614 J. Nat. Prod. 2005, 68, 614-616 
Leptolide, a New Furanocembranolide Diterpene from Leptogorgia alba 
Marcelino Guti?rrez,^ Todd L. Capson,* H?ctor M. Guzman,* Jos? Gonz?lez,^ Eduardo Ortega-Barr?a,^ 
Emilio Qui?o?,'f and Ricardo Riguera*'f 
Departamento de Qu?mica Org?nica, Instituto de Acuicultura y Unidad de RMN de Biomol?culas Asociada al CSIC, 
Universidad de Santiago de Compostela, E-15782 Santiago de Compostela, Spain, Smithsonian Tropical Research Institute, 
2072 Balboa, Anc?n, Republic of Panama, and Institute of Advanced Scientific Investigations and High Technology Services, 
Secretariat for Science and Technology, Clayton, Panama 
Received August 3, 2004 
Six furanocembranolides (1?6) and one pseudopterolide (7) have been isolated from the octocorals 
Leptogorgia alba and Leptogorgia r?gida, collected on the Pacific coast of Panama. Compound 1, named 
leptolide, has a new structure closely related to the neurotoxin lophotoxin (3). The X-ray structures of 
1?3 were determined, and the absolute configurations of 2?7 are discussed. Compounds 1?7 were 
evaluated in vitro against drug-resistant Plasmodium falciparum. 
Gorgonian octocorals are marine invertebrates that are 
a well-known source of novel natural products, ^-^ including 
highly functionalized diterpenes such as the furanocem- 
branolides.^ Thought to be used as chemical defenses 
against fish pr?dation,^* the furanocembranolides display 
a range of biological activities.^^^ 
In the course of a joint program between the University 
of Santiago de Compostela and the Panama International 
Cooperative Biodiversity Groups program (Panama ICBG), 
several specimens of octocorals were collected by scuba in 
the Coiba National Park, a marine protected area located 
in the Gulf of Chiriqui, in the southwest Pacific of the 
Republic of Panama.^ Among the octocorals collected were 
two species in the genus Leptogorgia (Gorgoniidae), L. alba 
and L. r?gida, whose crude extracts showed antiplasmodial 
activity in a novel in vitro fluorescence-based bioassay 
developed by the Panama ICBG.^ The crude extracts were 
subjected to bioassay-guided fractionation, yielding diter- 
penes 1?7. Compound 1 is a novel natural product, and 
its structure was deduced by ID and 2D NMR spectrometry 
and HREIMS and confirmed by single-crystal X-ray crys- 
tallography. Diterpenes 1?7 were evaluated for activity 
against a chloroquine-resistant strain o? Plasmodium fal- 
ciparum. 
The MeOH extracts of L. alba and L. r?gida showed 
antiplasmodial activity and were subjected to activity- 
guided fractionation. L. alba was subjected to liquid?liquid 
partition, and the active CH2Cl2-soluble material was 
fractionated using silica gel vacuum liquid chromatography 
(VLC) followed by silica gel flash chromatography, affording 
the new compound 1 and two known compounds identified 
as pukalide aldehyde (2)? and lophotoxin (3).^ The MeOH 
extract of L. r?gida was fractionated following the same 
protocol described for L. alba, resulting in the isolation of 
four known compounds identified as pukalide (4),^" lop- 
holide (5),11 13a-acetoxypukalide (6),^^ and deoxypseudo- 
pterolide (7).12 
Compound 1 showed a molecular ion at m/z 358 in the 
EIMS. The i^C NMR spectrum showed resonances for 20 
carbon atoms, while DEPT and HSQC experiments re- 
* To whom correspondence should be addressed. Tel/Fax: +34981591091. 
E-mail: ricardo@usc.es. 
^ University of Santiago de Compostela. 
* Smithsonian Tropical Research Institute. 
^ Institute of Advanced Scientific Investigations and High Technology 
Services. 
1 Rl = R2 = H 
3 Ri = H; R2 = OAc 
5 Ri = OMe; R2 = OAc 
2 Ri = R2 = H 
4 Ri = OMe; R2 = H 
6 Ri = OMe; R, = OAc 
vealed seven quaternary carbons, six methines, five m?th- 
yl?nes, and two methyl groups. These data and the HRE- 
IMS indicated the molecular formula, C20H22O6, requiring 
10 degrees of unsaturation. Detailed examination of ^^C, 
^H, and DEPT NMR spectra and comparison with litera- 
ture data indicated that compound 1 is a diterpene with a 
structure similar to ll/3,12/3-epoxypukalide,i^ with the 
primary difference being an aldehyde substituent bonded 
to C-4 of the furan ring. Evidence of the aldehyde function 
at C-4 was provided by correlations observed in 2D NMR 
experiments (Figure 1). The structure and relative stereo- 
chemistry of 1 were confirmed by single-crystal X-ray 
crystallography. 
Compounds 2, 4, 6, and 7 have an a,/3-unsaturated 
y-lactone moiety as common feature, and although they 
have been previously described in the literature,^!" ^^ ^j-^g 
absolute stereochemistry of these compounds had not been 
established. Previous studies with the a,/3-unsaturated 
y-lactone chromophore showed that the sign of the Cotton 
effect due to the n?jr* and Ji?Ji* transitions may be 
correlated directly with the absolute configuration of the 
stereogenic center at the y-carbon of the butenolide moi- 
ety.^*-^^ Interpretation of the CD data for compounds 2, 4, 
6, and 7 (Table 1) following the butenolide configurational 
rule^^ and the exciton chirality method^^ revealed that the 
configuration at C-10 in compounds 2, 4, and 6 is S, while 
10.1021/np049745z CCC: $30.25 ) 2005 American Chemical Society and American Society of Pharmacognosy 
Published on Web 04/05/2005 
Notes Journal of Natural Products, 2005, Vol. 68, No. 4    615 
? TOCSY(120ms) 
??HMBC 
Figure 1.   Selected 2D NMR correlations of compound 1. 
Table 1.  CD Data of Compounds 2, 4,6, and 7? 
CD, Ae (nm) 
compound n?ji* jt- -JT* 
-0.8 (276) 
-0.7 (262) 
-0.6 (263) 
0.6 (274) 
7.4 (218) 
9.3 (226) 
8.3 (226) 
8.7 (224) 
? Measured in MeCN at c =1 x 10"" M. 
Figure 2. ORTEP diagrams for compounds 1-3 with 30% thermal 
probability ellipsoids. Absolute configuration is implied only in the case 
of compound 2. 
the configuration at C-8 in compound 7 is R. Monocrystals 
suitable for X-ray analysis of compounds 1?3 were ob- 
tained and examined (Figure 2). Compound 2 crystallized 
in association with a molecule of chloroform (C2oH2205- 
CHCI3), and the anomalous dispersion of the chlorine atoms 
allowed us to assign its absolute configuration as IR, IS, 
8S, IOS (Figure 2), a result in agreement with the data 
obtained from the CD experiments. 
While Fenical and colleagues carried out a series of 
chemical interconversions to show that compounds 3, 4, 5, 
and 6 belong to the same stereochemical series, their 
absolute configurations remained unknown.^ The determi- 
nation of the absolute configuration of 4 and 6 in the 
present study allowed us to use the interconversion data 
to assign the absolute configuration of 3 and 5 as depicted 
in the structure block. 
Compounds 1?7 were tested in vitro against a chloro- 
quine-resistant strain of P. falciparum? Compounds 2, 5, 
and 6 exhibited moderate activity (IC50 = 134, 105, 109 
fjM., respectively), whereas compounds 3 and 7 showed 
more significant activity against the parasite (ICso = 74 
and 50 ?JM., respectively). Compounds 1 and 4 showed no 
measurable activity against the parasite. This is the first 
report of activity against P. falciparum for any member of 
the furanocembranolide class of compounds. 
Experimental Section 
General Experimental Procedures. Optical rotations 
were measured on a JASCO DIP-360 polarirtieter using a 1 
dm path length cell. UV spectra were measured in ethanol in 
a Hewlett-Packard 8452A diode array spectrophotometer. CD 
spectra were obtained in MeCN in a JASCO J-720 spectropo- 
larimeter. IR spectra were recorded in a Bruker IFS-66v (film, 
KBr). NMR spectra were recorded in d-chloroforrti at 250, 400, 
or 500 MHz using a Bruker DPX-250, Varian Inova-400, and 
Bruker AMX-500, respectively. HREIMS were measured on a 
Micromass Autospec mass spectrometer. X-ray crystallo- 
graphic data collection was carried out with a Bruker Nonius 
FR591-Kappa-CCD-2000 diffractometer or with a Bruker 
Smart-CCD-1000 diffractometer. Column chromatography was 
performed on silica gel 60 (70-230 mesh for VLC and 230- 
400 mesh for flash columns). Semipreparative HPLC purifica- 
tion was carried out on a Waters apparatus equipped with a 
refractive index detector and a Spherisorb 10 /im silica 
semiprep column (10 x 250 mm). 
Animal Material. Specimens of L. alba and L. r?gida 
(vouchers C7 and S3, respectively) were collected by scuba in 
the Coiba National Park at 3-10 m depth in August 2002. 
The samples were frozen immediately after collection and 
stored at ?80 ?C until extraction. Voucher specimens are 
maintained for reference at the Smithsonian Tropical Research 
Institute. Specimen C7 was consistent with the original 
description for L. alba (Duchassaing and Michelotti 1864).^^ 
Colonies are white or light pink in color. Colonies may grow 
up to 40 cm in height and 40 cm in width, flabelliform in 
nature, with long, slender pinnate branches, short stems, and 
thin holdfasts. The specimen S3 is consistent with the descrip- 
tion for L. r?gida (Verrill, 1868).^^ Colonies are upright, 
measuring up to 40 cm in height. Stems may reach 30 mm in 
width. Branches are mostly cylindrical, especially at the ends. 
The color is deep purple, but in some cases, yellowish rings of 
sclerites surrounding the polyp apertures are observed. 
Extraction and Isolation. The octocoral L. alba (121.9 g, 
fresh weight) was cut into small pieces and extracted with 
MeOH and CH2CI2. The extracts were pooled and concentrated 
under reduced pressure in a rotary evaporator to yield 3.4 g 
of crude extract that was subjected to solvent partition, 
affording four fractions (re-hexane, CH2CI2, re-butanol, and 
water). The CH2CI2 fraction (350 mg) was active against P. 
falciparum and was subjected to VLC on silica gel eluting with 
CH2Cl2-MeOH mixtures (from 5% to 100% MeOH), affording 
five fractions. Fraction 2 (174 mg) showed the most significant 
antiplasrtiodial activity and was subjected to silica gel flash 
chromatography eluting with re-hexane?EtOAc mixtures (33%, 
50%, and 100% EtOAc), jdelding 10 fractions. Fractions 4 and 
6 yielded compounds 1 (42 mg) and 2 (11 mg), respectively, in 
pure form. Fraction 7 (37 mg) was subjected to silica gel flash 
chromatography eluting with CHCls-MeOH (0.30% of MeOH), 
yielding 25 mg of compound 3. 
The octocoral L. r?gida was extracted and partitioned in the 
same way as described for L. alba, of which the CH2CI2 fraction 
(744 mg) showed activity in the antiplasmodial bioassay. The 
CH2CI2 fraction was subjected to VLC on silica gel, eluting with 
CH2Cl2-MeOH mixtures (from 3% to 100% MeOH), affording 
six fractions. Significant antiplasmodial activity was located 
in fraction 2 (382 mg), which was fractionated on a silica gel 
flash column eluting with mixtures of CH2CI2?MeOH (from 
0.5%) to 3.0% MeOH), yielding compounds 4 (105 mg) and 5 
(15 mg) from fractions 3 and 5, respectively. Fraction 6 (94 
mg) was chromatographed using silica gel flash column eluting 
with 0.5% MeOH in CH2CI2, affording compound 6 (23 mg). 
616    Journal of Natural Products, 2005, Vol. 68, No. 4 Notes 
Fraction 1 (10 mg) was subjected to purification by HPLC 
eluting with 50% EtOAc?isooctane, yielding compound 7 (2 
mg). 
Leptolide (1): red glassy solid; [a]% +10.4? (c 1.9, CHCI3); 
UV (EtOH) Amax (log e) 234 (3.34), 262 (3.38), 292 (3.36) nm; 
IR (KBr) Vmax 3489, 2932, 1778, 1680, 1084 cm^i; ^H NMR 
(CDCI3, 500 MHz) ? 9.82 (IH, s, H-18), 6.50 (IH, s, H-5), 5.06 
(IH, s, H-16a), 4.96 (IH, s, H-16b), 4.73 (IH, dd, J = 3.0, 4.0 
Hz, H-10), 4.04 (IH, s, H-7), 4.05 (IH, s, H-11), 3.49 (IH, dddd, 
J = 3.0, 8.5, 11.0, 11.0 Hz, H-1), 3.05 (IH, dd, J = 3.0, 18.0 
Hz, H-2a), 2.97 (IH, dd, J = 11.0, 18.0 Hz, H-2b), 2.55 (IH, 
ddd, J = 1.5, 11.0, 15.0 Hz, H-14a), 2.50 (IH, dd, J = 3.0, 15.5 
Hz, H-9a), 2.03 (IH, dd, J = 4.0, 15.5 Hz, H-9b), 1.75 (3H, s, 
H-17), 1.66 (IH, m, H-13a), 1.48 (IH, ddd, J = 1.5, 8.5, 15.0 
Hz, H-14b), 1.37 (IH, m, H-13b), 1.14 (3H, s, H-19); ^^C NMR 
(CDCI3, 125 MHz) ? 184.4 (CH, C-18), 171.2 (C, C-20), 162.2 
(C, C-3), 149.8 (C, C-6), 144.8 (C, C-15), 123.1 (C, C-4), 113.8 
(CH2, C-16), 105.1 (CH, C-5), 76.8 (CH, C-10), 63.2 (CH, C-11), 
62.4 (C, C-12), 56.3 (C, C-8), 55.3 (CH, C-7), 40.6 (CH, C-1), 
39.1 (CH2, C-9), 30.7 (CH2, C-2), 26.5 (CH2, C-13), 23.6 (CH2, 
C-14), 20.2 (CH3, C-19), 18.9 (CH3, C-17); EIMS m/z 358 [M] + 
(9), 329 (5), 301 (3), 231 (5), 203 (10), 189 (15), 175 (19), 149 
(53), 137 (81), 121 (24), 107 (37), 91 (54), 79 (52), 77 (47), 55 
(78), 43 (94), 39 (53), 28 (36), 18 (100); HREIMS m/z 358.1424 
(caled for C20H22O6, 358.1416). 
X-ray Crystal Structure of Leptolide (1).^^ A red prism 
was obtained by slow evaporation fi-om methanol. Crystal data. 
Empirical formula = C20H22O6, Mr = 358.38, T = 120(2) K, 
crystal system = orthorhombic, space group = P2i2i2i, unit 
cell dimensions: a = 10.091(5) A, b = 11.064(5) ?, c = 
16.970(5) A, y = 1894.6(14) ?^, Z = 4, fleaic= 1-256 Mg/m^, A 
= 0.7107 ?, /<(Mo Ka) = 0.093 mm"\ Fooo = 760, crystal size 
= 1.00 X 0.58 X 0.53 mm, theta range for data collection: 0 = 
2.19-24.04?, index ranges = -12 < /i < 12, 0 < * < 13, 0 < Z 
< 21, reflections collected = 9896, independent reflections = 
3734 (i?int = 0.0635), completeness to theta = 26.37?, 97.3%, 
absorption correction = semiempirical from equivalents, maxi- 
mum and minimum transmission = 0.9525 and 0.9131, 
respectively. Refinement method: full-matrix least-squares on 
F^, data/restraints/parameters = 3734/0/250, S = 1.003, final 
R indices [7 > 20(7)]: Rl = 0.0571, wR2 = 0.1430, R indices 
(all data): Rl = 0.1079, wR2 = 0.1646, Flack parameter = 
?0.5(18), extinction coefficient = 0.007(2), largest difference 
peak and hole = 0.212 and -0.180 e ?"^'. 
X-ray Crystal Structure of Pukalide Aldehyde (2).i^ A 
colorless needle was obtained by slow evaporation from 
chloroform. Crystal data. Empirical formula = C2oH2205- 
CHCI3, Mr = 461.74, T = 293(2) K, crystal system = hexagonal, 
space group = 7'65, unit cell dimensions: a = b = 20.6319(12) 
A, c = 9.8314(7) ?, y = 3624.3(4) ?, Z = 6, Dcaic = 1.269 Mg/ 
m^ A = 1.54184 ?, /<(Cu Ka) = 3.666 mm^i, Fooo = 1440, crystal 
size = 0.55 X 0.88 x 0.03 mm, theta range for data collection: 
e = 4.29-50.71?, index ranges = 0 < /i < 20, 0 < ? < 20, -9 
< Z < 9, reflections collected = 10 161, independent reflections 
= 2491 (7?int = 0.0702), completeness to theta = 50.71? 99.3%, 
absorption correction = semiempirical from equivalents, maxi- 
mum and minimum transmission = 0.775 and 0.376, respec- 
tively. Refinement method: full-matrix least-squares on F^, 
data/restraints/parameters = 2491/39/278, S = 1.111, final R 
indices [7 > 20(7)]: Rl = 0.0992, wR2 = 0.2659,7? indices (all 
data): Rl = 0.1225, wR2 = 0.2904, Flack parameter = 0.02(7), 
extinction coefficient = 0.0110(18), largest difference peak and 
hole = 0.425 and -0.322 e ?-=*. 
X-ray Crystal Structure of Lophotoxin (3).^^ A colorless 
plate was obtained by slow evaporation of chloroform. Crystal 
data. Empirical formula = C22H2408-H20, M^ = 432.41, T = 
293(2) K, A = 1.54184 A, crystal system = monoclinic, space 
group = P2i, unit cell dimensions: a = 9.696(5) A,b = 6.921(4) 
A, c = 17.237(8) k, ? = 91.18(4)?, V = 1156.4(11) ?^, Z = 2, 
?eaic = 1-242 Mg/m!?, fiiCu Ka) = 0.818 mui-\ Fooo = 456, 
crystal size = 0.75 x 0.10 x 0.03 mm, theta range for data 
collection:  9 = 5.19-50.48?, index ranges = -9 < Z? < 9, -6 
< ? < 6, 0 < Z < 17, reflections collected = 8270, independent 
reflections = 2366 (7?int = 0.0924), completeness to theta = 
50.48?, 99.3%, absorption correction = semiempirical from 
equivalents, maximum and minimum transmission = 1.053 
and 0.692, respectively. Refinement method: full-matrix least- 
squares on F^, data/restraints/parameters: 2366/1/283, S = 
1.105, final7? indices [7 > 20(7)]: Rl = 0.0847, wR2 = 0.2368, 
R indices (aU data): Rl = 0.1009, wR2 = 0.2666, Flack 
parameter = 0.0(7), largest difference peak and hole = 0.375 
and -0.239 e ?"^. 
Biological Evaluation. Antiplasmodial activity was de- 
termined in a chloroquine-resistant P. falciparum strain (W2) 
utilizing a novel microfluorimetric assay to measure the 
inhibition of the parasite growth based on the detection of the 
parasitic DNA by intercalation with PicoGreen.^ P. falciparum 
was cultured according to the methods described by Tr?ger 
and Jensen.^" The parasites were mantained at 2% haemat- 
ocrit in flat-bottom flasks (75 mL) with RMPI 1640 medium 
(GibcoBRL) supplemented with 10% human serum. 
Acknowledgment. We thank the Ministerio de Educaci?n 
y Ciencia (MEC) and the Xunta de Galicia for financial support 
for this research (grant numbers BQU2002-01195, SAF2003- 
08765-C03-01, PGIDT02BTF20902PR, PGIDT03PXIC20908PN, 
PGIDIT04PXIC20903PN). Funding was also provided by the 
Smithsonian Tropical Research Institute, the AVINA Founda- 
tion, and the Iternational Cooperative Biodiversity Groups 
program (grant numbers lUOl TWO 1021-01 and lUOl 
TW006634-01) from the National Institutes of Health, Na- 
tional Science Foundation, and the United State Department 
of Agriculture. We thank C. Guevara for field assistance, O. 
Breedy for assistance with the taxonomy, and the crew 
members from the R/V Urraca for logistical support. 
Supporting Information Available: CD spectra of compounds 
2, 4, 6, and 7, crystallographic information of compounds 1?3, and 
photos of the collected organisms are available free of charge via the 
Internet at http://pubs.acs.org. 
References and Notes 
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allocated the deposition numbers CCDC 246192 for compound 1, 
CCDC 246195 for compound 2, and CCDC 246262 for compound 3. 
Copies of the data can be obtained, free of charge, on application to 
the Director, CCDC, 12 Union Road, Cambridge CB2 lEZ, UK (fax: 
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NP049745Z