Synthesis of cytotoxic cembranolide analogues via acid-induced opening of oxiranes

Article abstract of DOI:10.1016/S0040-4020(00)01000-0

A large series of analogues of Eunicea cembranolides 1-3 were synthesized with the purpose of evaluating their cytotoxicity against 60 human cancer cell-lines. Most of the analogues were as active as the lead compounds and a few displayed increased cytotoxicity. Their syntheses were based on the specific reactivity and stereochemistry of the epoxide substructure and involved highly regio- and diastereoselective acid-induced chemical transformations.

Full text of DOI:10.1016/S0040-4020(00)01000-0

TETRAHEDRON
Pergamon
Tetrahedron 57 (2001) 93±107
Synthesis of cytotoxic cembranolide analogues via acid-induced
opening of oxiranesq
a,
Abimael D. Rodrõguez, Ivette C. PinÄa, Ana L. Acosta and Charles L. Barnes
a,²
a,³
b
*
Â
aDepartment of Chemistry, University of Puerto Rico, P.O. Box 23346, U.P.R. Station, San Juan, 00931-3346, Puerto Rico
bDepartment of Chemistry, 123 Chemistry Building, University of Missouri-Columbia, Columbia, MO 65211, USA
Received 17 August 2000; accepted 24 October 2000
AbstractÐA large series of analogues of Eunicea cembranolides 1±3 were synthesized with the purpose of evaluating their cytotoxicity
against 60 human cancer cell-lines. Most of the analogues were as active as the lead compounds and a few displayed increased cytotoxicity.
Their syntheses were based on the speci®c reactivity and stereochemistry of the epoxide substructure and involved highly regio- and
diastereoselective acid-induced chemical transformations. q 2000 Elsevier Science Ltd. All rights reserved.
1. Introduction
lute con®gurations of the synthetic analogues are as
depicted. Finally, the molecular structures of cembranolide
analogues 13, 17, and 32 were con®rmed by single-crystal
X-ray crystallography.
Gorgonian cembranolides belonging to the genus Eunicea
are easily accessible and contain highly reactive functions
which make them suitable templates for the synthesis of
analogues possessing uncommon structural features and
interesting biological properties.1,2 Naturally occurring
Eunicea cembranoids such as euniolide (1),3 12,13-bi-
sepieupalmerin (2),4 and eupalmerin (3)5 have shown strong
in vitro antitumor activity (Fig. 1). We have synthesized a
large series of unusual analogues of cembranolides 1±3 with
the purpose of evaluating their cytotoxicity against 60
human cancer cell-lines. Our studies with a-methylene-g-
lactones 1±3 aimed at establishing the feasibility of obtain-
ing unusual cembranoid systems of increased cytotoxicity
using simple functional group interconversions in concise
fashion. The complete structural assignment of all the
synthetic cembranoid analogues described in the present
work was accomplished on the basis of comprehensive 1D
and 2D NMR experiments involving 1H±1H COSY, DEPT,
NOESY, 1H±13C COSY (CSCMBB), selective INEPT,
HMQC and HMBC measurements. In most cases, these
2D NMR spectra provided both the structure and the
complete and unambiguous proton and carbon atom assign-
ments. Since starting materials 1±3 were optically pure
without ambiguity in the absolute con®guration, the abso-
2. Results and discussion
2.1. Transformations of euniolide (1)
This abundant compound is generally regarded as the
biosynthetic precursor of cembranolides 2 and 3.2 Our ®rst
attempt at structural modi®cation of euniolide (1) was to
rearrange the epoxide functionality to form a carbonyl
group at the C-3 position (Eq. (1)). To arrive at ketone 4
advantage was taken of the known chemoselectivity with
which a C-3,4 epoxide function such as that of 1 reacts with
various Lewis acid promoters to give predominantly the
C-4(R) diastereomer.6 Thus, treating euniolide (1) with
phosphorus pentoxide in re¯uxing benzene for 1 h delivered
a 37:22:1 mixture of ketones 4(R)-euniolone (4), 4(S)-
euniolone (5), and lactone 6 in 54% overall yield. Surpris-
ingly, under these reaction conditions the C-7,8 double bond
isomerized in lactone 6 giving the (Z)-D7 ole®n in 1%
isolated yield. These reaction conditions also led to a
large amount of an intractable mixture of highly polar
compounds accounting for the remainder of the starting
material 1. A combination of coupling constant analysis
and NOESY spectra established the relative stereochemistry
of 4(R)-euniolone as shown in structure 4, which showed
that it had the same relative stereochemistry as 5 at all
centers except at C-4. The large coupling (11.7 Hz)
observed between H-1 (dH 3.62) and H-2 (dH 2.51) suggests
that they are anti-parallel; therefore H-2 is b-oriented in 4.
In addition to producing NOEs at H-5 (dH 1.51) and H-50
(dH 1.85), Me-18 (dH 0.94) produced NOEs at H-2 also.
q Taken in part from the graduation theses of I. C. PinÄa (1995) and A. L.
Acosta (1997).
Keywords: coelenterates; epoxides; ethers; cytotoxins.
* Corresponding author. Tel.: 1787-764-0000 Ext. 4799; fax: 1787-751-
0625; e-mail: arodrig@goliath.cnnet.clu.edu
²
Visiting graduate student from the Universidad Central de Venezuela,
Caracas.
Graduate student sponsored by the NIH-MBRS and NSF-MRCE
Programs of the University of Puerto Rico.
³
0040±4020/01/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)01000-0

Â
A. D. Rodrõguez et al. / Tetrahedron 57 (2001) 93±107
94
Figure 1. Absolute structures of euniolide (1), 12,13-bisepieupalmerin (2), and eupalmerin (3).
This NOE between Me-18 and H-2 indicates that the Me-18
is on the same side as H-2, making the former b-oriented in
4. The NOESY spectrum of 4 also showed key correlations
between H-1/H-11, H-1/H-14, H-1/H-17, H-20/H-11, H-11/
H-13, and H-14/Me-20. From the foregoing discussion,
4(R)-euniolone has the relative stereochemistry depicted
in 4. An NOE between H-7 and Me-19 in lactone 6 estab-
lished that the D7 double bond has (Z)-geometry. When the
epoxide ring opening in 1 was tried with phosphorus pent-
oxide in the presence of triphenylphosphine the formation of
ketones 4±6 was completely suppressed, but the reaction
could not be driven to completion (Eq. (2)). In this way,
an easily separable 1.5:1 mixture of tetraene alcohols 7 and
8, respectively, was obtained in 37% yield. The HREI-MS,
IR, and NMR spectral data of 7 and 8 indicated that these
compounds possess similar molecular formula and function-
ality. The 13C NMR spectrum of 7, which also revealed the
lack of an epoxide moiety, displayed signals for three
methyl groups with the methyl resonance ascribed to
Me-18 occurring down®eld of 20 ppm. This indicated the
(Z)-con®guration of the D4 trisubstituted double bond.
Signals for only two methyl groups were observed in the
13C NMR spectrum of 8. The ole®nic methylene carbon
resonating at 112.2 ppm correlated to the two broad singlets
at d 4.99 and 4.94 assigned as the C-18 exo-methylene. The
relative stereochemistry of C-3 in alcohols 7 and 8 was
again determined with the aid of NOESY spectra. The
doublet of doublet absorbed at d 4.05 in 7 correlated to
the oxygenated methine carbon at 71.2 ppm and thus was
assigned as H-3. The strong NOESY correlation between
H-3 and the Me-18 group suggested a similar relative
stereochemistry for C-3 as euniolide (1). Likewise, the
doublet of doublet resonating at d 4.20, which was assigned
as the C-3 oxymethine in 8, showed a strong NOESY corre-
lation with one of the C-17 (dH 5.69) and one of the C-18
(dH 4.94) exo-methylene protons which suggested the same
relative stereochemistry at that position as 7. A more
promising result was achieved when 1 was treated with
BF3´O(C2H5)2 in benzene at 08C for 1.5 h. Under these
conditions, the desired euniolones 4 and 5 were formed in
reasonable yield (62%) as a 3:1 mixture, respectively.
Exposure of 1 to a mixture of triphenylphosphine and iodine
in CH2Cl2 at rt led to a complex product mixture from
which the main product (1S,3E,7E,11E,14S)-cembra-
3,7,11, 15(17)-tetraen-16,14-olide (9) could be isolated in
modest yield (33%), (Eq. (3)). The HREI-MS data for 9
suggested the molecular formula C20H28O2 and seven
degrees of unsaturation. The IR and NMR spectra indicated
the lack of hydroxyl and epoxy functionalities. On the other
1
hand, the H and 13C NMR showed the presence of three
methyl substituted trisubstituted double bonds. In the 13C
NMR spectrum these methyl resonances occur up®eld of
20 ppm indicating the (E)-con®guration of the trisubstituted
double bonds. These conditions also led to the formation of
lactones 10±12 in 18, 15, and 6% isolated yields, respec-
tively.
Further experimentation revealed that the introduction of
CHCl3 as co-solvent, after 4.5 h at 08C, suppressed the
formation of tetraene 9 giving instead a 7:1:1:1.5:1:1
mixture of products 4, 7, 8, and 12±14 in 81% overall
yield (Eq. (4)).
The formation of an oxa bridge function at the C-4/C-8
position of 10, 11, and 13 and at the C-4/C-7 position of

Â
A. D. Rodrõguez et al. / Tetrahedron 57 (2001) 93±107
95
Figure 2. Crystalline structure of 4, 8-oxabridged cembranolide 13.
ꢀ3†
1
product 14 was a pleasant surprise. Another surprise was
that certain analogues (i.e. 10 and 12±14) contained iodine.
The formation of the bridged ether cores of 13 and 14 must
proceed via epoxide ring opening in 1 upon nucleophilic
attack at C-3 with inversion of con®guration of an iodide
anion to give iodohydrin 12. The latter undergoes con-
version to diiodides 13 and 14 via intramolecular nucleo-
philic attack, from the a and b face, respectively, of the C-4
hydroxyl group on an activated D7 ole®n at either position.
Consecutive C±I bond reductions of 13 should furnish pyra-
nethers 10 and 11, respectively. While these conversions are
likely to involve solvent deprotonations by free radical
intermediates the mechanistic details of the conversions
1!12!13!10!11 were not probed further. The structural
assignments for 9±14 rest ®rmly on spectroscopic grounds
particularly informative (Fig. 2).7 Unfortunately, the H
NMR spectrum of 11 in CDCl3 solution gave signals with
co-incident chemical shift at d 1.13 (s, 6H) for Me-18 and
Me-19, thus the relative stereochemistry at C-4 and C-8 of
pyranethers 10 and 11 was established from the 2D-NOE
spectrum of 10. This experiment, which revealed the Me-18/
Me-19 connectivity, indicated that these methyl groups had
the same b-orientation as in cembranolide analogue 13.
2.2. Transformations of 12,13-bisepieupalmerin (2)
Conversion of 2 to diepoxide 15 was accomplished in 81%
isolated yield with m-CPBA in benzene at 258C for 1.5 h
(Eq. (5)). The oxidation proceeded to completion with no
traces of the alternate C-7(R),8(R) diastereomer detected.
Cembranolide 15, having the 7(S),8(S) con®guration, was
identical in all respects to the natural product 12,13-
bisepieupalmerin epoxide isolated previously from the
1
(1H and 13C/DEPT, H±1H COSY, CSCMBB, 2D-NOE,
selective INEPT, and HREI-MS studies) with the X-ray
crystallographic analysis of tricyclic lactone 13 proving
ꢀ4†

Â
A. D. Rodrõguez et al. / Tetrahedron 57 (2001) 93±107
96
Figure 3. ORTEP drawing of inolide-A (17).
gorgonian E. succinea.8 It should be noted that the peracid
oxidation of 2 in CH2Cl2 at 258C for 3 h produced roughly a
2:1 mixture of diepoxides the major one of which, after
separation by HPLC, was identical to the natural product.
Following epoxidation to generate 15, the fate of this diep-
oxide in strongly acidic media was explored. Thus, exposure
of 2 to m-CPBA in hot benzene, followed by treatment with
p-toluenesulfonic acid hydrate (PTSA´H2O) for 1 h, gave a
complex product mixture from which only pyranether 16
could be isolated (Eq. (6)). In this instance, protic acid-
catalyzed rearrangement of the epoxide groups followed
by intramolecular ring closure and concomitant loss of
water generated the 2,3-dihydro-4H-pyran moiety. While
we were unable to isolate additional diastereomers of 16,
their formation is highly probable and may explain why the
isolated yield of this reaction amounted to only 24%. In
agreement with the proposed structure, the HREI-MS data
for 16 suggested the molecular formula C20H28O4 and seven
the presence of three secondary methyl groups at d 1.09,
0.98, and 0.95 (each d, 3H) and a high®eld-shifted ole®nic
proton [d 4.85 (d, 1H, Jˆ4.5 Hz, H-2)] which, according to
a 1H±13C HETCOR experiment, correlated with the carbon
resonance at d 93.2 (d, C-2). The 13C NMR spectrum
revealed the presence of ketone and lactone carbonyls at d
215.8 (s) and 169.9 (s), respectively, and a down®eld-
shifted quaternary ole®nic carbon bearing oxygen [d
162.1 (s, C-3)] that showed through selective INEPT
1
NMR experiments two- and three-bond H±13C couplings
to H-2 and Me-18. The latter data suggested that C-3 partici-
pated in the formation of an ethereal linkage. Unfortunately,
the absence of signi®cant NOEs at Me-18 and Me-19 (or
H-4 and H-8) in pyranether 16 precluded our assignment of
the relative stereochemistry at C-4 and C-8.
In accord with our previous results, treatment of 12,13-bise-
pieupalmerin (2) with iodine in CH2Cl2 at 258C for
1 h yielded a complex mixture of products from which
1
degrees of unsaturation. The H NMR spectrum indicated

Â
A. D. Rodrõguez et al. / Tetrahedron 57 (2001) 93±107
97
tetracyclic furanethers inolide-A (17) and inolide-B (18)
during these I2-mediated cyclizations. On the other hand,
the formation of diiodide 19 is fully compatible with the
corresponding reaction in the euniolide series (1!14).
were isolated in 7 and 3% yields, respectively (Eq. (7)).9
Exposure of 2 to perchloric acid in chloroform at 258C for
2 h led, after acidolytic cleavage of the epoxide, to complex
product mixtures (Eq. (8)) from which inolide-A (17) was
obtained as the major product but in slightly higher isolated
yield (20%). The separation of cembranolides 20±25 was
achieved successfully by silica gel column chromatography
followed by normal-phase HPLC. As in the case of our
previous reactions with iodine, cyclic ether formation was
the chief reaction pathway here, but in sharp contrast, ole®n
isomerization was a major reaction pathway as well. The
spectroscopic properties for analogue 20 were identical with
those reported for eunicin, a major secondary metabolite
isolated from E. succinea.10 Additional information on the
relative position of the trisubstituted double bonds in
lactones 21±24 were obtained from HMQC and HMBC
NMR experiments. For instance, in (Z)-D7 eunicin (24),
ꢀ7†
This time, however, tricyclic diiodide 19 was also isolated
in 5% yield. The HREI-MS and NMR data of 19 indicated
similar functionality as furanether 14. The down®eld-
shifted chemical shifts of carbons C-3 (d 42.5, d) and C-8
(d 52.7, s) in the 13C NMR spectrum suggested the location
of the iodine atoms. NOEs were detected in CDCl3 solution
between H-7, H-3, and H-1 of furanether 19, which indi-
cated that these protons were all on the same (a) face of the
molecule. The structure of inolide-A (17) including its abso-
lute con®guration was con®rmed by X-ray crystallography
and is presented in Fig. 3. Interestingly, in the 13C NMR
spectra of cyclic ethers 18 and 19, many of the resonances
were quite broad signals of very low intensity. This
peculiarity, which was not observed in inolide-A (17), is a
consequence of slowly interconverting conformers. In
1
the methylene protons H-5 (dH 1.60) showing J-HMQC
correlations with the methylene carbon resonating at
3
41.6 ppm (C-5) displayed J-HMBC correlations with the
ole®n carbon resonated at 125.4 ppm (d, C-7). In turn, C-5
correlated to the C-18 methyl group absorbed at d 1.14. This
was further supported by the HMBC correlation of the C-3
proton signal (dH 3.46) with C-5. The Me-19 signals in the
13C NMR spectra of cembranolide analogues 22±24 (d 26.0,
25.4, and 23.8, respectively) were at low ®eld which indi-
cated that the trisubstituted double bonds are cis with
respect to the continuous chain of carbons. The Me-19
signal of (E)-D8 eunicin (21), on the other hand, was at
high ®eld (d 17.3) indicating that the D8 ole®n has the
(E)-con®guration.
1
comparison to 17, the most obvious difference in the H
NMR spectrum of 18 (in CDCl3 solution) was that the signal
for H-7 had shifted from d 3.74 in inolide-A (17) to d 4.12
in inolide-B (18). In view of these observations we conclude
that the C-7(S) con®guration we reported earlier for inolide-
B should be revised to the C-7(R) con®guration shown in
18.9 In sharp contrast to our previous ®ndings with euniolide
(1), none of the corresponding C-4,8 pyranethers are formed
The cyclization of 2 also proceeds with phosphorus pent-
oxide, as reported in our earlier work,9 but the current pro-
cedure furnishes a larger diversity of cembranolide
analogues albeit in slightly lower overall yield (71%),
(Eq. (9)). Besides the cyclization substrates 20, 24, and 25
ꢀ8†

Â
A. D. Rodrõguez et al. / Tetrahedron 57 (2001) 93±107
98
Figure 4. The X-ray structure of cembranolide 32.
ꢀ9†
which we had obtained previously, this reaction also
delivered lower yields of cyclized materials 26 and 27. As
was the case for eunicin (20), the spectroscopic properties
for analogue 26 were identical with those reported for
jeunicin, a minor secondary metabolite isolated from E.
succinea.11 The physical and spectral data of 27 were some-
what reminiscent of those of pyranether 25. The HREI-MS
spectral data for 27 suggested the molecular formula
C20H30O5 and six degrees of unsaturation. The 13C NMR
spectrum exhibited key signals at d 173.8 (s, C-16), 163.9
(s, C-1), 123.2 (s, C-15), 77.8 (d, C-14), and 54.9 (t, C-17)
whose chemical shift values and multiplicity con®rmed the
presence of a butenolide moiety bearing an a-hydroxyl-
methyl group.
C-12,14 bisepimer of (13aH, 14bH)-jeunicin isolated
from specimens of E. succinea collected in Bimini.13 The
relative position of the trisubstituted double bond in each of
these analogues was established from extensive NMR spec-
tral comparisons with cembranolide analogues 20±24. The
Me-19 signal of cyclic ethers 28 and 29 was at high ®eld
(each at d 15.6) whereas that of 30 and 31 resonated at low
®eld (d 24.2 and 23.7, respectively). This indicated that the
trisubstituted ole®n in each pair of isomers had the (E)- and
(Z)-con®guration, respectively. The proton on the oxy-
genated carbon C-3 of 28, 30 and 31 did not produce
NOESY correlations with the neighboring Me-18 a-methyl
group nor the H-13 oxymethine signal indicating the
b-orientation of the former proton.
The epoxide ring opening in eupalmerin (3) was likewise
affected with PTSA´H2O in benzene at 258C for 6 h, giving a
16:1:1:1 mixture of 12-epicueunicin (28) and a-methylene
lactones 32±34 in 42% overall yield (Eq. (11)). By using
PTSA´H2O the formation of cembranolide analogues
containing the oxepane ring is completely suppressed and
pyranethers such as 28 and 32 thrive as the predominant
cyclized products. The HREI-MS and IR data of 32
indicated similar molecular formula and functionality as
bisepimer 25. This was further supported by the NMR
data that displayed signals for only two methyl groups and
suggested the lack of epoxide functionality. The ole®nic
methylene carbon resonating at 113.5 ppm correlated to
the two broad singlets at d 4.94 and 4.86 assigned as the
2.3. Transformations of eupalmerin (3)
Analogues of eupalmerin (3), which has the same consti-
tution as 12,13-bisepieupalmerin (2) but opposite stereo-
chemistry at C-12 and C-13, were prepared next.
Treatment of eupalmerin (3) with phosphorus pentoxide in
benzene at 258C for 12 h afforded a 3:1:2:2 mixture of cyclic
ethers 28±31 in 61% overall yield favoring the pyranethers
in a 7:1 ratio (Eq. (10)). The HREI-MS, NMR, and IR data
of analogues 28±31 indicated similar molecular formula
and functionality as 20±24. Compound 28 was shown to
be the C-12 epimer of cueunicin,12 a minor secondary
metabolite isolated from E. succinea from CuracËao, whereas
12,13-bisepijeunicin (29) was likewise identi®ed as the

Â
A. D. Rodrõguez et al. / Tetrahedron 57 (2001) 93±107
99

Â
A. D. Rodrõguez et al. / Tetrahedron 57 (2001) 93±107
100
ꢀ10†
C-19 exo-methylene. This was con®rmed through the
2J-HMBC coupling of the C-19 methylene protons with
the quaternary ole®nic carbon at 148.0 ppm.
methylene carbons [d 37.6 (C-9), 36.6 (C-5), 29.4 (C-2),
29.2 (C-11), 23.3 (C-10), and 22.3 (C-6)], and three methyl
groups [d 25.8 (C-18), 16.5 (C-19), and 15.7 (C-20)]. Most
likely, its formation entails tandem acid-mediated ester
hydrolysis and nucleophilic attack at C-3 by the C-14
hydroxyl followed by lactonization of the ensuing
d-hydroxy carboxylic acid to give the d-lactone array.
The stereochemistry of cembranolide 32 was con®rmed by
X-ray analysis and is shown in Fig. 4. Interestingly, acid-
promoted rearrangement of the epoxide produced allylic
alcohol 33 as the only isomer of this class. The IR and
NMR data of 33 indicated the lack of epoxide functionality
and displayed signals for a similar functionality as alcohol 8,
namely, hydroxyl and a nearby exo-methylene group. The
ole®nic methylene carbon at 113.9 ppm correlated to a two
proton broad singlet at d 4.95 assigned as the C-18 exo-
methylene. The C-18 methylene protons also showed
2.4. Biological studies
The compounds listed in Table 1 were tested in the NCI's 60
cell-line human tumor screen. The results with 14 repre-
sentative cell-lines (CCRF-CEM, MOLT-4, PC-3,
DU-145, LOX-IMV1, MALME-3M, MCF7, MCF7 ADR-
RES, SNB-75, IGROV1, OVCAR-3, HCT-116, COLO 205,
and NCI-H522) are shown. In comparative testing against
these human tumor cell-lines, euniolide (1) and 12,13-bi-
sepieupalmerin (2) exhibited IC50 values of 0.1±43 mg/mL.
The acetate derivative of eupalmerin (3) exhibited IC50
values from 0.3±16 mg/mL. A cursory run through Table
1 indicates that almost all of the synthetic analogues are
strongly cytotoxic suggesting that chemical modi®cations
involving the epoxide substructure of cembranolides 1±3
are not generally deleterious to their cytotoxicity. The
notable exception to this is 27, which does not feature an
a-methylene-g-lactone functionality, and thus is less toxic
than its prototype in this bioassay. Moreover, several ana-
logues (13, 17, and 19) showed a characteristic pattern of
differential cytotoxicity and were approximately equipotent
(e.g. mean panel GI50'st1±2 nM). COMPARE pattern-
recognition analyses of the mean-graph pro®les of these
3
2J-HMBC and J-HMBC correlations with the quaternary
ole®nic carbon at 152.4 ppm (C-4) and the oxygenated
methine carbon at 71.2 ppm (C-3), respectively. The rela-
tive stereochemistry of C-3 in 33 was assigned similar to
that of 8 on the basis of a comparison of the splitting and
coupling of H-3. A most intriguing ®nding of the present
investigation was the isolation, albeit in rather low isolated
yield (2%), of known eupalmerolide (34).14 This structurally
complex analogue analyzed for C20H30O4 and showed bands
for a-methylene-d-lactone and hydroxyl groups in its IR
spectrum. The 13C NMR spectrum showed the presence of
a lactone carbonyl [d 163.8 (C-16)], one quaternary carbon
bearing oxygen [d 72.9 (C-4)], three oxymethine carbons [d
88.4 (C-3), 84.1 (C-13), and 78.7 (C-14)], two double
bonds: one trisubstituted [d 133.3 (s, C-8), 128.4 (d, C-7)]
and one terminal [d 136.9 (s, C-15), 126.0 (t, C-17)], two
methine carbons [d 40.7 (C-1) and 35.9 (C-12)], six
ꢀ11†

Â
A. D. Rodrõguez et al. / Tetrahedron 57 (2001) 93±107
101
analogues did not reveal any signi®cant correlations to the
pro®les of reference antitumor compounds contained in the
NCI's standard agent database.15 This suggests that these
modi®ed cembranolide analogues may act by a novel
mechanism of action since their activity pro®les did not
match compounds whose mechanism of action are known.
Among the 12,13-bisepieupalmerin (2) series, for instance,
a signi®cantly enhanced cytotoxic activity was generally
obtained by the introduction of cyclic ether linkages across
the cembrane skeleton. Thus, compound 17 is up to 15 times
more cytotoxic against the T-cell leukemia (CCRF-CEM)
cells than prototype 2 (compounds 2 and 17, IC50'sˆ0.15
and 0.01 mg/mL, respectively). Furthermore, lactone diio-
dide 19, having undergone preliminary evaluation in the
NCI's in vitro antitumor screening system, has been
selected by the Biological Evaluation Committee for further
in vivo anticancer studies which begin with the Hollow
Fiber Assay.16 Considering these early observations, ana-
logues of this series appear to be attractive targets for the
development of antitumor drugs. While further pharmaco-
logical evaluation of this series of compounds in relevant in
vitro tests is still in progress at this stage, already some
active analogues have displayed more potency than their
precursors.
large silica gel column (3 kg) using 30% EtOAc in hexane.
From this column 14 fractions were obtained the less polar
of which consisted of complex mixtures of unidenti®ed
sterols and fatty acid derivatives, and the known cembrano-
lide diterpene euniolide (1) (24 g). The more polar portion
of the extract was divided roughly into fractions 6±14 on the
basis of TLC analyses. From some of these fractions several
known cembranolides such as 12,13-bisepieupalmerin (2)
(5.0 g) and eupalmerin (3) (2.5 g) were identi®ed. The
identi®cation of 1±3 was accomplished through detailed
comparisons with the physical and chemical data previously
reported for these compounds.3±5 A similar extraction
procedure was followed with dried E. mammosa specimens.
3.3. Reaction of euniolide (1) with phosphorus pentoxide
3.3.1. Path A. A mixture of 1 (863 mg, 2.73 mmol) and
P2O5 (442 mg, 3.11 mmol) in dry benzene (50 mL) was
re¯uxed gently for 1 h before it was quenched with saturated
NaHCO3 (50 mL). The reaction mixture was extracted with
benzene (3£50 mL), concentrated, and puri®ed by column
chromatography on silica gel (22 g, 1:9 (v/v) ethyl acetate in
hexane) to give 4(R)-euniolone (4, 294 mg, 34%), 4(S)-
euniolone (5, 173 mg, 20%), lactone 6 (8 mg, 1%), and
370 mg of an intractable mixture of polar compounds.
3.3.2. Data for 4. White crystalline solid; [a]2D5ˆ221.78 (c
3. Experimental
4.4, CHCl3); IR (neat) 2967, 2929, 2858, 1755, 1703, 1665,
;
1392, 1375, 1273, 1247, 1168, 1099, 969, 946, 815 cm21
3.1. General experimental procedures
1
UV (MeOH) lmax 208 nm (e 10900); H NMR (CDCl3,
300 MHz) d 3.62 (ddd, 1H, Jˆ1.8, 6.9, 11.7 Hz, H-1),
2.51 (dd, 1H, Jˆ11.7, 18.6 Hz, H-2), 3.13 (dd, 1H, Jˆ2.1,
18.6 Hz, H-20), 2.49 (m, 1H, H-4), 1.51 (m, 1H, H-5), 1.85
(m, 1H, H-50), 1.91 (m, 1H, H-6), 2.03 (m, 1H, H-60), 4.87
(br t, 1H, Jˆ6.3 Hz, H-7), 2.13 (m, 2H, H-9), 2.16 (m, 2H,
H-10), 5.04 (br t, 1H, Jˆ7.5 Hz, H-11), 2.25 (dd, 1H, Jˆ
12.3, 15.0 Hz, H-13), 2.69 (br d, 1H, Jˆ15.0 Hz, H-130),
4.70 (ddd, 1H, Jˆ4.2, 6.9, 12.3 Hz, H-14), 5.71 (br t, 1H,
Jˆ1.2 Hz, H-17), 6.14 (dd, 1H, Jˆ0.9, 1.8 Hz, H-170), 0.94
(d, 3H, Jˆ6.9 Hz, Me-18), 1.46 (s, 3H, Me-19), 1.64 (s, 3H,
Me-20); 13C NMR (CDCl3, 75 MHz) d 36.1 (d, C-1), 41.9 (t,
C-2), 212.0 (s, C-3), 45.1 (d, C-4), 31.9 (t, C-5), 25.6 (t,
C-6), 124.7 (d, C-7), 135.5 (s, C-8), 39.0 (t, C-9), 24.6 (t,
C-10), 128.5 (d, C-11), 129.2 (s, C-12), 40.3 (t, C-13), 76.6
(d, C-14), 139.7 (s, C-15), 169.9 (s, C-16), 124.2 (t, C-17),
17.4 (q, C-18), 14.3 (q, C-19), 15.9 (q, C-20); HREI-MS m/z
[M]1 calcd for C20H28O3 316.2038, found 316.2034, 316
(3), 301 (1), 298 (3), 197 (5), 121 (5), 107 (14), 84 (78),
67 (14), 49 (100).
Infrared spectra were determined as thin ®lms and were
referenced to polystyrene. 1H NMR, 13C NMR, DEPT,
HMQC, HMBC, 1H±1H COSY, 1H±13C COSY
(CSCMBB), 2D-NOESY, and selective INEPT spectra
1
were recorded either at 300 and 500 MHz for H or at 75
and 125 MHz for 13C. Column chromatography was carried
out with silica gel (35±75 mesh). HPLC was performed
using a 10 mm silica gel Partisil 10 semipreparative column
(9.4 mm£50 cm). Reactions were monitored by TLC on
silica gel plates (0.25 mm) and visualized using UV light
and I2 vapors. Reagents from commercial suppliers were
used as provided. Yields refer to chromatographically and
spectroscopically pure materials.
3.2. Extraction and isolation of Eunicea g-cembranolides
1±3
The Caribbean gorgonian octocorals E. succinea and
E. mammosa were collected at 25 m depth by SCUBA
from Mona Island, Puerto Rico. The gorgonians were stored
at 08C immediately after collection, then were frozen at
2108C upon arrival, freeze-dried, and kept frozen until
extraction. The dried E. succinea (2.5 kg) was blended
with MeOH:CHCl3 (1:1) and after ®ltration the crude
extract was evaporated under vacuum to yield a residue
(322.9 g) which was partitioned between hexane and H2O.
The hexane extract was concentrated to yield 170.9 g of a
dark green oily residue, which was later dissolved in toluene
and ®ltered. The resulting ®ltrate was concentrated
(168.9 g), loaded onto a large size exclusion column (Bio-
Beads SX-3), and eluted with toluene. The combined
terpenoid-rich fractions (TLC guided) were concentrated
to a dark yellow oil (118.6 g) and chromatographed over a
3.3.3. Data for 5. White crystalline solid; [a]2D5ˆ24.18 (c
4.6, CHCl3); IR (neat) 2962, 2929, 2856, 1755, 1693, 1660,
1460, 1365, 1273, 1169, 1005, 946 cm21; UV (MeOH) lmax
207 nm (e 15600); 1H NMR (CDCl3, 300 MHz) d 3.61 (m,
1H, H-1), 2.49 (dd, 1H, Jˆ11.4, 16.8 Hz, H-2), 2.98 (dd,
1H, Jˆ2.7, 16.8 Hz, H-20), 2.43 (m, 1H, H-4), 1.51 (m, 1H,
H-5), 1.70 (m, 1H, H-50), 1.90 (m, 1H, H-6), 2.25 (m, 1H,
H-60), 5.01 (br t, 1H, Jˆ7.5 Hz, H-7), 1.94 (m, 1H, H-9),
2.21 (m, 1H, H-90), 2.25 (m, 2H, H-10), 5.04 (br t, 1H,
Jˆ7.8 Hz, H-11), 2.28 (m, 1H, H-13), 2.68 (br d, 1H,
Jˆ15.0 Hz, H-130), 4.76 (ddd, 1H, Jˆ3.9, 6.9, 11.4 Hz,
H-14), 5.59 (d, 1H, Jˆ1.5 Hz, H-17), 6.16 (d, 1H, Jˆ
1.8 Hz, H-170), 1.01 (d, 3H, Jˆ7.2 Hz, Me-18), 1.54 (s,

Â
A. D. Rodrõguez et al. / Tetrahedron 57 (2001) 93±107
102
3H, Me-19), 1.65 (s, 3H, Me-20); 13C NMR (CDCl3,
75 MHz) d 36.8 (d, C-1), 36.7 (t, C-2), 212.4 (s, C-3),
47.9 (d, C-4), 34.3 (t, C-5), 26.4 (t, C-6), 124.2 (d, C-7),
135.7 (s, C-8), 39.3 (t, C-9), 24.5 (t, C-10), 127.9 (d, C-11),
129.6 (s, C-12), 39.8 (t, C-13), 77.2 (d, C-14), 139.2 (s,
C-15), 169.8 (s, C-16), 123.8 (t, C-17), 17.2 (q, C-18),
15.0 (q, C-19), 16.4 (q, C-20); HREI-MS m/z [M]1 calcd
for C20H28O3 316.2038, found 316.2040, 316 (14), 301 (3),
298 (14), 283 (5), 192 (11), 179 (13), 167 (14), 164 (20), 163
(19), 134 (25), 119 (32), 107 (56), 81 (65), 69 (73), 53 (78),
41 (100).
23.1 (q, C-18), 14.8 (q, C-19), 15.6 (q, C-20); HREI-MS
m/z [M]1 calcd for C20H28O3 316.2038, found 316.2032,
316 (2), 298 (30), 278 (40), 277 (100), 201 (15), 157 (39),
145 (39), 133 (88), 119 (65), 107 (77), 93 (73), 91 (59).
3.3.7. Data for 8. Colorless oil; [a]2D5ˆ20.438 (c 0.74,
CHCl3); IR (neat) 3456, 3008, 2960, 2924, 2853, 1766,
1662, 1437, 1261, 1163, 1093, 1026, 815, 802 cm21; UV
(CHCl3) lmax 246 nm (e 1000); 1H NMR (CDCl3,
300 MHz) d 3.44 (m, 1H, H-1), 1.54 (m, 1H, H-2), 1.94
(m, 1H, H-20), 4.20 (dd, 1H, Jˆ1.8, 10.8 Hz, H-3), 2.08
(m, 1H, H-5), 2.28 (m, 1H, H-50), 2.10 (m, 1H, H-6), 2.25
(m, 1H, H-60), 5.22 (br t, 1H, Jˆ3.9 Hz, H-7), 2.08 (m, 2H,
H-9), 2.13 (m, 1H, H-10), 2.22 (m, 1H, H-100), 5.03 (br t,
1H, Jˆ6.0 Hz, H-11), 2.33 (dd, 1H, Jˆ10.8, 15.3 Hz, H-13),
2.52 (br d, 1H, Jˆ15.3 Hz, H-130), 4.74 (m, 1H, H-14), 5.69
(d, 1H, Jˆ2.1 Hz, H-17), 6.25 (d, 1H, Jˆ2.4 Hz, H-170),
4.94 (br s, 1H, H-18), 4.99 (br s, 1H, H-180), 1.56 (s, 3H,
Me-19), 1.65 (s, 3H, Me-20); 13C NMR (CDCl3, 75 MHz) d
39.6 (d, C-1), 32.9 (t, C-2), 72.5 (d, C-3), 151.6 (s, C-4),
31.6 (t, C-5), 26.8 (t, C-6), 125.2 (d, C-7), 134.9 (s, C-8),
38.8 (t, C-9), 24.7 (t, C-10), 128.9 (d, C-11), 129.7 (s, C-12),
40.8 (t, C-13), 78.4 (d, C-14), 139.3 (s, C-15), 170.4 (s,
C-16), 121.7 (t, C-17), 112.2 (t, C-18), 15.7 (q, C-19),
16.2 (q, C-20); HREI-MS m/z [M]1 calcd for C20H28O3
316.2038, found 316.2049, 316 (2), 278 (6), 277 (14), 221
(12), 149 (28), 111 (30), 97 (46), 81 (60), 69 (95), 57 (100).
3.3.4. Data for 6. Yellowish oil; [a]2D5ˆ250.78 (c 1.3,
CHCl3); IR (neat) 2954, 2925, 2855, 1763, 1709, 1456,
1376, 1261, 1165, 1001, 816, 757 cm21; UV (MeOH)
1
lmax 216 nm (e 12100); H NMR (CDCl3, 300 MHz) d
3.57 (ddd, 1H, Jˆ1.0, 3.9, 7.0 Hz, H-1), 2.54 (dd, 1H,
Jˆ7.1, 11.1 Hz, H-2), 3.01 (dd, 1H, Jˆ1.2, 11.1 Hz,
H-20), 2.46 (m, 1H, H-4), 1.49 (m, 1H, H-5), 1.80 (m, 1H,
H-50), 1.81 (m, 1H, H-6), 1.93 (m, 1H, H-60), 5.15 (br d, 1H,
Jˆ4.4 Hz, H-7), 1.19 (m, 1H, H-9), 2.21 (m, 1H, H-90), 1.94
(m, 1H, H-10), 2.33 (m, 1H, H-100), 5.25 (br d, 1H,
Jˆ6.4 Hz, H-11), 2.44 (m, 1H, H-13), 2.73 (br d, 1H,
Jˆ8.6 Hz, H-130), 4.74 (ddd, 1H, Jˆ2.4, 3.9, 7.1 Hz,
H-14), 5.77 (br t, 1H, Jˆ0.7 Hz, H-17), 6.19 (dd, 1H,
Jˆ0.6, 0.9 Hz, H-170), 1.05 (d, 3H, Jˆ4.3 Hz, Me-18),
1.67 (s, 3H, Me-19), 1.67 (s, 3H, Me-20); 13C NMR
(CDCl3, 75 MHz) d 36.1 (d, C-1), 34.8 (t, C-2), 212.0 (s,
C-3), 48.7 (d, C-4), 34.9 (t, C-5), 26.6 (t, C-6), 125.4 (d,
C-7), 133.6 (s, C-8), 31.0 (t, C-9), 24.2 (t, C-10), 128.6 (d,
C-11), 128.9 (s, C-12), 40.8 (t, C-13), 76.8 (d, C-14), 139.8
(s, C-15), 169.9 (s, C-16), 124.3 (t, C-17), 17.9 (q, C-18),
22.3 (q, C-19), 15.7 (q, C-20); HREI-MS m/z [M]1 calcd for
C20H28O3 316.2038, found 316.2032, 316 (21), 301 (4), 298
(17), 284 (13), 192 (10), 179 (15), 164 (20), 163 (21), 134
(28), 119 (35), 107 (61), 95 (50), 81 (69), 69 (87), 55 (100).
3.4. Reaction of euniolide (1) with BF3´O(C2H5)2
Freshly distilled BF3´O(C2H5)2 (0.2 mL, 1.28 mmol) was
added under N2 to a solution of 1 (368 mg, 1.16 mmol) in
dry benzene (10 mL) kept at 08C. After stirring for 1.5 h at
08C the reaction was quenched with H2O (2 mL) and
concentrated. The residue obtained was puri®ed by silica
gel chromatography (18 g, 1:9 (v/v) ethyl acetate in hexane)
to yield 4 (169 mg, 46% yield) and 5 (59 mg, 16% yield).
3.3.5. Path B. A mixture of 1 (181 mg, 0.57 mmol) and
(Ph)3P (150 mg, 0.57 mmol) in dry benzene (15 mL) was
treated with anhydrous P2O5 (300 mg, 2.1 mmol), allowed
to stir at 808C for 4 h, and concentrated. The residue
obtained was chromatographed on silica gel (25 g, 1:4
(v/v) ethyl acetate in hexane) to give 40 mg (22%) of 7
and 27 mg (15%) of 8.
3.5. Reaction of euniolide (1) with (Ph)3P/I2
3.5.1. Path A. To a solution of Ph3P (247 mg, 0.94 mmol)
and iodine (239 mg, 0.94 mmol) in CH2Cl2 (25 mL) at 258C
was added 1 (298 mg, 0.94 mmol) dissolved in 10 mL of
CH2Cl2, at once. After 20 min at 258C the mixture was
poured over aqueous NaHCO3, extracted with CHCl3
(3£50 mL), and concentrated. The residue was puri®ed by
silica gel chromatography (10 g, 3:7 (v/v) ethyl acetate in
hexane) followed by normal-phase HPLC (Partisil 10,
elution with 1:99 (v/v) 2-propanol in hexane) to give
93 mg (33%) of (1S,3E,7E,11E,14S)-cembra-3,7,11,
15(17)-tetraen-16,14-olide (9), 75 mg (18%) of 10, 45 mg
(15%) of 11, and 25 mg (6%) of 12.
3.3.6. Data for 7. Colorless oil; [a]2D5ˆ21.48 (c 1.7,
CHCl3); IR (neat) 3478, 2962, 2912, 2853, 1761, 1661,
1436, 1261, 1161, 1086, 1019, 802, 757 cm21; UV
(CHCl3) lmax 246 nm (e 1000); 1H NMR (CDCl3,
300 MHz) d 3.31 (m, 1H, H-1), 1.39 (m, 1H, H-2), 2.07
(m, 1H, H-20), 4.05 (dd, 1H, Jˆ2.7, 11.1 Hz, H-3), 5.54
(br t, 1H, Jˆ9.3 Hz, H-5), 2.39 (m, 1H, H-6), 3.64 (m,
1H, H-60), 5.06 (br d, 1H, Jˆ9.6 Hz, H-7), 2.12 (m, 2H,
H-9), 2.03 (m, 1H, H-10), 2.35 (m, 1H, H-100), 5.12 (br d,
1H, Jˆ8.7 Hz, H-11), 2.38 (m, 1H, H-13), 2.67 (br d, 1H,
Jˆ15.3 Hz, H-130), 4.71 (ddd, 1H, Jˆ4.2, 6.3, 11.1 Hz,
H-14), 5.71 (br s, 1H, H-17), 6.26 (br s, 1H, H-170), 1.59
(s, 3H, Me-18), 1.55 (s, 3H, Me-19), 1.65 (s, 3H, Me-20);
13C NMR (CDCl3, 75 MHz) d 39.0 (d, C-1), 30.6 (t, C-2),
71.2 (d, C-3), 128.4 (s, C-4), 126.1 (d, C-5), 26.2 (t, C-6),
125.1 (d, C-7), 138.7 (s, C-8), 38.6 (t, C-9), 24.4 (t, C-10),
129.7 (d, C-11), 133.1 (s, C-12), 39.7 (t, C-13), 78.2 (d,
C-14), 138.8 (s, C-15), 170.4 (s, C-16), 122.6 (t, C-17),
3.5.2. Data for 9. Colorless oil; [a]2D5ˆ131.48 (c 8.5,
CHCl3); IR (neat) 2961, 2925, 2852, 1768, 1666, 1441,
1329, 1265, 1158, 1100, 983, 936, 813 cm21; UV (MeOH)
1
lmax 208 nm; H NMR (CDCl3, 300 MHz) d 3.09 (m, 1H,
H-1), 2.22 (m, 2H, H-2), 4.90 (br t, 1H, Jˆ6.6 Hz, H-3),
1.97 (m, 1H, H-5), 2.13 (m, 1H, H-50), 2.12 (m, 2H, H-6),
4.83 (br t, 1H, Jˆ6.0 Hz, H-7), 2.14 (m, 2H, H-9), 2.12 (m,
2H, H-10), 4.99 (br t, 1H, Jˆ6.6 Hz, H-11), 2.24 (m, 1H,
H-13), 2.35 (dd, 1H, Jˆ6.9, 15.6 Hz, H-130), 4.67 (m, 1H,
H-14), 5.53 (d, 1H, Jˆ2.4 Hz, H-17), 6.18 (d, 1H,

Â
A. D. Rodrõguez et al. / Tetrahedron 57 (2001) 93±107
103
Jˆ2.4 Hz, H-170), 1.67 (s, 3H, Me-18), 1.56 (s, 3H, Me-19),
1.59 (s, 3H, Me-20); 13C NMR (CDCl3, 75 MHz) d 45.1 (d,
C-1), 27.8 (t, C-2), 126.4 (d, C-3), 133.3 (s, C-4), 38.8 (t,
C-5), 24.3 (t, C-6), 124.9 (d, C-7), 136.2 (s, C-8), 39.1 (t,
C-9), 24.5 (t, C-10), 121.5 (d, C-11), 130.5 (s, C-12), 39.3 (t,
C-13), 80.6 (d, C-14), 140.4 (s, C-15), 170.3 (s, C-16), 120.0
(t, C-17), 16.7 (q, C-18), 15.8 (q, C-19), 15.5 (q, C-20);
HREI-MS m/z [M]1 calcd for C20H28O2 300.2089, found
300.2094, 300 (5), 285 (17), 192 (10), 147 (16), 135 (18),
121 (35), 108 (34), 107 (45), 93 (55), 81 (51), 68 (100).
(s, 3H, Me-18), 1.73 (s, 3H, Me-19), 1.55 (s, 3H, Me-20);
13C NMR (CDCl3, 75 MHz) d 39.6 (d, C-1), 33.2 (t, C-2),
44.1 (d, C-3), 76.0 (s, C-4), 40.9 (t, C-5), 24.0 (t, C-6), 126.7
(d, C-7), 138.3 (s, C-8), 37.8 (t, C-9), 22.7 (t, C-10), 124.4
(d, C-11), 131.8 (s, C-12), 39.4 (t, C-13), 79.7 (d, C-14),
139.3 (s, C-15), 170.1 (s, C-16), 120.4 (t, C-17), 23.4 (q, C-
18), 15.4 (q, C-19), 15.1 (q, C-20); HREI-MS m/z [M2HI]1
calcd for C20H28O3 316.2038, found 316.2027, 316 (3), 299
(11), 254 (100), 217 (3), 177 (5), 128 (24), 127 (29), 107
(23), 93 (26), 81 (32), 67 (21).
3.5.3. Data for 10. Colorless oil; [a]2D1ˆ240.08 (c 6.8,
CHCl3); IR (neat) 2960, 2934, 2870, 1770, 1669, 1436,
1377, 1292, 1105, 1040, 974 cm21; UV (MeOH) lmax
3.5.6. Path B. To a mixture of iodine (351 mg, 1.38 mmol)
and Ph3P (363 mg, 1.38 mmol) stirred in CH2Cl2 at 08C
(30 mL) for 0.5 h was added dropwise a solution of 1
(0.44 g, 1.38 mmol) in a mixture of 1:1 CH2Cl2/CHCl3
(30 mL) over 5 min. After stirring at 08C for 4.5 h the
reaction mixture was poured over 0.1N NaHSO3 (40 mL),
extracted with CHCl3 (3£30 mL), and concentrated. The
resulting oil was chromatographed on silica gel (20 g,
elution with 15% ethyl acetate in hexane) to provide
93 mg (15%) of 12, 26 mg (6%) and 27 mg (6%), respec-
tively, of alcohols 7 and 8, and 295 mg of a mixture of 4, 13
and 14. Subsequent puri®cation of the mixture by reversed-
phase HPLC (silica gel ODS, elution with 15% water in
MeOH, ¯ow rate 1.5 mL/min, l 220 nm) furnished
185 mg (42%) of ketone 4, 48 mg (6%) of pyranether 13,
and 47 mg (6%) of furanether 14.
1
208 nm; H NMR (C6D6, 300 MHz) d 3.63 (m, 1H, H-1),
3.72 (d, 1H, Jˆ12.6 Hz, H-3), 1.00 (m, 1H, H-5), 2.10 (m,
1H, H-50), 5.14 (br d, 1H, Jˆ9.3 Hz, H-11), 4.81 (m, 1H,
H-14), 5.49 (d, 1H, Jˆ3.3 Hz, H-17), 6.24 (d, 1H,
Jˆ3.3 Hz, H-170), 1.35 (s, 3H, Me-18), 1.17 (s, 3H,
Me-19), 1.67 (s, 3H, Me-20); 13C NMR (CDCl3, 75 MHz)
d 46.9 (d, C-1), 29.4 (t, C-2), 50.5 (d, C-3), 74.8 (s, C-4),
39.4 (t, C-5), 16.6 (t, C-6), 32.3 (t, C-7), 74.6 (s, C-8), 42.8
(t, C-9), 22.8 (t, C-10), 135.3 (d, C-11), 122.4 (s, C-12), 43.4
(t, C-13), 76.5 (d, C-14), 137.9 (s, C-15), 170.2 (s, C-16),
119.6 (t, C-17), 19.5 (q, C-18), 27.9 (q, C-19), 15.5 (q,
C-20); HREI-MS m/z [M]1 calcd for C20H29IO3 444.1117,
found 444.1164, 444 (8), 318 (12), 317 (51), 299 (36), 271
(6), 191 (12), 121 (40), 93 (52), 81 (100), 69 (70).
1
3.5.7. Data for 13. Crystalline solid; H NMR (CDCl3,
3.5.4. Data for 11. White solid; [a]2D4ˆ233.08 (c 1.7,
CHCl3); IR (neat) 2966, 2935, 2866, 1765, 1456, 1373,
1290, 1261, 1120, 1042, 963 cm21; UV (MeOH) lmax
206 nm; 1H NMR (CDCl3, 300 MHz) d 2.96 (m, 1H,
H-1), 1.60 (m, 1H, H-2), 2.05 (m, 1H, H-20), 1.23 (m, 2H,
H-3), 1.10 (m, 1H, H-5), 1.41 (m, 1H, H-50), 1.50 (m, 1H,
H-6), 1.78 (m, 1H, H-60), 1.05 (m, 1H, H-7), 1.46 (m, 1H,
H-70), 1.35 (m, 1H, H-9), 1.51 (m, 1H, H-90), 1.78 (m, 1H,
H-10), 2.20 (m, 1H, H-100), 5.17 (br d, 1H, Jˆ9.3 Hz,
H-11), 2.28 (m, 2H, H-13), 4.82 (m, 1H, H-14), 5.51 (d,
1H, Jˆ2.7 Hz, H-17), 6.21 (d, 1H, Jˆ3.0 Hz, H-170), 1.13
(s, 3H, Me-18), 1.13 (s, 3H, Me-19), 1.67 (s, 3H, Me-20);
13C NMR (CDCl3, 75 MHz) d 45.8 (d, C-1), 19.3 (t, C-2),
43.8 (t, C-3), 72.5 (s, C-4), 32.2 (t, C-5), 16.1 (t, C-6), 37.5
(t, C-7), 71.4 (s, C-8), 42.9 (t, C-9), 23.1 (t, C-10), 134.7 (d,
C-11), 121.8 (s, C-12), 43.2 (t, C-13), 77.9 (d, C-14), 138.8
(s, C-15), 170.8 (s, C-16), 120.2 (t, C-17), 24.1 (q, C-18),
28.4 (q, C-19), 15.5 (q, C-20); HREI-MS m/z [M]1 calcd for
C20H30O3 318.2195, found 318.2204, 318 (5), 300 (5), 285
(3), 217 (4), 147 (9), 121 (22), 95 (35), 81 (48), 69 (40), 55
(48), 43 (100).
300 MHz) d 6.25 (d, 1H, Jˆ3.3 Hz), 5.48 (d, 1H,
Jˆ3.1 Hz), 5.10 (br d, 1H, Jˆ9.6 Hz), 4.80 (dd, 1H,
Jˆ7.3, 11.6 Hz), 4.20 (dd, 1H, Jˆ3.7, 12.9 Hz), 3.68 (d,
1H, Jˆ12.5 Hz), 3.58 (m, 1H), 1.69 (s, 3H), 1.49 (s, 3H),
1.42 (s, 3H); 13C NMR (CDCl3, 75 MHz) d 170.0, 137.6,
134.8, 122.8, 119.9, 77.6, 76.4, 75.4, 48.7, 46.8, 43.6, 43.4,
40.9, 33.0, 30.9, 29.5, 26.0, 22.5, 19.3, 15.5; HREI-MS m/z
[M]1 calcd for C20H28I2O3 570.0119, found 570.0100, 570
(3), 443 (22), 425 (9), 316 (14), 315 (13), 298 (16), 297 (20),
253 (100), 221 (6), 177 (7), 149 (20), 127 (34), 109 (49), 81
(58), 55 (63). The structural assignment to 13 was corrobo-
rated by X-ray crystallographic analysis (Fig. 2).
3.5.8. Data for 14. White solid; [a]2D4ˆ121.18 (c 0.9,
CHCl3); IR (neat) 2952, 2924, 2853, 1770, 1455, 1380,
1296, 1254, 1100, 1074, 1023, 930, 811 cm21; UV
(MeOH) lmax 208 nm (e 26100); 1H NMR (CDCl3,
300 MHz) d 3.56 (m, 1H, H-1), 2.02 (m, 1H, H-2), 2.30
(m, 1H, H-20), 3.80 (d, 1H, Jˆ7.6 Hz, H-3), 2.02 (m, 1H,
H-5), 2.17 (m, 1H, H-50), 1.97 (m, 1H, H-6), 2.13 (m, 1H,
H-60), 4.18 (dd, 1H, Jˆ3.2, 5.3 Hz, H-7), 1.71 (dd, 1H,
Jˆ5.4, 9.2 Hz, H-9), 2.27 (dd, 1H, Jˆ6.1, 9.2 Hz, H-90),
2.12 (m, 1H, H-10), 2.46 (m, 1H, H-100), 5.31 (br d, 1H,
Jˆ5.9 Hz, H-11), 2.34 (m, 2H, H-13), 4.85 (t, 1H,
Jˆ5.4 Hz, H-14), 5.51 (d, 1H, Jˆ1.8 Hz, H-17), 6.27 (d,
1H, Jˆ2.0 Hz, H-170), 1.40 (s, 3H, Me-18), 1.89 (s, 3H,
Me-19), 1.79 (s, 3H, Me-20); 13C NMR (CDCl3, 75 MHz)
d 47.0 (d, C-1), 30.7 (t, C-2), 44.8 (d, C-3), 87.4 (s, C-4),
41.0 (t, C-5), 29.9 (t, C-6), 81.9 (d, C-7), 58.2 (s, C-8), 44.7
(t, C-9), 26.6 (t, C-10), 132.1 (d, C-11), 126.5 (s, C-12), 43.8
(t, C-13), 76.2 (d, C-14), 137.5 (s, C-15), 169.9 (s, C-16),
120.2 (t, C-17), 21.7 (q, C-18), 28.5 (q, C-19), 15.6 (q,
C-20); HREI-MS m/z [M2I]1 calcd for C20H28IO3
443.1083, found 443.1077, 443 (6), 316 (6), 315 (5), 314
3.5.5. Data for 12. Yellowish oil; [a]2D4ˆ251.38 (c 2.6,
CHCl3); IR (neat) 3431, 2957, 2924, 2854, 1761, 1666,
1455, 1378, 1261, 1099, 984, 800, 757 cm21; UV (MeOH)
1
lmax 208 and 268 nm; H NMR (CDCl3, 300 MHz) d 3.33
(m, 1H, H-1), 1.97 (m, 1H, H-2), 2.56 (dd, 1H, Jˆ5.8,
9.5 Hz, H-20), 3.59 (d, 1H, Jˆ6.9 Hz, H-3), 1.88 (m, 1H,
H-5), 2.07 (m, 1H, H-50), 2.08 (m, 1H, H-6), 2.26 (m, 1H,
H-60), 4.88 (br d, 1H, Jˆ5.7 Hz, H-7), 2.11 (m, 2H, H-9),
2.02 (m, 1H, H-10), 2.39 (m, 1H, H-100), 5.34 (br d, 1H,
Jˆ5.7 Hz, H-11), 2.16 (m, 1H, H-13), 2.50 (dd, 1H, Jˆ3.7,
9.3 Hz, H-130), 4.58 (dd, 1H, Jˆ3.7, 6.7 Hz, H-14), 5.62 (d,
1H, Jˆ1.3 Hz, H-17), 6.19 (d, 1H, Jˆ1.4 Hz, H-170), 1.31

Â
A. D. Rodrõguez et al. / Tetrahedron 57 (2001) 93±107
104
(9), 279 (7), 254 (29), 167 (16), 149 (60), 95 (34), 81 (47),
71 (57), 57 (100).
1H NMR (CDCl3, 300 MHz) d 3.37 (m, 1H, H-1), 3.28 (d,
1H, Jˆ11.7 Hz, H-3), 4.12 (m, 1H, H-7), 2.96 (d, 1H,
Jˆ9.9 Hz, H-13), 4.45 (t, 1H, Jˆ8.7 Hz, H-14), 5.53 (d,
1H, Jˆ2.7 Hz, H-17), 6.38 (d, 1H, Jˆ3.0 Hz, H-170), 1.16
(s, 3H, Me-18), 0.77 (d, 3H, Jˆ7.2 Hz, Me-19), 1.08 (d, 3H,
Jˆ7.2 Hz, Me-20); 13C NMR (CDCl3, 75 MHz) d 38.2 (d,
C-1), 27.2 (t, C-2), 81.3 (d, C-3), 84.1 (s, C-4), 30.4 (t, C-5),
30.5 (t, C-6), 85.0 (d, C-7), 35.2 (d, C-8), 26.9 (t, C-9), 32.4
(t, C-10), 34.8 (t, C-11), 31.4 (d, C-12), 82.8 (d, C-13), 73.5
(d, C-14), 136.5 (s, C-15), 170.2 (s, C-16), 121.2 (t, C-17),
25.7 (q, C-18) (the signals for C-19 and C-20 were not
detected); HREI-MS m/z [M]1 calcd for C20H30O4
334.2145, found 334.2147, 334 (50), 316 (6), 278 (7), 182
(13), 153 (34), 107 (26), 95 (43), 85 (77), 81 (90), 69 (100),
55 (85).
3.6. Reaction of 12,13-bisepieupalmerin (2) with m-chloro-
perbenzoic acid
3.6.1. Path A. A solution of 12,13-bisepieupalmerin
(202 mg, 0.60 mmol) in dry benzene (30 mL) was stirred
vigorously with m-CPBA (150 mg, 1.3 meq) at rt for 1.5 h
and concentrated to leave a residue that was chromato-
graphed on silica gel (10 g). Elution with 30% ethyl acetate
in hexane yielded 171 mg (81%) of diepoxide 15 as a color-
less oil. Compound 15 was identical in all respects to natural
product 12,13-bisepieupalmerin epoxide.8
3.6.2. Path B. A solution of 2 (411 mg, 1.23 mmol) and
m-CPBA (213 mg, 1.23 mmol) in dry benzene (25 mL)
was warmed to 708C, stirred for 1 h, treated with p-toluene-
sulfonic acid hydrate (10 mg), and stirred for another hour
before being cooled and diluted with saturated NaHCO3
3.7.2. Data for 19. Colorless oil; [a]2D1ˆ10.28 (c 6.1,
CHCl3); IR (neat) 2964, 2947, 2924, 2871, 1755, 1668,
1446, 1378, 1282, 1147, 1075, 1019, 927, 804 cm21; UV
(MeOH) lmax 214 nm; 1H NMR (CDCl3, 300 MHz) d 3.33
(m, 1H, H-1), 1.95 (m, 1H, H-2), 2.65 (dd, 1H, Jˆ10.5,
15.9 Hz, H-20), 4.05 (d, 1H, Jˆ11.1 Hz, H-3), 4.38 (dd,
1H, Jˆ4.5, 11.1 Hz, H-7), 4.21 (br s, 1H, H-13), 4.44 (t,
1H, Jˆ6.3 Hz, H-14), 5.62 (d, 1H, Jˆ1.2 Hz, H-17), 6.16
(d, 1H, Jˆ1.8 Hz, H-170), 1.29 (s, 3H, Me-18), 1.97 (s, 3H,
Me-19), 0.89 (d, 3H, Jˆ6.6 Hz, Me-20); 13C NMR (CDCl3,
75 MHz) d 41.5 (d, C-1), 39.3 (t, C-2), 42.5 (d, C-3), 86.2 (s,
C-4), 41.2 (t, C-5), 31.0 (t, C-6), 82.7 (d, C-7), 52.7 (s, C-8),
42.0 (t, C-9), 23.7 (t, C-10), 31.1 (t, C-11), 31.8 (d, C-12),
82.2 (d, C-14), 140.7 (s, C-15), 170.0 (s, C-16), 120.4 (t,
C-17), 21.4 (q, C-18), 29.0 (q, C-19), 14.9 (q, C-20) (the
resonance line ascribable to C-13 could not be detected);
HREI-MS m/z [M2I]1 calcd for C20H30IO4 461.1189, found
461.1180, 461 (32), 443 (12), 334 (16), 333 (16), 316 (13),
315 (21), 297 (10), 249 (5), 189 (9), 109 (54), 95 (44), 81
(81), 69 (68), 55 (100).
solution.
Following
extraction
with
chloroform
(3£30 mL), the organic layers were dried and concentrated
to leave a crude oil (461 mg) that was puri®ed by means of
normal-phase HPLC (Partisil-10, elution with 7% 2-propa-
nol in hexane) to give 16 (98 mg, 24%).
3.6.3. Data for 16. Colorless oil; IR (neat) 2965, 2929,
2918, 2877, 1761, 1707, 1462, 1381, 1318, 1251, 1221,
1171, 1118, 1030, 1018 cm21; UV (MeOH) lmax 208 and
302 nm; 1H NMR (CDCl3, 300 MHz) d 3.57 (m, 1H, H-1),
4.85 (d, 1H, Jˆ4.5 Hz, H-2), 2.35 (dd, 1H, Jˆ3.6, 10.5 Hz,
H-6), 2.42 (m, 1H, H-60), 2.77 (m, 1H, H-8), 3.27 (dd, 1H,
Jˆ2.1, 9.9 Hz, H-13), 4.60 (dd, 1H, Jˆ8.1, 9.9 Hz, H-14),
5.61 (d, 1H, Jˆ3.3 Hz, H-17), 6.32 (d, 1H, Jˆ3.6 Hz,
H-170), 1.09 (d, 3H, Jˆ6.9 Hz, Me-18), 0.98 (d, 3H,
Jˆ6.9 Hz, Me-19), 0.95 (d, 3H, Jˆ7.5 Hz, Me-20); 13C
NMR (CDCl3, 75 MHz) d 36.3 (d, C-1), 93.2 (d, C-2),
162.1 (s, C-3), 34.4 (d, C-4), 31.1 (t, C-5), 41.1 (t, C-6),
215.8 (s, C-7), 43.6 (d, C-8), 33.7 (t, C-9), 21.9 (t, C-10),
32.6 (t, C-11), 29.9 (d, C-12), 80.5 (d, C-13), 72.4 (d, C-14),
138.0 (s, C-15), 169.9 (s, C-16), 123.1 (t, C-17), 19.4 (q,
C-18), 18.2 (q, C-19), 11.0 (q, C-20); HREI-MS m/z [M]1
calcd for C20H28O4 332.1988, found 332.2000, 332 (26), 261
(23), 256 (4), 219 (2), 191 (8), 149 (6), 137 (14), 121 (9), 95
(17), 81 (100).
3.8. Reaction of 12,13-bisepieupalmerin (2) with
perchloric acid
A solution of 2 (968 mg, 2.9 mmol) in chloroform (50 mL)
containing perchloric acid (6 drops) was stirred vigorously
at rt for 2 h, treated with saturated NaHCO3 solution, and
extracted with CHCl3 (3£30 mL). The combined organic
layers were dried and concentrated to leave a residue that
was chromatographed on silica gel (40 g, elution with
1:8:11 (v/v) 2-propanol/CHCl3/hexane) followed by
normal-phase HPLC (Partisil-10, elution with 1:19 (v/v)
2-propanol in hexane) giving 194 mg (20%) of inolide-A
(17),9 58 mg (6%) of eunicin (20),10 60 mg (6%) of (E)-D8
eunicin (21), 21 mg (2%) of (Z)-D8 jeunicin (22), 19 mg
(2%) of (Z)-D7 jeunicin (23), 78 mg (8%) of (Z)-D7 eunicin
(24), and 117 mg (12%) of 25.
3.7. Reaction of 12,13-bisepieupalmerin (2) with iodine9
A solution of iodine (246 mg, 0.97 mmol) in CH2Cl2
(30 mL) was added dropwise over 45 min to a magnetically
stirred solution of 2 (300 mg, 0.90 mmol) in CH2Cl2
(50 mL). The mixture was stirred at 258C for 1 h, concen-
trated, and the resulting oil was chromatographed on silica
gel (20 g, elution with chloroform) followed by normal-
phase HPLC (Partisil-10, elution with 1:19 (v/v) 2-propanol
in hexane) to give 22 mg (7%) of inolide-A (17) and 9 mg
(3%) of inolide-B (18) as white solids, and 25 mg (5%) of
diiodide 19. The structure of 17 was corroborated by X-ray
crystallographic analysis (Fig. 3).
3.8.1. Data for 21. Colorless oil; [a]2D5ˆ220.18 (c 3.2,
CHCl3); IR (neat) 3505, 2965, 2916, 2849, 1749, 1458,
1227, 1127, 1072, 1037, 991, 936, 817 cm21; UV (MeOH)
l
max 222 nm (e 2800); 1H NMR (CDCl3, 300 MHz) d 3.41
(m, 1H, H-1), 3.21 (d, 1H, Jˆ12.0 Hz, H-3), 5.02 (t, 1H, Jˆ
7.8 Hz, H-9), 3.04 (d, 1H, Jˆ9.6 Hz, H-13), 4.50 (t, 1H,
Jˆ8.7 Hz, H-14), 5.62 (d, 1H, Jˆ3.3 Hz, H-17), 6.40 (d,
1H, Jˆ3.6 Hz, H-170), 1.13 (s, 3H, Me-18), 1.52 (s, 3H,
Me-19), 0.89 (d, 3H, Jˆ6.6 Hz, Me-20); 13C NMR
3.7.1. Data for 18. White solid; [a]2D1ˆ236.78 (c 1.8,
MeOH); IR (neat) 2982, 2899, 2873, 1762, 1461, 1296,
1114, 1097, 1005, 947 cm21; UV (MeOH) lmax 210 nm;

Â
A. D. Rodrõguez et al. / Tetrahedron 57 (2001) 93±107
105
(CDCl3, 75 MHz) d 38.2 (d, C-1), 24.0 (t, C-2), 73.2 (d,
C-3), 73.6 (s, C-4), 35.4 (t, C-5), 18.8 (t, C-6), 36.5 (t,
C-7), 130.3 (s, C-8), 128.2 (d, C-9), 24.5 (t, C-10), 33.7 (t,
C-11), 34.2 (d, C-12), 78.6 (d, C-13), 73.4 (d, C-14), 136.8
(s, C-15), 170.3 (s, C-16), 121.4 (t, C-17), 23.3 (q, C-18),
17.3 (q, C-19), 14.3 (q, C-20); HREI-MS m/z [M]1 calcd for
C20H30O4 334.2144, found 334.2144, 334 (43), 316 (19),
288 (6), 221 (9), 193 (33), 164 (15), 135 (18), 121 (33),
109 (45), 95 (63), 81 (100), 67 (47), 55 (79).
for C20H30O4 334.2144, found 334.2130, 334 (9), 316
(9), 248 (6), 193 (4), 164 (8), 135 (6), 121 (19), 109
(21), 95 (33), 85 (65), 83 (100), 81 (45), 70 (35), 55 (44).
3.8.5. Data for 25. Colorless oil; [a]2D5ˆ235.08 (c 1.9,
CHCl3); IR (neat) 3487, 2961, 2911, 2882, 2856, 1756,
1662, 1636, 1451, 1287, 1205, 1100, 999, 894, 815,
1
754 cm21; UV (MeOH) lmax 207 nm (e 5500); H NMR
(CDCl3, 300 MHz) d 3.41 (m, 1H, H-1), 3.20 (d, 1H,
Jˆ11.7 Hz, H-3), 3.05 (dd, 1H, Jˆ2.7, 9.6 Hz, H-13),
4.55 (t, 1H, Jˆ8.7 Hz, H-14), 5.61 (d, 1H, Jˆ3.6 Hz,
H-17), 6.38 (d, 1H, Jˆ3.6 Hz, H-170), 1.16 (s, 3H, Me-
18), 4.73 (br s, 1H, H-19), 4.74 (br s, 1H, H-190), 0.92 (d,
3H, Jˆ6.6 Hz, Me-20); 13C NMR (CDCl3, 75 MHz) d 38.2
(d, C-1), 24.3 (t, C-2), 74.9 (d, C-3), 73.8 (s, C-4), 38.1 (t,
C-5), 23.5 (t, C-6), 37.2 (t, C-7), 145.5 (s, C-8), 37.8 (t, C-9),
20.2 (t, C-10), 31.0 (t, C-11), 32.2 (d, C-12), 78.9 (d, C-13),
73.1 (d, C-14), 136.3 (s, C-15), 170.2 (s, C-16), 121.6 (t,
C-17), 23.9 (q, C-18), 115.8 (t, C-19), 14.9 (q, C-20); HREI-
MS m/z [M]1 calcd for C20H30O4 334.2144, found 334.2176,
334 (62), 316 (58), 301 (8), 288 (10), 221 (19), 193 (16), 182
(27), 164 (41), 121 (74), 109 (83), 107 (73), 93 (59), 79 (92),
69 (73), 67 (88), 55 (100).
3.8.2. Data for 22. Colorless oil; IR (neat) 3445, 1751,
1463, 1095, 758 cm21; UV (MeOH) lmax 222 nm (e
3200); H NMR (CDCl3, 300 MHz) d 3.59 (m, 1H, H-1),
1
3.59 (m, 1H, H-3), 5.13 (m, 1H, H-9), 3.59 (m, 1H, H-13),
4.62 (t, 1H, Jˆ9.3 Hz, H-14), 5.57 (d, 1H, Jˆ3.0 Hz, H-17),
6.27 (d, 1H, Jˆ3.3 Hz, H-170), 1.18 (s, 3H, Me-18), 1.70 (s,
3H, Me-19), 0.96 (d, 3H, Jˆ6.3 Hz, Me-20); 13C NMR
(CDCl3, 75 MHz) d 36.1 (d, C-1), 30.3 (t, C-2), 74.3 (d,
C-3), 79.8 (s, C-4), 35.8 (t, C-5), 23.2 (t, C-6), 32.2 (t,
C-7), 141.0 (s, C-8), 123.7 (d, C-9), 26.2 (t, C-10), 33.0 (t,
C-11), 33.1 (d, C-12), 69.1 (d, C-13), 79.4 (d, C-14), 138.9
(s, C-15), 170.4 (s, C-16), 121.7 (t, C-17), 20.9 (q, C-18),
26.0 (q, C-19), 14.1 (q, C-20); HREI-MS m/z [M]1 calcd for
C20H30O4 334.2144, found 334.2131, 334 (14), 316 (10),
193 (12), 164 (12), 133 (15), 126 (14), 121 (31), 119 (22),
111 (21), 109 (33), 107 (42), 95 (65), 93 (56), 85 (66), 83
(100), 81 (89).
3.9. Reaction of 12,13-bisepieupalmerin (2) with
phosphorus pentoxide
A solution of 2 (3.0 g, 9.1 mmol) in benzene (95 mL) was
stirred at rt with anhydrous phosphorus pentoxide (1.7 g,
12 mmol) for 12 h, treated with saturated NaHCO3 solution,
and extracted with benzene (3£100 mL). Puri®cation of the
residue by chromatography on silica gel (80 g, elution with
1:6:13 (v/v) 2-propanol/CH2Cl2/hexane) followed by
normal-phase HPLC (Partisil-10, elution with 15% 2-propa-
nol in hexane) provided 1.4 g (45%) of eunicin (20),10
476 mg (16%) of 24, 80 mg (3%) of 25, 173 mg (6%) of
jeunicin (26),11 and 27 mg (1%) of butenolide 27.
3.8.3. Data for 23. Colorless oil; [a]2D5ˆ221.68 (c 1.9,
CHCl3); IR (neat) 3435, 2956, 2923, 2854, 1751, 1664,
1462, 1378, 1261, 1095, 1035, 803, 758 cm21; UV
(MeOH) lmax 222 nm (e 2900); 1H NMR (CDCl3,
300 MHz) d 3.67 (m, 1H, H-1), 3.59 (m, 1H, H-3), 5.28
(m, 1H, H-7), 3.80 (dd, 1H, Jˆ1.8, 9.9 Hz, H-13), 4.64 (t,
1H, Jˆ9.3 Hz, H-14), 5.61 (d, 1H, Jˆ2.7 Hz, H-17), 6.28
(d, 1H, Jˆ3.0 Hz, H-170), 1.10 (s, 3H, Me-18), 1.72 (s, 3H,
Me-19), 0.95 (d, 3H, Jˆ6.6 Hz, Me-20); 13C NMR (CDCl3,
75 MHz) d 35.6 (d, C-1), 31.2 (t, C-2), 74.7 (d, C-3), 79.8 (s,
C-4), 34.4 (t, C-5), 25.5 (t, C-6), 124.9 (d, C-7), 139.6 (s,
C-8), 34.5 (t, C-9), 22.5 (t, C-10), 33.6 (t, C-11), 34.3 (d,
C-12), 69.2 (d, C-13), 79.8 (d, C-14), 139.2 (s, C-15), 169.9
(s, C-16), 121.9 (t, C-17), 21.7 (q, C-18), 25.4 (q, C-19),
14.5 (q, C-20); HREI-MS m/z [M]1 calcd for C20H30O4
334.2144, found 334.2133, 334 (2), 316 (2), 181 (3), 164
(3), 147 (4), 135 (4), 126 (9), 121 (11), 109 (11), 107 (12),
95 (25), 85 (65), 83 (100), 81 (42), 79 (18), 55 (31).
3.9.1. Data for 27. Colorless oil; [a]2D5ˆ116.98 (c 0.53,
CHCl3); IR (neat) 3489, 2959, 2922, 2854, 1748, 1681,
1458, 1382, 1261, 1097, 1023, 758 cm21; UV (MeOH)
1
lmax 230 nm; H NMR (CDCl3, 300 MHz) d 2.34 (t, 1H,
Jˆ7.5 Hz, H-2), 3.11 (dd, 1H, Jˆ1.3, 8.0 Hz, H-20), 3.21
(dd, 1H, Jˆ1.3, 6.6 Hz, H-3), 3.15 (dd, 1H, Jˆ1.9, 5.6 Hz,
H-13), 4.60 (d, 1H, Jˆ5.6 Hz, H-14), 4.42 (br s, 2H, H-17),
1.24 (s, 3H, Me-18), 4.76 (br s, 1H, H-19), 4.77 (br s, 1H,
H-190), 1.01 (d, 3H, Jˆ4.1 Hz, Me-20), 1.60 (br s,
exchangeable, ±OH); 13C NMR (CDCl3, 75 MHz) d 163.9
(s, C-1), 27.9 (t, C-2), 80.0 (d, C-3), 73.9 (s, C-4), 38.1 (t,
C-5), 23.3 (t, C-6), 36.9 (t, C-7), 145.3 (s, C-8), 37.5 (t, C-9),
19.9 (t, C-10), 30.9 (t, C-11), 33.2 (d, C-12), 82.6 (d, C-13),
77.8 (d, C-14), 123.2 (s, C-15), 173.8 (s, C-16), 54.9 (t,
C-17), 23.9 (q, C-18), 115.8 (t, C-19), 15.0 (q, C-20);
HREI-MS m/z [M]1 calcd for C20H30O5 350.2093, found
350.2089, 350 (8), 332 (20), 314 (6), 223 (19), 205 (15),
195 (15), 193 (18), 177 (33), 135 (37), 121 (33), 109 (67), 95
(70), 81 (100), 59 (57).
3.8.4. Data for 24. Colorless oil; [a]2D5ˆ215.58 (c 15.4,
CHCl3); IR (neat) 3450, 2964, 2934, 2879, 1759, 1681,
1461, 1384, 1303, 1104, 1038, 1002, 951, 755 cm21; UV
(MeOH) lmax 203 nm (e 30500); 1H NMR (CDCl3,
300 MHz) d 3.40 (m, 1H, H-1), 3.46 (dd, 1H, Jˆ1.2,
11.4 Hz, H-3), 1.60 (br m, 2H, H-5), 5.25 (dd, 1H, Jˆ5.0,
10.0 Hz, H-7), 3.12 (d, 1H, Jˆ9.6 Hz, H-13), 4.37 (t, 1H,
Jˆ8.7 Hz, H-14), 5.63 (d, 1H, Jˆ3.3 Hz, H-17), 6.39 (d,
1H, Jˆ3.6 Hz, H-170), 1.14 (s, 3H, Me-18), 1.61 (s, 3H,
Me-19), 0.89 (d, 3H, Jˆ7. 2 Hz, Me-20); 13C NMR
(CDCl3, 75 MHz) d 38.5 (d, C-1), 24.7 (t, C-2), 75.6 (d,
C-3), 74.3 (s, C-4), 41.6 (t, C-5), 21.2 (t, C-6), 125.4 (d,
C-7), 136.4 (s, C-8), 33.6 (t, C-9), 23.5 (t, C-10), 32.5 (t,
C-11), 34.6 (d, C-12), 82.2 (d, C-13), 74.2 (d, C-14), 136.7
(s, C-15), 170.9 (s, C-16), 122.3 (t, C-17), 24.5 (q, C-18),
23.8 (q, C-19), 13.6 (q, C-20); HREI-MS m/z [M]1 calcd
3.10. Reaction of eupalmerin (3) with phosphorus
pentoxide
A solution of 3 (616 mg, 1.84 mmol) in benzene (40 mL)
was stirred at rt with anhydrous phosphorus pentoxide

Â
A. D. Rodrõguez et al. / Tetrahedron 57 (2001) 93±107
106
(340 mg, 2.39 mmol) for 12 h, treated with saturated
NaHCO3 solution, and extracted with benzene (3£
100 mL). Puri®cation of the residue by chromatography
on silica gel (15 g, elution with 1:1 (v/v) ethyl acetate in
hexane) followed by reversed-phase HPLC (Zorbax C8,
elution with 30% water in MeOH) provided 142 mg
(23%) of 12-epicueunicin (28), 45 mg (7%) of 12,13-bi-
sepijeunicin (29), 102 mg (16%) of 30, and 91 mg (15%)
of 31.
136.9 (s, C-15), 169.8 (s, C-16), 122.7 (t, C-17), 22.8 (q,
C-18), 24.2 (q, C-19), 15.9 (q, C-20); HREI-MS m/z [M]1
calcd for C20H30O4 334.2144, found 334.2146, 334 (13), 316
(30), 193 (17), 182 (12), 177 (10), 164 (15), 153 (12), 147
(14), 133 (19), 119 (31), 109 (42), 107 (38), 95 (57), 91 (40),
82 (24), 79 (68), 69 (55), 55 (100).
3.10.4. Data for 31. Colorless oil; [a]2D1ˆ272.48 (c 2.1,
CHCl3); IR (neat) 3446, 2959, 2926, 2856, 1761, 1456,
1374, 1263, 1145, 1106, 1019 cm21; UV (MeOH) lmax
1
3.10.1. Data for 28. Colorless oil; [a]2D1ˆ289.08 (c 3.9,
CHCl3); IR (neat) 3299, 2954, 2914, 2853, 1748, 1660,
1446, 1284, 1149, 1090, 1027, 991, 934 cm21; UV
(MeOH) lmax 210 nm (e 15200); 1H NMR (CDCl3,
300 MHz) d 3.42 (m, 1H, H-1), 3.52 (dd, 1H, Jˆ4.2,
12.3 Hz, H-3), 5.19 (t, 1H, Jˆ6.3 Hz, H-7), 3.58 (br d,
1H, Jˆ3.6 Hz, H-13), 4.70 (dd, 1H, Jˆ0.9, 9.0 Hz, H-14),
5.66 (d, 1H, Jˆ2.4 Hz, H-17), 6.39 (d, 1H, Jˆ2.7 Hz,
H-17'), 1.13 (s, 3H, Me-18), 1.51 (s, 3H, Me-19), 0.94 (d,
3H, Jˆ7.2 Hz, Me-20); 13C NMR (CDCl3, 75 MHz) d 34.8
(d, C-1), 26.0 (t, C-2), 76.5 (d, C-3), 74.4 (s, C-4), 44.7 (t,
C-5), 24.2 (t, C-6), 129.3 (d, C-7), 129.7 (s, C-8), 39.4 (t,
C-9), 22.8 (t, C-10), 29.1 (t, C-11), 35.5 (d, C-12), 73.0 (d,
C-13), 76.3 (d, C-14), 137.8 (s, C-15), 170.3 (s, C-16), 122.9
(t, C-17), 20.5 (q, C-18), 15.6 (q, C-19), 16.2 (q, C-20);
HREI-MS m/z [M]1 calcd for C20H30O4 334.2144,
found 334.2133, 334 (29), 316 (40), 193 (29), 182
(19), 177 (13), 164 (26), 153 (38), 121 (34), 109 (38),
95 (61), 81 (96), 67 (65), 55 (100).
212 nm (e 12800); H NMR (CDCl3, 300 MHz) d 3.44
(m, 1H, H-1), 3.58 (dd, 1H, Jˆ3.0, 12.6 Hz, H-3), 5.17 (t,
1H, Jˆ7.5 Hz, H-7), 3.83 (dd, 1H, Jˆ1.2, 4.5 Hz, H-13),
4.72 (dd, 1H, Jˆ1.2, 8.7 Hz, H-14), 5.69 (d, 1H, Jˆ2.4 Hz,
H-17), 6.41 (d, 1H, Jˆ2.4 Hz, H-170), 1.10 (s, 3H, Me-18),
1.66 (s, 3H, Me-19), 1.01 (d, 3H, Jˆ7.2 Hz, Me-20); 13C
NMR (CDCl3, 75 MHz) d 35.4 (d, C-1), 27.4 (t, C-2), 76.8
(d, C-3), 73.5 (s, C-4), 42.7 (t, C-5), 22.6 (t, C-6), 125.2 (d,
C-7), 138.4 (s, C-8), 31.2 (t, C-9), 25.8 (t, C-10), 32.0 (t,
C-11), 35.0 (d, C-12), 73.3 (d, C-13), 76.4 (d, C-14), 136.3
(s, C-15), 170.3 (s, C-16), 123.5 (t, C-17), 20.8 (q, C-18),
23.7 (q, C-19), 17.1 (q, C-20); HREI-MS m/z [M]1 calcd for
C20H30O4 334.2144, found 334.2189, 334 (21), 316 (25),
193 (25), 182 (16), 177 (11), 164 (24), 153 (23), 149 (22),
133 (20), 119 (28), 109 (19), 105 (18), 95 (64), 91 (20), 79
(44), 69 (71), 55 (100).
3.11. Reaction of eupalmerin (3) with p-toluenesulfonic
acid hydrate
3.10.2. Data for 29. Colorless oil; [a]2D1ˆ2122.28 (c 2.3,
CHCl3); IR (neat) 3443, 2952, 2929, 2854, 1733, 1660,
1456, 1406, 1287, 1260, 1162, 1099, 1052, 988, 937,
After a solution of 3 (700 mg, 2.1 mmol) in 30 mL of
benzene was treated with 15 mg of PTSA´H2O and stirred
at rt for 6 h, the mixture was concentrated to afford a semi-
solid mass. The residue was layered on top of a 50£2.5 cm
silica gel column eluted with increasing concentrations of
ethyl acetate in hexane. Concentration of the major band
gave 237 mg (34%) of 12-epicueunicin (28). Subsequent
puri®cation of the minor band by normal-phase HPLC
(Partisil-10, elution with 20% 2-propanol in hexane) fur-
nished 20 mg (3%) of 32, 18 mg (3%) of 33, and 15 mg
(2%) of known eupalmerolide (34).14
1
812 cm21; UV (MeOH) lmax 210 nm (e 5600); H NMR
(CDCl3, 300 MHz) d 3.44 (m, 1H, H-1), 3.58 (m, 1H,
H-3), 5.22 (br d, 1H, Jˆ11.1 Hz, H-7), 3.40 (br d, 1H,
Jˆ5.1 Hz, H-13), 4.74 (d, 1H, Jˆ9.3 Hz, H-14), 5.56 (d,
1H, Jˆ3.0 Hz, H-17), 6.37 (d, 1H, Jˆ3.6 Hz, H-170), 1.12
(s, 3H, Me-18), 1.59 (s, 3H, Me-19), 0.90 (d, 3H, Jˆ6.9 Hz,
Me-20); 13C NMR (CDCl3, 75 MHz) d 37.6 (d, C-1), 32.3 (t,
C-2), 73.6 (d, C-3), 78.8 (s, C-4), 39.8 (t, C-5), 23.1 (t, C-6),
130.0 (d, C-7), 127.5 (s, C-8), 41.1 (t, C-9), 23.5 (t, C-10),
28.4 (t, C-11), 40.4 (d, C-12), 74.4 (d, C-13), 79.7 (d, C-14),
137.1 (s, C-15), 170.0 (s, C-16), 120.9 (t, C-17), 13.3 (q, C-
18), 15.6 (q, C-19), 17.1 (q, C-20); HREI-MS m/z [M]1
calcd for C20H30O4 334.2144, found 334.2158, 334 (21),
316 (14), 181 (12), 177 (12), 164 (17), 159 (12), 147 (18),
133 (21), 119 (30), 109 (31), 107 (41), 95 (62), 91 (42), 79
(61), 67 (57), 55 (100).
3.11.1. Data for 32. Crystalline white solid; [a]2D1ˆ245.08
(c 1.4, CHCl3); IR (neat) 3544, 2932, 2905, 2855, 1751,
1405, 1366, 1298, 1201, 1138, 1096, 1001, 886 cm21; UV
(MeOH) lmax 212 nm (e 4500); 1H NMR (CDCl3,
300 MHz) d 3.46 (m, 1H, H-1), 3.65 (dd, 1H, Jˆ5.7,
12.9 Hz, H-3), 3.83 (br s, 1H, H-13), 4.63 (dd, 1H, Jˆ1.2,
9.3 Hz, H-14), 5.61 (d, 1H, Jˆ3.0 Hz, H-17), 6.41 (d, 1H,
Jˆ3.0 Hz, H-170), 0.97 (s, 3H, Me-18), 4.86 (br s, 1H,
H-19), 4.94 (br s, 1H, H-190), 1.06 (d, 3H, Jˆ6.6 Hz, Me-
20); 13C NMR (CDCl3, 75 MHz) d 35.1 (d, C-1), 24.6 (t,
C-2), 77.8 (d, C-3), 74.5 (s, C-4), 32.8 (t, C-5), 22.3 (t, C-6),
33.8 (t, C-7), 148.0 (s, C-8), 38.1 (t, C-9), 22.1 (t, C-10),
33.5 (t, C-11), 35.3 (d, C-12), 70.4 (d, C-13), 79.4 (d, C-14),
136.4 (s, C-15), 169.7 (s, C-16), 122.3 (t, C-17), 20.3 (q,
C-18), 113.5 (t, C-19), 15.8 (q, C-20); HREI-MS m/z [M]1
calcd for C20H30O4 334.2144, found 334.2140, 334 (5), 316
(12), 195 (15), 193 (41), 182 (25), 177 (11), 164 (40), 153
(61), 149 (22), 135 (18), 121 (48), 119 (32), 109 (34), 107
(45), 95 (73), 93 (60), 81 (100), 79 (57), 67 (54), 55 (93).
The structural assignment to 32 was corroborated by X-ray
crystallographic analysis (Fig. 4).
3.10.3. Data for 30. Colorless oil; [a]2D1ˆ228.08 (c 2.5,
CHCl3); IR (neat) 3473, 2961, 2928, 2856, 1759, 1660,
;
1454, 1376, 1286, 1139, 1105, 1030, 934, 813, 756 cm21
1
UV (MeOH) lmax 212 nm (e 4500); H NMR (CDCl3,
300 MHz) d 3.49 (m, 1H, H-1), 3.69 (dd, 1H, Jˆ4.8,
12.9 Hz, H-3), 5.16 (t, 1H, Jˆ7.5 Hz, H-9), 3.87 (br s, 1H,
H-13), 4.69 (dd, 1H, Jˆ1.5, 9.3 Hz, H-14), 5.65 (d, 1H,
Jˆ2.7 Hz, H-17), 6.44 (d, 1H, Jˆ3.3 Hz, H-170), 1.03 (s,
3H, Me-18), 1.66 (s, 3H, Me-19), 1.05 (d, 3H, Jˆ6.6 Hz,
Me-20); 13C NMR (CDCl3, 75 MHz) d 35.0 (d, C-1), 25.1 (t,
C-2), 76.8 (d, C-3), 75.0 (s, C-4), 36.4 (t, C-5), 21.1 (t, C-6),
34.6 (t, C-7), 134.3 (s, C-8), 126.6 (d, C-9), 25.1 (t, C-10),
31.8 (t, C-11), 34.6 (d, C-12), 73.0 (d, C-13), 78.4 (d, C-14),

Â
A. D. Rodrõguez et al. / Tetrahedron 57 (2001) 93±107
107
3.11.2. Data for 33. Colorless oil; [a]2D1ˆ213.38 (c 10.0,
1124, 1022, 958, 755 cm21; UV (MeOH) lmax 210 nm; H
1990, 46, 5889±5894. (b) Marshall, J. A.; Karas, L. J.;
Coghlan, M. J. J. Org. Chem. 1982, 47, 699±701.
(c) Marshall, J. A.; Andrews, R. C.; Lebioda, L. J. Org.
Chem. 1987, 52, 2378±2388.
CHCl3); IR (neat) 3451, 2928, 1754, 1445, 1272, 1159,
1
NMR (CDCl3, 300 MHz) d 3.40 (m, 1H, H-1), 4.21 (dd, 1H,
Jˆ3.6, 9.6 Hz, H-3), 5.57 (br t, 1H, Jˆ6.6 Hz, H-7), 3.60
(dd, 1H, Jˆ1.5, 8.1 Hz, H-13), 4.43 (dd, 1H, Jˆ6.3, 8.1 Hz,
H-14), 5.68 (d, 1H, Jˆ0.9 Hz, H-17), 6.27 (d, 1H, Jˆ
1.5 Hz, H-170), 4.95 (br s, 2H, H-18), 1.56 (s, 3H, Me-19),
0.89 (d, 3H, Jˆ6.9 Hz, Me-20); 13C NMR (CDCl3, 75 MHz)
d 37.6 (d, C-1), 32.1 (t, C-2), 71.2 (d, C-3), 152.4 (s, C-4),
32.8 (t, C-5), 28.0 (t, C-6), 127.0 (d, C-7), 134.5 (s, C-8),
36.1 (t, C-9), 20.8 (t, C-10), 31.4 (t, C-11), 29.6 (d, C-12),
74.3 (d, C-13), 82.3 (d, C-14), 139.0 (s, C-15), 169.9 (s,
C-16), 122.1 (t, C-17), 113.9 (t, C-18), 14.6 (q, C-19),
10.9 (q, C-20); HREI-MS m/z [M]1 calcd for C20H30O4
334.2144, found 334.2143, 334 (6), 316 (17), 181 (22),
163 (11), 147 (13), 145 (11), 121 (32), 119 (26), 109 (23),
107 (38), 105 (37), 95 (59), 93 (80), 91 (44), 81 (94), 79
(57), 69 (49), 55 (100).
4. Gopichand, Y.; Ciereszko, L. S.; Schmitz, F. J.; Switzner, D.;
Rahman, A.; Hossain, M. B.; Van der Helm, D. J. Nat. Prod.
1984, 47, 607±614.
Â
5. Fontan, L. A.; Rodrõguez, A. D. J. Nat. Prod. 1991, 54, 298±
Â
301.
6. (a) Schauder, J. R.; Krief, A. Tetrahedron Lett. 1982, 23,
4389±4392. (b) Bowden, B. F.; Coll, J. C.; Hicks, W.;
Kazlauskas, R.; Mitchell, S. J. Aust. J. Chem. 1978, 31,
2707±2712.
Â
7. Rodrõguez, A. D.; Acosta, A. L.; Huang, S. D. J. Chem.
Crystallogr. 1998, 28, 17±21.
Â
8. Rodrõguez, A. D.; Acosta, A. L. J. Nat Prod. 1998, 61, 40±45.
Â
9. Rodrõguez, A. D.; PinÄa, I. C.; Barnes, C. L. J. Org. Chem.
1995, 60, 8096±8100.
10. (a) Weinheimer, A. J.; Middlebrook, R. E.; Bledsoe, J. O.;
Marsico, W. E.; Karns, T. K. B. Chem. Commun. 1968,
384±385. (b) Gampe, Jr., R. T.; Alam, M.; Weinheimer,
A. J.; Martin, G. E.; Matson, J. A.; Willcott, M. R.; Inners,
R. R.; Hurd, R. E. J. Am. Chem. Soc. 1984, 106, 1823±1826.
11. (a) Van der Helm, D.; Enwall, E. L.; Weinheimer, A. J.; Karns,
T. K. B.; Ciereszko, L. S. Acta Crystallogr. 1976, B32, 1558±
1560. (b) Sanduja, R.; Linz, G. S.; Alam, M.; Weinheimer,
A. J.; Martin, G. E.; Ezell, E. L. J. Heterocycl. Chem. 1986,
23, 529±535.
Acknowledgements
This work was supported by grants from the NSF-EPSCoR,
NSF-MRCE, and NIH-MBRS Programs of the University of
Puerto Rico. We thank Anthony Sostre for assistance during
the collection of Eunicea species and the crew of the R/V
Isla Magueyes for logistic support. We extend our sincere
appreciation to Drs V. L. Narayanan and Edward Sausville
(NCI) for the cytotoxicity assays on human cells. Special
thanks to Professor Reinaldo S. Compagnone (U. Central de
Venezuela) for his support of our work.
12. (a) Gross, R. A. Ph.D. thesis, University of Oklahoma,
Norman, 1974. (b) Ciereszko, L. S. Mar. Res. Indones.
1977, 113±118. (c) Gupta, P. K. S.; Hossain, M. B.; Van
der Helms, D. Acta Crystallogr. 1986, C42, 434±436.
13. Weinheimer, A. J.; Matson, J. A.; Poling, M.; Van der Helm,
D. Acta Crystallogr. 1982, B38, 580±583.
References
 Â
14. Rodrõguez, A. D.; PinÄa, I. C.; Acosta, A. L.; Ramõrez, C.; Soto,
J. J. J. Org. Chem. 2001, 66, in press.
1. Faulkner, D. J. Nat. Prod. Rep. 2000, 17, 7±55, and previous
reports in this series.
15. Boyd, M. R.; Paull, K. D. Drug Dev. Res. 1995, 34, 91±109.
16. Hollingshead, M. G.; Alley, M. C.; Camalier, R. F.; Abbott,
B. J.; Mayo, J. G.; Malspeis, L.; Grever, M. R. Life Sci. 1995,
57, 131±141.
2. Wahlberg, I.; Eklund, A.-M. Prog. Chem. Org. Nat. 1992, 59,
141±294.
Â
3. (a) Morales, J. J.; Espina, J. R.; Rodrõguez, A. D. Tetrahedron