Anti-inflammatory constituents of the Red Alga Gracilaria verrucosa and their synthetic analogues

Article abstract of DOI:10.1021/np070452q

A chemical study on the anti-inflammatory components of the red alga Gracilaria verrucosa led to the isolation of new 11-deoxyprostaglandins (1-4), a ceramide (5), and a C₁₆ keto fatty acid (6), along with known oxygenated fatty acids (7-14). Their structures were elucidated on the basis of NMR and MS data. The absolute configurations of compounds 1-5 were determined by Mosher's method. The anti-inflammatory activity of the isolated compounds (1-14) was evaluated by determining their inhibitory effects on the production of pro-inflammatory mediators (NO, IL-6, and TNF-α) in lipopolysaccharide (LPS)-activated RAW 264.7 murine macrophage cells. Compounds 9 and 10 exhibited the most potent activity. In the evaluation of these two compounds and derivatized analogues (15-40), the anti-inflammatory activity was enhanced in some synthetic analogues. These enone fatty acids were investigated as potential anti-inflammatory leads for the first time.

Full text of DOI:10.1021/np070452q

232
J. Nat. Prod. 2008, 71, 232–240
Anti-inflammatory Constituents of the Red Alga Gracilaria Werrucosa and Their Synthetic
Analogues
Hung The Dang,† Hye Ja Lee,‡ Eun Sook Yoo,‡ Pramod B. Shinde,† Yoon Mi Lee,† Jongki Hong,§ Dong Kyoo Kim, and
Jee H. Jung*,†
College of Pharmacy, Pusan National UniVersity, Busan, Korea, College of Medicine, Cheju National UniVersity, Jeju, Korea, College of
Pharmacy, Kyung Hee UniVersity, Seoul, Korea, and Department of Chemistry, Inje UniVersity, Gimhae, Korea
ReceiVed August 28, 2007
A chemical study on the anti-inflammatory components of the red alga Gracilaria Verrucosa led to the isolation of new
11-deoxyprostaglandins (1–4), a ceramide (5), and a C16 keto fatty acid (6), along with known oxygenated fatty acids
(7–14). Their structures were elucidated on the basis of NMR and MS data. The absolute configurations of compounds
1–5 were determined by Mosher’s method. The anti-inflammatory activity of the isolated compounds (1–14) was evaluated
by determining their inhibitory effects on the production of pro-inflammatory mediators (NO, IL-6, and TNF-R) in
lipopolysaccharide (LPS)-activated RAW 264.7 murine macrophage cells. Compounds 9 and 10 exhibited the most
potent activity. In the evaluation of these two compounds and derivatized analogues (15-40), the anti-inflammatory
activity was enhanced in some synthetic analogues. These enone fatty acids were investigated as potential
anti-inflammatory leads for the first time.
Regulation of the inflammatory response is an essential element
in the pathogenesis of chronic inflammation-related disorders. The
inflammatory response involves the sequential activation of various
signaling pathways, including cyclooxygenases, nitric oxide syn-
thase, cytokines, and many more.1 It is well-established that the
excessive production of several pro-inflammatory mediators, includ-
ing nitric oxide (NO), interleukins (IL-6 and IL-1ꢀ), and tumor
necrosis factor-R (TNF-R), is implicated in several inflammatory-
related diseases such as rheumatoid arthritis, inflammatory bowel
disease, osteoarthritis, psoriasis, endotoxemia, and toxic shock
syndrome.2–12 Therefore, inhibition of the overproduction of these
pro-inflammatory mediators should be a useful approach to treat
these conditions.
the structure-activity relationship (SAR) of this type of fatty acid,
compounds 9, 10, and their analogues (15–40) were synthesized.
In the present study, we report the isolation and structural
elucidation of the compounds 1–14 and the synthesis and biological
evaluation of the enone fatty acids 9 and 10 and their synthetic
analogues (15–40).
Results and Discussion
Compound 1 was isolated as a colorless oil. Its molecular formula
was defined as C20H32O4 on the basis of HRFABMS and NMR
data. The exact mass of the [M + Na]+ ion (m/z 359.2187) matched
well with the expected molecular formula C20H32O4Na (∆ -1.1
mmu). The 13C NMR spectrum exhibited 20 carbons that were
attributed to one methyl (δ 14.4), 10 sp3 methylenes (δ 38.7, 38.5,
35.2, 33.0, 28.9, 27.9, 26.4, 26.3, 25.8, and 23.8), three sp3 methines
(δ 73.6, 55.9, and 46.1), four olefinic carbons (δ 135.6, 134.4, 131.7,
and 128.0), and two carbonyl carbons (δ 221.7 and 178.5). The
downfield-shifted carbonyl carbon at δ 221.7 indicated the presence
of the characteristic ketone function of the cyclopentanone ring.24
The 1H NMR spectrum of 1 disclosed two disubstituted olefins (δ
5.61, 5.53, 5.40, and 5.33), an allylic oxymethine (δ 4.00), and a
The red alga Gracilaria Verrucosa has been the subject of
previous investigations due to the presence of prostaglandins such
as PGA2 and PGE2.13,14 It was also suggested that the high content
of these prostaglandins in this alga was responsible for an associated
food poisoning, known as Ogonori poisoning, in Japan.15
In our preliminary investigation of the alga for its biological
activities, the methanolic extract was found to have significant
inhibitory activity to the pro-inflammatory cytokines IL-6 and TNF-
R. Therefore, the extract was subjected to bioassay-guided separa-
tion and resulted in the isolation of four 11-deoxyprostaglandins
(1–4), a ceramide (5), and oxygenated fatty acids (6–14). 11-
Deoxyprostaglandins were previously reported as synthetic ana-
logues of natural prostaglandins and have various biological
activities in their own right, such as bronchodilating, diuretic, and
antigastric secretion activities.16,17 Therefore, they have been the
target for total synthesis dating back to the early days of prostag-
landin research.18–23 The anti-inflammatory activity of the isolated
compounds (1-14) was evaluated by determining their inhibitory
effects on the production of major pathophysiological mediators
(NO, IL-6, and TNF-R) in lipopolysaccharide (LPS)-activated RAW
264.7 murine macrophage cells. Compounds 9 and 10 exhibited
the most significant suppressive effect on the pro-inflammatory
mediators. To further investigate the anti-inflammatory activity and
1
terminal methyl group (δ 0.90). Analysis of the H-1H COSY
spectrum demonstrated sequential correlations from H-2 (δ 2.26)
to H-8 (δ 2.00) on the R-side chain, between H-8 and H-12, and
from H-12 (δ 2.51) to H-20 (δ 0.90) on the ω-side chain. In the
HMBC spectrum, key correlations from C-8 to H-7 and H-11, from
C-9 to H-7, H-10, and H-11, and from C-12 to H-7, H-10, and
H-14 were observed. Therefore, the planar structure of 1 was defined
as 11-deoxyprostaglandin E2. The double bond at C-5 was assigned
the Z configuration (δC 27.9 and 25.8 for C-4 and C-7, respec-
tively).25 The double bond at C-13 was assigned the E configuration
from the coupling constant between the corresponding protons
(J13, 14) 15.2 Hz). The trans stereochemistry of the R- and ω-side
chains on the cyclopentanone ring was established by a NOE
experiment.26,27 The NOE correlation between H-7 and H-12 was
observed in the NOESY spectrum. In the same manner, the
orientation of the two side chains on the cyclopentanone ring of
compounds 2–4 was also determined to be trans.
* To whom correspondence should be addressed. Tel: 82-51-510-2803.
Fax: 82-51-513-6754. E-mail: jhjung@pusan.ac.kr.
† Pusan National University.
The absolute configuration at C-15 of 1 was defined by the
Mosher’s ester method. Compound 1 was treated overnight with
(S)-(+)- and (R)-(-)-R-methoxy-R-(trifluoromethyl)phenylacetyl
chloride in CDCl3/C5D5N at room temperature to afford the (R)-
‡ Cheju National University.
§ Kyung Hee University.
Inje University.
10.1021/np070452q CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/26/2008

Anti-inflammatory Constituents of Gracilaria Verrucosa
Journal of Natural Products, 2008, Vol. 71, No. 2 233
Chart 1.
and (S)-MTPA esters, respectively (1r and 1s, respectively). Positive
determined on the basis of the similar 13C NMR shifts of the carbons
C-13 (δ 135.5), C-14 (δ 134.8), C-15 (δ 73.4), and C-16 (δ 38.7)
in comparison with those of 1. The synthetic (8R,12R,15S)-11-
deoxyprostaglandin E1 was reported to have a negative optical
rotation ([R]D -45.7).21 Compound 3 showed the same sign of
optical rotation ([R]D -23.0). Therefore, as demonstrated for 1 and
2, the absolute configuration of 3 was also defined as (8R,12R,15S).
Compound 4 was obtained as a colorless oil. The molecular
formula of 4 was found to be C20H32O4 by HRFABMS and NMR
data. The exact mass of the [M + Na]+ ion (m/z 359.2183) matched
well with the expected molecular formula C20H32O4Na (∆ -1.5
mmu). The carbon signal at δ 213.6 indicated the presence of an
additional keto group in 4. In the COSY spectrum, a sequence of
correlations from H-2 to H-8 on the R-side chain and another one
starting from H-20 (δ 0.90) to H-16 (δ 2.46) were observed,
indicating that the carbonyl carbon should be located at C-15. The
trans orientation of the side chains on the cyclopentanone ring in
conjunction with the negative optical rotation ([R]D -53.0)
confirmed the (8R,12S) configuration for 4, identical to those of
the reported compound ([R]D -36.6), previously obtained by
microbial transformation of PGA2.17h
Compound 5 was isolated as a white, amorphous solid. The
HRFABMS and NMR data revealed a molecular formula of
C19H39NO3. The exact mass of the [M + Na]+ ion (m/z 352.2834)
matched well with the expected molecular formula C19H39NO3Na
(∆ +0.6 mmu). The 1H NMR spectrum of 5 displayed a formamide
group (δ 8.11),28,29 an iminomethine (δ 3.90), a hydroxymethylene
(δ 3.70), a hydroxymethine (δ 3.62), an intense aliphatic methylene
band (δ 1.29), and a terminal methyl group (δ 0.90). The 13C and
DEPT spectra of 5 were supportive of the above analysis, showing
a carbonyl (δ 163.8), an iminomethine (δ 55.6), a hydroxylmeth-
ylene (δ 62.0), a hydroxylmethine (δ 72.2), aliphatic methylenes
(δ 23.7–34.8), and a terminal methyl carbon (δ 14.4). Analysis of
1D and 2D spectra disclosed a ceramide skeleton of 5.
∆
(∆S - ∆R) values were observed for H-13 (+0.01) and H-14
SR
(+0.01), while a negative value was observed for H-20 (-0.01),
indicating that the configuration of the hydroxy group of 1 is S
(Figure 1).
The trans orientation of the side chains in combination with the
absolute configuration at C-15 (S) suggested that the absolute
configuration of three chiral centers in 1 to be either (8R,12R,15S)
or (8S,12S,15S). The synthetic (8R,12R,15S)-11-deoxyprostaglandin
E2 was reported to have a negative optical rotation ([R]D -54.0),18
while the (8S,12S,15S)-11-deoxyprostaglandin E2 methyl ester was
reported with a positive optical rotation ([R]D +43.6).19 Compound
1 showed a negative optical rotation ([R]D-33.0), and therefore,
the absolute configuration of 1 was assigned as (8R,12R,15S), which
is identical with that of common natural prostaglandins.
Compound 2 was isolated as a colorless oil. The molecular
formula of C21H34O4 was assigned by HRFABMS and NMR
spectroscopic data. The exact mass of the [M + Na]+ ion (m/z
373.2347) matched well with the expected molecular formula
C21H34O4Na (∆ -0.8 mmu). The 1H and 13C NMR data of
compound 2 were similar to those of 1 except for an additional
methoxy group, showing the proton signal at δ 3.67 (3H, s) and
the corresponding carbon signal at δ 51.2. The correlation between
the methoxy group at δH 3.67 and the carbonyl carbon at δC 176.0
was observed in the HMBC spectrum, indicating that compound 2
is a methyl ester of 1. The 15S configuration in 2 was determined
on the basis of the similar 13C NMR shifts of C-13-C-17 and
optical rotation ([R]D -35.0) in comparison with those of 1. The
synthetic (8R,12R,15S)-11-deoxyprostaglandin E2 methyl ester and
its (8S,12S,15S)-isomer were reported to show a negative ([R]D
-41) and a positive optical rotation ([R]D +43.6), respectively.19
Thus, the (8R,12R,15S) configuration was also established for 2.
Compound 3 was isolated as a colorless oil. The HRFABMS
and NMR data established the molecular formula of C20H34O4 for
3. The exact mass of the [M + Na]+ ion (m/z 361.2358) matched
well with the expected molecular formula C20H34O4Na (∆ +0.2
The absolute configuration of the hydroxymethine in 5 was
defined by the Mosher’s ester method. A negative ∆SR (∆S - ∆R)
value was obtained for H-4 (-0.031), while a positive value was
obtained for H-2 (+0.026), indicating that the configuration at C-3
is S (Figure 1). The remaining chiral center at C-2 was defined as
R by comparison of the optical rotation of 5 with that of the
synthetic L-threo-sphinganine (5a).30 The synthetic model com-
1
mmu). The H NMR data of 3 were similar to those of 1, except
for the absence of the double bond in the R-side chain, leading to
the upfield shift of the H-8 signal (δ 1.88) compared to those of 1
and 2 (δ 2.00). The data allowed assignment of the planar structure
of 3 as 11-deoxyprostaglandin E1. The 15S configuration was also

234 Journal of Natural Products, 2008, Vol. 71, No. 2
Dang et al.
Figure 1. ∆SR (∆S - ∆R) values for the MTPA esters of compounds
1 and 5.
Figure 3. FAB-CID-MS/MS of [M + 2Na – H]+ of 10 and its
hydrogenated product 10a with relative intensity values in parentheses.
Figure 4. TOCSY correlations of compounds 9 and 10.
Figure 2. FAB-CID-MS/MS of [M + 2Na – H]+ of 9 and its
hydrogenated product 9a with relative intensity values in parentheses.
respectively. From the CID spectra of [M - H + 2Na]+ ions of
these hydrogenated products, the ꢀ ion was observed at m/z 173
for 6a and 10a and m/z 187 for 9a; whereas, the γ′ ion was found
at m/z 243 for 6a and 10a and m/z 257 for 9a. These diagnostic
ions corroborated the location and direction of the enone group of
6, 9, and 10. The CID spectra of [M - H + 2Na]+ ions of the
enone fatty acids (9 and 10) and their hydrogenated products (9a
and 10a, respectively) are shown in Figures 2 and 3.
Location of the two different enone moieties of compounds 6,
9, and 10 was further corroborated by TOCSY experiments. The
correlations from H-2 to H-8 and from H-12 to the terminal methyl
group were observed in the TOCSY spectra of 6 and 10, whereas
the correlation from H-18 (δ 0.92) to H-11 (δ 2.58) was observed
in the TOCSY spectrum of 9 (Figure 4). Thus, the position of the
double bond was located at C-10 for 6 and 10 and C-8 for 9. As a
result, compounds 6, 9, and 10 were defined as (E)-9-oxohexadec-
10-enoic acid, (E)-10-oxooctadec-8-enoic acid, and (E)-9-oxooc-
tadec-10-enoic acid, respectively.
pound 5a with (2S,3S) configuration was reported to have a positive
optical rotation ([R]D +2.6), whereas compound 5 showed a
negative optical rotation ([R]D -2.7). Therefore, compound 5 was
defined as (2R,3S)-2-formamido-1,3-dihydroxyoctadecane.
Compound 6 was obtained as a colorless oil. The molecular
formula of C16H28O3 was assigned for 6 on the basis of HRFABMS
1
and H NMR data. The exact mass of the [M + Na]+ ion (m/z
291.1943) matched well with the expected molecular formula
1
C16H28O3Na (∆ +0.7 mmu). The H NMR data of 6 were almost
identical to those of 9 and 10, which were defined as enone fatty
acids by a detailed analysis of 1D and 2D NMR data. Compounds
9 and 10 were previously produced by synthesis or biotransforma-
tion.31,32 These two compounds were also uniquely found in airway
cells of cotton workers and showed inhibitory effects on growth of
several cancer cell lines.33
To locate the keto group as well as the double bond, compound
6 and other oxygenated fatty acids were analyzed by tandem mass
spectrometry (FAB-CID-MS/MS). The structural determination of
saturated oxo fatty acids was previously reported by utilizing
charge-remote fragmentations (CRF) of “fixed” molecular ions such
as [M - H + 2Na]+.34 The cationized acids undergo CRF upon
high-energy activation, giving a product-ion pattern that has a gap
corresponding to the oxo position and bordered by two high-
intensity peaks. One of the peaks corresponds to an ion that is
formed by the cleavage of the C-C bond ꢀ to the oxo position
and proximal to the charge (ꢀ ion), whereas the other is formed
from the cleavage of the C-C bond γ to the oxo position and distal
to the charge (γ′ ion). The oxo postion is easily determined by
identifying the gap and the ꢀ and γ′ ions (Figure 2). Applying this
method to enone fatty acids (9 and 10), the CID spectra of [M -
H + 2Na]+ ions of these compounds also showed fragment ions
along the chain and around the enone position. However, the definite
location and direction of Figures 2 and 3 (either 9 or 10) of the
enone group could not be derived on the basis of these fragmenta-
tions. Therefore, to establish conclusively the location and direction
of the enone group of compounds 6, 9, and 10, these compounds
were hydrogenated under palladium-charcoal (10% Pd-C) catalysis
to give the corresponding keto fatty acids 6a, 9a, and 10a,
Compounds 7 and 12–14, identified as saturated keto fatty acids,
were previously prepared from biotransformation or isolated from
a few higher plants.35–39 From the CID spectra of [M - H + 2Na]+
ions of these compounds, the ꢀ ion of 7, 12, 13, and 14 was
observed at m/z 187, 187, 201, and 215, respectively, whereas the
γ′ ion of 7, 12, 13, and 14 was found at m/z 257, 257, 271, and
285, respectively. These diagnostic ions led to the identification of
the position of the keto group at C-10 for 7 and 12, C-11 for 13,
and C-12 for 14.
Compounds 8 and 11 were defined as monounsaturated mono-
hydroxy fatty acids on the basis of 1D and 2D NMR data. The
CID spectra of [M - H + 2Na]+ ions of both compounds 8 and
11 showed a gap of 70 amu between two prominent peaks (m/z
159 and 229). These two peaks in turn had to be caused by allylic
cleavage between C-6 and C-7 and cleavage adjacent to the hydroxy
group between C-10 and C-11, respectively. The TOCSY spectrum
of 11, showing correlations from H-2 (δ 2.26) and H-3 (δ 1.59) to
H-7 (δ 2.03), was supportive of this analysis. Therefore, the position
of the double bond and the hydroxy group was located at C-8 and
C-10, respectively. Compound 11 had an R configuration at C-10
since it showed the same sign of optical rotation ([R]D -3.8) as
that of the reported compound ([R]D -1.48).40 The absolute

Anti-inflammatory Constituents of Gracilaria Verrucosa
Journal of Natural Products, 2008, Vol. 71, No. 2 235
Figure 7. Inhibitory effects of the isolated compounds (1-14) on
the production of TNF-R in LPS-activated RAW264.7 cells. RAW
264.7 cells (1.5 × 106 cells/mL) were stimulated with LPS (1 µg/
mL) in the presence of test samples (20 µg/mL) for 6 h (BL, blank).
Values are the mean ( SEM of three independent experiments
performed in triplicate. *p < 0.05 compared with the LPS alone.
Figure 5. Inhibitory effects of the isolated compounds (1-14) on
the production of NO in LPS-activated RAW 264.7 cells. RAW
264.7 cells (1.5 × 105 cells/mL) were stimulated with LPS (1 µg/
mL) in the presence of test samples (20 µg/mL) for 24 h (BL,
blank). Cell toxicity was determined using the LDH release method.
Values are the mean ( SEM of three independent experiments
performed in triplicate. *p < 0.05, **p < 0.01 compared with the
LPS alone.
Scheme 1. Synthesis of Enone Fatty Acid Analogues (15–18)a
a Reagents and conditions: Mn3O(OAc)9, (CH3)3COOH, EtOAc, rt, 48 h.
tural modification of 9 and 10 was directed to the enone group
considering the electronic effect as well as the size and shape of
substituents at the R-carbon. In parallel, the position of the enone
group and the terminal functional group were modified.
Figure 6. Inhibitory effects of the isolated compounds (1-14) on
the production of IL-6 in LPS-activated RAW 264.7 cells. RAW
264.7 cells (8.0 × 105 cells/mL) were stimulated with LPS (1 µg/
mL) in the presence of test samples (20 µg/mL) for 24 h (BL,
blank). Values are the mean ( SEM of three independent
experiments performed in triplicate. *p < 0.05, **p < 0.01
compared with the LPS alone.
The synthetic paths are outlined in Schemes 1 and 2. Scheme 1
describes the procedure of manganese(III) acetate-catalyzed allylic
oxidation of methyl oleate to the corresponding enone fatty acid
analogues (15–18). The compounds in other groups, including acid
(19, 9, 10, and 20), hydroxy (21–24), and amide groups (25-28),
were prepared from different starting materials by the same process.
Previously, the use of manganese(III) acetate as the catalyst for
the allylic oxidation of cyclic alkenes to the corresponding enones
was reported.49 The simplicity and efficiency of this method in one
reaction step led us to apply this protocol to prepare enones from
acyclic alkenes such as oleic acid and its analogues. However, the
direct oxidation of oleyl amine did not produce the corresponding
enone products. This is probably because acidic condition are
needed for this reaction.49 Therefore, starting materials with
functional groups such as ester, hydroxy, amide, and acid are
suitable. From the oxidation of each starting material, four trans
enone fatty acid isomers were formed with near equal yields, which
were separated by a revered-phase HPLC (YMC ODS-H80) with
the eluting order of 15 to 18 (Scheme 1). The structures of all
synthetic enone fatty acids (9, 10, and 15–28) were secured mainly
configuration of the hydroxy group of 8,41 previously unassigned,
was defined as S because it showed a positive optical rotation ([R]D
+2.8).
Compounds 1–14 were investigated for their anti-inflammatory
activity through the evaluation of their inhibitory effects on the
production of major pathophysiological mediators (NO, IL-6, and
TNF-R) in murine macrophage cells (Figures 5, 6, and 7). The data
showed that, among the compounds tested, compounds 9 and 10
produced the most significant effects on the pro-inflammatory
mediators without notable cytotoxicity to the RAW 264.7 cells at
their effective concentration. In a more detailed study, compounds
9 and 10 clearly showed suppressive effects on NO production (IC50
< 33.8 µM) and the expression of iNOS mRNA in a dose-dependent
manner (data not shown). Therefore, to further investigate the anti-
inflammatory activity and the structure-activity relationship (SAR)
of this type of fatty acid, compounds 9 and 10 and their analogues
(15-40) were synthesized. Structurally, compounds 9 and 10
possess a distinguished feature, an R,ꢀ-unsaturated carbonyl group
(enone group), which has been encountered in a number of potential
anti-inflammatory and anticancer agents such as sesquiterpene
lactones (parthenolide),42 2-cyclopentenone-1-one and its deriva-
tives,43 and cyclopentenone prostaglandins.44–48 Hence, the struc-
1
on the basis of their H NMR and FAB-CID-MS/MS data.
To differentiate four enone fatty acid isomers in each group, all
synthetic enone fatty acids were hydrogenated under palladium-
charcoal (10% Pd-C) catalysis to furnish the corresponding keto
fatty acid analogue, which was subjected to FAB-CID-MS/MS in
order to identify the position of the keto group as described for the
natural compounds. However, the hydrogenated products of four
enone fatty acid isomers obtained from the oxidation of oleyl
alcohol did not show clear fragmentations in their CID spectra.

236 Journal of Natural Products, 2008, Vol. 71, No. 2
Scheme 2. Synthesis of Compounds 29 and 37a
Dang et al.
Experimental Section
General Experimental Procedures. Optical rotations were mea-
1
sured on a JASCO P-1020 digital polarimeter. H, 13C, and 2D NMR
experiments were recorded on Varian Inova 400 and Varian Inova 500
spectrometers. Chemical shifts were reported with reference to the
respective residual solvent or deuterated solvent peaks (δH 3.30 and
δC 49.0 for CD3OD, or δH 7.24 for CDCl3). FABMS data were obtained
on a JEOL JMS SX-102A spectrometer. HRFABMS data were obtained
on JEOL JMS SX-101A spectrometer. HPLC was performed with a
YMC ODS-H80 column (250 × 10 mm, 4 µm, 80 Å) and a C18-5E
Shodex packed column (250 × 10 mm, 5 µm, 100 Å) using a Shodex
RI-71 detector. Biological assays were performed at Cheju National
University.
Extraction and Isolation. The red alga Gracilaria Verrucosa (order
Gigarinales, family Gracilariaceae) was collected off the coast of Jeju
Island, South Korea. The alga was extracted with MeOH at room
temperature, and the extract was partitioned between H2O and CH2Cl2.
The latter portion was further partitioned between n-hexane and aqueous
MeOH. The aqueous MeOH fraction was subjected to a step-gradient
reversed-phase flash column chromatography (YMC Gel ODS-A, 60
Å, 220 mesh) with a solvent system of 50 to 100% MeOH/H2O to
afford 20 fractions. Fraction 5 (136 mg), one of the bioactive fractions
(5, 7, and 11) showing significant inhibitory activity against the pro-
inflammatory cytokines, was subjected to a reversed-phase HPLC (C18-
5E Shodex packed) eluting with 60% aqueous CH3CN to afford
compounds 1 (3.0 mg), 3 (1.0 mg), and 4 (7.0 mg). Fraction 7 (74 mg)
was subjected to a reversed-phase HPLC (C18-5E Shodex packed)
eluting with 81% aqueous MeOH to yield compounds 2 (2.0 mg), 6 (2
mg), 7 (1.5 mg), and 8 (4.0 mg). Compound 5 (1.2 mg) was obtained
by separation of fraction 11 (125 mg) on a reversed-phase HPLC (YMC
ODS-H80) eluting with 88% aqueous CH3CN. Fraction 9 (238 mg)
was subjected to a reversed-phase HPLC (C18-5E Shodex packed)
eluting with 81% aqueous MeOH to afford compounds 9 (3.0 mg), 10
(3.2 mg), 11 (5.0 mg), 12 (1.5 mg), 13 (1.6 mg), and 14 (1.8 mg).
a Reagents and conditions: (i) Br2, CCl4, rt, 3 h, then NH4OH/ethanol, 3 h;
(ii) C6H7BO2, Pd[(C6H5)3P]4, 2M Na2CO3, ethanol, toluene, reflux, 3 h.
Therefore, these hydrogenated products were further oxidized to
convert the primary hydroxy group to a carboxylic acid, which
enabled location of the keto group of these compounds. Location
of the two different enone moieties of the synthetic enone fatty
acids was also corroborated by TOCSY experiments.
Scheme 2 outlines the synthetic path of the brominated and
phenyl-substituted derivatives. The brominated analogues (29–36)
were formed by the bromination of the enone fatty acids (15–19,
9, 10, and 20) using Br2 in CCl4 followed by base-catalyzed
elimination. Subsequent Suzuki coupling of some brominated
compounds (29, 31, 35, and 36) with phenylboronic acid mediated
by tetrakis(triphenylphosphine)palladinium(0) afforded the corre-
sponding phenyl-substituted analogues (37-40).
All of the synthetic compounds (9, 10, and 15-40) were
examined for their anti-inflammatory activity through the evaluation
of their inhibitory effects on the production of NO (Figure 8). When
the activity of six synthetic groups was compared, the potency was
in the order brominated (29-36) > hydroxy (21-24) > amide
(25–28) > acid (19, 9, 10, and 20) > methyl ester (15-18) >
phenyl analogues (37-40). However, the increased activity of the
brominated analogues, which were prepared to enhance the elec-
trophilicity of the ꢀ-carbon,50 was due to their strong cytotoxicity
to the cells. Meanwhile, the phenyl-substituted analogues (37-40)
did not show activity. Therefore, the presence of a bromine or a
phenyl group at the R-carbon of the R,ꢀ-unsaturated carbonyl group
appeared to be detrimental for the anti-inflammatory activity.
Regarding the role of the terminal functional group, the results
indicated that the presence of a free acid group was also necessary
for activity. However, substitution of the acid group by other
functional groups such as hydroxy, amide, or amine might be
desirable to enhance the activity. This indicates that, like cyclo-
pentenone prostaglandins,51 the presence of moieties other than the
enone group might also be important in directing the enone
reactivity toward targets, rendering enone fatty acids with enhanced
selectivity.
Concerning the enone position, it was observed that the activity
of the compounds having the double bond of the enone moiety
distal to the terminal functional groups is stronger than that of those
having the enone group in opposite direction, e.g., 10 > 9; 20 >
19; 23, 24 > 21, 22; 27, 28 > 25, 26. Taken together, the
preliminary investigation of the structure-activity relationship of
these enone fatty acids revealed that the reactive enone moiety still
plays a vital role for the anti-inflammatory activity of these
compounds. However, other factors such as the length of chain,
the terminal functional group, and the position and direction of the
enone group also modulate the activity of this type of fatty acid.
Illustration of the structure-activity relationship of these enone fatty
acids is presented in Figure 9.
(5Z,13E)-(8R,12R,15S)-15-Hydroxy-9-oxoprosta-5,13-dienoic acid
1
(1): colorless oil; [R]24 -33.0 (c 0.09, MeOH); H NMR (CD3OD,
D
400 MHz) δ 5.61 (1H, dd, J ) 15.2, 7.6 Hz, H-13), 5.53 (1H, dd, J )
15.2, 6.8 Hz, H-14), 5.40 (1H, m, H-5), 5.33 (1H, m, H-6), 4.00 (1H,
q, J ) 6.8 Hz, H-15), 2.51 (1H, m, H-12), 2.33 (3H, m, H-7, H-10b),
2.26 (2H, t, J ) 6.8 Hz, H-2), 2.10 (4H, m, H-4, H-11), 2.00 (1H, m,
H-8), 1.65 (3H, m, H-3, H-10a), 1.54 (2H, m, H-16), 1.48–1.28 (6H,
m, H-17-H-19), 0.90 (3H, t, J ) 7.2 Hz, H-20); 13C NMR (CD3OD,
100 MHz) δ 221.7 (s, C-9), 178.5 (s, C-1), 135.6 (d, C-13), 134.4 (d,
C-14), 131.7 (d, C-5), 128.0 (d, C-6), 73.6 (d, C-15), 55.9 (d, C-8),
46.1 (d, C-12), 38.7 (t, C-10), 38.5 (t, C-16), 35.2 (t, C-2), 33.0 (t,
C-18), 28.9 (t, C-11), 27.9 (t, C-4), 26.4 (t, C-17), 26.3 (t, C-3), 25.8
(t, C-7), 23.8 (t, C-19), 14.4 (q, C-20); HRFABMS m/z 359.2187 [M
+ Na]+ (calcd for C20H32O4Na, 359.2198).
Preparation of the (R)- and (S)-MTPA Esters of 1. Two portions
(each 0.4 mg) of compound 1 were treated overnight with (S)-(+)-
and (R)-(-)-R-methoxy-R-(trifluoromethyl)phenylacetyl chloride (1 µL)
in CDCl3 (0.5 mL)/C5D5N (4 µL) at room temperature to afford the
(R)- and (S)-MTPA esters, respectively (1r and 1s, respectively).
(R)-MTPA ester (1r): 1H NMR (CDCl3, 500 MHz) δ 5.935 (1H,
dd, J ) 16.0, 6.5 Hz, H-13), 5.785 (1H, dd, J ) 16.0, 6.5 Hz, H-14),
5.42 (2H, m, H-5, H-6), 5.149 (1H, q, J ) 6.5 Hz, H-15), 2.52–1.26
(22H, m), 0.85 (3H, t, J ) 7.0 Hz, H-20).
(S)-MTPA ester (1s): 1H NMR (CDCl3, 500 MHz) δ 5.945 (1H,
dd, J ) 15.0, 6.5 Hz, H-13), 5.795 (1H, dd, J ) 15.0, 6.5 Hz, H-14),
5.44 (2H, m, H-5, H-6), 5.165 (1H, q, J ) 6.5 Hz, H-15), 2.48–1.21
(22H, m), 0.84 (3H, t, J ) 6.5 Hz, H-20).
Methyl-(5Z,13E)-(8R,12R,15S)-15-hydroxy-9-oxoprosta-5,13-di-
enoate (2): colorless oil; [R]25 –35.0 (c 0.05, MeOH); 1H NMR
D
(CD3OD, 500 MHz) δ 5.61 (1H, dd, J ) 15.5, 7.6 Hz, H-13), 5.51
(1H, dd, J ) 15.5, 6.5 Hz, H-14), 5.38 (1H, m, H-5), 5.32 (1H, m,
H-6), 4.00 (1H, q, J ) 6.5 Hz, H-15), 3.67 (3H, s, OCH3), 2.51 (1H,
m, H-12), 2.32 (3H, m, H-7, H-10b), 2.30 (2H, t, J ) 7.2 Hz, H-2),
2.10 (4H, m, H-4, H-11), 2.00 (1H, m, H-8), 1.65 (3H, m, H-3, H-10a),
1.53 (2H, m, H-16), 1.46–1.28 (6H, m, H-17-H-19), 0.90 (3H, t, J )
7.0 Hz, H-20); 13C NMR (CD3OD, 100 MHz) δ 221.0 (s, C-9), 176.0
(s, C-1), 135.6 (d, C-13), 134.4 (d, C-14), 131.4 (d, C-5), 128.2 (d,
C-6), 73.6 (d, C-15), 55.9 (d, C-8), 51.2 (q, OCH3), 46.1 (d, C-12),
38.7 (t, C-10), 38.5 (t, C-16), 34.2 (t, C-2), 33.0 (t, C-18), 28.9 (t,
C-11), 27.7 (t, C-4), 26.4 (t, C-17), 25.9 (t, C-3), 25.8 (t, C-7), 23.7(t,
The in vitro anti-inflammatory activity of this type of compound
has been investigated for the first time and suggests that enone fatty
acids may serve as potential anti-inflammatory leads.

Anti-inflammatory Constituents of Gracilaria Verrucosa
Journal of Natural Products, 2008, Vol. 71, No. 2 237
Figure 8. Inhibitory effects of the synthetic compounds (9, 10, and 15-40) on the production of NO in LPS-activated RAW 264.7 cells.
RAW 264.7 cells (1.5 × 105 cells/mL) were stimulated with LPS (1 µg/mL) in the presence of test samples at concentrations of 5, 10, and
20 µg/mL for 24 h (BL, blank). Cell toxicity was determined using the LDH release method. Values are the mean ( SEM of three independent
experiments performed in triplicate.
11.6, 3.2 Hz, H-1a), 4.217 (2H, m, H-2, H-3), 1.445 (2H, m, H-4),
1.225 (26H, brs, H-5-H-17), 0.849 (3H, t, J ) 6.8 Hz, H-18).
(S)-MTPA ester (5s). 1H NMR (CDCl3, 400 MHz) δ 6.303 (1H,
brs, NH), 4.588 (1H, dd, J ) 11.6, 6.4 Hz, H-1b), 4.430 (1H, dd, J )
11.6, 3.6 Hz, H-1a), 4.243 (2H, m, H-2, H-3), 1.414 (2H, m, H-4),
1.221 (26H, brs, H-5-H-17), 0.845 (3H, t, J ) 6.4 Hz, H-18).
(E)-9-Oxohexadec-10-enoic acid (6): colorless oil; 1H NMR
(CD3OD, 500 MHz) δ 6.94 (1H, dt, J ) 14.0, 7.0 Hz, H-11), 6.13
(1H, dt, J ) 14.0, 1.5 Hz, H-10), 2.60 (2H, t, J ) 7.5 Hz, H-8), 2.26
(4H, m, H-2, H-12), 1.60 (4H, m, H-3, H-7), 1.51 (2H, quint, J ) 7.0
Hz, H-13), 1.36–1.31 (10H, m, CH2), 0.94 (3H, t, J ) 7.0 Hz, H-16);
Figure 9. Structure–activity relationships of enone fatty acids. Anti-
inflammatory activity was measured as inhibition on NO production.
FAB-CID-MS/MS m/z 313 [M – H + 2Na]+ (100), 283 (5), 269 (29),
255 (20), 173 (52), 159 (31), 145 (12), 131 (7), 117 (33), 104 (33), 90
(29); HRFABMS m/z 291.1943 [M + Na]+ (calcd for C16H28NaO3,
291.1936).
C-19), 14.4 (q, C-20); HRFABMS m/z373.2347 [M + Na]+ (calcd for
C21H34O4Na, 373.2355).
9-Oxohexadecanoic acid (6a). To a stirred solution of 6 (0.6 mg)
in MeOH was added 10% Pd/C (0.6 mg), and the resulting mixture
was stirred at room temperature under an atmosphere of H2 gas for
2 h. The mixture was filtered through Celite. The filtrate was
concentrated under reduced pressure and purified by a reversed-phase
HPLC (YMC ODS-H80) eluting with 90% aqueous MeOH to yield
compound 6a (0.3 mg): white, amorphous powder; FABMS m/z293
[M + Na]+, m/z 315 [M – H + 2Na]+; FAB-CID-MS/MS m/z 315 [M
– H + 2Na]+(100), 285 (10), 271 (9), 257 (50), 243 (86), 173 (66),
159 (27), 145 (20), 131 (9), 117 (45), 104 (40), 90 (21).
10-Oxohexadecanoic acid (7): white, amorphous powder; negative-
ion FABMS m/z269 [M - H]-; positive-ion FABMS m/z 293 [M +
Na]+, 315 [M – H + 2Na]+; FAB-CID-MS/MS m/z 315 [M – H +
2Na]+ (100), 299 (9), 285 (14), 271 (23), 257 (75), 187 (77), 173 (30),
159 (34), 145 (18), 131 (14), 117 (61), 104 (69), 90 (69).
(E)-(8R,12R,15S)-15-Hydroxy-9-oxoprost-13-enoic acid (3): col-
orless oil; [R]27D -23.0 (c 0.04, MeOH); 1H NMR (CD3OD, 500 MHz)
δ 5.61 (1H, dd, J ) 15.5, 7.5 Hz, H-13), 5.51 (1H, dd, J ) 15.5, 6.5
Hz, H-14), 4.00 (1H, q, J ) 6.5 Hz, H-15), 2.48 (1H, m, H-12), 2.31
(1H, m, H-10b), 2.19 (2H, t, J ) 7.5 Hz, H-2), 2.10 (2H, m, H-11),
1.88 (1H, m, H-8), 1.61 (3H, m, H-3, H-10a), 1.52 (2H, m, H-16),
1.43–1.28 (14H, m, CH2), 0.90 (3H, t, J ) 6.5 Hz, H-20); HRFABMS
m/z 361.2358 [M + Na]+ (calcd for C20H34O4Na, 361.2356).
(Z)-(8R,12S)-9,15-Dioxoprost-5-enoic acid (4): colorless oil; [R]24
D
1
–53.0 (c 0.1, MeOH); H NMR (CD3OD, 500 MHz) δ 5.42 (1H, m,
H-5), 5.33 (1H, m, H-6), 2.59 (1H, m, H-10b), 2.50 (1H, m, H-10a),
2.46 (2H, t, J ) 7.2 Hz, H-16), 2.35 (2H, m, H-7), 2.28 (4H, t, J ) 7.6
Hz, H-2, H-14), 2.11 (2H, q, J ) 7.2 Hz, H-4), 2.02 (2H, m, H-11),
1.86 (2H, m, H-8, H-12), 1.64 (2H, quint, J ) 7.2 Hz, H-3), 1.55 (6H,
quint, J ) 7.2 Hz, H-17-H-19), 1.45 (2H, m, H-13), 0.90 (3H, t, J )
7.2 Hz, H-20); 13C NMR (CD3OD, 100 MHz) δ 222.5 (s, C-9), 213.6
(s, C-15), 177.6 (s, C-1), 131.8 (d, C-5), 128.3 (d, C-6), 56.0 (d, C-8),
43.5 (t, C-16), 42.0 (d, C-12), 41.0 (t, C-10), 38.7 (t, C-14), 34.5 (t,
C-2), 32.6 (t, C-18), 29.4 (t, C-13), 27.7 (t, C-11), 27.6 (t, C-4), 26.3
(t, C-7), 26.1 (t, C-3), 24.6 (t, C-17), 23.5 (t, C-19), 14.3 (q, C-20);
HRFABMS m/z 359.2183 [M + Na]+ (calcd for C20H32O4Na, 359.2198).
(2R,3S)-2-Formamido-1,3-dihydroxyoctadecane (5): white, amor-
phous solid; [R]27D –2.7 (c 0.1, MeOH); 1H NMR (CD3OD, 400 MHz)
δ 8.11 (1H, s, CHO), 3.90 (1H, q, J ) 6.4 Hz, H-2), 3.70 (2H, d, J )
6.4 Hz, H-1), 3.62 (1H, m, H-3), 1.54 (2H, m, H-4), 1.29 (26H, brs,
H-5-H-17), 0.90 (3H, t, J ) 6.4 Hz, H-18); 13C NMR (CD3OD, 100
MHz) δ 163.8 (d, CHO), 72.2 (d, C-3), 62.0 (t, C-1), 55.6 (d, C-2),
34.8 (t, C-16), 33.1 (t, C-4), 30.8–30.5 (CH2), 26.8 (t, C-15), 23.7 (t,
C-17), 14.4 (q, C-18); HRFABMS m/z 352.2834 [M + Na]+ (calcd
for C19H39NO3Na, 352.2828).
(E)-(S)-10-Hydroxyhexadec-8-enoic acid (8): colorless oil; [R]24
D
+2.8 (c 0.04, MeOH); negative-ion FABMS m/z 269 [M - H]-;
positive-ion FABMS m/z 293 [M + Na]+, 315 [M – H + 2Na]+; FAB-
CID-MS/MS m/z 315 [M – H + 2Na]+ (100), 271 (12), 257 (10), 243
(14), 229 (20), 159 (24), 145 (12), 131 (10), 117 (78), 104 (90), 90
(71).
(E)-10-Oxooctadec-8-enoic acid (9): colorless oil; 1H NMR
(CD3OD, 500 MHz) δ 6.94 (1H, dt, J ) 16.0, 6.5 Hz, H-8), 6.13 (1H,
dt, J ) 16.0, 1.5 Hz, H-9), 2.58 (2H, t, J ) 7.5 Hz, H-11), 2.27 (4H,
m, H-2, H-7), 1.62 (4H, m, H-3, H-12), 1.51 (2H, m, H-6), 1.40-1.33
(14H, m, CH2), 0.92 (3H, t, J ) 6.5 Hz, H-18); FABMS m/z 319 [M
+ Na]+, 341 [M – H + 2Na]+; FAB-CID-MS/MS m/z 341 [M – H +
2Na]+ (100), 297 (14), 283 (17), 269 (14), 255 (86), 159 (57), 145
(19), 131 (12), 117 (88), 104 (89), 90 (81).
10-Oxooctadecanoic acid (9a). The method of preparation of 9a
from 9 was similar to that used for the preparation of 6a: white,
amorphous powder; FABMS m/z 343 [M - H + 2Na]+; FAB-CID-
MS/MS m/z 343 [M – H + 2Na]+(100), 313 (19), 299 (13), 285 (7),
271 (25), 257 (90), 187 (85), 173 (31), 159 (33), 145 (16), 131 (11),
117 (45), 104 (42), 90 (34).
Preparation of the (R)- and (S)-MTPA Esters of 5. Two portions
(each 0.2 mg) of compound 5 were treated overnight with (S)-(+)-
and (R)-(-)-R-methoxy-R-(trifluoromethyl)phenylacetyl chloride (0.4
µL) in CDCl3 (0.5 mL)/C5D5N (2 µL) at room temperature to afford
the (R)- and (S)-MTPA esters, respectively (5r and 5s, respectively).
(R)-MTPA Ester (5r): 1H NMR (CDCl3, 400 MHz) δ 6.212 (1H,
brs, NH), 4.562 (1H, dd, J ) 11.6, 6.4 Hz, H-1b), 4.457 (1H, dd, J )
(E)-9-Oxooctadec-10-enoic acid (10): colorless oil; 1H NMR
(CD3OD, 500 MHz) δ 6.91 (1H, dt, J ) 14.0, 7.0 Hz, H-11), 6.10
(1H, dt, J ) 14.0, 1.5 Hz, H-10), 2.58 (2H, t, J ) 7.5 Hz, H-8), 2.25
(4H, m, H-2, H-12), 1.58 (4H, m, H-3, H-7), 1.49 (2H, m, H-13),

238 Journal of Natural Products, 2008, Vol. 71, No. 2
Dang et al.
1.33–1.28 (14H, m, CH2), 0.90 (3H, t, J ) 7.0 Hz, H-18); FABMS
m/z 319 [M + Na]+, 341 [M – H + 2Na]+; FAB-CID-MS/MS m/z
341 [M – H + 2Na]+ (100), 297 (15), 283 (20), 269 (78), 255 (32),
173 (53), 159 (19), 145 (20), 131 (15), 117 (75), 104 (90), 90 (58).
9-Oxooctadecanoic acid (10a). The method of preparation of 10a
from 10was similar to that used for the preparation of 6a: white,
amorphous powder; FABMS m/z343 [M – H + 2Na]+; FAB-CID-MS/
MS m/z 343 [M – H + 2Na]+ (100), 313 (12), 299 (14), 285 (12), 271
(9), 257 (52), 243 (85), 173 (66), 159 (27), 145 (19), 131 (9), 117
(40), 104 (40), 90 (25).
amorphous powder; FABMS m/z 335 [M + Na]+; FAB-CID-MS/MS
m/z 335 [M + Na]+(100), 305 (5), 291 (6), 277 (6), 263 (14), 249
(16), 235 (63), 165 (24), 151 (6), 135 (3), 122 (2), 109 (3), 96 (7),
80 (6).
1
Methyl-(E)-8-oxooctadec-9-enoate (18): colorless oil; H NMR
(CD3OD, 400 MHz) δ 6.92 (1H, dt, J ) 15.6, 6.8 Hz, H-10), 6.10
(1H, dt, J ) 15.6, 1.6 Hz, H-9), 3.64 (3H, s, OCH3), 2.58 (2H, t, J )
7.2 Hz, H-7), 2.31 (2H, t, J ) 7.6 Hz, H-2), 2.23 (2H, q, J ) 6.8 Hz,
H-11), 1.58 (4H, m, H-3, H-6), 1.48 (2H, m, H-12), 1.33–1.28 (14H,
m, CH2), 0.89 (3H, t, J ) 6.8 Hz, H-18); FABMS m/z 333 [M + Na]+.
Methyl-8-oxooctadecanoate (18a). The method of preparation of
18a from 18 was similar to that used for the preparation of 6a: white,
amorphous powder; FABMS m/z 335 [M + Na]+; FAB-CID-MS/MS
m/z 335 [M + Na]+(100), 305 (8), 291 (10), 277 (8), 263 (19), 249
(13), 235 (19), 221 (91), 151 (24), 135 (14), 122 (3), 109 (7), 96 (5),
80 (5).
(E)-(R)-10-Hydroxyoctadec-8-enoic acid (11): colorless oil; [R]24
D
-3.8 (c 0.07, MeOH); negative-ion FABMS m/z 297 [M - H]-;
positive-ion FABMS m/z 321 [M + Na]+, 343 [M – H + 2Na]+; FAB-
CID-MS/MS m/z 343 [M – H + 2Na]+ (100), 299 (14), 271 (18), 257
(11), 243 (18), 229 (32), 159 (26), 145 (19), 131 (18), 117 (86), 104
(93), 90 (68).
10-Oxooctadecanoic acid (12): white, amorphous powder; negative-
ion FABMS m/z 297 [M - H]-; positive-ion FABMS m/z 321 [M +
Na]+, 343 [M – H + 2Na]+; FAB-CID-MS/MS m/z 343 [M – H +
2Na]+ (100), 327 (13), 313 (19), 299 (22), 285 (13), 271 (25), 257
(66), 187 (66), 173 (25), 159 (31), 145 (19), 131 (16), 117 (66), 104
(66), 90 (78).
11-Oxooctadecanoic acid (13): white, amorphous powder; negative-
ion FABMS m/z 297 [M - H]-; positive-ion FABMS m/z 321 [M +
Na]+, 343 [M - H + 2Na]+; FAB-CID-MS/MS m/z 343 [M – H +
2Na]+(100), 313 (14), 299 (14), 285 (36), 271 (71), 201 (75), 187 (25),
173 (32), 159 (29), 145 (21), 131 (14), 117 (71), 104 (89), 90 (64).
12-Oxooctadecanoic acid (14): white, amorphous powder; FABMS
m/z 321 [M + Na]+, 343 [M – H + 2Na]+; FAB-CID-MS/MS m/z
343 [M – H + 2Na]+ (100), 299 (16), 285 (42), 215 (42), 201 (29),
187 (24), 173 (21), 159 (26), 145 (21), 131 (13), 117 (63), 104 (63),
90 (55).
(E)-11-Oxooctadec-9-enoic acid (19). The method of preparation
of the acid derivatives (19, 9, 10, and 20) from oleic acid was similar
to that used for the preaparion of the methyl ester analogues (15-18):
1
colorless oil; H NMR (CD3OD, 400 MHz) δ 6.92 (1H, dt, J ) 16.4,
7.2 Hz, H-9), 6.11 (1H, dt, J ) 16.4, 1.2 Hz, H-10), 2.57 (2H, t, J )
7.6 Hz, H-12), 2.25 (4H, m, H-2, H-8), 1.59 (4H, m, H-3, H-13), 1.49
(2H, m, H-7), 1.34–1.30 (14H, m, CH2), 0.89 (3H, t, J ) 6.8 Hz, H-18);
FABMS m/z 341 [M + 2Na – H]+.
11-Oxooctadecanoic acid (19a). The method of preparation of 19a
from 19 was similar to that used for the preparation of 6a: white,
amorphous powder; FABMS m/z 343 [M – H + 2Na]+; FAB-CID-
MS/MS m/z 343 [M – H + 2Na]+ (100), 313 (16), 299 (11), 285 (21),
271 (76), 201 (94), 187 (31), 173 (64), 159 (35), 145 (23), 131 (12),
117 (56), 104 (52), 90 (35).
(E)-8-Oxooctadec-9-enoic acid (20): colorless oil; 1H NMR
(CD3OD, 400 MHz) δ 6.91 (1H, dt, J ) 16.0, 7.6 Hz, H-10), 6.10
(1H, dt, J ) 16.0, 1.4 Hz, H-9), 2.58 (2H, t, J ) 7.2 Hz, H-7), 1.58
(4H, m, H-3, H-6), 1.46 (2H, m, H-12), 1.35–1.21 (14H, m, CH2), 0.89
(3H, t, J ) 6.8 Hz, H-18); FABMS m/z 341 [M + 2Na – H]+.
8-Oxooctadecanoic acid (20a). The method of preparation of 20a
from 20was similar to that used for the preparation of 6a: white,
amorphous powder; FABMS m/z 343 [M – H + 2Na]+; FAB-CID-
MS/MS m/z 343 [M - H + 2Na]+(100), 313 (11), 299 (11), 285 (12),
271 (37), 257 (7), 243 (16), 229 (92), 159 (52), 145 (17), 131 (11),
117 (48), 104 (42), 90 (33).
Preparation of 15–18. General Procedure. To a solution of methyl
oleate (1.77 mmol), which was prepared from oleic acid, in EtOAc (5
mL) was added tert-butyl hydroperoxide in decane (8.85 mmol) and 3
Å molecular sieves (300 mg). The mixture was stirred for 30 min at
room temperature. Manganese(III) acetate dihydrate (0.18 mmol) was
added, and the mixture was stirred for 48 h at room temperature. The
solution was diluted with ether and filtered through Celite. The filtrate
was concentrated under reduced pressure and purified by a reversed-
phase HPLC (YMC ODS-H80, 250 × 20 mm, 4 µm, 80 Å) eluting
with 95% aqueous CH3CN to yield compounds 15 (23 mg), 16 (25
mg), 17 (29 mg), and 18 (26 mg).
(E)-11-Oxooctadec-9-enol (21). The method of preparation of the
hydroxy analogues (21-24) from oleyl alcohol was similar to that used
for the preparation of the methyl ester analogues (15-18): colorless
1
Methyl-(E)-11-oxooctadec-9-enoate (15): colorless oil; H NMR
1
(CD3OD, 400 MHz) δ 6.91 (1H, dt, J ) 16.0, 6.8 Hz, H-9), 6.11 (1H,
dt, J ) 16.0, 1.2 Hz, H-10), 3.64 (3H, s, OCH3), 2.57 (2H, t, J ) 7.6
Hz, H-12), 2.31 (2H, t, J ) 7.6 Hz, H-2), 2.22 (2H, q, J ) 6.8 Hz,
H-8), 1.59 (4H, m, H-3, H-13), 1.49 (2H, m, H-7), 1.34–1.30 (14H, m,
CH2), 0.89 (3H, t, J ) 6.8 Hz, H-18); FABMS m/z 333 [M + Na]+.
Methyl-11-oxooctadecanoate (15a). The method of preparation
of 15a from 15 was similar to that used for the preparation of 6a: white,
amorphous powder; FABMS m/z335 [M + Na]+; FAB-CID-MS/MS
m/z 335 [M + Na]+(100), 305 (9), 291 (9), 277 (16), 263 (80), 193
(51), 177 (25), 164 (14), 151 (5), 135 (5), 122 (4), 109 (10), 96 (13),
80 (9).
oil; H NMR (CD3OD, 400 MHz) δ 6.92 (1H, dt, J ) 15.6, 6.4 Hz,
H-9), 6.10 (1H, dt, J ) 15.6, 1.2 Hz, H-10), 3.53 (2H, t, J ) 6.8 Hz,
H-1), 2.57 (2H, t, J ) 7.2 Hz, H-12), 2.25 (2H, q, J ) 6.4 Hz, H-8),
1.59 (2H, m, H-13), 1.50 (4H, m, H-2, H-7), 1.34–1.28 (16H, m, CH2),
0.89 (3H, t, J ) 6.8 Hz, H-18); FABMS m/z 281 [M – H]-.
1
(E)-10-Oxooctadec-8-enol (22): colorless oil; H NMR (CD3OD,
400 MHz) δ 6.92 (1H, dt, J ) 16.0, 6.8 Hz, H-8), 6.10 (1H, dt, J )
16.0, 1.6 Hz, H-9), 3.53 (2H, t, J ) 6.4 Hz, H-1), 2.58 (2H, t, J ) 7.2
Hz, H-11), 2.25 (2H, q, J ) 6.8 Hz, H-7), 1.58 (2H, m, H-12), 1.50
(4H, m, H-2, H-6), 1.37–1.30 (16H, m, CH2), 0.89 (3H, t, J ) 6.8 Hz,
H-18); FABMS m/z 281 [M - H]-.
1
1
Methyl-(E)-10-oxooctadec-8-enoate (16): colorless oil; H NMR
(E)-9-Oxooctadec-10-enol (23): colorless oil; H NMR (CD3OD,
(CD3OD, 400 MHz) δ 6.91 (1H, dt, J ) 16.4, 6.8 Hz, H-8), 6.11 (1H,
dt, J ) 16.4, 1.2 Hz, H-9), 3.64 (3H, s, OCH3), 2.57 (2H, t, J ) 7.6
Hz, H-11), 2.31 (2H, t, J ) 7.2 Hz, H-2), 2.23 (2H, q, J ) 6.8 Hz,
H-7), 1.58 (4H, m, H-3, H-12), 1.49 (2H, m, H-6), 1.35–1.30 (14H, m,
CH2), 0.89 (3H, t, J ) 6.8 Hz, H-18); FABMS m/z 333 [M + Na]+.
Methyl-10-oxooctadecanoate (16a). The method of preparation
of 16a from 16 was similar to that used for the preparation of 6a: white,
amorphous powder; FABMS m/z335 [M + Na]+; FAB-CID-MS/MS
m/z 335 [M + Na]+(100), 305 (8), 291 (9), 277 (5), 263 (23), 249
(89), 179 (52), 166 (10), 151 (7), 135 (5), 122 (3), 109 (7), 96 (8), 80
(7).
400 MHz) δ 6.93 (1H, dt, J ) 16.4, 6.8 Hz, H-11), 6.10 (1H, dt, J )
16.4, 1.6 Hz, H-10), 3.53 (2H, t, J ) 6.8 Hz, H-1), 2.58 (2H, t, J ) 7.2
Hz, H-8), 2.25 (2H, q, J ) 6.8 Hz, H-12), 1.58 (2H, m, H-7), 1.50
(4H, m, H-2, H-13), 1.33–1.29 (16H, m, CH2), 0.89 (3H, t, J ) 7.2
Hz, H-18); FABMS m/z 281 [M – H]-.
1
(E)-8-Oxooctadec-9-enol (24): colorless oil; H NMR (CD3OD,
400 MHz) δ 6.92 (1H, dt, J ) 16.0, 6.8 Hz, H-10), 6.10 (1H, dt, J )
16.0, 1.6 Hz, H-9), 3.53 (2H, t, J ) 6.4 Hz, H-1), 2.58 (2H, t, J ) 7.2
Hz, H-7), 2.24 (2H, q, J ) 6.8 Hz, H-11), 1.58 (2H, m, H-6), 1.50
(4H, m, H-2, H-12), 1.33–1.28 (16H, m, CH2), 0.89 (3H, t, J ) 7.2
Hz, H-18); FABMS m/z 281 [M – H]-.
Oxidation of the Hydrogenated Products of 21–24. The hydro-
genated product 21a or 22a-24a (each 1 mg), which was formed by
the hydrogenation of 21 or 22–24 by the same method described for
the preparation of 6a, was dissolved in acetone, and the solution was
cooled to 0 °C. A 2 µL amount of Jones’ reagent (6.68 g of CrO3 in
5.75 mL of concentrated H2SO4, then H2O until volume of 25 mL)
was added, and the reaction was stirred at 0 °C for 2 h. The mixture
was filtered and washed with diethyl ether. The filtrate was concentrated
1
Methyl-(E)-9-oxooctadec-10-enoate (17): colorless oil; H NMR
(CD3OD, 400 MHz) δ 6.92 (1H, dt, J ) 16.0, 6.8 Hz, H-11), 6.10
(1H, dt, J ) 16.0, 1.6 Hz, H-10), 3.64 (3H, s, OCH3), 2.57 (2H, t, J )
7.2 Hz, H-8), 2.31 (2H, t, J ) 7.2 Hz, H-2), 2.23 (2H, q, J ) 6.8 Hz,
H-12), 1.58 (4H, m, H-3, H-7), 1.48 (2H, m, H-13), 1.32–1.29 (14H,
m, CH2), 0.90 (3H, t, J ) 7.6 Hz, H-18); FABMS m/z 333 [M + Na]+.
Methyl-9-oxooctadecanoate (17a). The method of preparation of
17a from 17 was similar to that used for the preparation of 6a: white,

Anti-inflammatory Constituents of Gracilaria Verrucosa
Journal of Natural Products, 2008, Vol. 71, No. 2 239
under reduced pressure and purified by a reversed-phase HPLC (YMC
ODS-H80) eluting with 95% aqueous MeOH to furnish keto fatty acids
21b-24b. The 1H NMR and MS data of 21b, 22b, 23b, and 24b were
identical with those of 19a, 9a, 10a, and 20a, respectively.
(6H, m, H-3, H-6, H-12), 1.38–1.31 (14H, m, CH2), 0.89 (3H, t, J )
6.8 Hz, H-18); FABMS m/z 411/413 [M + Na]+.
Methyl-(Z)-10-bromo-9-oxooctadec-10-enoate (31). The method
of preparation of 31 from 17 was similar to that used for the preparation
1
(E)-N-Trifluoroacetyl-11-oxooctadec-9-enyl amide (25). The
method of preparation of the amide analogues (25-28) from (Z)-N-
trifluoroacetyl-octadec-9-enyl amide, which was prepared from oleyl
amine and trifluoroacetic anhydride, was similar to that used for the
of 29: colorless oil; H NMR (CD3OD, 400 MHz) δ 7.31 (1H, t, J )
6.8 Hz, H-11), 3.64 (3H, s, OCH3), 2.82 (2H, t, J ) 7.6 Hz, H-8), 2.43
(2H, q, J ) 6.8 Hz, H-12), 2.31 (2H, t, J ) 8.0 Hz, H-2), 1.60–1.51
(6H, m, H-3, H-7, H-13), 1.35–1.31 (14H, m, CH2), 0.90 (3H, t, J )
7.2 Hz, H-18); FABMS m/z 411/413 [M + Na]+.
1
preparation of the methyl ester analogues (15-18): colorless oil; H
NMR (CDCl3, 400 MHz) δ 6.79 (1H, dt, J ) 15.6, 6.8 Hz, H-9), 6.42
(1H, brs, NH), 6.06 (1H, d, J ) 15.6 Hz, H-10), 3.33 (2H, q, J ) 6.4
Hz, H-1), 2.49 (2H, t, J ) 7.2 Hz, H-12), 2.18 (2H, q, J ) 6.8 Hz,
H-8), 1.55 (4H, m, H-2, H-13), 1.43 (2H, m, H-7), 1.28–1.17 (16H, m,
CH2), 0.85 (3H, t, J ) 6.0 Hz, H-18); FABMS m/z 400 [M + Na]+.
N-Trifluoroacetyl-11-oxooctadecanyl amide (25a). The method
of preparation of 25a from 25 was similar to that used for the
preparation of 6a: white, amorphous powder; FABMS m/z 402 [M +
Na]+; FAB-CID-MS/MS m/z 402 [M + Na]+ (100), 372 (12), 358 (9),
344 (21), 330 (71), 260 (68), 247 (27), 233 (14), 218 (12), 204 (10),
190 (7), 177 (9), 163 (9), 149 (14), 135 (3).
Methyl-(Z)-9-bromo-8-oxooctadec-9-enoate (32). The method of
preparation of 32 from 18 was similar to that used for the preparation
1
of 29: colorless oil; H NMR (CD3OD, 400 MHz) δ 7.31 (1H, t, J )
6.8 Hz, H-10), 3.64 (3H, s, OCH3), 2.82 (2H, t, J ) 7.6 Hz, H-7), 2.43
(2H, q, J ) 6.8 Hz, H-11), 2.31 (2H, t, J ) 7.2 Hz, H-2), 1.63–1.53
(6H, m, H-3, H-6, H-12), 1.35–1.28 (14H, m, CH2), 0.89 (3H, t, J )
6.8 Hz, H-18); FABMS m/z 411/413 [M + Na]+.
(Z)-10-Bromo-11-oxooctadec-9-enoic acid (33). The method of
preparation of 33 from 19 was similar to that used for the preparation
1
of 29: colorless oil; H NMR (CD3OD, 400 MHz) δ 7.31 (1H, t, J )
7.2 Hz, H-9), 2.81 (2H, t, J ) 7.6 Hz, H-12), 2.43 (2H, q, J ) 7.2 Hz,
H-8), 2.27 (2H, t, J ) 7.6 Hz, H-2), 1.60–1.53 (6H, m, H-3, H-7, H-13),
1.37–1.31 (14H, m, CH2), 0.90 (3H, t, J ) 7.6 Hz, H-18); FABMS
m/z 397/399 [M + Na]+.
(E)-N-Trifluoroacetyl-10-oxooctadec-8-enyl amide (26): colorless
1
oil; H NMR (CDCl3, 400 MHz) δ 6.79 (1H, dt, J ) 15.6, 7.2 Hz,
H-8), 6.50 (1H, brs, NH), 6.05 (1H, dt, J ) 15.6, 1.2 Hz, H-9), 3.33
(2H, q, J ) 6.8 Hz, H-1), 2.49 (2H, t, J ) 7.6 Hz, H-11), 2.18 (2H, q,
J ) 7.2 Hz, H-7), 1.55 (4H, m, H-2, H-12), 1.43 (2H, m, H-6),
1.29–1.19 (16H, m, CH2), 0.85 (3H, t, J ) 6.8 Hz, H-18); FABMS
m/z400 [M + Na]+.
(Z)-9-Bromo-10-oxooctadec-8-enoic acid (34). The method of
preparation of 34 from 9 was similar to that used for the preparation
1
of 29: colorless oil; H NMR (CD3OD, 400 MHz) δ 7.31 (1H, t, J )
7.2 Hz, H-8), 2.81 (2H, t, J ) 7.2 Hz, H-11), 2.43 (2H, q, J ) 7.2 Hz,
H-7), 2.27 (2H, t, J ) 7.6 Hz, H-2), 1.62–1.53 (6H, m, H-3, H-6, H-12),
1.37–1.31 (14H, m, CH2), 0.90 (3H, t, J ) 7.6 Hz, H-18); FABMS
m/z 397/399 [M + Na]+.
N-Trifluoroacetyl-10-oxooctadecanyl amide (26a). The method
of preparation of 26a from 26 was similar to that used for the
preparation of 6a: white, amorphous powder; FABMS m/z 402 [M +
Na]+; FAB-CID-MS/MS m/z 402 [M + Na]+ (100), 372 (9), 358 (10),
344 (5), 330 (22), 316 (85), 246 (68), 233 (21), 218 (9), 204 (8), 190
(9), 177 (9), 163 (7), 149 (14), 135 (4).
(Z)-10-Bromo-9-oxooctadec-10-enoic acid (35). The method of
preparation of 35 from 10 was similar to that used for the preparation
1
of 29: colorless oil; H NMR (CD3OD, 400 MHz) δ 7.30 (1H, t, J )
(E)-N-Trifluoroacetyl-9-oxooctadec-10-enyl amide (27): colorless
oil; 1H NMR (CDCl3, 400 MHz) δ 6.79 (1H, dt, J ) 16.0, 6.8 Hz, H-11),
6.45 (1H, brs, NH), 6.06 (1H, d, J ) 16.0 Hz, H-10), 3.33 (2H, q, J ) 6.8
Hz, H-1), 2.49 (2H, t, J ) 7.2 Hz, H-8), 2.18 (2H, q, J ) 6.8 Hz, H-12),
1.56 (4H, m, H-2, H-7), 1.42 (2H, m, H-13), 1.28–1.18 (16H, m, CH2),
0.85 (3H, t, J ) 6.8 Hz, H-18); FABMS m/z400 [M + Na]+.
N-Trifluoroacetyl-9-oxooctadecanyl amide (27a). The method of
preparation of 27a from 27 was similar to that used for the preparation
of 6a: white, amorphous powder; FABMS m/z402 [M + Na]+; FAB-
CID-MS/MS m/z 402 [M + Na]+ (100), 372 (10), 358 (12), 344 (9),
330 (8), 316 (52), 302 (88), 232 (61), 218 (19), 204 (9), 190 (9), 177
(5), 163 (8), 149 (10), 135 (3).
(E)-N-Trifluoroacetyl-8-oxooctadec-9-enyl amide (28): colorless
oil; 1H NMR (CDCl3, 400 MHz) δ 6.80 (1H, dt, J ) 16.4, 6.8 Hz, H-10),
6.44 (1H, brs, NH), 6.06 (1H, d, J ) 16.4 Hz, H-9), 3.33 (2H, q, J ) 6.4
Hz, H-1), 2.50 (2H, t, J ) 7.2 Hz, H-7), 2.18 (2H, q, J ) 6.8 Hz, H-11),
1.56 (4H, m, H-2, H-6), 1.43 (2H, m, H-12), 1.30–1.20 (16H, m, CH2),
0.85 (3H, t, J ) 6.8 Hz, H-18); FABMS m/z 400 [M + Na]+.
N-Trifluoroacetyl-8-oxooctadecanyl amide (28a). The method of
preparation of 28a from 28 was similar to that used for the preparation
of 6a: white, amorphous powder; FABMS m/z402 [M + Na]+; FAB-
CID-MS/MS m/z 402 [M + Na]+ (100), 372 (8), 358 (10), 344 (11),
330 (28), 316 (11), 302 (17), 288 (90), 218 (47), 205 (15), 190 (5),
177 (4), 163 (5), 149 (11), 135 (4).
6.8 Hz, H-11), 2.81 (2H, t, J ) 7.2 Hz, H-8), 2.42 (2H, q, J ) 6.8 Hz,
H-12), 2.27 (2H, t, J ) 7.2 Hz, H-2), 1.63–1.53 (6H, m, H-3, H-7,
H-13), 1.34–1.31 (14H, m, CH2), 0.90 (3H, t, J ) 6.8 Hz, H-18);
FABMS m/z 397/399 [M + Na]+.
(Z)-9-Bromo-8-oxooctadec-9-enoic acid (36). The method of
preparation of 36 from 20 was similar to that used for the preparation
1
of 29: colorless oil; H NMR (CD3OD, 400 MHz) δ 7.31 (1H, t, J )
7.2 Hz, H-10), 2.82 (2H, t, J ) 7.2 Hz, H-7), 2.43 (2H, q, J ) 7.2 Hz,
H-11), 2.28 (2H, t, J ) 7.2 Hz, H-2), 1.63–1.51 (6H, m, H-3, H-6,
H-12), 1.36–1.31 (14H, m, CH2), 0.90 (3H, t, J ) 6.8 Hz, H-18);
FABMS m/z 397/399 [M + Na]+.
Methyl-(Z)-10-phenyl-11-oxooctadec-9-enoate (37). To a stirred
solution of 29 (5 mg) and 1.6 mg of phenylboronic acid in a mixture
solvent of 1 mL of toluene, 1 mL of EtOH, and 35 µL of 2 M Na2CO3
was added 1 mg of tetrakis(triphenylphosphine)palladium(0). After
refluxing with vigorous stirring for 3 h, the solvent was removed in
vacuo. The residue was purified by a reversed-phase HPLC (YMC
ODS-H80) eluting with 95% aqueous CH3CN to yield compound 37
1
(2 mg): colorless oil; H NMR (CD3OD, 400 MHz) δ 7.40–7.30 (4H,
m, Ar-H), 7.06 (1H, dd, J ) 6.8, 1.6 Hz, Ar-H), 6.94 (1H, t, J ) 7.2
Hz, H-9), 3.64 (3H, s, OCH3), 2.61 (2H, t, J ) 7.2 Hz, H-12), 2.27
(2H, t, J ) 7.6 Hz, H-2), 2.05 (2H, q, J ) 7.2 Hz, H-8), 1.55 (4H, m,
H-3, H-13), 1.43 (2H, m, H-7), 1.28–1.22 (14H, m, CH2), 0.88 (3H, t,
J ) 7.6 Hz, H-18); FABMS m/z 409 [M + Na]+.
Methyl-(Z)-10-bromo-11-oxooctadec-9-enoate (29). To a solution
of 15 (20 mg) in CCl4 (0.5 mL) was added bromine (4 µL), and the
reaction was stirred at room temperature for 3 h. The solvent was
removed at reduced pressure, and the residue was dissolved in 0.5 mL
of EtOH, to which was added 50 µL of 28% NH4OH. The resulting
mixture was stirred for a further 3 h. After removal of solvent, the
residue was subjected to a reversed-phase HPLC (YMC ODS-H80)
eluting with 95% aqueous CH3CN to yield compound 29 (6 mg):
colorless oil; 1H NMR (CD3OD, 400 MHz) δ 7.30 (1H, t, J ) 6.8 Hz,
H-9), 3.64 (3H, s, OCH3), 2.81 (2H, t, J ) 7.6 Hz, H-12), 2.42 (2H, q,
J ) 6.8 Hz, H-8), 2.31 (2H, t, J ) 7.6 Hz, H-2), 1.60–1.53 (6H, m,
H-3, H-7, H-13), 1.36–1.31 (14H, m, CH2), 0.89 (3H, t, J ) 6.8 Hz,
H-18); FABMS m/z 411/413 [M + Na]+.
Methyl-(Z)-10-phenyl-9-oxooctadec-10-enoate (38). The method
of preparation of 38 from 31 was similar to that used for the preparation
1
of 37: colorless oil; H NMR (CD3OD, 400 MHz) δ 7.40–7.30 (4H,
m, Ar-H), 7.07 (1H, dd, J ) 6.8, 1.2 Hz, Ar-H), 6.95 (1H, t, J ) 7.6
Hz, H-11), 3.64 (3H, s, OCH3), 2.62 (2H, t, J ) 7.2 Hz, H-8), 2.30
(2H, t, J ) 7.2 Hz, H-2), 2.05 (2H, q, J ) 7.6 Hz, H-12), 1.57 (4H, m,
H-3, H-7), 1.43 (2H, m, H-13), 1.29–1.22 (14H, m, CH2), 0.87 (3H, t,
J ) 6.8 Hz, H-18); FABMS m/z409 [M + Na]+.
(Z)-10-Phenyl-9-oxooctadec-10-enoic acid (39). The method of
preparation of 39 from 35 was similar to that used for the preparation
1
of 37: colorless oil; H NMR (CD3OD, 400 MHz) δ 7.40–7.30 (4H,
m, Ar-H), 7.07 (1H, dd, J ) 6.8, 1.6 Hz, Ar-H), 6.95 (1H, t, J ) 7.6
Hz, H-11), 2.62 (2H, t, J ) 7.6 Hz, H-8), 2.25 (2H, t, J ) 7.2 Hz,
H-2), 2.05 (2H, q, J ) 7.6 Hz, H-12), 1.57 (4H, m, H-3, H-7), 1.43
(2H, m, H-13), 1.30–1.21 (14H, m, CH2), 0.87 (3H, t, J ) 6.8 Hz,
H-18); FABMS m/z 395 [M + Na]+.
Methyl-(Z)-9-bromo-10-oxooctadec-8-enoate (30). The method
of preparation of 30 from 16 was similar to that used for the preparation
1
of 29: colorless oil; H NMR (CD3OD, 400 MHz) δ 7.30 (1H, t, J )
7.2 Hz, H-8), 3.64 (3H, s, OCH3), 2.81 (2H, t, J ) 7.2 Hz, H-11), 2.42
(2H, q, J ) 7.2 Hz, H-7), 2.32 (2H, t, J ) 7.6 Hz, H-2), 1.63–1.54
(Z)-9-Phenyl-8-oxooctadec-9-enoic acid (40). The method of
preparation of 40 from 36 was similar to that used for the preparation

240 Journal of Natural Products, 2008, Vol. 71, No. 2
Dang et al.
1
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of 37: colorless oil; H NMR (CD3OD, 400 MHz) δ 7.40–7.30 (4H,
m, Ar-H), 7.07 (1H, dd, J ) 6.8, 2.0 Hz, Ar-H), 6.95 (1H, t, J ) 7.6
Hz, H-10), 2.62 (2H, t, J ) 7.6 Hz, H-7), 2.25 (2H, t, J ) 7.2 Hz,
H-2), 2.04 (2H, q, J ) 7.6 Hz, H-11), 1.57 (4H, m, H-3, H-6), 1.43
(2H, m, H-12), 1.31–1.21 (14H, m, CH2), 0.88 (3H, t, J ) 6.8 Hz,
H-18); FABMS m/z 395 [M + Na]+.
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Cytotoxicity Assay. Lactate dehydrogenase leakage is a means of
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cytotoxicity detection kit for 30 min at room temperature in the dark.
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Nitrite Assay. The production of nitric oxide (NO) was measured,
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sulfanilamide, 2.5% H3PO4). This mixture was incubated for 10 min
at room temperature (light protected). Absorbance at 540 nm was
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and the results were compared against a calibration curve using sodium
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Measurement of the Production of Pro-inflammatory Cytokines
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compounds on IL-6 and TNF-R production were determined by the
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and diluted with DMEM. The final concentration of chemical solvents
did not exceed 0.1% in the culture medium. At these conditions, none
of the solubilized solvents altered IL-6 and TNF-R production in RAW
264.7 cells. Before stimulation with LPS (1 µg/mL) and test materials,
RAW 264.7 cells were incubated for 18 h in 24-well plates under the
same conditions. Lipopolysaccharide (LPS) and the test materials were
then added to the cultured cells. The medium was used for IL-6 and
TNF-R assay using mouse ELISA kits (R & D Systems Inc., MN).
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Acknowledgment. This work was supported by a grant from Marine
Bio 21, Ministry of Maritime Affairs and Fisheries, Korea.
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