The marine sponge Diacarnus bismarckensis as a source of peroxiterpene inhibitors of Trypanosoma brucei, the causative agent of sleeping sickness

Article abstract of DOI:10.1021/np800711a

Human African trypanosomiasis, also known as African sleeping sickness, is a neglected tropical disease with inadequate therapeutic options. We have launched a collaborative new lead discovery venture using our repository of extracts and natural product c

Full text of DOI:10.1021/np800711a

218
J. Nat. Prod. 2009, 72, 218–222
The Marine Sponge Diacarnus bismarckensis as a Source of Peroxiterpene Inhibitors of
Trypanosoma brucei, the Causative Agent of Sleeping Sickness
Brent K. Rubio,† Karen Tenney,† Kean-Hooi Ang,§ Maha Abdulla,‡ Michelle Arkin,§ James H. McKerrow,‡ and
Phillip Crews*,†
Department of Chemistry and Biochemistry and Institute for Marine Sciences, UniVersity of California Santa Cruz,
Santa Cruz, California 95064, Sandler Center for Basic Research in Parasitic Disease, UniVersity of California San Francisco,
San Francisco, California 94143, and Small Molecule DiscoVery Center, UniVersity of California San Francisco,
San Francisco, California 94158
ReceiVed NoVember 7, 2008
Human African trypanosomiasis, also known as African sleeping sickness, is a neglected tropical disease with inadequate
therapeutic options. We have launched a collaborative new lead discovery venture using our repository of extracts and
natural product compounds as input into our growth inhibition primary screen against Trypanosoma brucei. Careful
evaluation of the spectral data of the natural products and derivatives allowed for the elucidation of the absolute
configuration (using the modified Mosher’s method) of two new peroxiterpenes: (+)-muqubilone B (1a) and (-)-ent-
muqubilone (3a). Five known compounds were also isolated: (+)-sigmosceptrellin A (4a), (+)-sigmosceptrellin A methyl
ester (4b), (-)-sigmosceptrellin B (5), (+)-epi-muqubillin A (6), and (-)-epi-nuapapuin B methyl ester (7). The isolated
peroxiterpenes demonstrated activities in the range IC50 ) 0.2-2 µg/mL.
The search for chemotherapeutics to treat human African
trypanosomiasis (HAT), also known as African sleeping sickness,
has not been a major target of industry drug discovery campaigns,
even though the currently available therapeutics are inadequate.1
This is surprising, as there are 50 000 annual cases of infection,
HAT is the world’s third most devastating parasitic disease, and it
remains a major threat to more than 60 million people.2–4 Because
it is a serious health problem for resource-poor regions of Africa,
HAT is designated as a neglected tropical disease (NTD). The
causative agent is a protozoan parasite, Trypanosoma brucei,5
subdivided in two subgenera, T. brucei gambiense in West Africa
and T. brucei rhodesiense in East Africa.1 Most of the four current
chemotherapeutics to combat these parasites at their different
development stages are very antiquated. The drugs (by registration
date for HAT treatment) consist of suramin4 (1922), pentamidine1
(1941), melarsoprol3 (1949), and eflorithine1 (1990). Another
relevant point is that new strains of T. brucei are showing cross-
resistance to some of these agents.6 In recent years, only one
compound, DB2897 (a synthetic analogue of pentamidine), received
serious clinical evaluation against HAT, but further development
was discontinued in 2008.3
The quest to discover antiparasitic lead structures has rarely
focused on the chemical diversity inherent in marine natural
products. Alternatively, recent studies have used combinatorial
chemistry libraries of purine-2 and thiosemicarbazone5-derived
structures to seek inhibitors of different T. brucei targets.2,5 In the
early 1990s the University of California Santa Cruz group
participated in a collaborative effort with Syntex (with Dr. T.
Matthews) to explore marine natural products for their potential to
provide anthelminthic lead structures,8 but unfortunately these
efforts were prematurely suspended. In this context there is a rather
attention-grabbing report showing that psammaplin A,9 first isolated
from the marine sponge Psamaplysilla in 1987 and found to possess
anthelminthic activity (against Nippostrongylus brasiliensis),8 was
recently found to be active against T. brucei (EC50 ) 2.6 µM).10
Inspired by this recent development, we have launched a new
collaborative lead discovery venture using our repository of extracts
and natural product compounds as input into our growth inhibition
primary screen against T. brucei. We were encouraged to continue
this effort because of an assay hit from the sponge Diacarnus
bismarckensis, which was available in large amounts. The current
literature shows over two dozen peroxiterpenes11 reported from
Diacarnus taxon, and these have been primarily evaluated in
anticancer screens.12 Reported in this account are our efforts to
isolate and define the total structures of the active principles,
presumed to be peroxiterpenes, followed by further biological
activity study.
Results and Discussion
Once the decision was made to select the T. brucei assay active
crude extracts from D. bismarckensis (coll. no. 03512), the next
step was to employ bioassay-guided fractionation. Although the
methanol crude extract partition (XFM, 2.06 g, IC50 ) 5.0 µg/mL)
showed good activity, the hexanes (XFH, 8.80 g, IC50 ) 0.02 µg/
mL) and dichloromethane (XFD, 0.66 g, IC50 ) 0.3 µg/mL)
partitions were selected for further study due to their higher
potencies. Analysis of their respective 13C NMR spectra confirmed
the presence of peroxiterpenes because of prominent resonances
in the region δC 80-82. Orthogonal chromatography facilitated the
isolation work, beginning with silica gel flash chromatography
followed by reversed-phase HPLC, which eventually afforded seven
peroxiterpene compounds. The XFH provided a mixture of 1a,
related to (+)-muqubilone/aikupikoxide A (2),13,14 and (-)-ent-
muqubilone (3a), an unreported enantiomer of 2. Preparation of
their respective methyl esters was required in the final purification
of these compounds. Five additional compounds came from the
XFH including (+)-sigmosceptrellin A (4a),15,16 (+)-sigmoscep-
trellin A methyl ester (4b),15,16 (-)-sigmosceptrellin B (5),16 (+)-
epi-muqubilin A (6),12a and (-)-epi-nuapapuin B methyl ester
(7).12a The XFD was also a source of four pure compounds: 1a,
4a, 5, and 6. We obtained all compounds as free carboxylic acids,
with the exception of minor quantities of methyl ester 4b and 7,
which are most likely artifacts of isolation. The known compounds
(4-7) have also been previously obtained from sponges classified
as Diacarnus sp.17
* To whom correspondence should be addressed. E-mail: phil@
chemistry.ucsc.edu. Tel: (831) 459-2603. Fax: (831) 459-2935.
† University of California, Santa Cruz.
The structure elucidation of the new compound (+)-muqubilone
B (1a), C24H40O6, was initiated by using the characteristic 13C NMR
shifts at δC ) 81.2 (CH) and 80.0 (C) to indicate the presence of
‡ Sandler Center BRPD, University of California, San Francisco.
§ SMDC, University of California, San Francisco.
10.1021/np800711a CCC: $40.75
2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/21/2009

Peroxiterpene Inhibitors from Diacarnus bismarckensis
Journal of Natural Products, 2009, Vol. 72, No. 2 219
a
Table 1. NMR Data for (+)-Muqubilone B (1a) in CDCl3
HMBC
position
δC, type
δH, mult. (J in Hz)
(H to C)
COSY
1
2
3
4ax
4eq
5
178.6, C
42.4, CH
81.2, CH
23.8, CH2
2.55, quint. (7.2)
3, 20
3, 20
4.25, ddd (8.4, 7.2, 4.2)
1.77, dtd (12.0, 8.4, 4.2) 21
2, 4
5
1.70, dtd (12.0, 4.2, 3)
1.66, m
3
3
4
32.5, CH2
80.0, C
6, 21
6
7
34.7, CH2
22.0, CH2
1.49, m
2.04, m
1.94, m
21
8
7, 9
8a
8b
9
124.6, CH 5.16, td (7.2, 1.2)
134.4, C
33.4, CH2
35.6, CH2
215.4, C
47.5, C
8
10
11
12
13
14
15a
15b
16
17
18
19
20
21
22
23,24
OH
2.21, t (7.8)
2.56, t (7.8)
9, 10, 12, 22 12
10, 11, 13
11
16
39.1, CH2
1.51, m
1.46, m
1.42, m
2.42, t (7.2)
16
18.9, CH2
43.9, CH2
209.1, C
29.8, CH3
12.7, CH3
22.6, CH3
16.0, CH3
24.3, CH3
15
16, 18
15, 17
16
a peroxide functionality with trisubstitution (Table 1).18 Distinct
NMR signals for a carboxylic acid (δC ) 178.6), an acetyl group
(δC ) 209.1, 29.8), an isolated ketone (δC ) 215.4), and a
trisubstituted double bond (δC ) 124.6, 134.4), the molecular
formula obtained from the HRESIMS, and dereplication insights
all supported that 1 was a norsesterterpene containing an endop-
eroxide ring. Another diagnostic NMR signal was that of an
isochronous gem-dimethyl functionality (δC ) 24.3; δH ) 1.13).
At this point it was evident that the C3-containing carboxylate and
the C16 acyclic side chains were similar to that of 2.13,14 The side-
by-side comparison of the similarities and differences in the NMR
data between 1a and 2 plus the 2D NMR data of the former (Figure
1) unequivocally confirmed that this pair had identical planar
structures and must be diastereomers.
2.14, s
1.20, d (7.2)
1.12, s
1.62, d (1.2)
1.13, s
3.71, s
17, 18
1, 2, 3
4, 5, 6, 8
9, 10, 11
13, 14, 15
1
2
a Measured at 600 MHz (1H) and 150 MHz (13C).
Establishing the complete absolute configuration of 1a, while
reasonably straightforward, required a multistep analysis. The E
double-bond geometry was confirmed by the NOE data shown in
Figure 1 (correlation from H-9 to H2-11) and the characteristic C-22
shift.19 We have previously used the empirical observations laid
out by Capon and MacLeod18 to set the relative configuration at
each of the three chiral centers of the molecular fragment present
in Diacarnus-derived peroxiterpenes and repeated this process
here,12a but it first required the preparation of the methyl ester 1b
to obtain a data set consistent with the models.20 The carbon
substituent at C-3 was set as equatorial because H-3 exhibited
vicinal axial coupling to H-4, J3,4 ) 8.0 Hz (J ) 3-4 Hz is expected
for equatorial). The methyl at C-6 (δC ) 24.2) was next assigned
Figure 1. Key 2D NMR correlations for (+)-muqubilone B (1a).
in view of their identical chirality. Our further discussion on the
absolute configuration of 2 shown here as 2S, 3R, 6S will
accompany the characterization of 3, which follows next.
Noted above was that the XFH provided a mixture of 1a and an
additional new compound, (-)-ent-muqubilone (3a). The separation
of these compounds was accomplished by first subjecting the
mixture to methylation using TMS-diazomethane followed by
isocratic HPLC purification to cleanly afford 1b ([R] ) +58) and
3b ([R] ) -47) in a 2:1 ratio. The absolute stereostructure assigned
for 3a was based on data obtained for 3b using the process parallel
to that described above for 1. Both 1b and 3b possessed identical
molecular formulas and afforded nearly identical NMR shifts with
2.13 The following observations of H-3, Jax ) 8.0 Hz; CH3-21, δC,ax
) 20.9; CH3-20, δH,threo ) 1.25 indicated that the relative configura-
tions reported for 3b were equivalent with that of 2, but these
compounds possessed opposite rotation data (see above) and must
be enantiomers. A review of the current literature (see Table S1,
Supporting Information)16,18,23 affirms that the variations in the
absolute configurations at C-2 and C-6 within the peroxide-
containing substructure comprised of the chain from C-1 to C-6
do not influence the rotation sign. Additionally, the sign of optical
rotation does not change upon substitution of the free carboxylate.
Thus, the absolute configuration at C-3 can be used as an empirical
anchor point. For example, when H-3 is in the axial orientation, a
(-) [R]D is observed for the 3S configuration and the (+) rotation
is observed for 3R (in CHCl3). Consequently, the compound (-)-
3b can be concluded to have the stereostructure of 2R, 3S, 6R.
as equatorial because of its diagnostic 13C NMR shift (δC,eq
)
23.5-24.0; δC,ax ) 20.5-20.9). Finally the erythro (R*/R*)
configuration at C-2/C-3 was designated based on the proton
chemical shift position at H3-20, δH 1.14 (standard values: δH,erythro
) 1.13-1.14; δH,threo ) 1.22-1.24).18 The resulting all-R* con-
figuration at each of the chiral centers confirmed that 1a ([R] )
+60) was an unreported diastereomer of 2, whose relative config-
uration were previously independently reported as 2R*, 3S*, 6R*
for both (+)-muqubilone ([R] ) +48)13 and aikupikoxide A ([R]
) +81).14
We next concluded it was essential to push forward and ascertain
the absolute configuration of 1a with the expectation such findings
could provide similar insights for 2. Hydrogenation of 1b21 afforded
diol 8, which was further transformed into Mosher’s esters 9a and
9b as shown in Scheme 1.21,22 The circumstance that the ∆9 double
bond was also reduced during the hydrogenation did not affect the
outcome in evaluating the ∆δ values (δS - δR) to verify C-3 as R.
Thus, the 2R, 3R, 6R final absolute configuration is now assigned
for (+)-muqubilone B (1a). It is notable that (+)-2R,3R,6R-epi-
muqubilin A12a (6) ([R] ) +59) also isolated from this sponge
represents an obvious biosynthetic precursor of (+)-1a, especially

220 Journal of Natural Products, 2009, Vol. 72, No. 2
Rubio et al.
Scheme 1. Modified Mosher’s Method Analysis of (+)-Muqubilone B (1a)a
a (i) TMS-CHN2 (2 M in hexanes), CH3OH, rt, 30 min; (ii) H2 (1 atm), Pd/C, EtOH, rt, 1 h; (iii) (+)- or (-)-MTPA-Cl, pyr., rt, 18 h.
Furthermore (+)-muqubilone/aikupikoxide A13,14 (2), whose rota-
tion data were discussed above, can be assigned as 2S, 3R, 6S.
The peroxiterpenes isolated in this work and evaluated against
T. brucei can be grouped into four structural types based simply
on the side chain attached at C-6 of the 1,2-dioxane ring. The first
three types each have the C16 carbon side chain that is either acyclic
(1-3), monocyclic (6), or bicyclic (4, 5). The final type (7) has a
C11 monocyclic carbon side chain. The muqubilones (1a, 1b, 3b)
showed similar bioactivities independent of the nature of the
carboxylate: free acid (1a, IC50 ) 2 µg/mL) or methyl ester 1b
(IC50 ) 5 µg/mL) and 3b (IC50 ) 3 µg/mL). Consistent with the
supposition that bioactivity hinges on the presence of the 1,2-
dioxane ring was that diol 8 (IC50 > 25 µg/mL) prepared from 1b
was inactive. A change in the side chain to that containing either
a monocyclic or bicyclic ring imparts a barely observable change,
as shown by the data for sigmosceptrellin A (4a, IC50 ) 1 µg/mL)
and its methyl ester (4b, IC50 ) 2 µg/mL), sigmosceptrellin B (5,
IC50 ) 0.2 µg/mL), and epi-muqubilin A (6, IC50 ) 0.9 µg/mL).
Finally, the length of the side chain does not greatly influence the
activity, as shown by epi-nuapapuin B methyl ester (7, IC50 ) 2
µg/mL).
It is relevant to overlay our observations with those from recent
studies involving 1,2-dioxane ring containing terpenoids or polyketides
and somewhat analogous peroxide ring compounds studied by
others. An investigation of Diacarnus megaspinorhabdosa afforded
a series of peroxiterpenes and one diol product with structures
parallel to those reported here.12 There were varying EC50 cyto-
toxicity effects observed from the evaluation of these compounds
against three cancer cell lines, and those possessing a C16 side chain
at C-6 were generally more cytotoxic than those with a C11
appendage. Curiously, the one natural product evaluated where the
peroxide ring was not present, but replaced by a diol, displayed
mild activity against one cell line and inactivity against the other
two cell types. In another study, the cytotoxic and antiparasitic
properties were compared for 1,2-dioxane and furan ketides isolated
from the sponge Plakortis angulospiculatus.24 These results showed
that both heterocyclic ring types displayed modest cytotoxicity
effects, while one of the 1,2-dioxane structures potently inhibited
Leishmania chagasi and Trypanosoma cruzi. These preceding
literature findings, and those not discussed here showing that other
polyketide 1,2-dioxanes have antileishmanial activity,25 alongside
our results together suggest that the polyalkylated 1,2-dioxane
frameworks are of promise for further development as lead
structures. In addition, the therapeutic index of antiparasitic to
cytotoxicity action for these structures is in the correct direction.
The responses of T. brucei rhodesiense have been evaluated for
two other sets of peroxide-containing structures as follows. The
wormwood tree peroxisesquiterpene, artemisinin, exhibits nM action
against Plasmodium falciparum.26 This compound (T. b. rhod-
esiense IC50 ) 627 or 2526 µg/mL) and its close analogue
deoxyartemisinin (T. b. rhodesiense IC50 ) 3426) possess similar
mild activity against T. b. rhodesiense, and the latter, devoid of
the cyclic peroxide functionality, is inactive versus P. falciparum.
A similar SAR activity pattern is shown for another matched set.
This includes the 1,2,4-trioxolane-containing synthetic polycyclic
OZ277 (T. b. rhodesiense IC50 ) 0.6 µg/mL)26 versus the 1,3-
dioxolane-containing synthetic polycyclic carbaOZ277 (T. b. rh-
odesiense IC50 ) 0.9 µg/mL),26 which are equipotent against T. b.
rhodesiense (and have not been tested versus P. falciparum).
Overall, it would appear that structural features of 5 (T. brucei IC50
) 0.2 µg/mL) would be an excellent starting point for further SAR
modifications, as it showed similar potencies on par with a current
HAT therapeutic, pentamidine (T. b. rhodesiense IC50 ) 0.4 µg/
mL).27
Conclusions
This study has revealed new dimensions to the structural and
biological activity properties of peroxiterpenes isolated from
Diacarnus sponges. First, careful evaluation of the spectral data of
the natural products and derivatives allowed for the elucidation of
the absolute configuration of compounds we isolated including 1a
and 3a plus a proposal for the absolute configuration of 2. Second,
the observation of enantiomeric structures (2 versus 3) from the
same genus, Diacarnus, but different species from different oceans
(2 from a Red Sea specimen, D. erythaeanus, and 3 from the Indo-
Pacific specimen D. bismarckensis) is a stunning and rare observa-
tion in marine natural products chemistry. Third, we have further
demonstrated the value in vigorously considering peroxiterpenes,
especially the known compound (-)-sigmosceptrellin B (5), as a
template for the development of therapeutic leads against T. brucei.
Experimental Section
General Experimental Procedures. All NMR experiments were
run on Varian UNITY 500 (500 and 125 MHz for 1H and 13C,
respectively) and Varian INOVA 600 spectrometers (600 and 150 MHz
for 1H and 13C, respectively) using standard pulse sequences with
residual solvent protons and carbons as references (CDCl3: 7.27 and
77.23 ppm for 1H and 13C, respectively). Mass measurements were
obtained on a benchtop Mariner ESI-TOF-MS. Crude extractions were
obtained using an accelerated solvent extractor (1500 psi, 100 °C, 25
min). Flash chromatography was done on a CombiFlash, using
prepacked 40 g silica columns. HPLC was performed with Phenomenex
Synergi RP-Max preparative (10 µm, C18, 21.2 mm × 250 mm) and
Luna semipreparative (5 µm, C18, 10 mm × 250 mm) columns.
Collection and Identification. The sponge materials were collected
in December 2003 by scuba at a depth range of 31-40 ft near Sanaroa,
Papua New Guinea (GPS ) 9°37.214′ S: 150°57.332′ E). The sponge
samples were ramose in shape with very small oscules throughout and
had a purple-colored endosome with a gray-colored ectosome. The
collection was identified as Diacarnus bismarckensis (Kelly-Borges &
Vacelet, 1995) by Dr. R. W. M. van Soest. Voucher samples have been
deposited at UCSC and the Zoological Museum of Amsterdam (UCSC
coll. no. 03512 ) ZMAPOR 18576). Photographs are also available
from the Crews laboratory.
Extraction and Isolation of 03512. Following standard Crews
laboratory field protocol, the sponge was soaked for over 24 h in an
ethanol/seawater (1:1) preservative solution, which was discarded in
the field. The samples were then packed in bottles and shipped to UCSC,
where the sponge was cut into 1.5 in. segments and dried under air for
2 days. A 73.4 g amount of dried sponge was extracted using an

Peroxiterpene Inhibitors from Diacarnus bismarckensis
Journal of Natural Products, 2009, Vol. 72, No. 2 221
accelerated solvent extractor (100 °C, 1500 psi, 25 min) to yield hexanes
(XFH, 8.80 g), CH2Cl2 (XFD, 0.6615 g), and CH3OH (XFM, 2.06 g)
crude extracts. A bioassay-guided fraction then followed on the XFH
and XFD fractions. The large quantity of XFH crude warranted a further
Kupchan-like solvent partition to yield hexanes (XFHFH, 4.08 g),
CH2Cl2 (XFHFD, 4.08 g), and CH3OH (XFHFM, 0.518 g) fractions.
The XFHFH crude was subjected to normal-phase automated flash
chromatography using a linear gradient on silica gel (CH2Cl2 to CH3OH,
40 min), yielding six fractions (XFHFHC1-FHFHC6). An orthogonal
chromatographic approach was used on the C3 (1.10 g) and C4 (942
mg) fractions with reversed-phase preparative HPLC (80% aq to 100%
CH3CN linear gradient, 0.1% formic acid, 20 min, ELSD) followed
by semipreparative HPLC (70% aq CH3CN isocratic, 0.1% formic acid,
UV ) 200 nm). From these fractions, (-)-epi-nuapapuin B methyl
ester12a (7, 2.9 mg), (+)-sigmosceptrellin A15,16 (4a, 68.6 mg), (-)-
sigmosceptrellin B16 (5, 33.8 mg), (+)-epi-muqubillin A12a (6, 23.9
mg), and (+)-sigmosceptrellin A methyl ester15,16 (4b, 8.2 mg) were
isolated. The XFHFD was treated in a similar manner to yield a 2:1
mixture (21.4 mg) of (+)-muqubilone B (1a) and (-)-ent-muqubilone
(3a), which were separable after methyl esterification and HPLC
(isocratic 85% aq CH3CN, 0.1% formic acid, UV ) 200 nm). The XFD
crude fraction was subjected to orthogonal chromatography, as previ-
ously described. These fractions yielded (+)-muqubilone B (1a, 14.3
mg), (+)-sigmosceptrellin A15,16 (4a, 34.3 mg), (-)-sigmosceptrellin
B12a (5, 6.1 mg), and (+)-epi-muqubillin A12a (6, 6.9 mg).
Trypanosoma brucei brucei Assay. T. brucei brucei strain 221 was
grown in complete HMI-9 medium containing 10% FBS, 10% Serum
Plus medium (Sigma Inc., St. Louis, MO) and 1X penicillin/strepto-
mycin. The trypanosomes were diluted to 1 × 105 per mL in complete
HMI-9 medium. Then 95 µL per well of the diluted trypanosomes was
added to sterile Greiner 96-well flat white opaque culture plates that
contained 5 µL of test samples (in 10% DMSO). Control wells
contained 95 µL of the diluted trypanosomes and 5 µL of 10% DMSO,
while control wells for 100% inhibition contained 95 µL of the diluted
trypanosomes and 5 µL of 1 mM thimerosal (in 10% DMSO).
Trypanosomes were incubated with test samples for 48 h at 37 °C with
5% CO2 before monitoring viability. Trypanosomes were then lysed
in the wells by adding 50 µL of CellTiter-Glo (Promega Inc., Madison,
WI). Lysed trypanosomes were placed on an orbital shaker at room
temperature for 2 min. The resulting ATP-bioluminescence of the
trypanosomes in the 96-well plates was measured at room temperature
using an Analyst HT plate reader (Molecular Devices, Sunnyvale, CA).
All IC50 curve fittings were performed with Prism 4 software (GraphPad,
San Diego, CA).
(CH2-7), 32.7 (CH3-11), 33.7 (CH2-19), 35.0 (CH2-12), 35.9 (CH2-5),
39.4 (CH2-15), 42.9 (CH-2), 44.2 (CH2-17), 44.2 (C-14), 52.2 (O-CH3),
80.1 (C-6), 81.5 (CH-3), 124.8 (CH-9), 134.6 (C-10), 174.6 (C-1), 208.8
(C-18), 215.4 (C-13); HRESIMS m/z 461.2906 [M + Na]+ (calcd for
C25H42O6Na, 461.2874).
Hydrogenation of (+)-Muqubilone B Methyl Ester (1b). A 4 mg
amount of (+)-muqubilone B methyl ester (1b) was dissolved into 2
mL of ethanol. Then 10 mg of palladium on carbon was added, and
the slurry was stirred under 1 atm of H2 for 1 h at room temperature.
The reaction mixture was quenched with water, and the palladium
catalyst was filtered through Celite to afford 4 mg of (+)-muqubilone
1
B diol (8). 8: [R]28 +1.7 (c 0.017, CHCl3); H NMR (CDCl3, 500
D
MHz) δH 0.87 (3H, d, J ) 6.5 Hz, H3-22), 1.11 (6H, s, H3-23, H3-24),
1.16 (3H, s, H3-21), 1.21 (3H, d, J ) 7 Hz), 1.30 (2H, m, H2-8), 1.37
(1H, m, Hb-5), 1.40 (2H, m, H2-11), 1.43 (1H, m, Hb-15), 1.44 (2H, m,
H2-7), 1.45 (2H, m, H2-16), 1.46 (1H, m, Ha-15), 1.50 (1H, m, Ha-5),
1.59 (1H, m, Hb-9), 1.60 (1H, m, Ha-10), 1.66 (1H, m, Ha-9), 1.67
(1H, m, Ha-4), 2.13 (3H, s, H3-19), 2.41 (2H, t, J ) 6.5 Hz, H2-17),
2.46 (2H, t, J ) 8.5 Hz, H2-12), 2.56 (H, quint., J ) 7 Hz, H-2), 3.70
(H, ddd, J ) 8, 6, 2 Hz, H-3), 3.72 (3H, s, O-CH3); 13C NMR (CDCl3,
125 MHz) δC 14.5 (CH3-20), 19.2 (CH3-22), 19.8 (CH2-16), 21.5 (CH2-
8), 24.6 (CH3-23), 24.6 (CH3-24), 27.1 (CH3-21), 29.0 (CH2-4), 30.1
(CH3-19), 30.9 (CH-10), 32.6 (CH2-11), 34.7 (CH2-12), 37.6 (CH2-9),
39.5 (CH2-15), 42.3 (CH2-7), 42.6 (CH2-5), 44.1 (CH2-17), 45.5 (CH-
2), 47.7 (C-14), 52.0 (O-CH3), 72.6 (C-6), 74.0 (CH-3), 176.7 (C-1),
208.9 (C-18), 216.1 (C-13); HRESIMS m/z 465.3224 [M + Na]+ (calcd
for C25H46O6Na, 465.3192).
(R)- and (S)-MTPA Esterification of (+)-Muqubilone B Diol (8).
A 2 mg sample of (+)-muqubilone B diol (8) was dissolved in 200 µL
of dry pyridine to which 15 µL of (-)-(R)-MTPA-Cl was added. This
was stirred at room temperature, under nitrogen, overnight. After the
solvent was evaporated under nitrogen, the reaction mixture was purified
by HPLC (C18 column, 85% aq CH3CN isocratic, UV ) 254 nm) to
yield 1.8 mg of (S)-MTPA ester (9a). Similarly, 1.8 mg of the diol (8)
was derivatized with (+)-(S)-MTPA-Cl to afford 1.7 mg of the (R)-
MTPA ester (9b). 9a: 1H NMR (CDCl3, 500 MHz) δH 0.863 (3H, d, J
) 6 Hz, H3-22), 1.110 (3H, s, H3-21), 1.117 (6H, s, H3-23, H3-24),
1.126 (3H, d, J ) 6 Hz, H3-20), 2.123 (3H, s, H3-19), 2.410 (2H, t, J
) 6.5 Hz, H2-17), 2.855 (2H, t, J ) 6.5 Hz, H2-12), 2.855 (H, quint.,
J ) 7 Hz, H-2), 3.522 (3H, s, MTPA-OCH3), 3.586 (3H, s, O-CH3),
5.394 (H, td, J ) 7.5, 4 Hz, H-3), 7.425 (4H, m, MTPA-Ar), 7.561
(1H, m, MTPA-Ar); ESIMS m/z 681 [M + Na].+ 9b: 1H NMR (CDCl3,
500 MHz) δH 0.873 (3H, d, J ) 6.5 Hz, H3-22), 1.036 (3H, s, H3-21),
1.121 (6H, s, H3-23, H3-24), 1.186 (3H, d, J ) 7.5 Hz, H3-20), 2.125
(3H, s, H3-19), 2.413 (2H, t, J ) 6.5 Hz, H2-17), 2.467 (2H, t, J ) 6.5
Hz, H2-12), 2.865 (1H, quint., J ) 7.5 Hz, H-2), 3.540 (3H, s, MTPA-
O-CH3), 3.644 (3H, s, O-CH3), 5.382 (1H, td, J ) 7.5, 4 Hz, H-3),
7.428 (4H, m, MTPA-Ar), 7.560 (1H, m, MTPA-Ar); ESIMS m/z 681
[M + Na]+.
(+)-Muqubilone B (1a): clear oil; [R]28D +59.5 (c 0.046, CHCl3);
NMR in Table 1; HRESIMS 425.2934 [M + H]+ (calcd for C24H41O6,
425.2898).
(+)-Sigmosceptrellin A (4a): physical data in accordance with
published data.15,18
Methylation of (-)-Ent-muqubilone (3a). A 5 mg amount of an
inseparable mixture of (+)-muqubilone B (1a) and (-)-ent-muqubilone
(3a) (2:1) was dissolved into 1 mL of CH3OH to which 2 mL of
trimethylsilyldiazomethane (2 M in hexanes) was added, and the mixture
was stirred at room temperature for 30 min. The solvent was evaporated
under nitrogen, and the reaction mixture was purified by HPLC (C18
column, 85% aq CH3CN isocratic, UV ) 200 nm) to yield 3.2 mg of
(+)-muqubilone B methyl ester (1b) and 1.6 mg of (-)-ent-muqubilone
methyl ester (3b). 3b: [R]28D ) -47 (c 0.004, CHCl3); 1H NMR (CDCl3,
500 MHz) δH 1.11 (6H, s, H3-23, H3-24), 1.25 (3H, d, J ) 7.0 Hz,
H3-20), 1.29 (3H, s, H3-21), 1.41 (2H, m, H2-16), 1.44 (2H, m, H2-7),
1.47 (2H, m, H2-15), 1.60 (3H, s, H3-22), 1.63 (2H, m, H2-5), 1.71
(2H, m, H2-4), 2.00 (2H, m, H2-8), 2.13 (3H, s, H3-19), 2.20 (2H, t, J
) 8.0 Hz, H2-11), 2.41 (2H, t, J ) 7.0 Hz, H2-17), 2.54 (2H, t, J ) 8.0
Hz, H2-12), 2.66 (1H, q, J ) 7.5 Hz, H-2), 3.70 (3H, s, O-CH3), 4.12
(1H, ddd, J ) 8.0, 7.5, 4.5 Hz, H-3), 5.10 (1H, t, J ) 7.0 Hz, H-9);
13C NMR (CDCl3, 125 MHz) δC 13.8 (CH3-20), 16.3 (CH3-22), 19.2
(CH2-16), 20.9 (CH3-21), 21.9 (CH2-8), 23.7 (CH2-4), 24.5 (CH3-23),
24.5 (CH3-24), 30.1 (CH3-19), 32.2 (CH2-5), 33.7 (CH2-11), 35.8 (CH2-
12), 39.4 (CH2-15), 39.8 (CH2-7), 43.2 (CH-2), 44.1 (CH2-17), 47.7
(C-14), 52.7 (O-CH3), 80.3 (C-6), 81.6 (CH-3), 124.6 (CH-9), 134.8
(C-10), 174.5 (C-1), 208.8 (C-18), 215.3 (C-13); HRESIMS m/z
461.2898 [M + Na]+ (calcd for C25H42O6Na, 461.2874).
(+)-Sigmosceptrillin A methyl ester (4b): physical data in ac-
cordance with published data.15,18
(-)-Sigmosceptrellin B (5): physical data in accordance with
published data.16,18
(+)-Epi-muqubilin A (6): physical data in accordance with
published data.12a
(-)-Epi-nuapapuin B methyl ester (7): physical data in accordance
with published data.12a
Methylation of (+)-Muqubilone B (1a). A 6.5 mg sample of (+)-
muqubilone B (1a) was dissolved into 1 mL of CH3OH to which 2 mL
of trimethylsilyldiazomethane (2 M in hexanes) was added, and the
mixture was stirred at room temperature for 30 min. The solvent was
evaporated under nitrogen, and the reaction mixture was purified by
HPLC (C18 column, 85% aq CH3CN isocratic, UV ) 200 nm) to yield
6.2 mg of (+)-muqubilone B methyl ester (1b). 1b: [R]28 +57.6 (c
D
1
0.011, CHCl3); H NMR (CDCl3, 500 MHz) δH 1.11 (3H, s, H3-21),
1.12 (6H, s, H3-23, H3-24), 1.14 (3H, d, J ) 7.5 Hz, H3-20), 1.41 (2H,
m, H2-7), 1.47 (2H, m, H2-16), 1.55 (2H, m, H2-15), 1.62 (3H, d, J )
1 Hz, H3-22), 1.63 (2H, m, H2-5), 1.76 (1H, dtd, J ) 13, 4.5, 2.5 Hz,
Heq-4), 1.85 (1H, dtd, J ) 13, 8, 4.5 Hz, Hax-4), 1.94 (1H, m, Hb-8),
2.04 (1H, m, Ha-8), 2.13 (3H, s, H3-19), 2.21 (2H, t, J ) 8 Hz, H2-11),
2.41 (2H, t, J ) 7 Hz, H2-17), 2.55 (2H, t, J ) 8 Hz, H2-12), 2.58 (1H,
quint., J ) 7.5 Hz, H-2), 3.70 (3H, s, OCH3), 4.24 (1H, ddd, J ) 8.0,
7.5, 4.5 Hz, H-3), 5.16 (1H, tq, J ) 6.5 Hz, 1, H-9); 13C NMR (CDCl3,
125 MHz) δC 13.1 (CH3-20), 16.3 (CH3-22), 19.2 (CH2-16), 22.3 (CH2-
8), 22.9 (CH2-4), 24.2 (CH3-21), 24.6 (CH3-23), 24.6 (CH3-24), 30.2
Acknowledgment. Large thanks to our long-standing collaborator,
Dr. Rob W. M. van Soest, for his expertise with sponge taxonomy.

222 Journal of Natural Products, 2009, Vol. 72, No. 2
Rubio et al.
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Funding for these projects is provided by the Sandler Family Founda-
tion, the National Institutes of Health (RO1 CA052855 and U01
AI075641), the NIGMS (MBRS GM058903), and the California
Institute for Quantitative Biosciences. We also thank Dr. W. Inman
for his insightful conversations and support of this study, the Captain
and Crew of the M/V Golden Dawn for their assistance in sponge
collection, and Dr. T. Matainaho of the University of Papua New Guinea
for his assistance with collection permits.
Supporting Information Available: NMR and MS spectra, sponge
photographs, isolation scheme, and table of optical rotations and
absolute configurations of known norterpene peroxides. This material
is available free of charge via the Internet at http://pubs.acs.org.
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NP800711A