
D (4, 10.8 mg).
s (OAc); positive ion EIMS (M+) m/z 464.2590 (calcd for
C29H36O5, ∆ -2.7 mmu).
Preparation of Lehualide A Acetate (5). Lehualide A
(1, 4.1 mg) was treated with acetic anhydride (100 µL) and
pyridine (100 µL) and stirred at room temperature for 16 h to
yield 2.2 mg of lehualide A acetate (5).
Acknowledgment. We thank B. Baker and C. Cabral for
their assistance in collecting the biological material. We also
thank M. Burger (Department of Chemistry, University of
Hawaii) for recording mass spectral data. We are also grateful
to Pharmamar S.A. for the cytotoxicity bioassays and financial
support. The upgrade on the 500 MHz NMR spectrometer used
in this research was funded by grants from CRIF Program of
the National Science Foundation (CHE9974921). F.D.H. ac-
knowledges support from NCRR/NIH grant 2P20RR016467.
Lehualide A (1): clear oil; UV (MeOH) λmax (log ꢀ) 242
(3.77), 292 (3.63) nm; IR (neat) νmax 3300, 2927, 1720, 1687,
1650, 1439, 1093 cm-1 1H and 13C NMR data, see Table 1;
;
HREIMS (M+) m/z 422.2461 (calcd for C27H34O4, ∆ -0.4 mmu).
Lehualide B (2): clear oil; UV max (MeOH) λmax (log ꢀ)
292 (3.92) nm; IR (neat) νmax 2927, 1655, 1453, 1377, 1331,
1265, 1154, 1029 cm-1 1H and 13C NMR data, see Table 1;
;
HREIMS (M+) m/z 436.2640 (calcd for C28H36O4, ∆ -2.6 mmu).
Lehualide C (3): clear oil; UV (MeOH) λmax nm (log ꢀ) 240
(3.92) nm; IR νmax (film) 2927, 1690, 1657, 1465, 1335, 1156
cm-1; 1H and 13C NMR data, see Tables 2; HREIMS (M+) m/z
424.2266 (calcd for C23H36O5S, ∆ 1.7 mmu).
References and Notes
(1) (a) Stierle, D. B.; Faulkner, D. J. J. Org. Chem. 1980, 45, 3396-
3401. (b) Gunasekera, S. P.; Gunasekera, M.; Gunawardana, G.;
McCarthy, P.; Burres, N. J. Nat. Prod. 1990, 53, 669-674. (c)
Compagnone, R. C.; Pin˜a, I. C.; Rangel, H. R.; Dagger, F.; Sua´rez, A.
I.; Reddy, M. V. R.; Faulkner, D. J. Tetrahedron 1998, 54, 3057-
3068. (d) Rudi, A.; Kashman, Y. J. Nat. Prod. 1993, 56, 1827-1830.
(e) Fontana, A.; Ishibashi, M.; Kobayashi, J. Tetrahedron 1998, 54,
2041-2048.
Lehualide D (4): clear oil; UV (MeOH) λmax (log ꢀ) 252 (3.92)
nm; IR νmax (film) 2925, 1659, 1464, 1378, 1333, 1266, 1156,
1030 cm-1; 1H and 13C NMR data, see Tables 2; HREIMS (M+)
m/z 382.2190 (calcd for C21H34O4S, ∆ -1.2 mmu).
(2) Costantino, V.; Fattorusso, E.; Mangoni, A.; Di Rosa, M.; Ianaro, A.
J. Am. Chem. Soc. 1997, 119, 12465-12470.
Lehualide A acetate (5): clear oil; 1H NMR (300 MHz,
CDCl3) 3.20 (d, 2H, J ) 6.6 Hz, H-7), 5.18 (brt, 1H, J ) 6.6
Hz, H-8), 2.00 (m, 2H, H-10), 2.11 (m, 2H, H-11), 5.20 (br t,
1H, J ) 6.3 Hz, H-12), 2.74 (br s, 2H, H-14), 5.36 (tq, 1H, J )
7.2, 1.2 Hz, H-16), 3.37 (d, 2H, J ) 7.2 Hz, H-17), 7.16 (m, 3H,
H-19, 21, 23), 7.27 (m, 2H, H-20, 22), 1.85 (s, 3H, H-24), 1.68
(s, 3H, H-25), 1.59 (s, 3H, H-26), 1.64 (s, 3H, H-27), 3.87 (s,
3H, OMe), 2.34 (s, 3H, OAc); 13C NMR (300 MHz, CDCl3) 160.5
s (C-2), 134.0 s (C-3), 150.7 s (C-4), 107.8 s (C-5), 155.6 s (C-
6), 30.2 t (C-7), 117.2 d (C-8), 138.6 s (C-9), 39.7 t (C-10), 26.3
t (C-11), 125.9 d (C-12), 133.3 s (C-13), 41.8 t (C-14), 134.0 s
(C-15), 124.0 d (C-16), 34.2 t (C-17), 141.7 s (C-18), 128.3 d
(C-19, 20, 22, 23), 125.6 d (C-21), 10.0 q (C-24), 16.4 q (C-25),
23.3 q (C-26), 15.9 q (C-27), 59.2 q (OMe), 20.4 q (OAc), 166.8
(3) Patil, A. D.; Freyer, A. J.; Bean, M. F.; Carte, B. K.; Westley, J. W.;
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