Tedanol: A potent anti-inflammatory ent-pimarane diterpene from the Caribbean Sponge Tedania ignis

Article abstract of DOI:10.1016/j.bmc.2009.09.010

Tedanol, a new brominated and sulfated pimarane diterpene was isolated from the Caribbean sponge Tedania ignis. Structure of tedanol was elucidated by mass spectroscopy and extensive NMR studies (including spectral simulation), and its absolute configurat

Full text of DOI:10.1016/j.bmc.2009.09.010

Bioorganic & Medicinal Chemistry 17 (2009) 7542–7547
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Tedanol: A potent anti-inflammatory ent-pimarane diterpene
from the Caribbean Sponge Tedania ignis
a
a
a
b
Valeria Costantino a, , Ernesto Fattorusso , Alfonso Mangoni , Cristina Perinu , Giuseppe Cirino ,
*
Luana De Gruttola b, Fiorentina Roviezzo b
a Dipartimento di Chimica delle Sostanze Naturali, Università degli Studi di Napoli Federico II, via D. Montesano 49, 80131 Napoli, Italy
b Dipartimento di Farmacologia Sperimentale, Università degli Studi di Napoli Federico II, via D. Montesano 49, 80131 Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 June 2009
Revised 3 September 2009
Accepted 6 September 2009
Available online 13 September 2009
Tedanol, a new brominated and sulfated pimarane diterpene was isolated from the Caribbean sponge
Tedania ignis. Structure of tedanol was elucidated by mass spectroscopy and extensive NMR studies
(including spectral simulation), and its absolute configuration was determined using the Mosher method.
Tedanol showed a potent anti-inflammatory activity at 1 mg/kg evaluated in vivo in a mouse model of
inflammation. After a single intraperitoneal administration, tedanol significantly reduced both the acute
and the subchronic phases of carrageenan-induced inflammation. The anti-inflammatory activity was
coupled with a strong inhibition of COX-2 expression, inhibition of cellular infiltration measured as mie-
loperoxidase (MPO) levels, and inhibition of iNOS expression. These features make tedanol a promising
template for the development of new anti-inflammatory molecules with low gastrointestinal toxicity.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Diterpenes
Pimarane
Anti-inflammatory activity
COX-2 inhibitors
Marine sponges
Tedania ignis
1. Introduction
However, our re-examination of the organic extract of T. ignis
revealed the presence of tedanol (1), a new brominated and sul-
Tedania ignis is a widespread Caribbean sponge, also known as
‘fire sponge’ because it causes a severe rash when handled by hu-
mans. Dermatites have also been reported to occur as a conse-
quence of the contact with the sponge tissue.1 It has not been
determined so far which chemicals cause rash and dermatitis. Pre-
vious analyses of Caribbean samples of T. ignis led to the isolation
of a new atisane derivative,2 several diketopiperazines,2,3 and some
carbazole and carboline derivatives.4 Many sponges contain a rich
flora of symbiotic bacteria,5 and T. ignis is one of these ‘high-micro-
bial-abundance sponges’. From a Micrococcus sp. obtained from the
tissue of T. ignis, Cardellina et al. have isolated some of the diketo-
piperazines previously isolated from the sponge6 along with four
new benzothiazoles.7
fated diterpene alcohol having an ent-pimarane skeleton. Tedanol
showed a potent anti-inflammatory activity, evaluated as reduc-
tion of the carrageenan-induced mouse paw edema, coupled with
a strong inhibition of COX-2 expression. In this paper, we report
on the structure elucidation of tedanol and the study of its anti-
inflammatory properties (Fig. 1).
2. Results
2.1. Isolation and structural elucidation
Samples of T. ignis collected in the mangroves of Sweeting Cay
(Grand Bahama Island) during the 2007 Pawlik expedition were
Finally, in 1984 Schimtz’s group reported the presence in T. ignis
of tedanolide,8 a potent cytotoxic macrolide that has been the tar-
get of intensive synthetic efforts. Although re-isolation of tedano-
lide was one of the reasons for our analysis of T. ignis, and in
spite of our best efforts, we were not able to find any tedanolide
in the extracts of the three different specimens of T. ignis from
Bahamas that we examined.
* Corresponding author. Tel.: +39 25081 678504; fax: +39 081 678552.
E-mail address: valeria.costantino@unina.it (V. Costantino).
Figure 1. Structures of compounds 1–3.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.010

V. Costantino et al. / Bioorg. Med. Chem. 17 (2009) 7542–7547
7543
immediately chopped in small pieces and frozen, then shipped to
our laboratory, where the samples were extracted in sequence
with MeOH and CHCl3. The MeOH extract was dried and parti-
tioned between water and BuOH, and the combined BuOH and
CHCl3 extracts were subjected to reversed-phase column chroma-
tography. The fraction eluted with H2O/MeOH 2:8 showed to con-
tain tedanol, but we were not able to obtain the compound in the
pure form by HPLC only. Instead, pure tedanol (1) (6.5 mg) was ob-
tained by reversed-phase HPLC, followed by preparative TLC using
BuOH/AcOH/H2O (60:15:25) as eluent.
atoms) pointed to the presence of one hydroxyl, one sulfate,
and two bromide functions linked to the four deshielded carbon
atoms C-3, C-7, C-15, and C-16. Derivatization of 0.6 mg of teda-
nol with Ac2O in Py gave the monoacetyl derivative 2, which
showed a remarkable downfield shift of H-7 from d 3.02 in the
natural compound 1 to d 4.39 in the acetate 2, thus demonstrat-
ing that the hydroxyl function is located at position 7. The two
bromine atoms present in the molecular formula were located
at C-3 (d 69.6) and C-15 (d 61.8), because the chemical shift of
these carbon atoms were about 15 ppm lower than those ob-
served in pimaranes oxygenated at these positions,9 while are
close to those observed in a 3,15-dibromoisopimarane from Lau-
rencia perforata.13 Finally, the sulfate group was located at the
remaining position 16, thus completing the planar structure of
compound 1.
The negative-ion ESI mass spectrum of 1 showed three Mꢀ
molecular ion peaks in the 1:2:1 ratio at m/z 545, 543, and 541, sug-
gesting the presence of two bromine atoms in the molecule. A high-
resolution measurement performed on the lowest-mass ion at m/z
541 determined the formula C20H31Br2O5S (exp.: 541.0272, calcd
for C20H3179Br2O5S: 541.0258) for this ion.
A preliminary 1H NMR analysis of compound 1 showed four
methyl singlets between d 1.13 and d 1.01 indicatives of a polycy-
clic diterpene skeleton. The 13C spectrum showed 20 carbon reso-
nances, that were assigned with an HSQC experiment as four CH3,
six CH2, six CH (one of which is sp2, d 115.9, and four linked to het-
eroatoms, d 78.3, 70.8, 69.6, and 61.8), and four non-protonated
carbon atoms (one of which is sp2, d 148.1). On the basis of the
molecular formula and with the evidence of a double bond, it
was clear that tedanol is a tricyclic diterpene.
The correlation peaks of the angular methyl groups [d 1.06 (H3-
17), d 1.13 (H3-18), d 1.01 (H3-19), d 1.08 (H3-20)] in the HMBC
spectrum allowed us to delineate two substructures (Fig. 2, bold
bonds), which were connected thanks to the proton–proton vicinal
couplings evidenced by the COSY spectrum (Fig. 2, solid bonds).
At this point, a pimarane skeleton was already delineated, ex-
cept for the C-8/C-14 and C-8/C-9 bonds (Fig. 2, dashed bonds),
connecting ring B with ring C. The bond between C-8 and C-9
was demonstrated by the allylic coupling between H-8 and H-11
and the homoallylic coupling between H-8 and H-12eq evidenced
by the COSY spectrum. The coupling between H-8 and the two pro-
tons at C-14 could not be evinced from the COSY spectrum because
the chemical shifts of H-8 and H-14eq were almost identical, lead-
ing to non-first-order multiplets. However, this couplings could be
demonstrated by spectral simulation. The subspectrum of the 10-
proton spin system comprising all protons on rings B and C was
calculated, optimizing the unknown NMR parameters until the
simulated multiplets reproduced accurately the experimental
ones.
2.2. Relative and absolute configuration
The relative configuration of the seven stereogenic centers of
tedanol was assigned using coupling constant analysis combined
with ROESY data. The A/B ring junction was established as trans be-
cause of the presence of a W long-range coupling between H-5 and
H3-18, indicative of their anti relationship. Relative configurations
at C-3, C-7, and C-8 were established from the large vicinal
coupling constants shown by H-3, H-7, and H-8 (see Table 1), indi-
cating that they are all axial. This completed the relative stereo-
chemistry of the trans-decaline moiety of the molecule (rings A
and B). The ROESY correlation peaks of H-15 with H-8 and H3-18,
indicated that C-15 lies on the same side of the molecule as C-8
and C-18. This determined the relative configuration at C-13, and
established that tedanol has the pimarane (or the enantiomeric
ent-pimarane) skeleton.
The ROESY correlation peak between H-15 and H3-18 was par-
ticularly interesting, because C-15 and C-18 are topologically far in
the molecule. Analysis of a molecular model of tedanol easily dem-
onstrated that H-15 and H3-18 can be in close proximity to each
other only if (a) the cyclohexene ring adopts the half-chair confor-
mation such that C-15 is pseudo-axial and C-17 is pseudoequatori-
al and (b) the conformation around the C-13/C-15 bond is such that
H-15 is directed inward of the cyclohexene ring (Fig. 4). The long-
range W coupling between H-15 and H3-17 observed in the COSY
spectrum, indicative of their anti relationship, showed that this
conformation around the C-13/C-15 bond is indeed largely
predominant.
The results (Fig. 3) pointed to a 5.9 Hz coupling constant be-
tween H-8 and H-14eq and a 10.7 Hz coupling constant between
H-8 and H-14ax, and also evidenced a quite large W coupling
(3.1 Hz) between H-12eq and H-14eq. In addition, the bond be-
tween C-8 and C-14 bond was confirmed by the ROESY correlation
peak between H-14ax (d 1.37) and H-7 (d 3.02).
The torsional rigidity of the C-13/C-15 bond can be justified by
unfavorable steric interactions of either the bromine atom or the
CH2SO3 group with H-8 in the two alternative staggered confor-
mations. More importantly, it was the key for the assignment of
the relative configuration at C-15. In fact, the ROESY correlation
peak between H-14eq and one of the protons at C-16 indicated that
C-14 and C-16 are gauche oriented, and this, together with the anti
relationship between H-15 and C-17, defined the relative configu-
ration at C-15 as depicted in Figure 4.
ꢀ
The remaining atoms to be assigned from the molecular for-
mula (two bromine, one sulfur, one hydrogen, and five oxygen
As for the absolute configuration of tedanol, we used Mosher’s
method,10 a reliable tool to establish the absolute configuration
of secondary alcohols. Tedanol was allowed to react with (S)-(+)-
and (R)-(ꢀ)-
a-methoxy-a-trifluoromethylphenylacetyl chloride
[(S)-(+)- and (R)-(ꢀ)-MTPA-Cl] to give, respectively, the 7-O-(R)-
MTPA and 7-O-(S)-MTPA esters 3r and 3s, whose 1H NMR spectra
were acquired and assigned.
According to the Mosher model, the esters adopt a preferential
conformation with the eclipsed CF3 and C@O groups, and the phe-
nyl group has a shielding effect on the neighboring protons. Thus,
Figure 2. Elucidation of the pimarane skeleton of tedanol (1). Part structures
determined from HMBC correlations of angular methyl protons are denoted with
bold lines, and bond determined from COSY correlations are denoted with solid
lines. X’s represent heteroatoms.
the observed
Dd values are consistent with the R configuration at
C-7 (Fig. 5), and therefore indicate that tedanol has the ent-pimara-
ne, and not the pimarane, skeleton.

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V. Costantino et al. / Bioorg. Med. Chem. 17 (2009) 7542–7547
Figure 3. Simulated spectrum of the spin system of H-5 through H-14 (upper trace) compared to the experimental spectrum (lower trace). All the signals (including the non-
first order multiplets of H8/H14eq and H-14ax) are accurately reproduced. Simulation parameters are: d(H-5) 1.0846, d(H-6ax) 1.6345, d(H-6eq) 1.9834, d(H-7) 3.0197, d(H-8)
2.3699, d(H-11) 5.3095, d(H-12ax) 1.8184, d(H-12eq) 2.4609, d(H-14ax) 1.3731, d(H-14eq) 2.3593, J(H-5/H-6ax) = 12.7 Hz, J(H-5/H-6eq) = 2.5 Hz, J(H-6ax/H-6eq) = ꢀ12.7 Hz,
J(H-6ax/H-7) = 11.1 Hz, J(H-6eq/H-7) = 4.7 Hz, J(H-7/H-8) = 9.8 Hz, J(H-8/H-11) = 1.7 Hz, J(H-8/H-12ax) = 3.5 Hz, J(H-8/H-12eq) = 1.6 Hz, J(H-8/H-14ax) = 10.7 Hz, J(H-8/H-
14eq) = 5.9 Hz, J(H-11/H-12ax) = 2.0 Hz, J(H-11/H-12eq) = 6.3 Hz, J(H-12ax/H-12eq) = ꢀ17.3 Hz, J(H-12eq/H-14eq) = 3.1 Hz, J(H-14ax/H-14eq) = ꢀ13.9 Hz.
Table 1
1H and 13C NMR data of tedanol 1 (CD3OD)
Position
1
dH [mult., J (Hz)]
dC [mult.]
ax
eq
ax
eq
1.55 (ddd, 13.5, 13.3, 3.6)
1.70 (ddd, 13.3, 3.6, 3.4)
2.27 (dddd, 13.6, 13.5, 12.7, 3.4)
2.16 (dddd, 13.6, 4.2, 3.6, 3.6)
4.02 (dd, 12.7, 4.2)
—
1.08 (dd, 12.7, 2.5)
1.62 (ddd, 12.7, 12.7, 11.1)
1.98 (ddd, 12.7, 4.7, 2.5)
3.02 (ddd, 11.2, 9.7, 4.7)
2.37 (dddddd, 10.7, 9.8, 5.9, 3.5, 1.7, 1.6)a
—
39.6 (CH2)
2
32.4 (CH2)
3
4
5
6
69.6 (CH)
40.8 (C)
51.6 (CH)
33.8 (CH2)
ax
eq
7
8
9
10
11
12
78.3 (CH)
40.3 (CH)
148.1 (C)
40.1 (C)
115.9 (CH)
39.7 (CH2)
—
5.31(ddd, 6.3, 2.0, 1.7)
1.82 (ddd,17.3, 3.5, 2.0)
2.46 (ddd, 17.3, 6.3, 3.1, 1.6)
—
ax
eq
Figure 4. ROESY correlations used to determine relative configurations at C-13 and
C-15.
13
14
36.7 (C)
39.4 (CH2)
ax
eq
1.37(dd, 13.9, 10.7)a
2.36 (ddd, 13,9, 5.9, 3.1)a
4.37 (dd, 8.9, 3.4)
4.49 (dd,11.8, 3.4)
4.19 (dd, 11.8, 8.9)
1.06 (s)
15
16
61.8 (CH)
70.8 (CH2)
a
b
17
18
19
20
24.6 (CH3)
21.7 (CH3)
18.7 (CH3)
30.9 (CH3)
1.13 (s)
1.01 (s)
1.08 (s)
a
Non-first-order multiplet, coupling constants determined by spectral simula-
tion (see text and Fig. 3).
2.3. Anti-inflammatory activity
The anti-inflammatory activity was evaluated as inhibition of
the carrageenan-induced paw edema in mice.11,12 Intraperitoneal
administration of tedanol (0.1–1 mg/kg) to mice 30 min before
subplanatar carrageenan injection, causes a significant and dose
related reduction in edema (Fig. 6). Because edema evaluation
gives information relative to inflammatory swelling, due to vascu-
lar leakage, but not on cell infiltration, mieloperoxidase (MPO) lev-
els were also measured. MPO evaluation is a widely used indirect
index of cell infiltration. The systemic administration of tedanol
prior to induction of inflammation (1 mg/kg) significantly inhibits
MPO activity at 4 and 48 h after carrageenan injection (Fig. 7).
In order to further investigate on the mechanism underlying
tedanol anti-inflammatory activity, western blot analysis was per-
Figure 5. Preferred conformation of the (S)-MTPA ester of tedanol (3s) according to
the Mosher model, and observed
Dd of the neighboring protons.
formed on the paws harvested from mice pretreated with tedanol
at the highest dose. Tedanol (1 mg/kg) significantly inhibited COX-
2 expression both in the early phase (2 and 4 h) and in the second
edema phase (48 and 72 h). Conversely, tedanol did not affect COX-
1 expression in the early phase, but significantly inhibited its
expression in the second phase. Tedanol pretreatment significantly
inhibited iNOS expression at 2 h and 48 h only (Fig. 8). These data
suggest that tedanol exerts its anti-inflammatory activity by reduc-
ing both vascular permeability and cell migration that involves

V. Costantino et al. / Bioorg. Med. Chem. 17 (2009) 7542–7547
7545
Figure 6. Effect of systemic administration of tedanol (0.1–1 mg/kg) on carra-
***
geenan-induced mouse paw edema (* p <0.05;
p <0.001 vs vehicle).
Figure 8. Western blot analysis for COX-2 (panel A), COX-1 (panel B) and iNOS
(panel C) performed on inflamed mouse paws harvested from tedanol- (1 mg/kg) or
vehicle-treated mice. The blots are representative of three different experiments
reported as densitometry analysis in bar graphs (* p <0.05; ** p <0.01; *** p <0.001 vs
vehicle).
alcohol 1. To the best of our knowledge this is the first report of an
ent-pimarane diterpene from a sponge tissue. The known natural
metabolites most closely related to tedanol are a dibromoditerpene
isolated in 1984 from the alga L. perforata13 and its diacetate from
the sponge Spongia zymocca,14 but these are compounds based on
an isopimarane rather than an ent-pimarane skeleton.15
Tedanol turned out to be biologically attractive because it
showed a potent anti-inflammatory activity, evaluated as reduction
of the carrageenan-induced mouse paw edema. Another diterpene
with the pimarane skeleton, acanthoic acid,16 which is present in
the Korean plant Acanthopanax koreanum traditionally used in pop-
ular medicine for the cure of rheumatism, has been shown to possess
in vitro COX-2 inhibitory activity.17,18 However, no data are avail-
able to date on the in vivo anti-inflammatory activity of this com-
pound or other natural pimarane diterpenes. Indeed, in vivo anti-
inflammatory activity requires, in addition to COX-2 inhibition, a
favorable pharmacokinetic profile and lack of toxicity.
Figure 7. MPO evaluation performed on inflamed mouse paws harvested from mice
systemically treated with the higher dose of tedanol (1 mg/kg)- versus vehicle-
treated mice in the first (panel A) and second phase (panel B) of edema (** p <0.01 vs
vehicle).
mainly modulation of expression of COX-2 and, to a lesser extent,
of COX-1 and iNOS.
3. Discussion
The structure of the new diterpene tedanol from T. ignis was
established as the brominated and sulfated ent-pimarane diterpene

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V. Costantino et al. / Bioorg. Med. Chem. 17 (2009) 7542–7547
In the present study, we demonstrated that tedanol does pos-
to proceed for 18 h at room temperature, and the reaction mixture
was then dried under vacuum, giving 0.6 mg of tedanol 7-O-acetate
(2): colorless amorphous solid; 1H NMR (CD3OD): d 5.39 (1H, br d,
J = 6.3 Hz, H-11), 4.45 (1H, dd, J = 11.6 and 4.1 Hz, H-16a), 4.39 (1H,
ddd, J = 10.8, 10.6, and 4.7 Hz, H-7), 4.36 (1H, dd, J = 8.2 and 4.1 Hz,
-H-15), 4.20 (1H, dd, J = 11.6 and 8.2 Hz, H-16b), 4.04 (1H, dd,
J = 12.7 and 4.2 Hz, H-3), 2.66 (1H, m, H-8), 2.46 (1H, br d,
J = 17.4 Hz, H-12eq), 2.29 (1H, m, H-2ax), 2.17 (2H, overlapped,
H-2ax and H-14eq) , 2.03 (1H, ddd, J = 12.5, 4.6, and 2.5 Hz, H-
6eq), 1.84 (1H, ddd, J = 17.4, 3.5, and 2.2 Hz, H-12ax), 1.72 (2H,
overlapped, H-1eq and H-6ax), 1.58 (1H, ddd, 13.3, 13.4, and
3.6 Hz, H1ax), 1.31 (1H, m, H-14ax), 1.160 (3H, s, H3-18), 1.16
(1H, dd, J = 12.8 and 2.5 Hz, H-5), 1.055 (3H, s, H3-20), 1.046 (3H,
s, H3-17), 0.998 (3H, s, H3-19).
sess in vivo anti-inflammatory activity. After a single intraperito-
neal administration, tedanol significantly reduced both the acute
(4 h) and subchronic (48 h) phase of the carrageenan-induced
paw edema in mice. The anti-inflammatory activity was coupled
with a strong inhibition of COX-2 expression, inhibition of cellular
infiltration measured as mieloperoxidase (MPO) levels, and inhibi-
tion of iNOS expression. These features, together with its solubility
in water, not frequently encountered among natural diterpenes,
make tedanol a promising template for the development of new
anti-inflammatory molecules with low gastrointestinal toxicity.
4. Experimental section
4.1. General experimental procedures
4.3. MTPA derivatization of tedanol
High-resolution ESI-MS spectra were performed on a Bruker
APEX II FT-ICR mass spectrometer. ESI MS experiments were per-
formed on a Applied Biosystem API 2000 triple-quadrupole mass
spectrometer. The spectra were recorded by infusion into the ESI
source using MeOH as the solvent. Optical rotations were mea-
sured at 589 nm on a Perkin–Elmer 192 polarimeter using a 10-
cm microcell. 1H and 13C NMR spectra were determined on Varian
UnityInova spectrometers at 500 and 700 MHz; chemical shifts
were referenced to the residual solvent signal (CD3OD: dH = 3.31,
dC = 49.0). For an accurate measurement of the coupling constants,
the one-dimensional 1H NMR spectra were transformed at 128 K
points (digital resolution: 0.05 Hz). Homonuclear 1H connectivities
were determined by COSY experiments. Through-space 1H connec-
tivities were evidenced using a ROESY experiment with a mixing
time of 450 ms. The reverse single-quantum heteronuclear correla-
Tedanol (0.4 mg) was dissolved in 500
ll of pyridine, and (S)-
(+)-MTPA chloride (10 l) was added. The reaction was allowed
l
to proceed for 18 h at room temperature, and the reaction mixture
was then dried under vacuum, giving, the 7-O-(R)-MTPA ester 3r.
The same procedure, using (R)-(ꢀ)-MTPA chloride, gave the 7-O-
(S)-MTPA ester 3s.
4.3.1. Tedanol 7-O-(R)-MTPA ester (3r)
Colorless solid; 1H NMR (CD3OD): d 5.42 (1H, br d, J = 6.3 Hz, H-
11), 4.62 (1H, ddd, J = 10.9, 10.9, and 4.7 Hz, H-7), 4.36 (1H, dd,
J = 11.6 and 3.1 Hz, H-16a), 4.33 (1H, dd, J = 9.0 and 3.1 Hz, H-15),
4.11 (1H, dd, J = 11.6 and 9.0 Hz, H-16b), 4.05 (1H, dd, J = 12.6
and 4.2 Hz, H-3), 2.70 (1H, m, H-8), 2.47 (1H, br d, J = 17.4 Hz, H-
12eq), 2.26 (1H, dddd, J = 13.3, 13.3, 13.3, and 3.2 Hz, H-2ax),
2.17 (1H, dddd, J = 13.7, 3.9, 3.9, and 3.9 Hz, H-2eq), 2.01 (1H,
ddd, J = 12.1, 4.6, and 2.4 Hz, H-6eq), 1.95 (1H, ddd, J = 13.8, 5.9,
and 3.1 Hz, H-14eq), 1.85 (1H, ddd, J = 17.4, 3.1, and 2.4 Hz, H-
12ax), 1.71 (1H, ddd, J = 13.4, 3.4, and 3.4 Hz, H-1eq), 1.62 (quartet,
J = 12.2 Hz, H-6ax), 1.59 (1H, m, H-1ax) , 1.34 (1H, J = 13.8 and
10.7 Hz, H-14ax), 1.20 (1H, dd, J = 12.7 and 2.3 Hz, H-5), 1.11 (3H,
s, H3-18), 1.06 (3H, s, H3-20), 1.00 (3H, s, H3-17), 0.94 (3H, s, H3-
19).
1
tion (HSQC) spectra were optimized for an average JCH of 145 Hz.
The multiple-bond heteronuclear correlation (HMBC) experiments
3
were optimized for a JCH of 8 Hz. Spectral simulations were per-
formed using Bruker’s NMRSIM program. High performance liquid
chromatographies (HPLC) were achieved on a Varian Prostar 210
apparatus equipped with an Varian 350 refractive index detector.
4.2. Collection, extraction, and isolation
4.3.2. Tedanol 7-O-(S)-MTPA ester (3s)
Colorless solid; 1H NMR (CD3OD): d 5.37 (1H, br d, J = 6.3 Hz, H-
11), 4.52 (1H, ddd, J = 10.9, 10.9, and 4.7 Hz, H-7), 4.22 (1H, dd,
J = 9.8 and 2.7 Hz, H-15), 4.04 (2H, overlapped, H-3 and H-16a),
3.88 (1H, dd, J = 11.9 and 9.8 Hz, H-16b), 2.57 (1H, m, H-8), 2.42
(1H, br d, J = 17.4 Hz, H-12eq), 2.27 (1H, dddd, J = 13.3, 13.3, 13.3,
and 3.2 Hz, H-2ax), 2.17 (2H, overlapped, H-2eq and H-6eq), 1.80
(1H, quartet, J = 12.1 Hz, H-6ax), 1.77 (1H, ddd, J = 17.4, 3.3, and
2.4 Hz, H-12ax), 1.70 (1H, ddd, J = 13.4, 3.4, and 3.4 Hz, H-1eq),
1.57 (1H, ddd, J = 13.4, 13.4, and 3.5 Hz, H-1ax) , 1.37 (1H, m, H-
14eq), 1.21 (1H, dd, J = 12.7 and 2.4 Hz, H-5), 1.13 (3H, s, H3-18),
1.09 (3H, s, H3-20), 1.03 (1H, J = 13.9 and 10.9 Hz, H-14ax), 1.00
(3H, s, H3-19), 0.85 (3H, s, H3-17).
Specimens of T. ignis were collected in the Mangroves of Sweet-
ing Cay (Grand Bahama Island) during the 2007 Pawlik expedition.
They were frozen immediately after collection and kept frozen un-
til extraction. The sponge (58 g of dry weight after extraction) was
homogenized and extracted with MeOH (5 ꢁ 1 L) and then with
CHCl3 (2 ꢁ 1 L). The MeOH extracts were partitioned between
H2O and n-BuOH, and the BuOH layer was combined with the
CHCl3 extract and concentrated in vacuo. The organic extract
(12.8 g) was chromatographed on a column packed with RP-18 sil-
ica gel. A fraction eluted with MeOH/H2O 8:2 (195 mg) was sub-
jected to HPLC separation on an RP-18 column [MeOH/H2O
(7:3)], thus affording a fraction (6.5 mg) mainly composed of com-
pound 1. Final purification was achieved by preparative TLC (SiO2,
20 ꢁ 20 cm, 0.5 mm thick) using BuOH/AcOH/H2O (60:15:25) as
eluent, which gave 3.1 mg of pure tedanol 1.
4.4. Mouse paw edema
Male CD-1 mice weighing 23–27 g were separated in groups
(n = 6) and lightly anesthetized with enflurane. Each group of ani-
4.2.1. Tedanol (1)
Colorless amorphous solid, ½a D25
¼ þ6 (c 0.1 in MeOH); HRESIMS
ꢂ
mals received subplantar injection of 50 ll of carrageenan 1% w/
(negative ion mode, MeOH) 541.0272 (Mꢀ, C20H3179Br81BrO5S gives
541.0258); ESIMS (negative ion mode, MeOH) m/z 543, 541, and
539 (Mꢀ); 1H and 13C NMR: Table 1.
v.11,12 Paw volume was measured using a hydroplethismometer
specially modified for small volumes (Ugo Basile, Milan, Italy)
immediately before the subplantar injection and 2, 4,6, 24, 48, or
72 h thereafter. The increase in paw volume was evaluated as dif-
ference between the paw volume at each time point and the basal
paw volume. In another set of experiments, CD-1 were subjected to
4.2.2. Tedanol 7-O-acetate (2)
Tedanol 1 (0.6 mg) was dissolved in 500
ll of pyridine and
500 l of acetic anhydride were added. The reaction was allowed
l
a previous intraperitoneal injection of tedanol 1 (200 ll of water

V. Costantino et al. / Bioorg. Med. Chem. 17 (2009) 7542–7547
7547
solution, 0.1–1 mg/kg), and after 30 min each group of animals re-
ceived subplantar injection of 50 l of carrageenan. Data are ex-
pressed as mean SEM. The level of statistical significance was
determined by two-way analysis of variance (ANOVA) followed
by Bonferroni’s test for multiple comparisons, using the GraphPad
Prism software.
(Grant agreement no. 229893). A warm thank you is due to Pro-
fessor J. R. Pawlik, University of North Carolina at Wilmington,
for inviting them to participate to the ‘2007 Pawlik Expedition’
under the National Science Foundation project grant entitled
‘Assessing the chemical defenses of Caribbean Invertebrates’,
during which the material was collected, and Prof. Sven Zea
(Universidad Nacional de Colombia) for identifying the sponge.
Mass and NMR spectra were recorded at the ‘Centro di Servizi
Interdipartimentale di Analisi Strumentale’, Università di Napoli
‘Federico II’. The assistance of the staff is gratefully
acknowledged.
l
4.5. Western blot
Carragenan-injected paws from CD-1 mice sacrificed at 2, 4, 6
24, 48, and 72 h were homogenised in a 10 mM HEPES pH 7.4 buf-
fer containing saccharose (0.32 M), EDTA (100
(1 mM), phenylmethylsulfonyl fluoride (1 mg/ml), and leupeptin
(10
g/ml)11 using a Polytron homogenizer (3 cycles of 10 s at
lM), dithiothreitol
Supplementary data
l
maximum speed). After centrifugation at 3000 rpm for 15 min,
protein supernatant content was measured by Bradford reagent,
and protein concentration was adjust at 30 lg. Protein samples
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.09.010.
were loaded on 10% PAGE–SDS and transferred onto nitrocellulose
membranes for 45 min at 250 mA. Membranes were blocked in
PBS-Tween 20 (0.1%) containing 5% non-fat milk and 0.1% BSA
for 30 min at 4 °C. Membranes were washed with PBS-Tween 20
(0.1%) at 5 min intervals for 30 min, and incubated with anti-
COX-1, anti-COX-2, anti-iNOS, and anti-eNOS overnight at 4 °C.
Blots were washed with PBS-Tween 20 (0.1%) at 5 min intervals
for 30 min and incubated with HRP-anti-rabbit IgG (1:20,000) for
2 h at 4 °C. The immunoreactive bands were visualized using an
enhanced chemiluminiscence system (ECL; Amersham, USA).
References and notes
1. Yaffee, H. S.; Stargardter, F. Arch. Dermatol. 1963, 87, 601.
2. Schmitz, F. J.; Vanderah, D. J.; Hollenbeak, K. H.; Enwall, C. E. L.; Gopichand, Y. J.
Org. Chem. 1983, 48, 3941.
3. Dillman, R. L.; Cardellina, J. H., II J. Nat. Prod. 1991, 54, 1159.
4. Dillman, R. L.; Cardellina, J. H., II J. Nat. Prod. 1991, 54, 1056.
5. Hentschel, U.; Usher, K. M.; Taylor, M. W. FEMS Microbiol. Ecol. 2006, 55,
167.
6. Stierle, A. C.; Cardellina, J. H., II; Singleton, F. L. Experientia 1988, 44, 1021.
7. Stierle, A. A.; Cardellina, J. H., II Tetrahedron Lett. 1991, 32, 4847.
8. Schmitz, F. J.; Gunasekera, S. P.; Yalamanchili, G.; Hossain, M. B.; van der Helm,
D. J. Am. Chem. Soc. 1984, 106, 7251.
9. Chen, W.; Tang, W.; Zhang, R.; Lou, L.; Zhao, W. J. Nat. Prod. 2007, 70, 567.
10. Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1993, 95, 512; Ohtani, I.; Kusumi, T.;
Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113, 4092.
11. Posadas, I.; Bucci, M.; Roviezzo, F.; Rossi, A.; Parente, L.; Sautebin, L.; Cirino, G. J.
Pharmacol. 2004, 142, 331.
12. Henriques, M. G.; Silva, P. M.; Martins, M. A.; Flores, C. A.; Cunha, F. Q.;
Assreuy-Fiho, J.; Cordeiro, A. Braz. J. Med. Biol. Res. 1987, 20, 243–249.
13. Gonzalez, A. G.; Ciccio, J. F.; Rivera, A. P.; Martin, J. D. J. Org. Chem. 1985, 50,
1261.
14. Guella, G.; Mancini, I.; Pietra, F. Comp. Biochem. Physiol. 1992, 103B, 1019.
15. Although in Ref. 13 and in all the subsequent literature (including Ref. 14) the
compound is described as an isopimarane (i.e., 13-epi-pimarane), the structural
drawing on the paper shows the stereochemistry of a pimarane. Because the
stereochemical assignment was only based on analogy with known
compounds, the actual stereochemistry at C-13 of the bromoditerpene from
L. perforata remains undetermined.
4.6. MPO measurement
Mice from different groups were sacrificed with CO2 at 2, 4, 6
24, 48, and 72 h after carrageenan injection. Injected paws were
cut, weighted and homogenated in 1 ml of HTAB buffer using a
Polytron homogenizer (2 cycles of 10 s at maximum speed). After
centrifugation of homogenates at 10,000 rpm for 2 min, superna-
tant fractions were assayed for MPO activity using the method de-
scribed by Bradley et al.19 Briefly, samples were mixed with
phosphate buffer containing 1 mM O-dianisidine dihydrochloride
and 0.001% hydrogen peroxide in a microtiter plate reader. Absor-
bance was measured at 450 nm taking three reading at 30 s inter-
vals. Units of MPO were calculated considering that 1 U.
16. Kim, Y. H.; Chung, B. S.; Sankawa, U. J. Nat. Prod. 1988, 51, 1080.
17. Suh, Y. G.; Kim, Y. H.; Park, M. H.; Choi, Y. H.; Lee, H. K.; Moon, J. Y.; Min, K. H.;
Shin, D. Y.; Jung, J. K.; Park, O. H.; Jeon, R. O.; Park, H. S.; Kang, S. A. Bioorg. Med.
Chem. Lett. 2001, 11, 559.
MPO = 1 lmol H2O2 split , and 1 lmol H2O2 gives a change in
absorbance of 1.13 ꢁ 10ꢀ2 nm/min.
18. Suh, Y. G.; Lee, K. O.; Moon, S. H.; Seo, S. Y.; Lee, Y. S.; Kim, S. H.; Paek, S. M.;
Kim, Y. H.; Lee, Y. S.; Jeong, J. M.; Lee, S. J.; Kim, S. G. Bioorg. Med. Chem. Lett.
2004, 14, 3487.
19. Posadas, I.; Terencio, M. C.; Guillén, I.; Ferrándiz, M. L.; Coloma, J.; Payá, M.;
Alcaraz, M. J. Naunyn-Schmied. Arch. Pharmacol. 2000, 361, 98.
Acknowledgments
This research work was supported by MIUR PRIN 2007 (Grant
no. 2007KZ9W5J) and by the European Project NatPharma