Phytochemistry, Vol. 31, No. 12, pp. 41154118, 1992 CO31--9422/92$5.00+0.

00
Printedin Great Britain. Q 1992 PergamonPress Ltd

DEBROMOISOCYMOBARBATOL, A NEW CHROMANOL FEEDING

DETERRENT FROM THE MARINE ALGA CYMOPOLIA BARBATA

MINKYU PARK, WILLIAM FENICAL* and MARK E. HAYt

Scripps Institution of Oceanography, University of California-San Diego, La Jolla, CA 92093-0236,U.S.A.;

tuniversity of North Carolina at Chapel Hill, Institute of Marine Sciences, Morehead City, NC 28557, U.S.A.

(Received 16 March 1992)

Key Word Index-Cymopolia barbata; Dasycladaceae; Chlorophyta; bromochromanols; marine algae; feeding
deterrents; marine chemical ecology.

Abstract-A new bromochromanol, debromoisocymobarbatol, has been isolated from a Florida Keys collection of the
green marine alga Cymopolia barbata. The structure of this new compound was determined by spectral methods. In
field experiments and aquarium assays the new metabolite reduced grazing by an herbivorous fish, an omnivorous fish
and an amphipod, thus indicating that the compound may serve a defensive role in this alga.

INTRODUCTION In an attempt to evaluate more fully the biological

The importance of plant secondary metabolites in
reducing herbivory in terrestrial communities is well-
documented and generally accepted as one of the most
effective means of herbivore deterrence [l-3]. Recent
investigations in marine communities also document that
plant secondary metabolites can significantly diminish
feeding by herbivores [4], and that algal chemical de-
fences can be effective even on coral reefs where,rates of
herbivory appear to exceed those known from any other
habitat, either terrestrial or marine [S, 63. It is now
known that many chemically rich algae are relatively
resistant to herbivory and that many heavily calcified
species (an obvious structural defence) also contain biolo-
gically-active secondary metabolites [4].
Tropical green seaweeds of at least two orders are
known to be fertile sources for bioactive metabolites [7,
81. Members of the order Dasycladales are less-well
studied, but one member, Cymopolia barbata, was one of
the first green algae recognized to be chemically rich. In a
search for new antibiotics, Martinez Nadal et al., in
1964 [9] showed that the diethyl ether extract of
C. barbata contained substances with significant anti-
microbial activity. Subsequent chemical studies of
C. barbata collected in Bermuda [lo], Florida [ll], the
Canary Islands [la] and Puerto Rico [13] showed the
alga to contain complex mixtures of bromine-containing
prenylated hydroquinones. The major metabolite, cymo-
pol (2) , was found to act as an antifeedant against the
herbivorous gastropod mollusc Littorina littorea [14],
the sea urchin Lyteckinus uariegatus [15], and mixed
species of herbivorous fishes on Caribbean coral
reefs [16]. In contrast, this compound stimulated feeding
by the sea urchin Diadema antillarum [ 161.
*Author to whom correspondence should be addressed.
properties of the various prenylhydroquinones produced
by C. barbata, we investigated the chemistry of a Florida
Keys collection. In this paper we report the isolation and
structure determination of a new minor metabolite, de-
bromoisocymobarbatol (1) which possesses a chromanol
functionality. The new compound is closely related to
cymobarbatol(3) and 4-isocymobarbatol(4), antimutag-
enic hydroquinones recently reported from the same
source [ 131.
RESULTS AND DISCUSSION
Debromoisocymobarbatol (1) was isolated, as a vis-
cous oil, from the extract of C. barbata by standard
chromatographic methods involving silica vacuum flash
chromatography and HPLC. The molecular formula for
1, C,,H,,O,Br, was determined by a combination of
13C NMR and HRMS data ([Ml’ m/z=324.0756,
talc. 324.0725). Analysis of 13C and ‘H NMR, and UV
and IR data showed that a bromohydroquinone ring,
which is a common component of other metabolites
isolated from this alga, accounts for four of its six degrees
of unsaturation. The ‘H NMR spectrum of debromoiso-
cymobarbatol~ showed signals from a gem-dimethyl con-
stellation, a quaternary bridgehead methyl, and two para-
substituted aromatic protons. Since no other vinyl pro-
tons were present, the compound was concluded to be
tricyclic. The presence of two methylene protons at 6 2.52
and 2.63, coupled to a bridgehead methine proton at
6 1.62 forming an ABX pattern, along with the bridge-
head methyl singlet at 6 1.18, suggested the presence of a
six-membered chromanol ring. The second ring was then
also required to be six-membered and to possess the gem-
dimethyl functionality.
The full structure of 1 was assigned by comprehensive
NMR studies including a series of NOEDS experiments

4115
 M. PARK et al.

HO

Br
\

Fig. 1. Results of NOEDS ~x~~~ents with debromoi~cymobarbatol. Enha~~ments observed when the
folIowing were performed: (1) irradiation of the C-9 methyl protons; (2) irradiation of the C-10 equatorial proton;
(3) irradiation of the C-3 proton; (4) irradiation of the C-7 methyl protons.

which established the refative stereochemistry at all cen-
tres (Fig. 1). The ‘H NMR spectrum showed three fully
resolved methyl singlets. Irradiation of the C-9 methyl
signal (S 1.18) enhanced the C-8 methyl (60.88) and the C-
10 proton at 62.52, thus showing that these enhanced
protons are axial and on the same side of the six-
membered ring. Irradiation of the equatorial C-7 methyl
singlet enhanced the proton at 62.63 (C-10 eq) and the
bridgehead proton at S 1.62, thus establishing the stereo-
chemistry of the ring juncture.
Although C. barbata is a calcified alga and thus has
some structural defences, chemical defences also appear
to be important. Fig. 2 shows how this compound afIec-
ted feeding by two common herbivorous fishes and an
herbivorous amphipod. In field assays, debromoisocymo-
barbatol reduced feeding by herbivorous parrotfishes by
 A bromochromanol
AMOUNT EATEN (%I
0 20 40 60 80
P<WI
N=19
.005> P>.oo I
N=20
-CONTROL •=TREA~RENT
Fig. 2. The effect of debromoisocymobarbatol, at 1% of plant
dry mass, on feeding by coral reef parrotfishes in field assays (top
histogram) and by omnivorous pinfish Lagodon rhomboides
(middle histogram) and an herbivorous amphipcxt, Hyale macro-
dactyla (bottom histogram), in laboratory assays.
a significant 29% (P<O.O05, paired t-test) relative to
controls. This effect was more pronounced in aquarium
assays with the omnivorous pinfish Lagodon rhomboides;
consumption was reduced by 50% (P<O.OOl, paired t-
test). The compound also reduced grazing by 22%
(P <0.005, paired t-test) in assays using the Caribbean
amphipod Hyale macrodactyla. On the basis of these and
earlier observations, debromoisocymobarbatol, and sev-
eral other metabolites from Cymopolia barbata appear to
form the basis of an effective adaptation for chemical
defence.
EXPERIMENTAL
General. Proton NMR spectra were recorded in CDCl, at
360 MHz, using a spectrometer constructed from an Oxford
magnet and Nicolet-1180E Fourier transform data system, at the
UCSD NMR facility. NOEDS experiments were performed
essentially following the experiments outlined by Hall and
Sanders [17]. t3CNMR spectra were recorded in CDCI, solu-
tion at 50 MHz. High resolution mass measurements were
supplied by Dr Richard W. Kondrat, University of California,
Riverside.
Extraction and purijication of debromoisocymobarbatol. Cymo-
polia barbata was collected at Sawyer Key in the Florida Keys in
July, 1987. The alga (15 g dry wt) was kept frozen until it was
defrosted and extracted repeatedly with CH,Cf,-MeOH (2: 1).
Removal of the solvents under red. pres. left a dark green gum
(1.35 g). Preliminary flash chromatography of the extract on
silica gel, using EtOAc-isooctane mixtures, yielded mixtures
containing many known Cymopolia metaboliteq as well as a
fraction rich in compound 1. Final purification of this fraction by
silica HPLC (EtOAc-isooctane, 3: 17) yielded pure debromoiso-
cymobarbatol (l), 27 mg, as 2% of extract (0.2% dry wt_).
DebromoisocymobarbatoZ(1) HREIMS: [M] + m/z= 324.0756,
talc. 324.0725 for C,,HH,,O,Br; UV dzf” (log E):258 nm (3.67),
325 nm (3.82); IR VET:3430,2930,2&O, 1490, 1470, 1210, 1190,
1150, 1100 cm-‘; ‘H NMR (360 MHz, CDCI,): 6 0.88 (s, 3H,
C-8), 0.98 (s, 3H, C-7), 1.18 (s, 3H, C-9), 1.25 (m, 2H, C-l), 1.59 (m,
2H, C-6), 1.62 (dd, lH, 5=12.8, 5.0 Hz, C-3), 2.52 (lH, dd, J
=16.5, 12.8, H-lOax), 2.63 (lH, dd, 5=16.5, 5.0, H-1OeqX 6.73
(lH, s, C-12), 6.88 (iH, s, C-15); “C NMR (CDCI,): 19.72 (Me,
from Cymopolia barbata 4117
C-7 or C-8), 19.68 (Me, C-7 or C-8), 20.6 (CH,, C-l), 23.3 (CH,,
C-lo), 32.0 (C, C-9), 33.4 (C, C-2), 39.9 (CH,, C-5), 41.5 (CH,,
C-6), 48.0 (CH, C-3), 77.3 (C, C-4), 107.6 (C, C-14), 115.8 (CH,
C-15), 119.6(CH,C-12), 123.9(C,C-11), 145.4(CH,C-13), 147.6
(C, C-16).
Bioassays. Field bioassays to assess feeding by parrot-
fishes were conducted at a depth of 8 m on a reef at the northern
boundary of the Looe Key Sanctuary near Big Pine Key,
Florida. Compound 1 was dissolved in Et 2O and coated on to
blades of the palatable seagrass Z’halassia testudinum that had
been blotted dry. Final concn of the compound was 1% of plant
dry mass. Four 6-cm lengths of these blades were anchored
between the strands ofa three-strand polypropylene rope, and 26
of these ropes were placed on the reef along with paired control
ropes holding equivalent blades that had been treated with Et,0
alone. Ropes of a pair were within 1 m of each other. Pairs were
sepd by a minimum of 4 m. After 2.5 hr, all ropes were collected
from the reef and grazing was measured as the area of T’halassia
blades missing from each rope. Further details of this method
have appeared elsewhere [16].
Lab. assays with the omnivorous pinfish Lagodon rhomboides
and the herbivorous amphipod Hyale macrodactyla were con-
ducted by coating 1 in Et,0 on to the palatable green alga Ulna
sp. at a concn of 1% of plant dry mass. Controls were coated
with Et,0 alone. Nineteen Lagodon were confined in separate
38 I aquaria and given equal masses of treatment and control
algae. Similar aquaria held equivalent masses of these same
pieces of algae but no fish; these served as controls for changes in
mass unrelated to grazing. When approximately half of either the
treatment or control alga had been eaten, all algae were removed
from the tank and reweighed. Changes in mass of algae exposed
to fish grazing were corrected by changes of portions of those
same individuals that were not subjected to fish grazing See
Renaud et al. [18] for a discussion of these methods and
statistical procedures. Assays with amphipods were similar,
however, they were conducted in smaller containers (200 ml) and
consumption was measured as area, rather than mass, eaten.
Acknowledgements-This research is a result of generous finan-
cial support from the National Science Foundation under grants
OCE 89-11872 (to M.E.H.), OCE 89-12600 and CHE 90-08621
(to W.F.).
REFERENCES
1. Rosenthal, G. A. and Janzen, D. H. (eds) (1979) Herbivores:
Their Interactions with Secondary Plant Metabolites. Aca-
demic Press, Berkeley.
2. Crawley, M. J. (1983) Herbiuory: 7’he Dynamics of
Animal-Plant Interactions. Studies in Ecology, Vol. 10,
437 pp. Univ. of California Press, Berkeley.
3. Coley, P. D., Bryant, J. B. and Chapin, F. S. 3rd (1985)
Science 230, 895.
4. Hay, M. E. and Fenical , W. (1988) Ann. Rev. Ecol. Syst. 19,
111.
5. Carpenter, R. C. (1986) Ecol. Monogr. 56, 343.
6. Hay, M. E. (1991) in The Ecology of Fishes on Coral Reefs
(Sale, P. F., ed.), pp. 96-119. Academic Press, San Diego.
7. Paul, V. J. and Fenical, W. (1987) in Bioorganic Marine
Chemistry, Vol. 1, pp. l-29. Springer, Berlin.
8. Paul, V. J. and Fenical, W. (1987) Mar. Ecol. Prog. Ser.
34, 157.
9. Martinez Nadal, N. G., Rodriguez, L. V. and Casillas, C.
M. (1964) Antimicrob. Agents Chemother. 1964, 131.
10. Hogberg, H. E., Thompson, R. H. and King, T. J. (1976)
J. Chem. Sot. Perkins I 1696.
4118 M.
11. McConnell, 0. J., Hughes, P. A. and Targett, N. M. (1982)
P~ytoc~~~iry 21,2139.
12. Estrada, D. M., Martin, J. D. and Perez, C. (1987) J. Nat.
Prod. 50,135.
13. Wall, M. M., Wani, M. C., Manikumar, G., Taylor, H.,
Hughes, T. J., Gaetano, K., Gerwick, W. H., McPhail, A.
T. and McPhail, D. R. (1989) J. Nat. Prod. 52, 1092.
14. Targett, N. M. and McConnell, 0. J. (1982) J. Chem. Ecol.
8. 115.
PARK et al.
15. McConnell, 0. J., Hughes, P. A., Targett, N. M. and
Daley, J. (1982) J. Chem. Ecof. 8, 1427.
16. Hay, M. E., Fenical, W. and Gustafson, K. (1987) Ecology
68,1581.
17. Half, D. and Sanders, J. K. M. (1980) J. Am. Chem. Sot.
102, 5703.
18. Renaud, P. E., Hay, M. E. and Schmitt, T. M. (1990)
Oecokogia 82, 217.