Eunicidiol, an anti-inflammatory dilophol diterpene from Eunicea fusca

Article abstract of DOI:10.1021/np300149y

A new dilophol diterpene, eunicidiol (1), has been isolated from the crude extract of Eunicea fusca, a gorgonian coral collected from Hillsboro Ledge, Florida. This compound was purified, along with fuscol (2) and eunicol (3), using a combination of normal- and reversed-phase chromatography methods. The structure of eunicidiol (1) was elucidated by 1D and 2D NMR spectroscopic analysis, and the absolute configuration was assigned using Moshers method. The anti-inflammatory activity of 1-3 was evaluated by measuring their ability to reduce phorbol myristate acetate (PMA)-induced edema in a mouse ear model. Topical application of a 100 μg/ear dose of diterpenes 1-3 significantly reduced edema by 44%, 46%, and 54%, respectively. This activity was superior to indomethacin, a known anti-inflammatory used as a control.

Full text of DOI:10.1021/np300149y

Article
pubs.acs.org/jnp
Eunicidiol, an Anti-inflammatory Dilophol Diterpene from Eunicea
fusca
Douglas H. Marchbank,† Fabrice Berrue,†,‡ and Russell G. Kerr*,†,‡
†Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown,
Prince Edward Island, C1A 4P3, Canada
‡Department of Chemistry, University of Prince Edward Island, Charlottetown, Prince Edward Island, C1A 4P3, Canada
S
* Supporting Information
ABSTRACT: A new dilophol diterpene, eunicidiol (1), has been
isolated from the crude extract of Eunicea fusca, a gorgonian coral
collected from Hillsboro Ledge, Florida. This compound was
purified, along with fuscol (2) and eunicol (3), using a combination
of normal- and reversed-phase chromatography methods. The
structure of eunicidiol (1) was elucidated by 1D and 2D NMR
spectroscopic analysis, and the absolute configuration was assigned
using Mosher’s method. The anti-inflammatory activity of 1−3 was
evaluated by measuring their ability to reduce phorbol myristate
acetate (PMA)-induced edema in a mouse ear model. Topical application of a 100 μg/ear dose of diterpenes 1−3 significantly
reduced edema by 44%, 46%, and 54%, respectively. This activity was superior to indomethacin, a known anti-inflammatory used
as a control.
arine corals belonging to the order Gorgonacea have
provided a wealth of structurally diverse diterpenes
vivo anti-inflammatory activity by assessing their ability to reduce
PMA-induced edema in a mouse ear model.
M
with potential application as therapeutic agents.1 The Caribbean
gorgonian Eunicea fusca is a source of the anti-inflammatory
fuscosides A (4) and B (5), diterpene arabinose glycosides that
reduce phorbol myristate acetate (PMA)-induced mouse ear
edema with activities comparable to indomethacin.2,3 Fusco-
side B is known for its selective inhibition of 5-lipoxygenase
and negligible effect on cyclooxygenases and prostaglandin
biosynthesis.4 As part of our interest in this glycoside, we
recently synthesized a small library of 1,2-trans glycoside
analogues of fuscoside B, including a novel class of dilophol
glycosides called the eunicosides, by glycosylating the tertiary
hydroxy groups of fuscol (2) and eunicol (3).5 In addition we
have also investigated the biosynthesis of 2−4 by examining the
metabolism of 3H-geranylgeranyl diphosphate by E. fusca cell-
free extracts.6
We have continued our search for new anti-inflammatory
compounds from E. fusca by examining minor constituents
displaying structural homology to 2, 4, and 5. We report herein
the characterization of eunicidiol (1) using NMR spectroscopy
and mass spectrometry. This diterpene is composed of an
uncommon dilophol carbon skeleton that consists of a
cyclodecane ring as well as an eight-carbon side chain, which is
analogous to the structure of fuscol (2) and eunicol (3). While
the relative configuration of 1 was obtained by NOESY
correlations, the absolute configuration was solved by Mosher’s
method using α-methoxyphenylacetic acid (MPA). Further-
more, an enantiospecific Cope rearrangement of the cyclo-
decadiene moiety of 3 to produce 2 has firmly established the
absolute configuration of 3. Diterpenes 1−3 were evaluated for in
RESULTS AND DISCUSSION
■
Eunicidiol (1) was obtained, along with known compounds
fuscol (2) and eunicol (3), from the crude extract of E. fusca and
purified by semipreparative reversed-phase high-performance
liquid chromatography (RP-HPLC) using a combination of C18
and phenylhexyl stationary phases. The molecular formula of 1
was established by HRESIMS analysis (m/z 327.2286 [M + Na]+),
Received: February 24, 2012
Published: June 26, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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indicating the molecular formula C20H32O2 and an index of
hydrogen deficiency of five. The existence of hydroxy groups was
reflected by the IR spectrum (3357 cm−1) and the two oxygen-
bearing carbon resonances (δC 76.2, 70.4). The NMR
spectroscopic data for 1 were acquired using benzene-d6, as
other solvents, including methanol-d4, dichloromethane-d2, and
acetone-d6, produced poorly resolved NMR spectra with broader
and weaker signals. Notably, such signal broadening was also
observed in the 1H and 13C NMR spectra of 3, as a consequence
of conformational isomerism of the cyclodecane ring (Figures
S17, S18).
Figure 1. Selected HMBC (1H → 13C) and COSY correlations (bold
bonds) of partial structures A−C as well as the assembled structure of
eunicidiol (1).
1
The H NMR spectrum of 1 indicated a conjugated diene
system, identical to that of 2 and 3, comprising the olefinic
resonances of H-13 [δH 6.60 (1H, dd, J = 15.1, 10.8 Hz)], H-12
[δH 5.99 (1H, d, J = 10.8 Hz)], and H-14 [δH 5.72 (1H, d, J =
15.1 Hz)] (Table 1). The HMBC spectrum showed correlations
Owing to the lack of unambiguous coupling of partial structure A
to other nuclei, additional partial structures were elucidated
independently.
The C-3 (δC 150.0) and C-16 (δC 114.7) resonances were
assigned by HMBC and HSQC coupling with terminal olefinic
methylene protons H-16a/b (δH 5.04, 4.94). Long-range
heteronuclear correlations between H-16b and C-4 (δC 25.7)
and C-2 (δC 76.2) established the 1,1-disubstituted double bond.
The incorporation of a secondary hydroxy group at C-2 was
established on the basis of the deshielded proton (δH 3.87) and
carbon chemical shifts. This analysis provided a four-carbon
segment referred to as partial structure B. Partial structure C was
composed of a trisubstituted double bond according to long-
range HMBC correlations between the remaining olefinic C-17
methyl group (δH 1.53) and multiple carbon resonances [C-5 (δC
25.8), C-9 (δC 31.2), C-6 (δC 125.9), C-7 (δC 133.5), and C-8 (δC
40.6)]. The COSY spectrum indicated a vicinal correlation
between the C-6 olefinic methine (δH 5.45) and C-5 methylene
(δH 2.29) as well as between H-8a (δH 1.93) and H-9 (δH 1.29).
The three partial structures must assemble to provide a 10-
membered ring to satisfy the required index of hydrogen
deficiency of five. The HMBC coupling between H-10 and C-2
provided the basis for connecting partial structures A and B at
C-1 and C-2. In addition, the C-10 methine proton of partial
structure A displayed a COSY correlation with the C-9
methylene protons of partial structure C to signify the
attachment of C-9 and C-10. Moreover, the HMBC correlations
between H-10 and C-8 and C-9 confirmed this assembly. It was
not possible to establish the attachment of C-4 and C-5 via
COSY correlations as a result of the inability to discern between
cross-peaks caused by the vicinal correlation between H-4a/H-5b
and the geminal correlation between H-4a/H-4b. Instead, this
assignment was made on the basis of an HMBC correlation
between H-4 and C-5. The results of a TOCSY experi-
ment agreed with the assignment of the methylene proton
resonances to the two separate spin systems of the cyclodecane
ring (Figure S8).
Table 1. NMR Spectroscopic Data (600 MHz, Benzene-d6) for
Eunicidiol (1) [δ in ppm Relative to Residual Solvent Signal]
a
δH,
multiplicity
b
c
b
position
δC, type
(J in Hz)
COSY
10
HMBC
NOESY
2
1
2
36.9, CH2 1.60, br m
76.2, CH
3.87, br d
(7.7)
1, 10, 12,
16a
3
150.0, C
4a
25.7, CH2 2.20, app. td
(12.3, 3.9)
4b, 16b
2, 3, 5, 16 16b, 17
16b
4b
5a
5b
6
2.05, m
25.8, CH2 2.29, m
2.08, m
4a, 5a, 16b
4b, 5b, 6
5a
125.9, CH
133.5, C
5.45, m
5a
8a, 10
7
8a
8b
9
40.6, CH2 1.93, m
2.01, m
9
6
9
31.2, CH2 1.29, m
8a, 10
1, 9
8b
10
44.8, CH
1.86, app. t
(8.5)
2, 8, 9, 11, 2, 6, 12
12, 18
11
12
13
141.9, C
124.8, CH
123.2, CH
5.99, d (10.8) 13, 18
10, 14, 18 1, 10, 14
11, 15, 19, 18, 19,
6.60, dd
12, 14
(15.1, 10.8)
20
20
14
140.1, CH
5.72, d (15.1) 13
12, 15, 19, 12, 19,
20
20
15
70.4, C
16a
16b
17
114.7, CH2 5.04, br s
4.94, s
4a, 16b
4b, 16a
2
2, 3, 4
4a, 4b
15.7, CH3 1.53, s
13.9, CH3 1.67, s
30.2, CH3 1.24, s
5, 6, 7, 8, 9 4a
10, 11, 12 13
18
12
19/20
13, 14, 15, 13, 14
19, 20
a
b
See Figures S1−S8 for NMR spectra. COSY and NOESY correlations
c
The large vicinal coupling constant (J = 15.1 Hz) across the
are from proton(s) stated to the indicated proton. HMBC correlations
Δ
13,14 double bond indicated the E configuration. The Δ11,12 and
are from proton(s) stated to the indicated carbon.
Δ6,7 double bonds were also assigned E configurations given
the observed NOESY correlations between H-18/H-13 and
H-6/H-8a. The determination of relative configuration was also
enabled by interpretation of NOESY correlations. The observed
NOESY correlation between H-6 and H-10 suggested that both
protons occupied the same side of the average plane of the ring.
Meanwhile, the apparent lack of NOESY correlations between
H-2/H-4b and H-2/H-6 implied the opposite orientation for
H-2. This configuration was upheld by the NOESY correlation
between H-2/H-12. The observed NOESY correlations were in
agreement with the MM2-minimized model of eunicidiol (1)
between H-14 and C-19/20 (δC 30.2) and C-15 (δC 70.4),
indicating the presence of geminal dimethyl groups and a tertiary
hydroxy group. The C-20 methyl protons (δH 1.24) displayed
HMBC correlations with C-19 and C-15 to further establish this
moiety. The olefinic proton H-12 exhibited HMBC coupling
with C-10 (δC 44.8) and C-18 (δC 13.9), confirming that the side
chain of 1 was the same as that of 2 and 3. The C-10 methine
proton showed COSY correlations with the C-1 methylene
protons (δH 1.60), thus providing partial structure A (Figure 1).
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Scheme 2. Semisynthesis of Fuscol (2) by Cope
a
Rearrangement of Eunicol (3)
a
Reagents and conditions: (a) Δ toluene, 2.5 h (38%).
having the 10S configuration, and for that reason the absolute
configuration of eunicol was established as such. We also suggest
that the configurations of fuscoside A (4) and eunicene A (8) are
consistent with their biosynthetic relatives 2 and 3 and therefore
have the absolute configurations displayed in Scheme 3.
We have a long-standing interest in the biosynthesis of
marine diterpenes and have previously shown that in E. fusca
the diterpene cyclase product is eunicene A (8) and the first
oxidation product is eunicol (3).6 Compound 3 leads to both
fuscoside A (4) and fuscol (2). It seems reasonable to suggest
that eunicidiol (1) is derived from an oxidation of 3 as outlined in
Scheme 3.
Fuscosides A (4) and B (5) are known to exhibit topical anti-
inflammatory activity, as they reduce PMA-induced mouse ear
edema by 85% and 52%, respectively, and are comparable to the
known anti-inflammatory agent indomethacin.3 Fuscoside B (5)
is further distinguished as a selective 5-lipoxygenase (5-LO)
inhibitor, having a negligible effect on cyclooxygenase and other
targets in the inflammatory cascade.4
Diterpenes 1−3 were evaluated for in vivo anti-inflammatory
activity in the mouse ear edema assay at a single dose (100 μg/ear)
(Table 2). Indomethacin was administered as a reference
anti-inflammatory agent (3 mg/ear). Topical application of
diterpenes 1−3 significantly reduced mouse ear edema at 24 h
post-treatment. Although comparable to indomethacin, the
reduction of edema by 1−3 was notably achieved with much
smaller doses. Eunicol (3) displayed a modest increase in
potency over fuscol (2) and eunicidiol (1) at 24 h; however the
difference between the diterpenes was not statistically significant.
In addition, compounds 1−3 displayed no signs of toxicity and
otherwise appeared to be safe at the administered dose. An
evaluation of structure−activity relationships has implicated the
tertiary hydroxy group and conjugated diene moiety as being
important for the observed anti-inflammatory activity, as this is
the most common structural feature of the diterpenes tested.
It remains to be determined whether diterpenes 1−3 operate
through a similar mechanism of action as fuscoside B (5) as
inhibitors of 5-LO.
1
Figure 2. Key NOESY correlations (1H ↔ H) for eunicidiol (1)
[ChemBioDraw 3D MM2-minimized model].
(Figure 2). Compound 1 and fuscol (2) likely have a common
biosynthetic precursor and, therefore, presumably have the same
configuration at C-10. As a result, the relative configuration of 1
was determined to be 2R*,10S*.
The absolute configuration of eunicidiol (1) was unambigu-
ously assigned by modifying the asymmetric secondary hydroxy
group with (R)- and (S)-α-methoxyphenylacetic acid (Scheme 1).
Scheme 1. Synthesis of the (R)- and (S)-MPA Esters of
Eunicidiol and 1H NMR Chemical Shift Differences ΔδR−S
a
(δR − δS) in ppm
a
Reagents and conditions: (a) (i) (R)-MPA, DCC, DMAP, DCM, rt,
(ii) MeOH. (b) (i) (S)-MPA, DCC, DMAP, DCM, rt, (ii) MeOH. See
1
Figures S11, S12 for H NMR spectra.
While the (R)- and (S)-MPA esters of eunicidiol (6 and 7) were
successfully synthesized using submilligram quantities of 1, the
tertiary allylic hydroxy group was transformed to its methyl ether
during the reaction workup. The scarcity of 1 precluded any
effort to synthesize MPA esters without methyl ethers; however
such efforts were deemed unnecessary, as substitution of the
achiral tertiary hydroxy group was not expected to affect the
analysis. Compared to 7, H-10, H-17, and H-18 of 6 experienced
greater shielding from the phenyl ring, while H-16a/b were
less shielded. As a result, the ΔδR−S values indicated the 2R
configuration, providing the absolute configuration of 2R,10S.
Cope rearrangements of germacrenes and germacrenolides to
elemenes and elemenolides, respectively, are known to proceed
with high stereospecificity and retention of the configuration
at C-10.7 While the absolute configuration of fuscol (2) has
been established,8 the configuration of eunicol (3) was assumed
to be 10S given its role as a biosynthetic precursor to 2.6
To unambiguously assign the absolute configuration of 3, we
enabled an enantiospecific Cope rearrangement of the cyclo-
EXPERIMENTAL SECTION
■
General Experimental Procedures. Medium-pressure liquid
chromatography (MPLC) was performed over RediSep columns
using a Teledyne Combiflash Rf, while all flash chromatography was
carried out on silica gel (230−400 mesh, Fisher). Separation of products
by HPLC was carried out using Phenomenex Luna Phenyl-Hexyl (250 ×
10 mm, 5 μm) or Phenomenex Gemini C18 (250 × 10 mm, 5 μm)
reversed-phase semipreparative columns. All 1H and 13C NMR spectra
were acquired on a Bruker 600 MHz NMR spectrometer operating at
600 and 150 MHz, respectively. All chemical shifts are reported in δ
units and were relative to the residual solvent signal of benzene-d6 (1H,
7.15 ppm; 13C, 128.02 ppm). Coupling constants are reported with the
abbreviations (s) singlet, (d) doublet, (t) triplet, (m) multiplet, (br)
broad, (app.) apparent. High-resolution mass spectra were measured on
a Thermo Scientific Exactive mass spectrometer with an electrospray
ionization source. Optical rotations were measured on a Rudolph
Autopol III polarimeter. Infrared spectra were acquired using attenuated
total reflectance on a Thermo Nicolet 6700 FT-IR spectrometer.
decadiene moiety of 3 (Scheme 2). The H and 13C NMR
1
spectra of the rearranged product were identical to those of 2
(Figure S13−S16), indicating the 2R,7R,10S configuration or
its enantiomer. The absolute configuration of 2R,7R,10S was
ascertained by comparing the specific rotation of the rearranged
product with that of 2. This observation is in agreement with eunicol
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Scheme 3. Biosynthesis of Diterpenes in E. fusca with Proposed Origin of Eunicidiol (1)
was added and the reaction was left to stir overnight at room
temperature. The reaction mixture was concentrated under a stream of
N2 and eluted through a plug of C18 using MeOH. The filtrate was
evaporated in vacuo to provide the reaction crude, which was separated
by C18 RP-HPLC using a gradient of MeOH/H2O (90:10 for 5 min,
increasing linearly to 100:0 at 30 min, 3.0 mL/min, 6/7 tR 15.2 min).
For 6: HRESIMS m/z 489.2980 [M + Na]+ (calcd for C30H42O4Na,
489.2975). For 7: HRESIMS m/z 489.2973 [M + Na]+ (calcd for
C30H42O4Na, 489.2975). See Figures S11 and S12 for 1H NMR spectra
of 6 and 7. Product yields could not be determined with accuracy owing
to the small scale of the reactions.
Table 2. Reduction of PMA-Induced Mouse Ear Edema by
Diterpenes 1−3
ab
,
group
dosage (mg/ear)
reduction of ear edema (%)
c
indomethacin
eunicidiol (1)
fuscol (2)
3.0
0.1
0.1
0.1
51.4 2.7
c
44.4 3.2
c
46.3 3.7
c
eunicol (3)
53.7 4.9
a
b
Data are presented as the mean SEM. Percent reduction of ear
c
edema is relative to the control group. Statistically significant difference
relative to the control group, unpaired Student’s t-test (p < 0.05).
Semisynthesis of Fuscol (2). A purified sample of eunicol (3)
(1.01 mg, 3.49 μmol) was refluxed in toluene for 2.5 h and evaporated in
vacuo. Analysis of the reaction crude by NMR revealed the formation of
fuscol (2) (1.28 μmol, 38%), which was later purified by C18 RP-HPLC
(MeOH/H2O, 88:12, 3.0 mL/min, tR 25.6 min). Semisynthetic fuscol
Analysis of samples by LC with hyphenated MS-ELSD-UV was
performed using a Finnigan LXQ ion trap mass spectrometer and an
analytical C18 RP-HPLC column.
(2): [α]25 = +28.8 (c 0.02 in CHCl3). Isolated fuscol (2): [α]25
=
D
D
Isolation of Fuscol (2) and Eunicol (3). E. fusca was collected from
Hillsboro Ledge, Florida by scuba, air-dried at the surface, and kept
frozen during transportation. The identity of each coral was confirmed
by comparing the TLC profile to that of a pure sample of 2. After
lyophilization, the gorgonians (124.6 g) were exhaustively extracted with
DCM, and the combined extracts were concentrated to provide a
viscous oil (11.47 g). The crude extract was initially partitioned between
hexanes and MeOH/H2O (9:1); afterward the hexanes layer was
evaporated (6.319 g) and separated by vacuum flash chromatography
using a stepwise gradient elution with hexanes/EtOAc. The hexanes/
EtOAc (4:1) fraction (592.0 mg) contained 2 and 3 according to LC-MS
analysis. This fraction was separated by C18 RP-MPLC (MeOH/H2O
gradient) to provide a mixture of 2 and 3 (289.9 mg, 2.3% of dry coral
weight). The diterpenes were separated by phenylhexyl RP-HPLC
(MeOH/H2O, 87:13, 3.0 mL/min, 2 retention time (tR) 24.8 min, 3 tR
26.7 min) and further purified by C18 RP-HPLC (MeOH/H2O, 83:17,
3.0 mL/min, 2 tR 37.6 min, 3 tR 39.6 min). For 2: HRESIMS m/z
311.2346 [M + Na]+ (calcd for C20H32ONa, 311.2345). For 3:
HRESIMS m/z 311.2346 [M + Na]+ (calcd for C20H32ONa, 311.2345).
All 1H and 13C NMR spectroscopic data were in total agreement with
the literature (Figures S13, S15, S17, and S18).6,9
+20.4 (c 0.06 in CHCl3). Fuscol (lit.): [α]D = +21.0 (c 0.9 in CHCl3).8b
See Figures S13−S16 for a comparison of 1H and 13C NMR spectra.
In Vivo Mouse Ear Edema Assay. The assay was completed by
Amplia PharmaTek Inc. as previously described by Martinez et al.10 The
6−7-week-old female CD-1 mice (Charles River Canada Inc.) were pro-
vided with food and water ad libitum and acclimatized for at least 5 days.
PMA (100 μg/mL, n = 6), indomethacin (150 mg/mL, n = 6), and
diterpenes 1−3 (5 mg/mL, n = 3) were formulated in acetone (PMA)
and DMSO (indomethacin and diterpenes). Initially, the right ears were
treated with DMSO (20 μL, vehicle control), indomethacin (3 mg in
20 μL of DMSO), or diterpene (100 μg in 20 μL of DMSO), while the
left ears received vehicle (20 μL of DMSO). One hour later, the
phlogistic agent, PMA (2 μg, 20 μL of acetone), was administered by
topical application to the right ears and the left ears received vehicle
(20 μL of acetone). Edema was measured at 6 and 24 h post-PMA
treatment using a digital caliper and calculated by subtracting the
thickness of the left ear from the right ear. The data are given as the
mean SEM in terms of percent reduction of edema. The statistical
significance of the comparison between test groups and the PMA
control was assessed by Student’s unpaired t-test. A p value < 0.05 was
considered to indicate statistical significance.
Isolation of Eunicidiol (1). Eunicidiol (1) was present in the
hexanes/EtOAc (4:1) fraction and collected alongside 2 and 3 during
the above-mentioned C18 RP-MPLC separation. The compound was
purified by a combination of phenylhexyl RP-HPLC (MeOH/H2O,
77:23, 3.0 mL/min, tR 31.1 min) and C18 RP-HPLC (MeOH/H2O,
80:20, 3.0 mL/min, tR 21.5 min) to provide a colorless oil (1, 1.5 mg).
Eunicidiol (1): [α]25D = −33.4 (c 0.02 in MeOH); IR νmax 3357, 3064,
3039, 2971, 2925, 2857, 1645, 1450, 1385, 1371, 1149, 1021, 968, 899;
HRESIMS m/z 327.2286 [M + Na]+ (calcd for C20H32O2Na,
327.2295). NMR spectroscopic data (Figures S1−S8) are summarized
in Table 1.
Synthesis of (R)- and (S)-MPA Esters of Eunicidiol (6 and 7). A
solution containing eunicidiol (1) (100 μg, 0.33 μmol), DMAP (3.8 mg,
31.1 μmol), DCC (20.0 mg, 96.9 μmol), and 3 Å molecular sieves was
stirred in freshly distilled DCM (3 mL) under an N2 atmosphere for
20 min. Subsequently, an excess of (R)-or (S)-MPA (13.8 mg, 83.0 μmol)
ASSOCIATED CONTENT
* Supporting Information
NMR spectra and MS data for diterpenes 1−3 and MPA esters 6
and 7 are available. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +1 902 566 0565. Fax: +1 902 566 7445. E-mail: rkerr@
upei.ca.
Notes
The authors declare no competing financial interest.
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Article
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge financial support from
Natural Sciences and Engineering Council of Canada (NSERC),
the Canada Research Chair program, the University of Prince
Edward Island, the Atlantic Innovation Fund, and the Jeanne and
́
Jean-Louis Levesque Foundation. The authors also acknowledge
assistance from Amplia PharmaTek Inc. as well as NMR services
provided by Dr. C. Kirby and L. Kerry (AAFC).
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