Bioactive cycloperoxides isolated from the Puerto Rican sponge Plakortis halichondrioides

Article abstract of DOI:10.1021/np100461t

Two new five-membered-ring polyketide endoperoxides, epiplakinic acid F methyl ester (1) and epiplakinidioic acid (3), and a peroxide-lactone, plakortolide J (2), were isolated from the Puerto Rican sponge Plakortis halichondrioides, along with two previo

Full text of DOI:10.1021/np100461t

1
694
J. Nat. Prod. 2010, 73, 1694–1700
Bioactive Cycloperoxides Isolated from the Puerto Rican Sponge Plakortis halichondrioides
†
†
†
†
,†
‡
Carlos Jim e´ nez-Romero, Idelisse Ortiz, Jan Vicente, Brunilda Vera, Abimael D. Rodr ´ı guez,* Sangkil Nam, and
Richard Jove
‡
Department of Chemistry, UniVersity of Puerto Rico, P.O. Box 23346, UPR Station, San Juan, Puerto Rico 00931-3346, and Department of
Molecular Medicine, Beckman Research Institute, City of Hope, 500 East Duarte Road, Duarte, California 91010, United States
ReceiVed July 7, 2010
Two new five-membered-ring polyketide endoperoxides, epiplakinic acid F methyl ester (1) and epiplakinidioic acid
(
3), and a peroxide-lactone, plakortolide J (2), were isolated from the Puerto Rican sponge Plakortis halichondrioides,
along with two previously reported cyclic peroxides, 4 and 5. The structures of the new metabolites were determined
by spectroscopic and chemical analyses. The absolute stereostructures of 1, 2, and 5 were determined by degradation
reactions followed by application of Kishi’s method for the assignment of absolute configuration of alcohols. Biological
screening of cycloperoxides 1-5 and semisynthetic analogues 7-12 for cytotoxic activity against various human tumor
cell lines revealed that compounds 3, 4, and 11 are very active. Upon assaying for antimalarial and antitubercular
activity, some of the compounds tested showed strong activity against the pathogenic microbes Plasmodium falciparum
and Mycobacterium tuberculosis.
Marine sponges of the family Plakinidae have been reported to
be a rich source of cyclic peroxides, many of which often exhibit
antimicrobial, ichthyotoxic, antineuroinflammatory, and antitumor
1
properties. Recently, a number of such bioactive polyketides have
also been shown to be active against the protozoan parasites
Plasmodium falciparum, Leishmania chagasi, Trypanosoma brucei
2
brucei, and Trypanosona cruzi. Many of these interesting natural
products contain either a six-membered (1,2-dioxane) or a five-
3
membered (1,2-dioxolane) peroxide ring. Representative examples
of these groups of secondary metabolites, most of which have been
isolated from sponges belonging to the two taxonomically related
genera Plakinastrella and Plakortis, include the plakinic acid and
4
,5
the plakortolide series of cyclic peroxides. Already in 2003, we
reported two strongly cytotoxic 1,2-dioxanes, plakortide O and
plakortide P, from the Caribbean marine sponge Plakortis hali-
6
chondrioides (Wilson, 1902). Since that investigation, we have
isolated three new endoperoxides from a recently re-collected
sponge specimen using a bioassay-guided purification protocol. The
first two compounds, trivially named epiplakinic acid F methyl ester
(
1) and plakortolide J (2), contain a conjugated triene along an
unbranched C16 alkyl side chain. The least abundant of these
metabolites, epiplakinidioic acid (3), is a rare dicarboxylic acid
derivative with a shortened side chain compared to compounds 1
and 2. In this account, we report the isolation, structure determi-
nation, and biological evaluation of these natural products and those
of several semisynthetic analogues.
of n-hexane-acetone of increasing polarity displaying e30% cells
viability were investigated further. Additional separation steps of
the active fractions by normal-phase column chromatography led
to the isolation of new polyketide derivatives epiplakinic acid F
methyl ester (1), plakortolide J (2), and epiplakinidioic acid (3), as
well as the known polyketides epiplakinic acid F (4) and plakor-
tolide F (5). As specific rotation data were lacking, identification
of known compounds 4 and 5 was based exclusively on comparison
of their spectroscopic properties (IR, MS, and NMR) with values
Results and Discussion
A freeze-dried specimen of the sponge P. halichondrioides (395
4
d,7
already described in the literature.
The 13C NMR spectrum and HREIMS data for 1 suggested a
molecular formula of C24 . Inspection of the NMR spectra
g) was extracted with a 1:1 mixture of CHCl
3 3
-CH OH, and after
concentration the organic extract was partitioned between n-hexane
and water. Significant antitumor activity was detected in the
n-hexane extract (16.4 g); upon treatment with 1.0 mg/mL extract,
reduction of cell viability was 70%, 90%, and 92% for DU-145
prostate cancer, A2058 melanoma, and MDA-MB-435 breast cancer
40 4
H O
suggested that the compound was closely related to epiplakinic acid
F (4) reported by Wright in 2001 from a Plakinastrella sponge
4
d
species from the Seychelle Islands. A comparison of the NMR
data of 1 (Table 1) with those of 4 quickly showed that 1 has the
same 1,2-dioxolane ring (with trans-oriented methyl substituents
at the C3 and C5 positions) and the same unbranched C16 alkyl
1
cells, respectively. Subsequent analysis of this material by H and
1
3
C NMR indicated the presence of polyketides; therefore a small
portion of the n-hexane extract was subjected to Si gel flash
chromatography. Only those compounds that eluted with mixtures
1
4
16
18
chain (including assignment of geometry of the ∆ , ∆ , and ∆
double bonds) of 4. Thus, the only difference between both
compounds arises from the replacement of the carboxylic acid
moiety at C2 in 4 by a methylcarboxylate group in 1. Methylation
of 4 at 25 °C with diazomethane in ether yielded an inseparable
mixture of compounds, the major one of which was shown to be
*
To whom correspondence should be addressed. Tel: (787)-764-0000,
ext. 4799. Fax: (787)-756-8242. E-mail: abrodriguez@uprrp.edu.
†
University of Puerto Rico.
Beckman Research Institute, City of Hope.
‡
1
0.1021/np100461t  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/05/2010

BioactiVe Cycloperoxides from Plakortis halichondrioides
Journal of Natural Products, 2010, Vol. 73, No. 10 1695
Table 1. 13C NMR (125 MHz), H NMR (500 MHz), HMBC,
1
Table 3. 13C NMR (125 MHz), H NMR (500 MHz), HMBC,
1
and NOESY Spectroscopic Data for Epiplakinic Acid F Methyl
and NOESY Spectroscopic Data for Plakortolide J (2) in
a
a
Ester (1) in CDCl
3
CDCl
3
b
c
b
c
atom
δ
C
, mult
δ
H
, mult (J in Hz)
HMBC
NOESY
atom
δ
C
, mult
174.3, C
34.3, CH
δ
H
, mult (J in Hz)
HMBC
NOESY
1
2
2
3
4
4
5
6
6
7
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
171.1, C
1
R
ꢀ
44.0, CH
2
2.76, d (14.5)
2.65, d (14.5)
1, 3, 4, 22
1, 3, 4, 22
H-4ꢀ, H
H-4ꢀ, H
3
-22
-22
2R
2ꢀ
3
4
5R
5ꢀ
2
2.62, d (15.0)
2.91, dd (15.0, 10.0) 1, 3
1, 3
H-2ꢀ
H-2R, H
3
3
-23
-23
83.9, C
81.1, CH 4.45, d (10.0)
1, 2, 3
H-5ꢀ, H
3
d
R
ꢀ
55.4, CH
2
2.22, d (12.5)
2.46, d (12.5)
2, 5, 6, 7, 22 H-6Rꢀ, H
2, 5, 6, 23
3
-22
-23
82.8, C
H-2Rꢀ, H
3
40.5, CH
2
2.16, d (15.0)
1.70, d (15.0)
3, 4, 23, 24 H-5ꢀ, H
6, 7, 23, 24 H-3, H-5R,
3
-24
86.5, C
R
ꢀ
R
ꢀ
39.6, CH
2
1.69, br t (12.2)
1.53, br t (12.2)
1.28, br s
1.37, br m
1.28, br s
1.28, br s
1.28, br s
1.28, br s
1.37, br m
2.05, m
H-4R
H-4R
H-7Rꢀ, H -23
3
6
80.1, C
24.5, CH
2
7R
7ꢀ
8
41.0, CH
2
1.50, br m
1.50, br m
1.37, br m
1.27, br m
1.27, br s
1.27, br s
1.27, br m
1.37, br m
2.06, br m
29.5, CH
29.4, CH
29.3, CH
30.0, CH
29.1, CH
32.8, CH
2
2
2
2
2
2
23.1, CH
29.5, CH
29.4, CH
29.3, CH
30.0, CH
29.1, CH
32.8, CH
2
2
2
2
2
2
2
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
10
11
12
13
14
10
12, 14, 15
134.5, CH 5.65, dt (14.2, 7.0) 13, 16
13, 15, 16
14, 17
17
130.4, CH 6.03, br m
130.9, CH 6.10, br m
130.8, CH 6.08, br m
129.5, CH 6.06, br m
16
18
18
17
15 134.4, CH 5.65, dt (14.2, 7.0)
16 130.4, CH 6.04, br m
17 130.9, CH 6.10, br m
18 130.8, CH 6.07, br m
19 129.4, CH 6.02, br m
20 135.9, CH 5.71, dt (14.5, 7.5)
19
19
18
135.9, CH 5.71, dt (14.5, 7.5) 17, 20, 21
25.8, CH
13.6, CH
24.1, CH
23.3, CH
51.7, CH
2
3
3
3
3
2.08, m
0.99, t (7.4)
1.43, s
1.28, s
3.69, s
18, 19, 21
19, 20
2, 3, 4
4, 5, 6, 7
1
18, 21, 22
19, 20, 22
20, 21
3, 4, 5
5, 6, 7
21
22
23
24
a
25.8, CH
13.6, CH
25.9, CH
22.4, CH
2
3
3
3
2.08, m
1.00, t (5.0)
1.38, s
H-2Rꢀ, H-4R
H-4ꢀ
H-2ꢀ, H-3, H-5ꢀ
H-5R
-
OCH
a
3
1.28, s
Chemical shift values are in parts per million relative to the residual
(7.26 ppm) or CDCl (77.0 ppm) signals. Assignments were
aided by 2D NMR experiments, spin-splitting patterns, number of
attached protons, and chemical shift values.
were obtained from a DEPT-135 experiment. Carbon atoms correlated
Chemical shift values are in parts per million relative to the residual
(7.26 ppm) or CDCl (77.0 ppm) signals. Assignments were
aided by 2D NMR experiments, spin-splitting patterns, number of
attached protons, and chemical shift values.
were obtained from a DEPT-135 NMR experiment. Carbon atoms
CHCl
3
3
CHCl
3
3
b
13
b
13
C NMR multiplicities
C NMR multiplicities
c
c
1
1
to proton resonances in the H column. Parameters were optimized for
correlated to proton resonances in the H column. Parameters were
2
,3
2,3
d
J
C-H ) 6 and 8 Hz.
optimized for
intensity.
JCH ) 6 and 8 Hz. Broad resonance line of low
Scheme 1. Conversion of Endoperoxide 1 to Derivatives 7-9
Scheme 2. Empiral Rules for the Determination of Absolute
Configuration of Secondary and Tertiary Alcohols
1
ester 1 by comparison of H NMR spectra. Compound 1, however,
was extremely unstable, decomposing in the presence of air over
absolute configuration from its methyl ester 1 by reductive cleavage
varying periods of time, to carboxylic acid 6, which was devoid of
significant biological activity. The co-isolation of 1 and 4 from
the same organism raises the possibility that the former compound
is an isolation artifact due to the extraction with MeOH.
As the absolute stereostructure of epiplakinic acid F (4) was not
determined during the 2001 investigation, we established the
2
of the peroxide ring with H and 10% Pd/C in EtOAc to afford the
8
diol-ester derivative 8 and a small amount of the separable peroxide-
ester derivative 7 (Scheme 1). The former intermediate, obtained
in 29% isolated yield, was intramolecularly transesterified to
1
3
δ-lactone 9. Upon analysis of the C NMR chemical shifts of the
carbons in 9 adjacent to the tertiary alcoholic center in the presence
a
3
Table 2. Assignment of Absolute Configuration of Compounds 9, 10, 12, and 13 at 15 mol % per OH of (R)- and (S)-Eu(tfc)
∆
δ
R
) δCX
R
- δCY
R
∆δ
S
S S
) δCX - δCY
compound
δCX
R
δCY
R
∆δ
R
δCX
S
δCY
S
∆δ
S
∆∆δ ) ∆δ
R
- ∆δ
S
9
44.16
90.41
90.53
46.01
44.73
38.87
38.15
43.39
-0.57
51.54
52.38
2.62
44.23
90.32
90.53
45.97
44.75
38.73
38.14
43.34
-0.52
51.59
52.39
2.63
-0.05
-0.05
-0.01
-0.01
1
1
1
0
2
3
a
13C NMR data of alcohols 9, 10, 12, and 13 were recorded in C
6 6
D , CDCl
3 6 3
, acetone-d , and CDCl , respectively.

1
696 Journal of Natural Products, 2010, Vol. 73, No. 10
Jim e´ nez-Romero et al.
9
of chiral lanthanide shift reagents, as described by Kishi et al.,
Scheme 3. Conversion of Plakortolide J (2) to Lactones 10 and
the absolute configuration at C-3 was found to be S (Table 2).
Coupled with the relative geometry derived from the above analyses,
this established the absolute configuration of the peroxide ring in
compound 1 as 3S, 5R.
11
Plakortolide J [(2), (3S,4S,6S,15E,17E,19E)-4,6-dimethyl-4-hy-
droxy-3,6-peroxydocosa-15,17,19-trienoic acid 1,4-lactone] was
isolated as an extremely unstable colorless oil, which rendered its
purification and characterization difficult. The molecular formula
of C24H
38
O
4
, which requires six degrees of unsaturation, was
established from HREIMS. The IR spectrum showed a strong
-1
absorbance at 1767 cm , suggestive of a γ-lactone, and no other
13
carbonyl or hydroxy band. The C NMR data, together with the
1
results of H NMR (Table 3) and HSQC experiments, indicated
2
3
the presence of six sp methine, 11 sp methylene, and three methyl
Scheme 4. Conversions of Plakortolide F (5) to Diol-Lactone
Derivative 12 and Diacetate 13
groups, two of which appeared as singlets (δ 1.38 and 1.28) and
one as a triplet (δ 1.00, J ) 5.0 Hz) in the H NMR spectrum. The
1
remaining carbons were assigned as a lactone carbonyl (δ 174.3,
C), two oxygenated quaternary carbons observed at δ 82.8 and 80.1,
and one oxygenated methine carbon at δ 81.1. Analysis of the
1
1
H- H COSY and HSQC spectra of 2 allowed us to establish partial
structures, which in turn were linked by the 2
,3
JC-H long-range
HMBC correlations and side-by-side comparisons to the spectro-
5
scopic data of related peroxide-lactones. In the HMBC spectrum,
H-3 (δ 4.45) was coupled to C-1 (δ 174.3); H
coupled to C-3 (δ 81.1), C-4 (δ 82.8), and C-5 (δ 40.5); and H
(
3
-23 (δ 1.38) was
-24
δ 1.28) was coupled to C-5 (δ 40.5), C-6 (δ 80.1), and C-7 (δ
3
4
1.0). While the carbon connectivity about the peroxide-lactone
moiety of 2, established by COSY, HSQC, and HMBC experiments,
was found to be identical to that of known plakortolide F (5), the
relative configuration of the peroxide ring was different. A NOESY
spectrum exhibited a strong correlation between the H-5R resonance
6
R by reductive cleavage of the peroxide ring in 5 to yield diol-lactone
derivative 12 as the sole detectable isomer (Scheme 4) followed by
application of Kishi’s method. Furthermore, as the ∆∆δ value
obtained for 12 was arguably low (Table 2), we confirmed the latter
assignment utilizing the diacetate 13, which was readily prepared from
9
observed at δ 2.16 and H
at δ 1.70 and both H -23 and H-3, suggesting that the relative
configuration of the CH -23 and CH -24 groups of plakortolide J
3
-24 and between the H-5ꢀ signal observed
3
1
2
2 after treatment with Ac O-pyridine. Thus, while the outcomes of
3
3
1
0
these measurements were comparable, insofar as both afforded the
is trans. This contention was supported by the shift differences
observed in the 13C NMR spectra of peroxide-lactones 2 and 5 for
the signals due to the angular methyls at C-6 (δ 22.4 in 2 vs 24.9
in 5) and the C-7 methylenes (δ 41.0 in 2 vs 37.0 in 5).
same absolute configuration for 5, we consider that the -0.01 value
for ∆∆δ in these instances is predictive (Scheme 2, Table 2).
Epiplakinidioic acid [3, (3S,5R)-3,5-dimethyl-3,5-peroxytetrade-
canedioic acid], C16 by HRESIMS, was isolated as a colorless
oil that showed a shortened side chain compared to compound 4.
13
28 6
H O
The remaining part of the molecule was established to be a
conjugated triene along the unbranched C16 alkyl chain on the basis
The IR spectrum revealed a broad hydroxy band centered at 3416
1
1
1
of interpretation of the UV, H NMR, and H- H COSY spectra.
-1
-1
1
cm and a carbonyl band at 1715 cm . Inspection of the H and
15
17
19
The position of the triene at ∆ , ∆ , and ∆ was determined from
the COSY, HSQC, and HMBC data. Long-range correlations were
observed in the HMBC experiment as follows: the terminal methyl
protons observed at δ 1.00 showed coupling to C-21 (δ 25.8) and
13
C NMR (see Experimental Section), COSY, HSQC, and HMBC
spectra of 3 indicated the presence of two carboxylic acid units,
located at the termini of the 14-carbon spin system starting from
C-1. A comparison of the H and C NMR spectra with those of
methyl ester 6 indicated that 3 was the corresponding diacid. All
1
13
C-20 (δ 135.9); the allylic protons H -21 (δ 2.08) showed coupling
2
1
to C-19 (δ 129.4), C-20 (δ 135.9), and C-22 (δ 13.6). The H NMR
spectrum indicated large J values (J ≈ 14 Hz) for the scalar coupling
between H-15 and H-16 and between H-19 and H-20, allowing for
other spectroscopic data, together with the [R]
D
value of +33.9 (c
1.2, CHCl ), support the absolute stereostructure assigned to 3. The
3
observation that compound 4 was also extremely unstable, decom-
posing rapidly over time, suggests that 3 might be formed during
the isolation process upon air oxidation of the double bond at C-14
15
19
the assignment of an E configuration to the ∆ and ∆ double
bonds. The configuration at ∆17 was deduced to be E, as the
13
C
chemical shift values for C-15-C-20 were found to be almost
identical to those of conjugated triene 1 in addition to the
remarkably similar absorption maxima observed in the UV spectra
of 1 and 2. Moreover, both C-16 and C-19 in 2 would be expected
to resonate at higher field (ca. 125 ppm) should the double bond at
14,15
of 4.
Naturally occurring cycloperoxides 1-5, as well as semisynthetic
derivatives 7-12, were evaluated using MTS assays in DU-145
prostate cancer and melanoma cells in the City of Hope (COH)
16
Comprehensive Cancer Center. Table 4 shows the behavior of
1
1,12
C-17 have the opposite Z configuration.
The absolute configuration of 2 was likewise determined upon
reductive cleavage of the peroxide-lactone ring with H and 10% Pd/C
these compounds in the DU-145 prostate cancer and A2058
melanoma cells. Fresh samples of 1 and 2, which might have
partially decomposed in transit, were not very active. Aged samples
of 1, which had previously rearranged to 6, were completely
2
in EtOAc to afford diol derivative 10 as the main product along with
minor peroxide-lactone derivative 11 (Scheme 3). Assignment of the
absolute configuration of 10 at C-3 following simple analysis of its
8
inactive. On the other hand, the prostate cancer and melanoma
cells were significantly more sensitive to compounds 3 and 11,
whereas epiplakininc acid F (4) was notably more toxic to the
prostate cancer cells. Also, Table 4 shows that semisynthetic
derivative 11 was surprisingly potent (less than 1 µM for the IC50
value) against the melanoma cells. The most abundant natural
1
3
C NMR behavior in CDCl
reagents established the absolute configuration of the peroxide ring as
S, 4S, 6S (Scheme 2, Table 2). In a similar manner, we also
determined the absolute configuration of plakortolide F (5) as 3S, 4S,
3
in the presence of chiral lanthanide shift
3

BioactiVe Cycloperoxides from Plakortis halichondrioides
Table 4. Biological Activities of Compounds 1-5 and 7-12
Journal of Natural Products, 2010, Vol. 73, No. 10 1697
cancer cell line
infectious microorganism
a
b
(
% growth)
(IC50 µM)
c
d
compound
DU-145
A2058
DU-145
A2058
P. falciparum
M. tuberculosis
1
2
3
4
5
7
8
9
92
112
31
15
62
38
81
102
79
13
86
106
23
51
71
69
79
60
75
8
>10
>10
4
1
>10
8
>10
>10
24
2
>10
>10
>10
3
>10
>10
>10
>10
>10
24
4
>10
0.3
3
62
71
N.T.
92
59
N.T.
e
>0
e
>10
>10
>10
0.6
0.3
7
e
N.T.
30
13
68
N.T.
0.05
1
1
1
0
1
2
0.8
>10
e
95
81
+
Ctrl
0.09
a
b
DU-145 prostate cancer and A2058 melanoma cells (5000 cells/well, 96-well plates) were treated with compounds at 10 µM for 48 h. Cells were
treated with compounds in a dose-dependent manner for 48 h. Then, the MTS assays assessed cell viability. Experiments in quadruplicate.
Chloroquine-resistant (CQ-R) W2 strains. IC50 in µg/mL. Chloroquine was used as a positive control. Mtb H37Rv strain. Minimum inhibitory
concentrations (MIC) in µg/mL. Rifampin was used as a positive control. N.T. ) not tested.
c
d
e
product, plakortolide F (5), was assayed in the NCI’s in Vitro
antitumor screen consisting of 60 human tumor cell lines, selectively
displaying potent cytotoxicity against LOX IMVI melanoma cancer,
IGROV1 ovarian cancer, and UO-30 renal cancer (the percent of
growth of the treated cells when compared to the untreated cells
was approximately 0, 0, and 17%, respectively). Of the compounds
tested for the inhibition of Plasmodium falciparum, the parasite
responsible for the most severe forms of malaria, compounds 3,
Nonetheless, many triods had rounded edges with a thick center. Triods
and diods were variable in size. Minimum diod length varied from
10 to 160 µm, and the maximum actine length of triod length varied
1
between 20 and 60 µm. The ectosome measured between 450 and 650
µm thick. A high density of unusual cavities formed a mesh that ran
perpendicular to the surface of the ectosome. An underwater photograph
of one of the sponge specimens is available as Supporting Information.
Collection, Extraction, and Isolation. Fresh specimens of the
sponge Plakortis halichondrioides (Wilson, 1902) (phylum Porifera;
class Demospongiae; subclass Homoscleromorpha; order Homoscle-
rophorida; family Plakinidae) were collected by hand using scuba at
depths of 90-100 ft off Mona Island, Puerto Rico, in July 2006. A
voucher specimen (No. IM06-09) is stored at the Chemistry Department
of the University of Puerto Rico, R ´ı o Piedras Campus. The organism
was frozen and lyophilized prior to extraction. The dry specimens (395
g) were cut into small pieces and blended in a mixture of
1
0, and 11 demonstrated the most toxic effects (IC50’s < 1 µg/mL)
(
Table 4). Interestingly, the in Vitro antimarial activity of 1,2-
dioxanes 2 and 5 was quite modest compared to that of 1,2-
dioxolanes 1 and 4. When taken together, these results alone cannot
account for the strong antimalarial activity shown by 3, 10 and 11.
In Vitro antituberculosis screening of compounds 1, 2, 4, 5, and 11
against Mycobacterium tuberculosis H37Rv showed moderate to
weak inhibitory activity (MIC g 50 µg/mL), whereas hydroxylac-
tones 9 and 10 significantly inhibited bacterial growth.
CHCl
concentrated and stored under vacuum to yield a dark gum (100 g),
which was suspended in H
O (2 L) and extracted with n-hexane (3 ×
L). Concentration under reduced pressure yielded 16.4 g of the
3
-MeOH (1:1) (11 × 1 L). After filtration, the crude extract was
2
2
Experimental Section
n-hexane extract as a dark brown oil, a portion of which (3.7 g) was
chromatographed over Si gel (130 g) using mixtures of
n-hexane-acetone of increasing polarity (0-100%). A total of 11
General Experimental Procedures. Optical rotations were obtained
with an Autopol IV automatic polarimeter. Infrared and UV spectra
were obtained with a Nicolet Magna FT-IR 750 spectrometer and a
Shimadzu UV-2401 PC UV-visible spectrophotometer, respectively.
1
fractions (I-XI) were generated on the basis of TLC and H NMR
analysis. Further purification of fraction II (1.3 g) by Si gel (20.0 g)
column chromatography in 2% acetone-n-hexane afforded eight
subfractions, denoted as A-H. Subfraction II(B) consisted of pure
epiplakinic acid F methyl ester (1) (153.4 mg, 0.17% yield), and
subfractions II(F) (47.0 mg) and II(G) (68.3 mg) were pooled together
1
D- and 2D-NMR spectra were recorded with a Bruker DRX-500 FT-
NMR spectrometer. Mass spectrometric data were generated at the Mass
Spectrometry Laboratory of the University of Illinois at Urbana-
Champaign. Column chromatography was performed using Si gel
(
35-75 mesh). TLC analysis was carried out using glass precoated Si
gel plates, and the spots were visualized using a UV lamp at λ ) 254
nm or by exposure to I vapor. All solvents used were either spectral
grade or distilled from glass prior to use. Commercially available chiral
shift reagents (R)-Eu(tfc) and (S)-Eu(tfc) were purchased from Sigma
and rechromatographed over Si gel (8.0 g) in 70% CHCl
containing several drops of glacial AcOH to yield pure plakortolide J
2) (18.6 mg, 0.02% yield). Purification of subfraction II(H) (659.1
mg) by Si gel (13.0 g) column chromatography using CHCl as eluant
3
-n-hexane
2
(
3
3
3
17
afforded epiplakinidioic acid (3) (4.6 mg, 0.005% yield) along with
known epiplakinic acid F (4) (16.3 mg, 0.02% yield). Further scrutiny
revealed that fraction IV consisted of pure plakortolide F (5) (196.3
Aldrich Co. and dried at 130 °C for 48 h prior to use. The percentage
yields of compounds 1-5 are based on the weight of the dry sponge
specimen. The trivial names assigned to compounds 1-3 are based on
7
4
a,5a
mg, 0.22% yield).
previous work by Rinehart and Davidson, respectively.
Methyl (3S,5R,14E,16E,18E)-3,5-dimethyl-3,5-peroxyeneicosa-
Animal Material. All individuals, found along the ceiling of cave
overhangs with a drop shape, were massively encrusting with irregular
conulose surface. Most specimens collected measured up to 20 cm long
and 5 cm thick. All Plakortis halichondrioides colonies were overgrown
with the thinly-encrusting sponge Xestospongia deweerdtae, which
provides a lavender pink crust over the olive green color of P.
halichondroides. However, specimens turned to a brownish color,
producing a dark exudate when brought to the surface. Individuals were
easily broken, with firm consistency. Noticeable oscules along the
surface were circular and measured 2.0-10.0 mm in diameter. The
choanosome was compact with many cavities. Both choanosome and
ectosome formed by high abundance of diods that were arranged
homogenously and densely over the sponge body. Diods were curved
with sharp edges. Straight triods with sharp edges were highly abundant.
1
4,16,18-trienoate (epiplakinic acid F methyl ester) (1): pale
2
0
yellowish oil; [R]
D
3 3
+34.4 (c 1.0, CHCl ); UV (CH OH) λmax (log ε)
2
1
57 (4.54), 267 (4.66), 278 (4.57) nm; IR (neat) νmax 3013, 2928, 2854,
-
1 1
3
740, 1455, 1437, 1375, 1346, 1209, 996 cm ; H NMR (CDCl , 500
1
3
+
MHz) and C NMR (CDCl
3
3
, 125 MHz), see Table 1; EIMS m/z [M]
92 (3), 173 (65), 141 (42), 117 (43), 99 (68), 81 (100), 57 (83), 55
+
(
96); HREIMS m/z [M] 392.2918 (calcd for C24
3S,4S,6S,15E,17E,19E)-4,6-Dimethyl-4-hydroxy-3,6-peroxydocosa-
15,17,19-trienoic acid 1,4-lactone (plakortolide J) (2): colorless oil;
40 4
H O , 392.2927).
(
2
0
[R]
D
+29.1 (c 1.1, CHCl
3 3
); UV (CH OH) λmax (log ε) 258 (4.62),
267 (4.75), 278 (4.67) nm; IR (neat) νmax 3011, 2963, 2924, 2851, 1767,
-
1 1
3
1456, 1267, 1175, 1159, 993, 916, 725 cm ; H NMR (CDCl , 500
1
3
+
3
MHz) and C NMR (CDCl , 125 MHz), see Table 3; EIMS m/z [M]

1
698 Journal of Natural Products, 2010, Vol. 73, No. 10
Jim e´ nez-Romero et al.
3
(
90 (5), 262 (5), 222 (4), 177 (22), 149 (84), 109 (25), 85 (65), 83
100); HREIMS m/z [M]+ 390.2756 (calcd for C24
, 390.2770).
3S,5R)-3,5-Dimethyl-3,5-peroxytetradecanedioic acid (epipla-
1115, 1036, 800 cm-1; 1H NMR (CDCl
3
, 500 MHz) δ 2.63 (dd, 1H, J
H
38
O
4
) 16.5, 2.0 Hz, H-2R), 2.51 (d, 1H, J ) 16.5 Hz, H-2ꢀ), 2.08 (dd, 1H,
(
J ) 14.5, 2.0 Hz, H-4R), 1.73 (d, 1H, J ) 14.5 Hz, H-4ꢀ), 1.80 (br m,
20
kinidioic acid) (3): colorless oil; [R]
D
+33.9 (c 1.2, CHCl
3
); IR (neat)
2H, H
28H), 0.89 (t, 3H, J ) 6.5 Hz, H
δ 170.3 (C, C-1), 83.7 (C, C-5), 69.3 (C, C-3), 45.1 (CH
(CH , C-2), 42.5 (CH , C-6), 31.9 (CH , C-7), 30.9 (CH
29.9-29.3 (11 × CH , C-8-C-18), 28.9 (CH
22.7 (CH , C-20), 14.1 (CH
2
-6), 1.41 (s, 6H, H -22 and H -23), 1.30-1.20 (br envelope,
3
3
-
1
13
ν
max 3416, 2930, 2855, 1715, 1456, 1376, 1306, 1217, 1091, 756 cm
H NMR (CDCl
;
3
-21); C NMR (CDCl
3
, 125 MHz)
, C-4), 43.9
, C-23),
2
, C-22), 24.1 (CH , C-19),
1
3
, 500 MHz) δ 2.78 (d, 1H, J ) 15.0 Hz, H-2R), 2.71
2
(
2
d, 1H, J ) 15.0 Hz, H-2ꢀ), 2.45 (d, 1H, J ) 12.5 Hz, H-4ꢀ), 2.34 (t,
2
2
2
3
H, J ) 7.5 Hz, H-13Rꢀ), 2.24 (d, 1H, J ) 12.5 Hz, H-4R), 1.70-1.49
-15), 1.29 (br envelope,
, 125 MHz) δ 172.2 (C,
, C-4),
, C-8-C-13),
, C-16); HRESIMS m/z
2
3
+
(
1
br m, 4H, H-6Rꢀ and H-12Rꢀ), 1.46 (s, 3H, H
3
2
3
, C-21); EIMS m/z [M - H
2
O] 350 (4),
1
3
3
35 (3), 290 (4), 266 (10), 143 (100), 125 (52), 103 (45), 101 (90), 85
3 3
3H, H-7-H-11 and H -16); C NMR (CDCl
(
15), 83 (20).
C-14), 172.1 (C, C-1), 86.6 (C, C-5), 83.9 (C, C-3), 55.6 (CH
4
2
2
Reduction of Plakortolide J (2): A mixture of plakortolide J (28.0
4.0 (CH
4.4 (CH
2
, C-2), 39.6 (CH
, C-7), 23.9 (CH
2
, C-6), 29.9-28.9 (6 × CH
, C-15), 23.2 (CH
Na, 339.1784).
2
mg, 0.071 mmol) and 10% Pd on charcoal in EtOAc (15 mL) was
stirred under H2 (1 atm) at 25 °C for 24 h. The reaction mixture
was filtered and concentrated in Vacuo to afford an oil, which was
chromatographed through a short plug of Si gel (0.8 g) using a 4:1
2
3
3
+
18
[
M + Na] 339.1777 (calcd for C16
Plakortolide F (5): yellowish oil; [R]
CH OH) λmax (log ε) 224 (3.82), 269 (3.43) nm; IR (neat) νmax 3417,
925, 2853, 1763, 1614, 1515, 1464, 1263, 1218, 1172, 955 cm
28 6
H O
2
0
D
3
+5.7 (c 1.1, CHCl ); UV
(
2
3
-
1
mixture of CHCl -EtOAc as eluant, thus providing pure 10 and 11.
.
3
20
Compound 10 (14.0 mg, 49%): white solid; [R]
D
3
+3.7 (c 0.8, CHCl );
Neither the specific rotation nor the UV data for 5 were previously
published. In addition, the original IR spectral data lack some of the
IR (neat) νmax 3487, 3345, 2915, 2850, 1767, 1470, 1379, 1292, 1201,
-
1 1
4
d,7
3
1170, 1070, 941 cm ; H NMR (CDCl , 500 MHz) δ 4.20 (d, 1H, J
key absorption bands described here.
Spontaneous Decomposition of Epiplakinic Acid F Methyl Ester
1). Compound 1 was very unstable, decomposing rapidly in CDCl
)
10.0 Hz, H-3), 2.92 (dd, 1H, J ) 20.0, 10.0 Hz, H-2ꢀ), 2.55 (d, 1H,
J ) 20.0 Hz, H-2R), 2.17 (d, 1H, J ) 15.0 Hz, H-5R), 2.09 (d, 1H, J
-23), 1.35
-24), 1.28-1.25 (br envelope, 28H), 0.88 (t, 3H, J ) 5.0 Hz,
(
3
)
15.0 Hz, H-5ꢀ), 1.60 (br m, 2H, H-7Rꢀ), 1.44 (s, 3H, H
3
solution in the presence of air and light. Following purification of the
decomposition mixture by Si gel column chromatography using 10%
EtOAc in hexane as eluant, compound 6 was identified as the sole
(
s, 3H, H
3
3
1
H
3
-22); C NMR (CDCl
3
, 125 MHz) δ 175.3 (C, C-1), 90.1 (C, C-4),
, C-5), 43.6 (CH , C-7), 38.1
, C-24), 29.7-29.3 (11 × CH
, C-8), 22.7 (CH , C-21), 14.1
365 (3), 309 (17), 292
5), 269 (14), 173 (38), 155 (87), 130 (13), 113 (100), 95 (19), 71
2
0
73.8 (CH, C-3), 73.0 (C, C-6), 43.9 (CH
CH , C-2), 31.9 (CH , C-20), 30.0 (CH
C-9-C-19), 26.8 (CH , C-23), 24.4 (CH
, C-22); EIMS m/z [M - H O - CH
2
2
product: colorless oil; [R]
D
+32.3 (c 1.6, CHCl
3
); IR (neat) νmax
(
2
2
3
2
,
3
1
700-3000 (br), 2926, 2854, 1732, 1715-1682 (br), 1456, 1374, 1260,
-
1
1
3
2
2
091 cm ; H NMR (CDCl
3
3
, 300 MHz) δ 3.69 (s, 3H, -OCH ), 2.77
+
(
CH
3
2
3
]
(
d, 1H, J ) 14.5 Hz, H-2R), 2.65 (d, 1H, J ) 14.5 Hz, H-2ꢀ), 2.47 (d,
(
1
1
1
H, J ) 12.3 Hz, H-4ꢀ), 2.34 (t, 2H, J ) 7.8 Hz, H-13Rꢀ), 2.23 (d,
+
(
26); HRESIMS m/z [M + H] 399.3483 (calcd for C24H O ,
47 4
H, J ) 12.3 Hz, H-4R), 1.72-1.48 (br m, 4H, H-6Rꢀ and H-12Rꢀ),
20
3
1
1
1
1
1
1
H
8
(
99.3474). Compound 11 (5.0 mg, 18%): white solid; [R]
.2, CHCl ); IR (film) νmax 2920, 2851, 1769, 1468, 1379, 1268, 1176,
080, 1057, 954, 917 cm ; H NMR (CDCl
D
+27.5 (c
.43 (s, 3H, H
3
-15), 1.28 (br envelope, 13H, H-7-H-11 and H
C NMR (CDCl
, 75 MHz) δ 171.1 (2 × C, C-1 and C-14), 86.5 (C,
C-5), 83.9 (C, C-3), 55.4 (CH , C-4), 51.7 (CH , -OCH ), 44.0 (CH
C-2), 39.6 (CH , C-6), 30.9 (CH , C-8-C-
, C-13), 29.9-29.4 (5 × CH
2), 24.5 (CH , C-7), 24.1 (CH , C-15), 23.2 (CH , C-16); HRESIMS
m/z [M + Na] 353.1930 (calcd for C17 Na, 353.1940).
3
-16);
13
3
3
-
1 1
3
, 500 MHz) δ 4.46 (d,
2
3
3
2
,
H, J ) 5.0 Hz, H-3), 2.91 (dd, 1H, J ) 20.0, 5.0 Hz, H-2ꢀ), 2.62 (d,
2
2
2
H, J ) 20.0 Hz, H-2R), 2.16 (d, 1H, J ) 15.0 Hz, H-5R), 1.70 (d,
1
2
+
3
3
H, J ) 15.0 Hz, H-5ꢀ), 1.51 (m, 2H, H-7Rꢀ), 1.39 (s, 3H, H
3
-23),
-24), 1.25 (br envelope, 28H), 0.88 (t, 3H, J ) 5.0 Hz,
-22); C NMR (CDCl , 125 MHz) δ 174.3 (C, C-1), 82.8 (C, C-4),
1.1 (CH, C-3), 80.1 (C, C-6), 41.0 (CH , C-7), 40.5 (CH , C-5), 34.3
CH , C-2), 31.9 (CH , C-20), 30.0 (CH
, C-8), 29.7-29.4 (11 × CH
C-9-C-19), 25.9 (CH , C-23), 23.1 (CH , C-21), 22.7 (CH , C-24), 14.1
171 (50), 155 (48), 101 (42),
30 6
H O
.28 (s, 3H, H
3
3
Reduction of Epiplakinic Acid F Methyl Ester. A mixture of
compound 1 (52.8 mg, 0.134 mmol) and 10% Pd on charcoal in EtOAc
13
3
2
2
(
15 mL) was stirred under H
mixture was filtered and concentrated in Vacuo, and the residue obtained
was eluted through a short plug of Si gel (1.0 g) with 100% CHCl
2
(1 atm) at 25 °C for 24 h. The reaction
2
2
2
2
,
3
2
+
3
3
,
(
8
CH
3
, C-22); EIMS m/z [M - C16
5 (68), 83 (100), 71 (88); HRESIMS m/z [M + H] 397.3316 (calcd
, 397.3318).
33
H ]
affording two fractions. The least polar fraction (23.9 mg) was purified
further by flash CC over Si gel (0.7 g) using a 9:1 mixture of
hexane-EtOAc to afford peroxide ester derivative 7 (3.8 mg, 7%):
+
45 4
for C24H O
Reduction of Plakortolide F (5). A mixture of plakortolide F (70.0
mg, 0.179 mmol) and 10% Pd on charcoal in EtOAc (15 mL) was
2
0
yellow oil; [R]
D
+36.0 (c 0.8, CHCl
738, 1463, 1375, 1220, 1163, 1097, 1011 cm ; H NMR (CDCl
00 MHz) δ 3.69 (s, 3H, -OCH ), 2.76 (d, 1H, J ) 15.0 Hz, H-2R),
3
); IR (neat) νmax 2917, 2849,
-
1 1
1
5
2
2
3
,
stirred under H (1 atm) at 25 °C for 24 h. The reaction mixture was
2
3
filtered and concentrated in Vacuo to afford a homogeneous oil that
.65 (d, 1H, J ) 15.0 Hz, H-2ꢀ), 2.46 (d, 1H, J ) 15.0 Hz, H-4ꢀ),
.22 (d, 1H, J ) 15.0 Hz, H-4R), 1.59 (br m, 2H, H-6Rꢀ), 1.43 (s, 3H,
was passed through a short plug of Si gel (1.0 g) using a 4.5:0.5 mixture
of CHCl -EtOAc to obtain diol-lactone derivative 12 (17.4 mg, 25%):
3
H
5
(
(
2
2
3
-22), 1.28 (s, 3H, H
3
-23), 1.25 (br envelope, 28H), 0.88 (t, 3H, J )
-21); C NMR (CDCl , 125 MHz) δ 171.1 (C, C-1), 86.5
C, C-5), 83.9 (C, C-3), 55.4 (CH , C-4), 51.8 (CH , -OCH ), 44.0
CH , C-2), 39.7 (CH , C-6), 31.9 (CH , C-19), 30.1 (CH , C-7),
9.7-29.4 (11 × CH , C-8-C-18), 24.2 (CH , C-22), 23.3 (CH , C-23),
2.7 (CH , C-20), 14.1 (CH , C-21); ESIMS m/z [M + H] 399.3 (calcd
, 399.3). The more polar fraction was identified as diol-
20
white solid; [R]
D
+31 (c 0.2, CHCl
3 3
); UV (CH OH) (log ε) λmax 224
1
3
.0 Hz, H
3
3
(
1
4.16), 279 (3.56) nm; IR (neat) νmax 3381, 2918, 2848, 1729, 1615,
-1 1
2
3
3
6
518, 1465, 1384, 1249, 1200, 1077, 934 cm ; H NMR (acetone-d ,
2
2
2
2
5
00 MHz) δ 7.00 (d, 2H, J ) 5.0 Hz, H-17), 6.72 (d, 2H, J ) 5.0 Hz,
2
3
3
H-18), 5.05 (br s, 1H, exchangeable, 3-OH), 4.39 (br s, 1H, exchange-
+
2
3
able, 6-OH), 4.22 (d, 1H, J ) 10.0 Hz, H-3), 2.98 (dd, 1H, J ) 20.0,
for C24
ester derivative 8 (15.4 mg, 29%): colorless oil; [R]
CHCl ); IR (neat) νmax 3400, 2920, 2851, 1738, 1467, 1439, 1377, 1342,
47 4
H O
5
2
1
3
.0 Hz, H-2ꢀ), 2.49 (t, 2H, J ) 5.0 Hz, H-15Rꢀ), 2.25 (d, 1H, J )
0.0 Hz, H-2R), 2.22 (d, 1H, J ) 15.0 Hz, H-5R), 1.85 (d, 1H, J )
5.0 Hz, H-5ꢀ), 1.56-1.53 (br m, 4H, H-7R, H-8R, H-14Rꢀ), 1.47 (s,
2
0
D
+8.9 (c 1.4,
3
-1 1
1
2
1
1
206, 1013 cm ; H NMR (CDCl
3 3
, 500 MHz) δ 3.71 (s, 3H, -OCH ),
H, H
3
-20), 1.40 (br m, 2H, H-7ꢀ, H-8ꢀ), 1.35 (s, 3H, H
3
-21),
1
3
.83 (d, 1H, J ) 15.0 Hz, H-2R), 2.50 (d, 1H, J ) 15.0 Hz, H-2ꢀ),
1.30-1.28 (br envelope, 10H); C NMR (acetone-d , 125 MHz) δ
6
.80 (d, 1H, J ) 15.0 Hz, H-4ꢀ), 1.70 (d, 1H, J ) 15.0 Hz, H-4R),
175.0 (C, C-1), 156.0 (C, C-19), 134.1 (C, C-16), 129.8 (2 × CH,
.52 (br m, 2H, H-6Rꢀ), 1.35 (s, 3H, H
3
-22), 1.27 (s, 3H, H
3
-23), 1.25
C-17), 115.7 (2 × CH, C-18), 90.6 (C, C-4), 74.0 (CH, C-3), 72.0 (C,
13
(
br envelope, 28H), 0.87 (t, 3H, J ) 5.0 Hz, H
25 MHz) δ 173.4 (C, C-1), 73.4 (C, C-5), 72.3 (C, C-3), 51.7 (CH
OCH ), 49.0 (CH , C-4), 46.1 (CH , C-2), 44.8 (CH
C-19), 30.2 (CH , C-8-C-18), 29.6 (CH
, C-7), 29.7-29.3 (11 × CH
C-22), 28.8 (CH , C-23), 22.7 (CH , C-20), 14.1 (CH , C-21); ESIMS
m/z [M + H] 401.3 (calcd for C24 , 401.3).
3
-21); C NMR (CDCl
3
,
C-6), 47.0 (CH , C-7), 45.2 (CH , C-5), 38.2 (CH , C-2), 35.5 (CH ,
2
2
2
2
1
-
3
,
C-15), 32.5 (CH , C-14), 30.8 (CH , C-13), 29.0 (4 × CH , C-9-C-
2
2
2
3
2
2
2
, C-6), 31.9 (CH
2
,
12), 27.9 (CH , C-20), 25.5 (CH , C-21), 24.4 (CH , C-8); EIMS m/z
3 3 2
+
2
2
3
,
15 23
[M-C H O] 173 (6), 155 (100), 113 (20), 107 (98); ESIMS m/z [M
+
3
2
3
+ Li] 399.3 (calcd for C H O Li, 399.3).
2
3
36
5
+
H
49
O
4
Acetylation of Diol-Lactone Derivative 12. A mixture of diol-
lactone 12 (40.0 mg, 0.102 mmol), dry pyridine (2.0 mL), and acetic
anhydride (0.2 mL) was stirred at 25 °C for 24 h. The reaction mixture
was concentrated in Vacuo, and the oily residue obtained was passed
through a short plug of Si gel (0.7 g) using a 7:3 mixture of
n-hexane-acetone to afford diacetate 13 (38 mg, 95%): yellowish oil;
Synthesis of δ-Lactone 9. A solution of diol ester derivative 8 (15.4
mg, 0.038 mmol) in hexane (15 mL) was treated with glacial acetic
acid (3 drops) and heated to 60 °C for 2 h. Evaporation in Vacuo
afforded pure δ-lactone 9 (8.0 mg, 56%): white solid; [R]
1
2
0
D
+14.6 (c
.0, CHCl ); IR (neat) νmax 3470, 2920, 2851, 1703, 1468, 1379, 1145,
3

BioactiVe Cycloperoxides from Plakortis halichondrioides
Journal of Natural Products, 2010, Vol. 73, No. 10 1699
2
0
[
(
1
R]
D
+8.2 (c 1.2, CHCl
3
); UV (CH
3
OH) (log ε) λmax 202 (3.78), 265
HSQC, HMBC, NOESY, and MS spectra of cycloperoxides 1, 2, and
6. This material is available free of charge via the Internet at http://
pubs.acs.org.
2.58), 271 (2.55) nm; IR (neat) νmax 3516, 2926, 2854, 1747, 1508,
464, 1372, 1234, 1046, 944, 913 cm ; H NMR (CDCl , 500 MHz)
3
-
1 1
δ 7.16 (d, 2H, J ) 5.0 Hz, H-17), 6.97 (d, 2H, J ) 5.0 Hz, H-18), 5.18
(
(
(
1
3
d, 1H, J ) 10.0 Hz, H-3), 3.03 (dd, 1H, J ) 20.0, 5.0 Hz, H-2ꢀ), 2.57
References and Notes
t, 2H, J ) 5.0 Hz, H-15Rꢀ), 2.45 (d, 1H, J ) 20.0 Hz, H-2R), 2.27
(
1) (a) Casteel, D. A. Nat. Prod. Rep. 1992, 9, 289–312. (b) Casteel, D. A.
Nat. Prod. Rep. 1999, 16, 55–73. (c) Rahm, F.; Hayes, P. Y.; Kitching,
W. Heterocycles 2004, 64, 523–575. (d) Dembitsky, V. M.; Glorio-
zova, T. A.; Poroikov, V. V. Mini-ReV. Med. Chem. 2007, 7, 571–
s, 3H, OCOCH
3
), 2.09 (s, 3H, OCOCH
5.0 Hz, H-5Rꢀ), 1.58 (s, 3H, H -20), 1.47 (m, 2H, H-7Rꢀ), 1.31 (s,
H, H -21), 1.29-1.25 (br envelope, 14H); C NMR (CDCl
), 169.6 (C, OCOCH
3
), 1.92 (dd, 2H, J ) 20.0,
3
1
3
3
3
, 125
),
MHz) δ 173.7 (C, C-1), 169.8 (C, OCOCH
1
CH, C-18), 88.1 (C, C-4), 75.8 (CH, C-3), 72.2 (C, C-6), 45.8 (CH
C-7), 43.2 (CH , C-5), 35.7 (CH , C-2), 35.3 (CH , C-15), 31.4 (CH
, C-13), 29.6-29.2 (4 × CH
C-14), 30.0 (CH , C-9-C-12), 27.9 (CH
C-20), 24.6 (CH , C-21), 23.7 (CH , C-8), 21.1 (CH , OCOCH
3
3
5
89. (e) Thomas, T. R. A.; Kavlekar, D. P.; LokaBharathi, P. A. Mar.
48.5 (C, C-19), 140.4 (C, C-16), 129.2 (2 × CH, C-17), 121.1 (2 ×
Drugs 2010, 8, 1417–1468.
2
,
2
,
3
,
(2) (a) Taglialatela-Scafati, O.; Fattorusso, E.; Romano, A.; Scala, F.;
Barone, V.; Cimino, P.; Stendardo, E.; Catalanotti, B.; Persico, M.;
Fattorusso, C. Org. Biomol. Chem. 2010, 8, 846–856. (b) Feng, Y.;
Davis, R. A.; Sykes, M.; Avery, V. M.; Camp, D.; Quinn, R. J. J.
Nat. Prod. 2010, 73, 716–719. (c) Kossuga, M. H.; Nascimento, A. M.;
Reim a˜ o, J. Q.; Tempone, A. G.; Taniwaki, N. N.; Veloso, K.; Ferreira,
A. G.; Cavalcanti, B. C.; Pessoa, C.; Moraes, M. O.; Mayer, A. M. S.;
Hajdu, E.; Berlinck, R. G. S. J. Nat. Prod. 2008, 71, 334–339.
(3) Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.; Prinsep,
M. R. Nat. Prod. Rep 2010, 27, 165–237, and previous articles in this
series.
2
2
2
2
2
3
2
3
3
), 20.9
+
(
CH
3
, OCOCH
3
); ESIMS m/z [M - H
2
O + Na] 481.3 (calcd for
O + H] 459.3 (calcd for
+
C
C
27
H
38
O
O
6
Na, 481.3) and m/z [M - H
2
27
H
39
6
, 459.3).
Determination of Absolute Configuration of Alcohols 9, 10, 12,
and 13. Samples were prepared individually in an oven-dried vial with
3.5-4.5 mg of alcohols 9, 10, 12, and 13 in 0.5 mL of C , CDCl
acetone-d , and CDCl , respectively, containing 15% per OH of chiral
shift reagent. The samples were transferred to oven-dried NMR tubes
∼
6
D
6
3
,
(
4) (a) Phillipson, D. W.; Rinehart, K. L., Jr. J. Am. Chem. Soc. 1983,
6
3
1
05, 7735–7736. (b) Davidson, B. S. J. Org. Chem. 1991, 56, 6722–
6724. (c) Horton, P. A.; Longley, R. E.; Kelly-Borges, M.; McConnell,
1
7
(
cooled to room temperature under a stream of nitrogen) via syringe.
O. J.; Ballas, L. M. J. Nat. Prod. 1994, 57, 1374–1381. (d) Chen, Y.;
Killday, K. B.; McCarthy, P. J.; Schimoler, R.; Chilson, K.; Selitren-
nikoff, C.; Pomponi, S. A.; Wright, A. E. J. Nat. Prod. 2001, 64,
Following acquisition, each substrate was recovered by filtration through
a pipet column of Si gel (Sep-Pak Si gel cartridge). For each filtration
CH Cl was used as the solvent. To determine the absolute configuration
2 2
of the alcohols in 9, 10, 12, and 13 according to a method described
by Kishi and co-workers, the NMR behaviors of the carbons adjacent
to the alcoholic center (CX and CY) were measured in the presence of
2
62–264. (e) Dalisay, D. S.; Quach, T.; Nicholas, G. N.; Molinski,
T. F. Angew. Chem. Int. Ed. 2009, 48, 4367–4371. (f) Dalisay, D. S.;
Quach, T.; Molinski, T. F. Org. Lett. 2010, 12, 1527–1527.
(
(
5) (a) Davidson, B. S. Tetrahedron Lett. 1991, 32, 7167–7170. (b)
Varoglu, M.; Peters, B. M.; Crews, P. J. Nat. Prod. 1995, 58, 27–36.
(c) Qureshi, A.; Salv a´ , J.; Harper, M. K.; Faulkner, D. J. J. Nat. Prod.
1998, 61, 1539–1542. (d) Perry, T. L.; Dickerson, A.; Khan, A. A.;
Kondru, R. K.; Beratan, D. N.; Wipf, P.; Kelly, M.; Hamann, M. T.
Tetrahedron 2001, 57, 1483–1487. (e) Rudi, A.; Afanii, R.; Gravalos,
L. G.; Aknin, M.; Gaydou, E.; Vacelet, J.; Kashman, Y. J. Nat. Prod.
9
,19
(
R)- and (S)-Eu(tfc)
3
(see Table 2).
Cytotoxicity Assay. DU-145 human prostate cancer, A2058
malanoma, and MDA-MB-435 breast cancer cell lines were obtained
from ATCC. These cells were cultured in RPMI-1640 or DMEM
medium containing 10% fetal bovine serum (FBS), 100 units/mL
penicillin, and 100 µg/mL streptomycin. All cells were maintained
in a 5% CO atmosphere at 37 °C. To determine the viability of the
cells, Promega CellTiter 96 aqueous nonradioactive cell proliferation
assays (MTS) were performed as described by the supplier (Promega;
2
003, 66, 682–685.
6) (a) del Sol Jim e´ nez, M.; Garz o´ n, S. P.; Rodr ´ı guez, A. D. J. Nat. Prod.
2003, 66, 655–661. (b) del Sol Jim e´ nez, M.; Garz o´ n, S. P.; Rodr ´ı guez,
A. D. J. Nat. Prod. 2003, 66, 1404.
2
(7) The trivial name plakortolide F was claimed simultaneously by
Hamann (ref 5d) and Wright (ref 4d) for two peroxide-lactones isolated
from distinct Plakinastrella sponge species. The compound herein
referred to as plakortolide F (i. e., 5) was identical to the material
reported by the Wright group.
2
0
Madison, WI). Briefly, cells (5000/well) were seeded in 96-well
plates and incubated overnight at 37 °C in 5% CO . Cells were
treated for 48 h with each compound. The concentration used was
0 µM. Dimethyl sulfoxide (DMSO) was used as the vehicle control.
2
1
(
8) Compound 6 showed no cytotoxicity against DU-145 prostate cancer
or A2058 melanoma cells at a concentration of 10 µM, nor was it
active against Mycobacterium tuberculosis H37Rv at concentrations
g 64 µg/mL.
IC50 values of compounds were determined in a dose-dependent
manner (0.1, 0.5, 1, 5, 10, 20, and 50 µM). Cell viability was
determined by tetrazolium conversion to its formazan dye, and
absorbance of formazan was measured at 490 nm using an automated
ELISA plate reader. The production of formazan dye was directly
proportional to the number of living cells. Each experiment was
done in quadruplicate in the absence of a positive control.
Antituberculosis and Antiplasmodial Assays. Biological activity
tests against the pathogenic microbes Mycobacterium tuberculosis and
Plasmodium falciparum were performed as previously described.
(
9) (a) Ghosh, I.; Zeng, H.; Kishi, Y. Org. Lett. 2004, 6, 4715–4718. (b)
Adams, C. M.; Ghosh, I.; Kishi, Y. Org. Lett. 2004, 6, 4723–4726.
(
10) For related peroxide-lactones having trans methyl substituents at the
C-4 and C-6 positions, see refs 5b and 5d.
(11) Yasuda, I.; Takeya, K.; Itokawa, H. Phytochemistry 1982, 21, 1295–
1298.
(12) Guella, G.; Mancini, I.; Pietra, F. HelV. Chim. Acta 1989, 72, 1121–
2
1
1124.
(
(
13) The lowest value for ∆∆δ in the article by Kishi and coworkers (ref
Rifampin and chloroquine were used as positive controls, respectively.
9
a) is-0.029.
14) The conjugated polyene chain of compounds 1, 2, and 4 is a
reactive, electron-rich system that is likely susceptible to attack
by electrophilic reagents such as hydroxy and peroxy radicals and
thus is responsible for the instability of these compounds toward
oxidation.
Acknowledgment. We thank J. Marrero, J. C. Asencio, R. Rios,
J. J. La Clair, M. J. Lear, and the crew of the R/V Pez Mar for
providing logistic support during the sponge collection. The National
Cancer Institute (NCI), the Institute for Tuberculosis Research at
the University of Illinois at Chicago, and the INDICASAT (Clayton,
Panama) provided in Vitro cytotoxicity, antituberculosis, and anti-
malarial activity data, respectively. Mass spectral determinations
were provided by the Mass Spectrometry Laboratory of the
University of Illinois at Urbana-Champaign. The expert assistance
of Dr. V. Vicente for sponge taxonomy determination is gratefully
acknowledged. Financial support to C.J.-R. was provided by the
IFARHU-SENACYT Program of the Republic of Panama. This work
was supported by a grant from the NIH-SC1 Program (Grant
(
15) Given their extreme chemical instability, we speculate that Nature
uses unsaturated conjugated polyenes such as 1, 2, and 4 as
biogenetic precursors to the phenyl polyketide peroxide series of
Plakortis/Plakinastrella metabolites (i.e., the plakortolides) via a
cascade of efficient E/Z-isomerizations, electrocyclizations, and
further dehydrogenations to account for aromatic ring formation.
For articles suggesting that some of these conversions are chemi-
cally viable, see: (a) Jacobsen, M. F.; Moses, J. E.; Adlington,
R. M.; Baldwin, J. E. Tetrahedron 2006, 62, 1675–1689. (b)
Rodr ´ı guez, R.; Adlington, R. M.; Eade, S. J.; Walter, M. W.;
Baldwin, J. E.; Moses, J. E. Tetrahedron 2007, 63, 4500–4509.
16) Wright and co-workers reported that free acid 4 showed moderate
antifungal activity against Candida albicans, whereas 5 was inactive
against both C. albicans and Aspergillus fumigatus; see ref 4d.
17) In order to obtain reliable results, it is essential that both R- and
S-enantiomers of the shift reagent be of similar quality (purity and
(
(
1
SC1GM086271-01A1) awarded to A.D.R.
Supporting Information Available: An underwater photograph of
1
13
Plakortis halichondrioides and copies of the H NMR, C NMR,

1
700 Journal of Natural Products, 2010, Vol. 73, No. 10
Jim e´ nez-Romero et al.
moisture content) and that NMR data with both the R- and S-shift
(20) (a) Malich, G.; Markovic, B.; Winder, C. Toxicology 1997, 124, 179–
192. (b) Nam, S.; Williams, A.; Vultur, A.; List, A.; Bhalla, K.;
Smith, D.; Lee, F. Y.; Jove, R. Mol. Cancer Ther. 2007, 6, 1400–
reagent be collected on the same instrument.
(
18) The 13C NMR chemical shifts assigned to the carbonyl carbons of 3
are not very accurate, as these signals were weak and broad. The
assignments shown can be interchanged.
1
405.
21) Wei, X.; Rodr ´ı guez, A. D.; Baran, P.; Raptis, R. G. J. Nat. Prod.
010, 73, 925–934.
(
(
19) It has been noted that ∆δ are always greater with chiral Pr-based shift
reagents than with the corresponding Eu-based shift reagents. Thus,
the former lanthanide shift reagents have a superior capacity for
enantiotopic discrimination of carbons within a molecule; see ref 9a.
2
NP100461T