Fijiolides A and B, inhibitors of TNF-α-induced NFκB activation, from a marine-derived sediment bacterium of the genus Nocardiopsis

Article abstract of DOI:10.1021/np100087c

Fijiolide A, a potent inhibitor of TNF-α-induced NFκB activation, along with fijiolide B, were isolated from a marine-derived bacterium of the genus Nocardiopsis. The planar structures of fijiolides A (1) and B (2) were elucidated by interpretation of 2D NMR spectroscopic data, while the absolute configurations of these compounds were defined by interpretation of circular dichroism and 2D NMR data combined with application of the advanced Mosher's method. Fijiolides A and B are related to several recently isolated chloroaromatic compounds, which appear to be the Bergman cyclization products of enediyne precursors. Fijiolide A reduced TNF-α-induced NFκB activation by 70.3%, with an IC₅₀ value of 0.57 μM. Fijiolide B demonstrated less inhibition, only 46.5%, without dose dependence. The same pattern was also observed with quinone reductase (QR) activity: fijiolide A was found to induce quinone reductase-1 (QR1) with an induction ratio of 3.5 at a concentration of 20 μg/mL (28.4 μM). The concentration required to double the activity was 1.8 μM. Fijiolide B did not affect QR1 activity, indicating the importance of the nitrogen substitution pattern for biological activity. On the basis of these data, fijiolide A is viewed as a promising lead for more advanced anticancer testing.

Full text of DOI:10.1021/np100087c

1080
J. Nat. Prod. 2010, 73, 1080–1086
Fijiolides A and B, Inhibitors of TNF-r-Induced NFKB Activation, from a Marine-Derived
Sediment Bacterium of the Genus Nocardiopsis
Sang-Jip Nam,† Susana P. Gaudeˆncio,† Christopher A. Kauffman,† Paul R. Jensen,† Tamara P. Kondratyuk,‡ Laura E. Marler,‡
John M. Pezzuto,‡ and William Fenical*,†
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, UniVersity of CaliforniasSan Diego,
La Jolla, California 92093-0204, and College of Pharmacy, UniVersity of Hawaii at Hilo, Hilo, Hawaii 96720
ReceiVed February 6, 2010
Fijiolide A, a potent inhibitor of TNF-R-induced NFκB activation, along with fijiolide B, were isolated from a marine-
derived bacterium of the genus Nocardiopsis. The planar structures of fijiolides A (1) and B (2) were elucidated by
interpretation of 2D NMR spectroscopic data, while the absolute configurations of these compounds were defined by
interpretation of circular dichroism and 2D NMR data combined with application of the advanced Mosher’s method.
Fijiolides A and B are related to several recently isolated chloroaromatic compounds, which appear to be the Bergman
cyclization products of enediyne precursors. Fijiolide A reduced TNF-R-induced NFκB activation by 70.3%, with an
IC50 value of 0.57 µM. Fijiolide B demonstrated less inhibition, only 46.5%, without dose dependence. The same pattern
was also observed with quinone reductase (QR) activity: fijiolide A was found to induce quinone reductase-1 (QR1)
with an induction ratio of 3.5 at a concentration of 20 µg/mL (28.4 µM). The concentration required to double the
activity was 1.8 µM. Fijiolide B did not affect QR1 activity, indicating the importance of the nitrogen substitution
pattern for biological activity. On the basis of these data, fijiolide A is viewed as a promising lead for more advanced
anticancer testing.
Nature is an important resource for the discovery of anticancer
drugs. The relevance of the marine environment as a source of novel
anticancer compounds has been validated by the discovery of ca.
2500 new metabolites with antiproliferative activity.1 Bacteria
belonging to the order Actinomycetales, commonly known as
actinomycetes, have provided nearly 50% of the bioactive microbial
natural products discovered as of 2002.2 Although most microbial
small molecule discovery efforts have focused on actinomycetes
from terrestrial environments, marine-derived actinomycetes are
proving to be an important resource that has led to the discovery
of biologically active molecules with unique chemical structures.3
Excellent examples are the salinosporamides4a and marinomycins.4b
Salinosporamide A, isolated from Salinispora tropica, is a potent
20S proteasome inhibitor that recently completed phase I clinical
trials for treatment of solid tumors, lymphomas, and multiple
myeloma. The marinomycins isolated from a “Marinispora” sp.
showed significant antimicrobial activity against drug-resistant
bacterial pathogens and selective cancer cell cytotoxicity against
melanoma cell lines.4b
a focal point for drug discovery from different sources, including
the marine environment. Over the past decade, several compounds
have been discovered that selectively interfere with the NFκB
pathway. Examples include phenols and polyphenols (e.g., cur-
cumin, resveratrol, caffeic acid phenethyl ester, quercetin, epigal-
locatechin-3-gallate), lignans, sesquiterpenes, diterpenes, and trit-
erpenes.8 The majority of these compounds are antioxidants, which
act by blocking the protein kinase-mediated IκB degradation,
thereby preventing NFκB activation.
An additional strategy for protecting cells from cancer initiation
involves decreasing metabolic enzymes responsible for generating
reactive species (phase I enzymes) while increasing phase II
enzymes that can deactivate radicals and electrophiles known to
intercede in normal cellular processes. Reduction of electrophilic
quinones by quinone reductase (QR) is an important detoxification
pathway, and induction of this enzyme often correlates with
induction of other phase II enzymes such as glutathione S-
transferase. Based on the evaluation of approximately 3800 marine
organism extracts, the percentage characterized as “active” QR
activators was established in the range of 17% of the total. About
60 QR inducers have been isolated from a diversity of sources,
and substantial molecular diversity has been observed. These active
compounds comprise ceramides, terpenoids, withanolides, fla-
vonoids, chalcones, alkaloids, and diarylheptanoids.8
As part of a program to explore marine bacterial metabolites as
inhibitors of tumor initiation and promotion, we have targeted
nuclear factor kappa B (NFκB), a transcription factor that regulates
the expression of more than 400 different genes. NFκB is an
inducible, well-characterized protein, responsible for the regulation
of complex phenomena, with a pivotal role in controlling cell
signaling.5 Many genes activated by NFκB encode proteins that
are involved in tumorigenesis, including cyclooxygenase (COX)-2
and matrix metalloproteinase (MMP)-9, adhesion molecules, chemok-
ines, inflammatory cytokines, inducible nitric oxide synthase
(iNOS), and antiapoptosis proteins bcl-2 and bcl-xl.6 Constitutive
expression of NFκB is a frequent occurrence in tumor cells, and
inhibitors of NFκB have the potential of suppressing carcinogen-
esis.7 Since NFκB represents an important and attractive therapeutic
target for drugs to treat many disorders, including arthritis, asthma,
autoimmune diseases, and different types of cancer, it has become
As part of our collaborative program to define new modulators
of NFκB and QR, the marine-derived actinomycete strain CNS-
653, isolated from a marine sediment sample collected from the
Beqa Lagoon, Fiji, was evaluated. Analysis of the 16S rRNA gene
sequence places this strain within the genus Nocardiopsis
(GQ374440). Strain CNS-653 shares a nearly identical (99.8%) 16S
sequence with another marine-derived strain, CNR-712 (EF392847),
which produces the modified peptides lucentamycins A-D, and
was isolated from a marine sediment sample collected in the
Bahamas.9 LC-MS analysis of a whole culture extract of this strain
revealed peaks illustrating the characteristic MS isotopic patterns
of dichlorinated secondary metabolites ([M + H]+:[M + H + 2]+:
[M + H + 4]+ ) 10:6:1). Fractionation of a 30 L culture extract
of this strain by standard silica column chromatography, followed
by reversed-phase HPLC separation, yielded fijiolides A (1, 31.0
* Corresponding author. Tel: 1-858-534-2133; 1-858-534-1318. E-mail:
wfenical@ucsd.edu.
† Scripps Institution of Oceanography.
‡ University of Hawaii.
10.1021/np100087c  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/18/2010

Fijiolides A and B from a Nocardiopsis Bacterium
Journal of Natural Products, 2010, Vol. 73, No. 6 1081
mg) and B (2, 3.0 mg) as colorless noncrystalline solids. Herein,
we report details of the isolation and structure elucidation of
fijiolides A and B and their effects on TNF-R-induced NFκB activity
and QR induction.
Figure 1. CD spectra for fijiolides A (1) and B (2) in MeOH (7.1
× 10-4 M).
The molecular formula of 1 was deduced as C34H3935Cl2N2O10,
on the basis of analysis of HRESIMS data (a pseudomolecular ion
peak at m/z 705.1997 [M + H]+) and interpretation of 13C NMR
data. The 1H NMR spectrum of 1 displayed ortho-coupled aromatic
protons [δH 7.08 (d, J ) 8.0 Hz), 6.64 (d, J ) 8.0 Hz)], meta-
coupled aromatic protons [δH 6.52 (d, J ) 2.0 Hz), 5.98 (d, J )
2.0 Hz)], three olefinic protons [δH 6.76 (d, J ) 2.1 Hz), 6.65 (dd,
J ) 5.3, 2.1 Hz), 6.62 (d, J ) 5.3 Hz)], and three downfield methine
protons [δH 5.87 (s), 4.66 (dd, J ) 11.0, 4.0 Hz), 4.61 (ddd, J )
of the spin system from the anomeric proton H-1′ through H-4′
[H1′ (δH 4.45, d, J ) 8.0 Hz)-H-2′ (δH 2.92, dd, J ) 8.0, 3.4
Hz)-H-3′ (δH 4.06, d, J ) 8.0, 3.4 Hz)-H-4′ (δH 2.98)] of the
aminosugar. Long-range HMBC correlations from the N-dimethyl
protons (δH 2.79) to C-4′ (δC 69.6) and from the dimethyl protons
(δH 1.46, 1.43) to C-4′ (δC 69.6) and C-5′ (δC 75.0) allowed
placement of these protons at C-4′ and C-5′, respectively. A long-
range HMBC correlation from the anomeric proton H-1′ (δH 4.45)
to C-9 (δC 99.8) provided evidence for placing the sugar at C-9.
The three-bond HMBC correlations from the methylene protons
H-14 (δH 4.23, 3.64) to the carbonyl carbon C-15 (δC 170.0) and
from the methine proton H-17 (δH 4.61) to the carbonyl carbon
C-24 (δC 168.4) permitted the ester linkage between C-14 and C-15
and the amide linkage between C-17 and C-24, respectively. Lastly,
placement of the chlorine at C-3 completed the structure assignment
of 1.
1
13.4, 13.4, 3.8 Hz)]. The H NMR and 13C NMR spectra also
revealed two N-methyl groups [δH 2.79 (δC 44.4 and 42.9)], three
methyl singlets [δH 1.46 (δC 20.6), 1.43 (δC 31.5), 1.70 (δC 22.6)],
and six exchangeable protons [(4-OH, 2-NH); δH 9.09, 8.45, 8.41,
5.86, 5.73, 4.95]. Interpretation of 2D NMR spectoscopic data
allowed three partial structures to be constructed: a benzodihydro-
pentalene, a 3′-chloro-5′-hydroxy-ꢀ-tyrosine, and an aminosugar
unit. COSY NMR data, including cross-peaks from H-11 (δH 6.65,
dd, J ) 5.3, 2.1 Hz) to H-10 (δH 6.62, d, J ) 5.3 Hz) and H-12
(δH 6.76, d, J ) 2.1 Hz), along with the 1D proton coupling
constants of these three protons, indicated that they belonged to a
cyclopentadienyl spin system. The presence of ortho-coupled
aromatic protons [H-5 (δH 6.64, d, J ) 8.0 Hz) and H-6 (δH 7.08,
d, J ) 8.0 Hz)] indicated a 1,2,3,4-tetrasubstituted benzene ring.
The connection between the cyclopentadienyl moiety and the
1,2,3,4-tetrasubstituted benzene ring was established by long-range
HMBC correlations observed from H-12 (δH 6.76) to C-1 (δC
148.8), C-2 (δC 135.7), C-9 (δC 99.8), and C-10 (δC 135.5); from
H-8 (δH 5.87) to C-1 (δC 148.8), C-2 (δC 135.7), C-6 (δC 125.3),
C-7 (δC 147.7), C-9 (δC 99.8), and C-10 (δC 135.5); and from H-6
(δH 7.08) to C-2 (δC 135.7), C-7 (δC 147.7), and C-8 (δC 83.2).
The oxygenated methine proton H-13 (δH 4.66), which was coupled
to the methylene protons H2-14 (δH 4.23, and δH 3.64), also
displayed long-range HMBC correlations to carbons C-3 (δC 126.7),
C-4 (δC 139.3), and C-5 (δC 129.1). These data allowed construction
of the benzodihydropentalene unit in 1 (C-1 to C-12).
The 3′-chloro-5′-hydroxy-ꢀ-tyrosine amino acid component was
established by the observation of COSY NMR cross-peaks between
a downfield methine proton H-17 (δH 4.61) and the methylene
protons H2-16 (δH 2.57, and δH 2.07). In addition, these assignments
were also confirmed by long-range HMBC NMR correlations from
H-17 to C-15 (δC 170.0), C-18 (δC 138.8), C-19 (δC 114.2), C-23
(δC 113.9), and C-24 (δC 168.4) and from the phenol proton 22-
OH (δH 8.45) to C-21 (δC 138.3), C-22 (δC 151.0), and C-23 (δC
113.9). The connection between C-8 and C-21 was established by
a three-bond HMBC correlation from H-8 (δH 5.87) to C-21 (δC
138.3).
The molecular formula of 2 was assigned as C32H3635Cl2N2O9,
on the basis of the pseudomolecular ion peak at m/z 663.1871 [M
+ H]+ observed in the HRESIMS spectrum. The 1H NMR spectrum
of 2 was almost identical to that of 1 except for an upfield shifted
methine proton H-17 (δH 4.09) and the absence of one methyl
singlet. This difference indicated 2 possessed a primary amine at
C-17 instead of an acetamide group. Interpretation of 2D COSY,
HSQC, and HMBC NMR spectroscopic data allowed the planar
structure of 2 to be assigned as shown.
The relative and absolute configurations of 1 and 2 were
determined by analysis of circular dichroism (CD) spectroscopic
data, application of the advanced Mosher’s method, and combined
spectroscopic methods. Fijiolides A (1) and B (2) were assigned
identical absolute configurations on the basis of the virtually
identical CD curves observed for these two metabolites (Figure 1).
The relative configuration of the ribopyranose ring of 1 was
determined by analysis of vicinal coupling constants and by analysis
of NOESY spectroscopic data. The magnitude of the 1JCH coupling
constant for the anomeric proton (H-1′, 162 Hz) indicated that this
center possesses a ꢀ-configuration.10 A large diaxial coupling
constant (8.0 Hz) between H-1′ and H-2′ indicated an axial
configuration for H-2′, while a small vicinal coupling constant (3.4
Hz) between H-2′ and H-3′ and strong NOESY correlations between
H-2′ and H-3′ and between H-3′ and H-4′ established that H-2′
and H-3′ are in the equatorial and axial positions, respectively
(Figure 2). Determination of the absolute configuration of the
ribopyranose of 1 was achieved using the advanced Mosher method
involving NMR analysis of MTPA ester pairs produced by
derivatization with R- and S-MTPA-Cl (R-methoxy-R-(trifluorom-
ethyl)phenylacetyl chloride).11 Calculation of ∆δS-R values of bis-
MTPA esters (3a,b) clearly established the absolute configuration
of C-2′ as R and allowed the absolute configuration of the
ribopyranose to be assigned as 1′S, 2′R, 3′R, 4′S (Figure 3a).
The 4-deoxy-4-dimethylamino-5,5-dimethylribopyranose sugar
was constructed on the basis of analysis of COSY and HMBC NMR
spectroscopic data. COSY NMR correlations permitted construction

1082 Journal of Natural Products, 2010, Vol. 73, No. 6
Nam et al.
Figure 2. Relative configuration of the ribopyranose based on key
NOESY correlations (dashed arrows).
Figure 4. Two possible stereoisomers (a, b) and selected key
NOESY correlations for determination of the configurations at C-8
and C-9 for fijiolides (a).
Figure 3. ∆δS-R of 1H for S- and R-bis-MTPA esters of 1 (a), ∆δS-R
of 1H for S- and R-mono-MTPA amides of 2 (b), ∆δS-R of 1H for
S- and R-mono-MTPA esters of 1 (c).
The absolute configurations at C-17 and C-13 in 1 and 2 were
determined as S and R, respectively, also by the application of the
advanced Mosher’s method. Treatment of 2 in pyridine R-(-)-
MTPA-Cl and (S)-(+)- MTPA-Cl yielded the MTPA amides 4a
and 4b. Positive ∆δS-R values for H-19 and H-23 and negative
∆δS-R values for H2-16 allowed the absolute configuration of C-17
to be assigned as S (Figure 3b). To assign the absolute configura-
tions at C-13, the 1,2-diol groups of the ribopyranose in 1 were
first protected by conversion to the acetonide 5 using 2,2-
dimethoxypropane and pyridinium p-toluenesulfate in methanol
(Scheme 1). Treatment of 5 in pyridine with R-MTPA-Cl and
S-MTPA-Cl yielded the S-MTPA ester 6a and R-MTPA ester 6b,
respectively. Calculation of ∆δS-R values of MTPA esters of 6a,b
established the absolute configuration of C-13 as R (Figure 3c).
The absolute configurations of the remaining components of 1
were determined by interpretation of NOESY NMR correlations.
A NOESY correlation between H-8 (δH 5.87) and the anomeric
proton H-1′ (δH 4.45) of the ribopyranose suggested a trans-
orientation of the C-9 glycoside and C-8 ether bonds.12 There were
only two possible stereoisomers, (8R, 9R) or (8S, 9S), for fijiolides,
as shown in Figure 4. A NOESY correlation between H-12 and
22-OH revealed that ester and ether linkages between the two
aromatic rings of 1 restrict free rotation of the ꢀ-tyrosine moiety
in fijiolides (Figure 4a,b). The strong NOESY correlations between
Figure 5. Chemical structures of cyanosporasides (7 and 8),
sporolides (9 and 10), and aromatization products of C-1027 (11)
and C-1027 (12).
H-5 and H-13 and between H-17 and H-23 suggested that C-8 and
C-9 could be assigned R and R configurations, respectively (Figure
4a).13
The chlorocyclopenta[a]indene carbon skeleton of the fijiolides
resembles that of the cyanosporasides14a and sporolides,14b isolated
from strains of the marine actinomycete genus Salinispora (Figure
5). Both cyanosporasides A and B (7, 8) and sporolides A and B
(9, 10) are equally represented in the respective fermentation
extracts of the producing strains. This is what would be expected
for the cycloaromatization of the enediyne precursors to Bergman
diradical intermediates followed by nonspecific quenching with
hydrogen15a or chloride, a process that has been experimentally
Scheme 1. Production of Acetonide 5 from Fijiolide A (1)

Fijiolides A and B from a Nocardiopsis Bacterium
Journal of Natural Products, 2010, Vol. 73, No. 6 1083
and is therefore useful in the study of chemopreventive agents. Hepa
1c1c7 (mouse hepatoma) cells were used to assess quinone
reductase activity via a color change as MTT is reduced to a blue
formazan.23 Fijiolide A (1) was found to enhance QR1 activity with
an induction ratio of 3.5 at 20 µg/mL (28.4 µM). The concentration
required to double induction (CDI) was 1.8 µM (the CDI value of
4-bromoflavone, as a positive control, is 0.013 µM). Fijiolide B
did not demonstrate QR1 induction activity, indicating that substitu-
tions on the nitrogen atom affect activity. Differential activity of
this type was also observed with the NFκB assay.
QR1 is well established to undergo transcriptional activation
through NFκB signaling pathways.24 Although NFκB alone seems
to account for only 20-30% of QR promoter activity, revealed by
mutational analysis, NFκB is responsible for a proportion of the
immediate response of the QR gene promoter.25 Significant
inhibition of luciferase NFκB activity and QR activation was
detected with fijiolide A at reasonably low concentrations (IC50 for
NFκB, 0.57 µM; CDI for QR1, 1.8 µM). It is likely that QR
activation is NFκB independent. In summary, fijiolide A modulates
distinct and important mechanisms, and this would be worthy of
more detailed investigation.
Figure 6. Energy-minimized model of fijiolide A (1) (calculated
by ChemBio 3D (ver. 11.0), red ) oxygen, green ) chlorine, white
) hydrogen, blue ) nitrogen, gray ) carbon atom).
verified.15b Interestingly, chloride addition occurred primarily at
the C-3 position in fijiolides A and B. Energy-minimized modeling
of fijiolides revealed that C-6 in the benzene ring is in closer
proximity to the chlorine atom of ꢀ-tyrosine than C-3 is to the 22-
OH group of ꢀ-tyrosine (Figure 6). The ribopyranose is also located
near C-6 in the fijiolides, further restricting chloride addition to
that site.
With the exception of the chlorine substituents, the fijiolides are
nearly identical to the aromatization product from the bacterial
metabolite C-1027 chromophore (11), derived from an antitumor
antibiotic enediyne, C-1027 (12), isolated in 1988 by Marunaka
et al. from the culture broth of Streptomyces globisporus.16 C-1027
is responsible for the activity of the complete C-1027 natural
product, which consists of an apoprotein with a single polypeptide
chain of 110 amino acid residues and a chromophore with the nine-
membered enediyne and benzoxazine moieties. In 1995, Yu et al.
demonstrated that loss of the benzoxazine moiety in the aromati-
zation product of C-1027 resulted in a substantial decrease of DNA
binding affinity (∼400 fold).17 Fijiolides A and B, which resemble
the aromatization product of the C-1027 chromophore without its
benzoxazine moiety, do not display significant activity against either
HCT-116 or methicillin-resistant Stapyhlococcus aureus (MRSA)
(data not shown).
NFκB plays a central role in a variety of disease states, including
cancer. As a transcription factor, activated NFκB regulates the
expression of numerous genes, which subsequently influence cell
survival, proliferation, differentiation, and inflammation.18,19 Nu-
merous studies demonstrating the effects of different compounds
on NFκB activity have been reported. However, the results of these
studies are often inconsistent, which is likely due to the use of
diverse cell types and assay methods. For assessment of NFκB
activity, we used stably transfected 293 human embryonic kidney
cells that demonstrated NFκB activation by treatment with 12-O-
tetradecanoylphorbol-13-acetate, as well as TNF-R. We observed
a 5- to 15-fold NFκB activation by treatment with 0.14 nM TNF-
R, high values for luciferase luminescence, and good reproduc-
ibility.20 With this assay, fijiolide A inhibited TNF-R-induced NFκB
activity by 70.3%, and dose dependence demonstrated an IC50 value
of 0.57 µM. Fijiolide B was less active, showing only 46.5% without
a dose-dependent response.
An established mechanism of cancer chemoprevention involves
the stimulation of metabolic detoxification activity. The detoxifi-
cation enzyme quinone reductase 1 (QR1) is an important phase II
enzyme (one that deactivates reactive and potentially carcinogenic
species) that converts quinones to hydroquinones, reducing oxidative
cycling.21 The enzyme exhibits broad specificity, reducing a wide
range of hydrophobic quinones of varying structure.22 The induction
of QR often coincides with induction of other phase II enzymes
Experimental Section
General Experimental Procedures. The optical rotations were
measured using a Rudolph Research Autopol III polarimeter with a 10
cm cell. UV spectra were recorded in a Varian Cary UV-visible
spectrophotometer with a path length of 1 cm, and IR spectra were
recorded on a Perkin-Elmer 1600 FT-IR spectrometer. CD spectra were
collected with an AVIV model 215 CD spectrometer with a 0.5 cm
1
long cell. H and 2D NMR spectral data were recorded at 500 or 600
MHz in DMSO-d6, CDCl3, or methanol-d4 solution containing Me4Si
as internal standard on Varian Inova spectrometers. 13C NMR spectra
were acquired at 150 MHz on a Varian Inova spectrometer. High-
resolution ESI-TOF mass spectra were provided by The Scripps
Research Institute, La Jolla, CA, or by the mass spectrometry facility
at the Department of Chemistry and Biochemistry at the University of
California, San Diego, La Jolla, CA. Low-resolution LC/MS data were
measured using a Hewlett-Packard series 1100 LC/MS system with a
reversed-phase C18 column (Phenomenex Luna, 4.6 mm ×100 mm, 5
µm) at a flow rate of 0.7 mL/min.
Collection and Phylogenetic Analysis of Strain CNS-653. The
marine-derived actinomycete, strain CNS-653, was isolated from a surf
zone sediment sample collected near Beqa Island in Beqa Lagoon in
Fiji, July 2006. NCBI BLAST analysis of the partial 16S rDNA
sequence of CNQ-653 (deposited with GenBank as accession number
GQ374440) indicates that this strain is affiliated with the genus
Nocardiopsis.
Cultivation and Extraction. The bacterium (strain CNS-653) was
cultured in 30 2.8 L Fernbach flasks each containing 1 L of a seawater-
based medium (10 g starch, 4 g yeast extract, 2 g peptone, 1 g CaCO3,
40 mg Fe2(SO4)3 ·4H2O, 100 mg KBr) and shaken at 230 rpm at
27 °C. After seven days of cultivation, sterilized XAD-16 resin (20
g/L) was added to adsorb the organic products, and the culture and
resin were shaken at 215 rpm for 2 h. The resin was filtered through
cheesecloth, washed with deionized water, and eluted with acetone.
The acetone was removed under reduced pressure, and the resulting
aqueous layer was extracted with EtOAc (3 × 500 mL). The EtOAc-
soluble fraction was dried in Vacuo to yield 3.5 g of extract.
Isolation of Fijiolides A and B. The extract (3.5 g) was fractionated
by open column chromatography on silica gel (25 g), eluted with a
step gradient of CH2Cl2 and MeOH. The CH2Cl2/MeOH (5:1) fraction
contained a mixture of both fijiolides, which were purified by reversed-
phase HPLC (Phenomenex Ultracarb C30, 250 × 100 mm, 2.0 mL/
min, 5 µm, 100 Å, UV ) 210 nm) using a gradient solvent system
from 5% to 100% CH3CN (0.05% TFA) over 60 min to afford fijiolides
A (1, 30.0 mg) and B (2, 3.0 mg), as pale yellow oils.
Fijiolide A (1): pale yellow oil; [R]2D1 -440 (c 0.5, MeOH); UV
(MeOH) λmax (log ε) 213 (4.4), 285 (3.7), 330 (2.7) nm; IR (KBr) νmax
3380, 1725, 1677, 1512, 1190, 1128 cm-1; 1H and 2D-NMR (600 MHz,
DMSO-d6), see Table 1; HRESIMS [M + H]+ m/z 705.1997 (calcd
for C34H3935Cl2N2O10, 705.1976).

1084 Journal of Natural Products, 2010, Vol. 73, No. 6
Nam et al.
Table 1. NMR Spectroscopic Data for Fijiolide A (1) (DMSO-d6)a
no.
δC, mult.b
δH (J in Hz)
COSY
HMBCc
1
2
3
4
5
6
7
8
148.8, C
135.7, C
126.7, C
139.3, C
129.1, CH
125.3, CH
147.7, C
83.2, CH
99.8, C
6.64, d (8.0)
7.08, d (8.0)
6
5
3, 4, 6, 7, 13
2, 4, 5, 7, 8
5.87, s
1, 2, 6, 7, 9, 10, 21
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
1′
2′
3′
4′
5′
135.5, CH
138.0, CH
129.4, CH
73.1, CH
65.9, CH2
170.0, C
42.9, CH2
49.0, CH
138.8, C
114.2, CH
129.2, C
138.3, C
151.0, C
113.9, CH
168.4, C
22.6, CH3
93.1, CH
69.6, CH
67.5, CH
69.6, CH
75.0, C
6.62, d (5.3)
6.65, dd (5.3, 2.1)
6.76, d (2.1)
4.66, dd (11.0, 4.0)
11
10, 12
11
14, 13-OH
13
1, 8, 9, 11, 12
1, 9, 10, 12,
1, 2, 9, 10, 11
3, 4, 5, 14
4.23, dd (11.0, 4.0), 3.64, dd (11.0, 4.0)
4, 13, 15
2.57, dd (13.4, 3.8), 2.07, t (13.4)
4.61, ddd (13.4, 13.4, 3.8)
17
15, 17, 18
15, 16, 18, 19, 23, 24
16, 17-NH
6.52, d (2.0)
23
19
17, 18, 20, 21, 23
17, 18, 21, 22
5.98, d (2.0)
1.70, s
24
4.45, d (8.0)
2.92, dd (8.0, 3.4)
4.06, dd (8.0, 3.4)
2.98d
2′
9, 2′, 3′, 5′
1′, 3′
2′, 4′
3′
1′, 4′
1′, 5′
4′-NMe2
4′-NMe2
6′-MeR
6′-Meꢀ
13-OH
17-NH
22-OH
2′-OH
42.9, CH3
44.4, CH3
31.5, CH3
20.6, CH3
2.79, br s
2.79, br s
1.43, s
1.46, s
5.86
8.41, d (8.0)
8.45, s
4.95, br s
5.73, br s
9.09, br s
4′
4′
4′, 5′, 6′-Meꢀ
4′, 5′, 6′-MeR
13
16, 17, 24
21, 22, 23
2′
3′
3′-OH
4′-NH+
4′-NMe2
a 600 MHz for 1H NMR and 150 MHz for 13C NMR. b Numbers of attached protons were determined by analysis of 2D spectra. c HMBC
correlations are from the stated protons to the indicated carbons. d Coupling constant could not be determined because of overlapping signals.
Fijiolide B (2): pale yellow oil; [R]2D1 -360 (c 0.2, MeOH); UV
(MeOH) λmax (log ε) 213 (4.3), 285 (3.8), 330 (2.7) nm; IR (KBr) νmax
3383, 1678, 1512, 1201, 1134, 1048 cm-1; 1H and 2D-NMR (600 MHz,
DMSO-d6), see Table 2; HRESITOFMS [M + H]+ m/z 663.1871 (calcd
for C32H3735Cl2N2O9, 663.1871).
Hz), 6.59 (dd, 1H, J ) 5.9, 3.0 Hz), 5.86 (s, 1H), 5.856 (dd, 1H, J )
11.0, 4.0 Hz), 4.857 (d, 1H, J ) 11.0 Hz), 4.72 (dd, 1H, J ) 11.0, 4.0
Hz), 4.680 (dd, 1H, J ) 11.0, 4.0 Hz), 4.492 (t, 1H, J ) 11.0 Hz),
4.376 (t, 1H, J ) 1.2 Hz), 3.864 (dd, 1H, J ) 11.0, 4.0 Hz), 3.52 (s,
3H), 3.263 (d, 1H, J ) 1.2 Hz), 2.95 (s, 3H), 2.899 (s, 6H), 2.62 (t,
1H, J ) 11.0 Hz), 2.20 (t, 1H, J ) 11.0 Hz), 1.822 (s, 3H), 1.499 (s,
3H), 1.493 (s, 3H); ESIMS m/z 1137 [M + H]+, 1159 [M + Na]+.
MTPA Amides of 2 (4a, 4b). Fijiolide B (1.3 mg) was dissolved in
freshly distilled dry pyridine (2 mL), several dry crystals of dimethy-
laminopyridine were added, and the mixture was stirred for 15 min at
RT. Treatment with R-(-)-MTPA-Cl yielded the S-MTPA amide at
room temperature after 11 h. The reaction was quenched by 1 mL of
MeOH. After removal of solvent under vacuum, the residue was purified
by reversed-phase HPLC (Phenomenex Luna 5u C18 (2) 100 Å, 250
× 100 mm, 2.0 mL/min, UV detection at 210 nm) using a gradient
solvent system from 5% to 100% CH3CN (0.05% TFA) over 60 min.
The S-MTPA amide was obtained at 41 min. The R-MTPA amide was
prepared with S-MTPA-Cl in the same manner. ∆δS-R values for the
S- and R-MTPA amides of 2 were recorded in ppm in DMSO-d6.
Bis-MTPA Esters of 1 (3a, 3b). Fijiolide A (7.0 mg) was dissolved
in freshly distilled dry pyridine (2 mL), and several dry crystals of
dimethylaminopyridine were added. The mixture was stirred for 15
min at RT. Treatment with R-(-)-MTPA-Cl yielded the bis-S-MTPA
ester at 60 °C after 12 h. The reaction was quenched by 1 mL of MeOH.
After removal of solvent under vacuum, the residue was purified by
reversed-phase HPLC (Phenomenex Luna 5u C18 (2) 100 Å, 250 ×
100 mm, 2.0 mL/min, UV detection at 210 nm) using a gradient solvent
system from 5% to 100% CH3CN (0.05% TFA) over 50 min. The bis-
S-MTPA ester was obtained at 36 min. The bis-R-MTPA ester was
prepared with S-MTPA-Cl in the same manner. ∆δS-R values for the
S- and R-MTPA esters of 1 were recorded in ppm in methanol-d4.
Bis-S-MTPA ester of fijiolide A (3a): 1H NMR (600 MHz,
methanol-d4) δ 7.55 (m, 2H), 7.45-7.30 (m, 8H), 6.89 (d, 1H, J ) 1.5
Hz), 6.63 (d, 1H, J ) 5.9 Hz), 6.589 (dd, 1H, J ) 5.9, 3.0 Hz), 6.484
(d, 1H, J ) 8.0 Hz), 6.42 (d, 1H, J ) 3.0 Hz), 6.292 (d, 1H, J ) 8.0
Hz), 5.802 (dd, 1H, J ) 11.0, 4.0 Hz), 5.54 (s, 1H), 4.717 (d, 1H, J )
11.0 Hz), 4.69 (dd, 1H, J ) 11.0, 4.0 Hz), 4.678 (dd, 1H, J ) 11.0,
4.0 Hz), 4.465 (t, 1H, J ) 11.0 Hz), 4.39 (t, 1H, J ) 1.2 Hz), 3.71 (s,
1H), 3.646 (dd, 1H, J ) 11.0, 4.0 Hz), 3.37 (s, 3H), 3.270 (d, 1H, J )
2.0 Hz), 2.910 (s, 6H), 2.60 (t, 1H, J ) 11.0 Hz), 2.55 (s, 3H), 2.14 (t,
1H, J ) 11.0 Hz), 1.822 (s, 3H), 1.449 (s, 3H), 1.443 (s, 3H); ESIMS
m/z 1137 [M + H]+, 1159 [M + Na]+.
1
S-MTPA amide of fijiolide B (4a): H NMR (600 MHz, DMSO-
d6) δ 9.96 (d, 1H, J ) 7.4 Hz), 8.98 (br s, 1H), 7.36-7.25 (m, 5H),
7.08 (d, 2H, J ) 8.0 Hz), 6.738 (m, 1H), 6.64-6.59 (m, 4H), 6.040
(m, 1H), 6.03 (m, 1H), 5.89 (m, 1H), 5.80 (m, 1H), 4.99 (m, 1H), 4.73
(m, 1H), 4.640 (m, 1H), 4.48 (d, 1H, J ) 10.0 Hz), 4.27 (dd, 1H, J )
10.4, 10.4 Hz), 4.06 (m, 1H), 3.78 (s, 3H), 3.62 (dd, 1H, J ) 11.0, 6.0
Hz), 3.00 (s, 6H), 2.92 (br s, 1H), 2.91 (m, 1H), 2.840 (m, 1H), 2.110
(dd, 1H, J ) 11.0, 2.0 Hz), 1.37 (br s, 6H); ESIMS m/z 879 [M +
H]+, 901 [M + Na]+.
Bis-R-MTPA ester of fijiolide A (3b): 1H NMR (600 MHz,
methanol-d4): δ 7.74 -7.30 (m, 12H), 7.021 (d, 1H, J ) 8.0 Hz), 6.83
(d, 1H, J ) 1.5 Hz), 6.736 (d, 1H, J ) 8.0 Hz), 6.67 (d, 1H, J ) 5.9
R-MTPA amide of fijiolide B (4b): H NMR (600 MHz, DMSO-
1
d6) δ 8.98 (br s, 1H), 8.91 (d, 1H, J ) 7.4 Hz), 7.36-7.25 (m, 5H),
7.17 (d, 2H, J ) 8.0 Hz), 6.718 (m, 1H), 6.711 (d, 1H, J ) 2.0),

Fijiolides A and B from a Nocardiopsis Bacterium
Journal of Natural Products, 2010, Vol. 73, No. 6 1085
Table 2. NMR Spectroscopic Data for Fijiolide B (2) (DMSO-d6)a
no.
δC, mult.b
δH (J in Hz)
COSY
HMBCc
1
2
3
4
5
6
7
8
148.2, C
135.7, C
126.9, C
139.8, C
129.5, CH
125.5, CH
147.8, C
83.9, CH
100.4, C
135.8, CH
138.5, CH
129.9, CH
72.7, CH
65.5, CH2
6.66, d (8.0)
7.16, d (8.0)
6
5
3, 4, 6, 7, 13
2, 4, 5, 7, 8
5.88, s
1, 2, 6, 7, 9, 10, 21
9
10
11
12
13
14
6.62, d (5.3)
6.65, dd (5.3, 2.1)
6.75, d (2.1)
4.66, dd (11.0, 4.0)
4.28, dd (11.0, 4.0),
3.68, dd (11.0, 4.0)
11
10, 12
11
14, 13-OH
13
1, 8, 9, 11, 12
1, 9, 10, 12,
1, 2, 9, 10, 11
3, 4, 5, 14
4, 13, 15
15
16
168.4, C
41.7, CH2
2.85, dd (13.4, 1.8),
2.20, t (13.4)
4.09, dd (13.4, 1.8)
17
16
23
15, 17, 18
17
18
19
20
21
22
23
1′
51.2, CH
133.1, C
114.4, CH
130.1, C
139.9, C
151.7, C
115.2, CH
93.4, CH
69.6, CH
67.8, CH
69.6, CH
75.3, C
15, 16, 18, 19, 23
17, 18, 20, 21, 23
6.76, d (2.0)
6.06, d (2.0)
4.44, d (8.0)
2.91, br s
4.05, br s
2.99, br s
19
2′
1′, 3′
2′, 4′
3′
17, 18, 21, 22
9, 2′, 3′, 5′
1′, 4′
1′, 5′
4′-NMe2
2′
3′
4′
5′
4′-NMe2
4′-NMe2
6′-MeR
6′-Meꢀ
13-OH
17-NH2
22-OH
2′-OH
3′-OH
42.9, CH3
44.6, CH3
31.9, CH3
21.2, CH3
2.79, br s
2.79, br s
1.37, s
1.41, s
5.86
8.58
8.84, s
4.97, br s
5.72, br s
9.13, br s
4′
4′
4′, 5′, 6′-Meꢀ
4′, 5′, 6′-MeR
13
21, 22, 23
2′
3′
4′-NH+
4′-NMe2
600 MHz for H NMR. b Numbers of attached protons were determined by analysis of 2D spectra. c HMBC correlations are from the stated protons
a
1
to the indicated carbons.
6.67-6.62 (m, 3H), 6.10 (d, 1H, J ) 2.0), 6.020 (m, 1H), 5.87 (m,
1H), 5.77 (m, 1H), 4.99 (m, 1H), 4.68 (m, 1H), 4.644 (m, 1H), 4.46
(d, 1H, J ) 10.0 Hz), 4.24 (dd, 1H, J ) 10.4, 10.4 Hz), 4.06 (m, 1H),
3.82 (s, 3H), 3.64 (dd, 1H, J ) 11.0, 6.0 Hz), 2.92 (br s, 1H), 2.91 (m,
1H), 2.79 (s, 6H), 2.841 (m, 1H), 2.116 (dd, 1H, J ) 11.0, 2.0 Hz),
1.40 (br s, 6H); ESIMS m/z 879 [M + H]+, 901 [M + Na]+.
Acetonide 5. Fijiolide A (1, 5.0 mg) was dissolved in 4 mL of dry
MeOH solution, along with 5 mg of pyridinium-p-toluenesulfonate and
5 mg of p-toluenesulfonic acid. The solution was cooled in an ice water
bath, and 2 mL of 2,2-dimethoxypropane was added. The solution was
stirred at 60 °C for 12 h. The reaction was quenched with 5% aqueous
NaHCO3, and the product was extracted with EtOAc. The EtOAc was
removed in Vacuo, and the residual material was purified by reversed-
phase HPLC (Phenomenex Ultracarb C30 100 Å, 250 × 100 mm, 2.0
mL/min, UV detection at 210 nm) using a gradient solvent system from
5% to 100% CH3CN (0.05% TFA) over 60 min. The acetonide 5 (4.8
mg) was isolated with a retention time of 31 min.
of methanol. After removal of solvent under vacuum, the residue was
purified by reversed-phase HPLC (Phenomenex Luna 5u C18 (2) 100
Å, 250 × 100 mm, 2.0 mL/min, UV detection at 210 nm) using a
gradient solvent system from 5% to 100% CH3CN (0.05% TFA) over
40 min. The S-MTPA ester was eluted at 30 min. The R-MTPA ester
was prepared with S-MTPA-Cl in the same manner. ∆δS-R values for
the S- and R-MTPA esters of acetonides were recorded in ppm in
CDCl3.
S-MTPA ester of acetonide 5 (6a): 1H NMR (600 MHz, CDCl3) δ
7.428 (d, 1H, J ) 8.0 Hz), 7.390 (d, 1H, J ) 8.0 Hz), 7.34-7.28 (m,
5H), 6.93 (s, 1H), 6.85 (d, 1H, J ) 8.0 Hz), 6.79 (s, 1H), 6.40 (s, 1H),
6.20 (d, 1H, J ) 2.0 Hz) 5.855 (dd, 1H, J ) 12.1, 4.0 Hz), 5.73 (m,
1H), 4.85 (m, 1H), 4.605 (t, 1H, J ) 12.1 Hz), 4.48 (d, 1H, J ) 2.0
Hz), 4.16 (d, 1H, J ) 11.0 Hz), 3.774 (dd, 1H, J ) 11.0, 4.0 Hz), 3.47
(m, 1H), 3.43 (s, 3H), 3.27 (m,1H), 2.90 (m, 2H), 2.85 (s, 6H), 2.11 (t,
1H, J ) 11.0 Hz), 1.91 (s, 3H), 1.56 (s, 3H), 1.51 (s, 3H), 1.18 (s,
3H), 1.07 (s, 3H); ESIMS m/z 961 [M + H]+, 983 [M + Na]+.
Acetonide 5: 1H NMR (500 MHz, methanol-d4) δ 7.35 (d, 1H, J )
8.0 Hz), 6.99 (d, 1H, J ) 1.5 Hz), 6.82 (dd, 1H, J ) 5.8, 3.0 Hz), 6.78
(d, 1H, J ) 5.8 Hz), 6.59 (d, 1H, J ) 3.0 Hz), 6.15 (d, 1H, J ) 1.5
Hz), 5.94 (s, 1H), 4.83 (m, 2H), 4.65 (m, 1H), 4.55 (dd, 1H, J ) 10.4,
10.4 Hz), 4.31 (d, 1H, J ) 11.0 Hz), 3.79 (dd, 1H, J ) 10.4, 3.5 Hz),
3.62 (t, 1H, J ) 3.5 Hz), 3.55 (m, 1H), 3.44 (m, 1H), 3.04 (br s, 6H),
2.72 (dd, 1H, J ) 12.4, 2.8 Hz), 2.26 (t, 1H, J ) 12.4 Hz), 1.93 (s,
3H), 1.60 (s, 3H), 1.54 (s, 3H), 1.35 (s, 3H), 1.21 (s, 3H); ESIMS m/z
745 [M + H]+, 767 [M + Na]+.
MTPA Esters of 5 (6a, 6b). The acetonide 5 (2.4 mg) was dissolved
in freshly distilled dry pyridine (2 mL), and several dry crystals of
dimethylaminopyridine were added. The mixture was stirred for 15
min at RT. Treatment with R-(-)-MTPA-Cl yielded the S-MTPA ester
at room temperature after 11 h. The reaction was quenched by 1 mL
R-MTPA ester of acetonide 5 (6b): H NMR (600 MHz, CDCl3)
1
δ 7.428 (d, 1H, J ) 8.0 Hz), 7.390 (d, 1H, J ) 8.0 Hz), 7.34-7.28
(m, 5H), 6.90 (s, 1H), 6.84 (d, 1H, J ) 8.0 Hz), 6.79 (s, 1H), 6.40 (s,
1H), 6.20 (d, 1H, J ) 2.0 Hz), 5.845 (dd, 1H, J ) 12.1, 4.0 Hz), 5.70
(m, 1H), 4.85 (m, 1H), 4.660 (t, 1H, J ) 12.1 Hz), 4.46 (d, 1H, J )
2.0 Hz), 4.15 (d, 1H, J ) 11.0 Hz), 3.884 (dd, 1H, J ) 11.0, 4.0 Hz),
3.47 (m, 1H), 3.44 (s, 3H), 3.27 (m, 1H), 2.90 (m, 2H), 2.87 (s, 6H),
2.12 (t, 1H, J ) 11.0 Hz), 1.91 (s, 3H), 1.61 (s, 3H), 1.51 (s, 3H), 1.18
(s, 3H), 1.07 (s, 3H); ESIMS m/z 961 [M + H]+, 983 [M + Na]+.
NFKB Assay. Panomics Inc. (Fremont, CA) has established NFκB
reporter stable cells in a number of popular cell lines suitable for specific
applications in the study of NFκB. We employed human embryonic
kidney cells 293 (Panomics Inc., Fremont, CA) for monitoring any
changes occurring along the NFκB pathway. Stably transfected cells

1086 Journal of Natural Products, 2010, Vol. 73, No. 6
Nam et al.
were seeded into sterile 96-well plates at a density of 20 × 103 cells
per well. Cells were maintained in Dulbecco’s modified Eagle’s medium
(Invitrogen, Carlsbad, CA), supplemented with 10% FBS, 100 units/
mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine. After
an incubation period of 48 h, the medium was replaced and various
concentrations of test chemicals (dissolved in PBS) were added. Human
recombinant TNF-R (Calbiochem, Gibbstown, NJ) was used as an
activator (2 ng/mL). Following a 6 h incubation period, the medium
was discarded and the cells were washed once with PBS. The cells
were then lysed by adding 50 µL/well (diluted with water five times)
of Reporter Lysis Buffer (Promega, Madison, WI) and incubating for
5 min with shaking. The plates were then stored at -80 °C until the
luciferase assay was performed using the Luc assay system from
Promega.20 The gene product, luciferase enzyme, reacts with luciferase
substrate, emitting light, which was detected in a luminometer (LU-
MIstar Galaxy BMG). Data are expressed as IC50 values (i.e.,
concentration required to inhibit TNF-R-activated NFκB activity by
50%).
Quinone Reductase 1 Assay. QR1 activity was assessed in 96-
well plates using Hepa 1c1c7 murine hepatoma cells as previously
described.26 Briefly, cells were grown to a density of 2 × 104 cells/
mL in 200 µL of MEM-R containing 5% antibiotic-antimycotic
(Gibco) and 10% fetal bovine serum at 37 °C in a 5% CO2 atmosphere.
After preincubation for 24 h, the media was changed and test
compounds were added to a final concentration of 20 µg/mL. For dose
dependence, samples were added in five serial 3-fold dilutions, starting
at 20 µg/mL. The cells were incubated with test samples for an
additional 48 h. Quinone reductase activity was measured as a function
of the NADPH-dependent menadiol-mediated reduction of 3-(4,5-
dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a blue
formazan.23 Cytotoxicity was determined via crystal violet staining of
identical plates. Specific activity is defined as nmol of formazan formed
per mg protein per min.27 The induction ratio of QR activity represents
the specific enzyme activity of agent-treated cells compared with a
DMSO-treated control. The concentration to double activity (CDI) was
determined through a dose-response assay for active substances
(induction ratio > 2).
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Acknowledgment. Financial support was provided by the National
Cancer Institute, NIH (under grant P01 CA048112), and U01-
TW007401-O1, the latter under the Fogerty Center’s International
Cooperative Biodiversity Groups program. We thank the people of Fiji
in the Beqa Lagoon region and W. Aalbersberg (University of the South
Pacific) for providing laboratory space and facilitating field collections.
We also thank the government of Fiji for permission to export samples
and work in their territorial waters. S.P.G. is grateful to the Fundac¸a˜o
para a Cieˆncia e Tecnologia, Portugal, for a postdoctoral fellowship.
We thank Dr. C. C. Hughes for his generous advice in the determination
of the configurations of fijiolides A and B.
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Supporting Information Available: 1H, 13C, and 2D NMR spectra
of fijiolide A (1) and H spectra of fijiolide B (2), MTPA esters 3a,
3b, 4a, 4b, 6a, 6b, and acetonide 5 are available free of charge via the
Internet at http://pubs.acs.org.
1
(22) Su, B.-N.; Gu, J.-Q.; Kang, Y.-H.; Park, E. J.; Pezzuto, J. M.; Kinghorn,
A. D. Mini-ReV. Org. Chem. 2004, 1, 115–123.
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