Bioactive Chromodorolide Diterpenes from an Aplysillid Sponge
ABSTRACT Abstract: The known chromodorolides A (1) and B (2), and a new derivative chromodorolide C (3) have been isolated from an Australian sponge and characterized by 1D and 2D NMR experiments. The chromodorolides exhibited significant cytotoxicity against the P388 mouse leukaemia cell line, and also showed activity against the freeliving larval stages of the parasitic nematodes Haemonchus contortus and Trichostrongylus colubriformis.
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ABSTRACT: Efficient routes towards the austrodorane skeleton from the labdane diterpene (−)-sclareol (22) are described. The processes, based on pinacol rearrangement, take place with complete diastereoselectivity. Utilizing these, the marine nor-sesquiterpenes (+)-austrodoral (1) and (+)-austrodoric acid (2) have been prepared from 22. Ketone 19, a key intermediate in the synthesis of rearranged cytotoxic diterpene lactones, such as norrisolide (3), has also been prepared in moderate yield.Tetrahedron 11/2007; . 63, no. 48, (2007):11943-11951. · 2.82 Impact Factor
Article: Spongiane Diterpenoids[Show abstract] [Hide abstract]
ABSTRACT: In this review we cover the structures, occurrence, biological activities and synthesis of all the spongianes and rearranged spongianes since their discovery in 1974. We have given special attention to structure revisions and biological properties of these polycyclic terpenoids of exclusive marine origin. However, an important part of the review describes the synthetic efforts in the field. Thus, this part has been subdivided into syntheses from other natural products, syntheses by biomimetic approaches and other approaches including enantioselective total syntheses.Current Bioactive Compounds 02/2007; 3(1):1-36.
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ABSTRACT: Chemical investigation of an Okinawan marine sponge resulted in the isolation of two new spongian-class diterpenes 1 and 2 together with two known compounds, chromodorolide B (3) and chromodorolide C (4). Compound 1, named chromodorolide D, is an example of a diterpenoid with a highly rearranged chromodorane carbon skeleton, while compound 2 retains the open side chains. The structures of the new compounds were elucidated on the basis of extensive spectroscopic analysis and chemical conversion. Compounds 1–4 exhibited significant cytotoxicity against NBT-T2 rat bladder epithelial cells.Chemistry of Natural Compounds 07/2012; 48(3). · 0.50 Impact Factor
Mar. Drugs 2004, 2, 101-107
Bioactive Chromodorolide Diterpenes from an Aplysillid
Wimolpun Rungprom1, Warinthorn Chavasiri1, Udom Kokpol1, Andrew Kotze2 and Mary J.
1 Natural Products Research Unit, Department of Chemistry, Chulalongkorn University, Bangkok
2 CSIRO Livestock Industries, Queensland Bioscience Precinct, Brisbane 4067, Australia
3 Department of Chemistry, School of Molecular and Microbial Sciences, The University of
Queensland, Brisbane 4072, Australia
* Author to whom correspondence should be addressed; Tel. (+61)-7-3365-3605, Fax (+61)-7-
3365-4273, E-mail: firstname.lastname@example.org
Received: 7 May 2004 / Accepted: 1 August 2004 / Published: 25 August 2004
Abstract: The known chromodorolides A (1) and B (2), and a new derivative
chromodorolide C (3) have been isolated from an Australian sponge and characterized by
1D and 2D NMR experiments. The chromodorolides exhibited significant cytotoxicity
against the P388 mouse leukaemia cell line, and also showed activity against the free-
living larval stages of the parasitic nematodes Haemonchus contortus and
Keywords: Marine sponges, secondary metabolites, diterpenes, chromodoranes,
Highly functionalized terpenoids with intact spongian carbon skeletons, or with rearranged
carbon skeletons that can derive from a hypothetical spongian diterpenoid precursor, have been
isolated from marine sponges and nudibranchs [1-4]. Nudibranchs belonging to the genus
Mar. Drugs 2004, 2
Chromodoris have proven to be an extremely rich source of diterpenoids related to compounds from
Aplysillid sponges [2,4-9]. The nudibranchs usually form a specific chemical association with the
sponge, on which they feed and from which they concentrate the diterpenes. The chromodorane
diterpenes, chromodorolides A and B are diterpenoid examples with a highly rearranged carbon
skeleton, and have previously been reported from the nudibranch Chromodoris cavae collected in
Sri Lanka [8,9]. In this paper we wish to describe the new chromodorane diterpene chromodorolide
C isolated together with chromodorolides A and B from an encrusting yellow sponge collected from
Mooloolaba, Sunshine Coast, Queensland. We also report bioactivity testing of chromodorolides A
Results and Discussion
The small scale extract of the sponge exhibited potent cytotoxicity (94 + 3% inhibition at 100
µg/mL) against a P388 mouse leukemia cell line. The frozen sponge (5.87 g) was then extracted by
dichloromethane/methanol (1:1) three times, and the resulting extract was further partitioned
between EtOAc and water. The ethyl acetate layer was subjected to silica gel flash column
chromatography using a step gradient from hexane to 100% EtOAc to yield two major fractions.
The first fraction contained chromodorolide B (2) and the second fraction was purified by NPHPLC
using hexane and EtOAc (7:2) to give chromodorolide A (1) and the new compound,
chromodorolide C (3). The two known compounds 1 and 2 were identified by comparison of their
spectroscopic data with those from the literature [8,9].
(2) R = OAc
(3) R = OH
The new chromodorane, compound 3 was isolated as a colorless oil and had a molecular peak at
m/z 473.2148 corresponding to C24H34O8Na indicated by HRESIMS. The 1H NMR spectrum
indicated the presence of three singlet methyls (δ 0.82, 0.83 and 0.76), and two additional singlet
methyls (δ 2.05 and 2.13) that could be assigned to acetate moieties. Two methine protons (δ 6.51
and 6.00) supported the presence of acetal functionalities. Numerous complex signals in the region
δ 0.93-1.48 were consistent with the compound having a terpene skeleton. In the 13C NMR
spectrum, signals at δ 174.8, 168.8 and 169.2 were consistent with a lactone carbonyl and two
acetate carbonyls respectively. The similarity of the 1H and 13C spectra of 3 to those of 2 identified
Mar. Drugs 2004, 2
the new compound as a chromodorane diterpene, possessing a 3,4 ring fused bisacetal-oxalone.
HMBC correlations connected the acetal methine proton H-15 at δ 6.51 to C-14 at δ 45.4, and H-14
at δ 2.98 to C-15 at δ 98.1. DQFCOSY correlations then linked H-14 to H-13 at δ 3.52 which was
further coupled to the second methine H-16 at δ 6.00. However, comparison of the spectral data
showed that 3 only differed from 2 in its acetylation pattern. The HMBC spectrum showed the
connectivity from H-15 at δ 6.51 to an acetate carbonyl at δ 169.2 and from H-17 at δ 5.19 to the
acetate carbonyl at δ 168.8, and from two methyl acetate signals at δ 2.05 and δ 2.13 to the acetate
carbonyls at δ 169.2 and δ 168.8 respectively, confirming the position of the acetate substituents.
Additionally, in 3 a signal at δ 78.1, assigned to C-12, was evident and this suggested a quaternary
carbon bearing a hydroxy group, whereas in compound 2, the corresponding signal resonated at δ
81.3. These data manifestly identified 3 as a new compound and allowed all resonances to be
assigned (Table 1). In particular, the bicyclic terpene framework of 3 was carefully assigned by
DQFCOSY and HMBC. The assignments for C-1 and C-3 were made on the basis of correlations to
nearby methyl groups as shown in Table 1, while assignments for C-2 and C-6 were confirmed by
HMBC correlations to H-3ax and to H-5 respectively. These data suggest that the NMR
assignments published by Andersen et al. for chromodorolide B  may need revision. Small scale
acetylation of 3 with acetic anhydride in pyridine provided an acetate product whose 1H NMR
spectrum was identical to that of chromodorolide B 2.
This is the first report of these chromodorane diterpenes from a sponge source. It has been
proposed by Andersen et al.  that the biosynthesis of the chromodorane skeleton starts from a
spongian diterpene followed by opening and contraction of the six membered ring to the five
membered ring [8,9]. Oxidative cleavage of a second six-membered ring, followed by lactonization
of the C-11 carboxyl group with the hydroxy group at C-15 or C-16 then leads to two different
bisacetal oxalone skeletons. The discovery of 3 indicates that acetylation could be a stepwise
process in the sponge.
Chromodorolide A has been reported to exhibit cytotoxic and antimicrobial activity . When
tested individually against the P388 cell line, chromodorolides A, B and C displayed significant
inhibition (66 (+ 3), 70 (+ 2) and 42 (+ 4) %, respectively) at concentrations of 10 µg/mL, but did
not show useful activity at concentrations of 1 µg/mL. Chromodorolide A 1 also showed
nematocidal activity against the larval stages of Haemonchus contortus and Trichostrongylus
colubriformis, two important pathogens of sheep and other ruminants. A concentration of 100
µg/mL caused 94 (± 3) % and 95 (± 4) % (Mean ± SE, n = 2 separate experiments) inhibition of the
development of H. contortus and T. colubriformis larvae, respectively. A concentration of 10
µg/mL did not affect development for H. contortus, however T. colubriformis development was
inhibited by 33 (± 1) %.
Mar. Drugs 2004, 2
Table 1. 1H (500 MHz) and 13C (125 MHz) data for chromodorolide C (3)
a Inverse detection at 500 MHz; solution in CDCl3; 13C = 77.0 ppm and 1H = 7.25 ppm.
b Inverse detection at 500 MHz; correlations observed when 1J13C-1H = 135 Hz and long range nJ13C-1H = 8 Hz.
1H-1H COSY HMBCb
ax 0.93, dt (J = 2.0, 12.0 Hz)
eq 1.53, m
2 19.9 ax 1.38, m
eq 1.55, m
H-2eq, H-3ax, H-3eq
40.9 ax 1.03, dt (J = 3.0, 12.0 Hz)
eq 1.38, m
H-2ax, H-2eq, H-3eq
4 33.0 - - H-5, Me-18, Me-19
5 57.0 1.09, dd (J = 6.5, 13.0 Hz) H-6ax, H-6eq Me-18, Me-19, Me-
21.10 ax 1.30, m
eq 1.55, m
H-5, H-6eq, H-7
H-5, H-6ax, H-7
25.2 ax 1.42, m
eq 1.55, m
H-6ax, H-6eq, H-9
H-6ax, H-6eq, H-9
8 47.5 2.58, ddd (J = 8.2, 12.0, 12.0
H-9, H-14, H-17 H-15, H-17
9 50.2 1.71, dd (J = 9.8, 12.0) H-7ax, H-7eq, H-8 H-8, H-17. Me-20
10 43.8 - - H-5, Me-20
11 174.8 - - H-16, H-17
12 78.1 - - H-13, H-14, H-16,
13 52.1 3.52, dd (J = 6.0, 8.2 Hz) H-14, H-16 H-15, H-16
14 45.4 2.98, t (J = 8.2 Hz) H-8, H-13 H-8, H-15
15 98.1 6.51, s - H-8, H-13, H-14
16 103.3 6.00, d (J = 6.0 Hz) H-13 H-15
17 75.3 5.19, d (J = 12.0 Hz) H-8 H-8, H-13, H-14
Me-18 33.4 0.82, s - Me-19
Me-19 21.0 0.83, s - H-5, Me-18,
Me-20 13.6 0.76, s - H-5, H-9
Mar. Drugs 2004, 2
Chromodorolide diterpenes have been isolated for the first time from an Australian marine
sponge and their cytotoxicity and also the anthelmintic activity of one of them against H. contortus
and T. colubriformis has been demonstrated.
We thank Dr David De Vries and Ms Linda Banbury, Centre for Phytochemistry, Southern Cross
University for P388 cytotoxicity screening. This research was funded by the Australia Research
Council. Wimolpun Rungprom thanks the Thailand Royal Golden Jubilee Fund for the award of a
1H and 13C NMR spectra were obtained in CDCl3 on a Bruker DRX spectrometer and were
recorded at 500 and 125 MHz respectively. Electrospray mass spectra were recorded on a P E
SCIEX API III triple quadropole mass spectrometer for solutions in methanol. Flash
chromatography was carried out using Kieselgel 60 (230-400 mesh), while high performance liquid
chromatography was carried out on a Waters µ-Porasil semi preparative column (300 mm x 7.8 mm)
connected to a Waters 515 HPLC pump and a Gilson 132 refractive index detector. All solvents
were freshly distilled or were of HPLC grade.
A single specimen of the sponge was collected from under a ledge at 10 m depth. The collection
was made at the Fishhole at the Inner Gneerings Reef, Mooloolaba South-East Queensland in
December 2002. The encrusting sponge was a bright lemon yellow colour underwater and
developed the blue coloration on exposure to air that is typical of Aplysilla sulphurea. A voucher
sample and photograph is held at the Department of Chemistry, The University of Queensland. A
small sample of animal material has been submitted to The Queensland Museum for taxonomic
Extraction and Purification
The frozen sponge (5.9 g) was cut into small pieces and extracted with 100 mL of DCM/MeOH
(1:1) three times. The organic layer was filtered through a plug of cotton wool and the solvent was
evaporated in vacuo to give the crude extract (624 mg) that was further partitioned with EtOAc and
water (100 mL). The EtOAc layer was concentrated and subjected to silica flash column
Mar. Drugs 2004, 2
chromatography using a step gradient from hexane to 100% EtOAc to give two major fractions. The
first fraction was identified as chromodorolide B (2) (5.7 mg)  and the second fraction was
purified by NPHPLC using hexane and EtOAc (7:2) as eluent to furnish chromodorolide A (1) (5.7
mg) [8,9] and the new compound, chromodorolide C (3) (1.7 mg).
Chromodorolide C (3): Colorless oil; [α]D -78o (c = 0.10, CH2Cl2); 1H-NMR (CDCl3; 500 MHz)
13C-NMR (CDCl3; 125 MHz) see Table 1, HRESIMS found 473.2148, C24H34O8Na (M+Na)+
requires 473.2151(+0.8 ppm).
Acetylation of Chromodorolide C (3): To a solution of chromodorolide C (3, 1.0 mg, 2.2x10-3
mmol) in dry pyridine (1.0 mL) was added acetic anhydride (0.1 mL). The mixture was stirred at
room temperature overnight. The reaction was quenched by adding 1 mL of water, and extracted
twice with 1 mL of EtOAc. The organic layer was blown down over N2 gas. The acetate product
(0.9 mg) was obtained as a colorless oil by purifying on a silica-seppak using hexane:EtOAc (7:2)
as eluent, and identified as (2) by comparison of 1H-NMR spectra.
P388D1 mouse lymphoblast cells were grown at 37oC, 5% CO2 in DMEM media containing
10% horse sera with 2mM L-glutamine and 100 U/mL penicillin and 100 µg/mL streptomycin.
Cells growing in log phase were diluted in this media and transferred (99 µL/well) to a 96-well
tissue culture plate. Samples were tested in triplicate and included the crude DCM/MeOH extract
from the sponge as well as purified samples of individual chromodorolide metabolites. Samples
were dissolved in DMSO at an initial concentration of 10 mg/mL and diluted by serial dilution in
DMSO. Samples were added to the wells (1 µL) so that final concentrations tested were 100, 10 and
1 µg/mL. Plates were incubated (37oC, 5% CO2) for 24 h. After this time, cytotoxicity was
determined using an ATPLite assay which measures the amount of ATP present in the cells. The
luminescence produced, proportional to the number of viable cells, was read on a Victor 2
multilabel plate reader (Wallac).
Nematocidal activity was determined using the method of Lacey et al . Nematode eggs were
placed into wells of a mictotitre plate containing chromodorolide A in 2% agar. The eggs hatched
overnight, a nutrient medium was added, and the larvae were held at 27oC for 6 days. The number
of larvae that developed to the L3 stage was counted and compared to control wells. For each
nematode species, the effect of chromodorolide C was examined in two separate experiments at
concentrations of 100 and 10 µg/mL (duplicate or triplicate assays wells at each concentration for
experiments one and two, respectively) and the percentage development was calculated for each
Mar. Drugs 2004, 2
assay relative to the mean development in twelve control assay wells. Data is therefore presented as
Mean ± SE, n = 2 separate experiments.
References and Notes
1. Tischler, M.; Andersen, R. J.; Choudhary, M. I.; Clardy. J. Terpenoids from the sponge
Aplysilla glacialis and specimens of the nudibranch Cadlina luteomarginata found on the
sponge. J. J. Org. Chem. 1991, 56, 42-47.
2. Hochlowski, J. E.; Faulkner, D. J.; Matsumoto, G. K.; Clardy. J. Norrisolide, a novel diterpene
from the dorid nudibranch Chromodoris norrisi. J. J. Org. Chem. 1983, 48, 1141-1142.
3. Molinski, T. F.; Faulkner, D.J. A new diterpene lactone from an Australian Aplysilla species. J.
Org. Chem. 1986, 51, 1144-1146.
4. Karuso, P.; Taylor, W. C.; White, A. H. The constituents of marine sponges. 11. Isolation of 8
diterpenes from Aplysilla rosea. Aust. J. Chem, 1986, 39, 1629-1641.
5. Hochlowski, J. E.; Faulkner, D. J. Chemical constituents of the nudibranch Chromodoris
marislae. Tetrahedron Lett. 1981, 22, 271-274.
6. Bobzin, S. C.; Faulkner, D. J. Diterpenes from the marine sponge Aplysilla polyrhapsis and the
dorid nudibranch Chromodoris norrisi. J. Org. Chem. 1989, 54, 3902-3907.
7. Cimino, G.; Fontana, A.; Gavagnin, M. Marine opistobranch mollusks: chemistry and ecology
in sacoglossans and dorids. Curr. Org. Chem. 1999, 3, 327-372.
8. Dumdei, E. J.; de Silva, E. D.; Andersen, R. J. Chromodorolide A, a rearranged diterpene with
a new carbon skeleton from the Indian ocean nudibranch Chromodoris cavae. J. Am. Chem
Soc. 1989, 111, 2712-2713.
9. Morris S. A.; Dumdei, E. J.; de Silva, E. D.; Andersen, R. J. Chromodorane diterpenes from the
tropical dorid nudibranch Chromodoris cavae. Can. J. Chem. 1991, 69, 768-771.
10. Lacey, E; Redwin, J. M.; Gill, J. H.; Demargheriti, V.M; Waller, P.J. In Resistance of Parasites
to Antiparasitic Drugs; Boray, J.C.; Martin, P.J.; Roush, R.P., Eds; MSD AGVET, Rahway, NJ,
1990, pp 177-184.
Samples Availability: Samples and copies of 1H and 13C NMR spectra are available from the
© 2004 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.