Scalarane sesterterpenoids: Semisynthesis and biological activity

Article abstract of DOI:10.1021/np900326a

Aiming to improve the potency and selectivity of scalarane sesterterpenoids, a series of natural and semisynthetic analogues, derived from the cytotoxic naturally abundant sesterterpene heteronemin (1), were evaluated for their in vitro antimicrobial and

Full text of DOI:10.1021/np900326a

1492
J. Nat. Prod. 2009, 72, 1492–1496
Scalarane Sesterterpenoids: Semisynthesis and Biological Activity
Haidy N. Kamel,*,† Young B. Kim,‡ John M. Rimoldi,‡ Frank R. Fronczek,§ Daneel Ferreira,† and Marc Slattery*,†
Departments of Pharmacognosy and Medicinal Chemistry, Research Institute of Pharmaceutical Sciences, UniVersity of Mississippi, UniVersity,
Mississippi 38677, and Department of Chemistry, Louisiana State UniVersity, Baton Rouge, Louisiana 70803
ReceiVed June 1, 2009
Aiming to improve the potency and selectivity of scalarane sesterterpenoids, a series of natural and semisynthetic
analogues, derived from the cytotoxic naturally abundant sesterterpene heteronemin (1), were evaluated for their
in vitro antimicrobial and cytotoxic properties. The new sesterterpenes 16-O-methylsesterstatin 4 (6c), 17, 24-
dihydroheteronemin (7a), 16, 25-deacetoxy-17, 24-dihydroheteronemin (7b), and 16-deacetoxy-25-methoxy-17,
24-dihydroheteronemin (7c) were structurally defined via physical data analyses. Scalarane sesterterpenes possessing
an unsaturated 1,4-dialdehyde moiety showed potent inhibitory activity against methicillin-resistant Staphylococcus
aureus at concentrations that are not significantly cytotoxic to mammalian cells. The structural features for the
cytotoxicity of scalarane sesterterpenoids are discussed.
Scalaranes constitute an important structural class of sester-
terpenes prominent in Dictyoceratida sponges, e.g., Hyrtios
erecta,1 Spongia spp.,2 Hyatella intestinalis,3 Phyllospongia
madagascarensis,4 and Cacospongia scalaris.5 These sesterte-
penes have also been isolated from nudibranchs associated with
the sponges, and hence, the sesterterpenoids in the nudibranch
were suggested to have a dietary origin.6 Several structural
classes of the scalarane sesterterpenoids were isolated including
C25 (scalarane), C26 (homoscalarane), and C27 (bishomoscalarane)
compounds containing tetra- and pentacyclic skeletons.14 The
structural diversity in the scalarane family mainly arises from
the different oxidation states at C-24 and C-25.15 The structural
types of natural scalarane sesterterpenes have been outlined by
Crews.14 All of them possess a conserved trans-configuration
of the A/B/C/D ring junctions, and the majority are characterized
by oxygenation at C-12. Natural scalaranes with structural
diversity in the A, D, and/or E ring systems have been
reported.10d,14,16 Additionally, a radical relay hydrogenation
synthetic method has recently been used to effect B ring
functionalization.15
Results and Discussion
In our semisynthetic approach, we used the sesterterpene
heteronemin (1), available in large quantities from the sponge
Hyrtios sp.,17 as a starting material for the synthesis of a series of
scalaranes including four new (6c, 7a-7c) and 15 known analogues
previously isolated in low yields from marine sponges. Heteronemin
(1) is an abundant sesterterpene first reported from the sponge
Heteronema erecta in 197617 and subsequently identified as an
antitubercular8b and a farnesyl transferase inhibitor.18 However, its
strong but nonselective cytotoxicity prevented further biological
studies.
Structurally, heteronemin (1) possesses a pentacyclic scalarane
skeleton including a dihydrofuran moiety. It has nine stereogenic
centers, a secondary hydroxy group at C-12, an acetal moiety at
C-25, and a trisubstituted C-17-C-24 double bond. The arrange-
ment of the various functionalities is unique and allows for
numerous chemical transformations. Treatment of heteronemin (1)
with acetic anhydride in pyridine afforded 16-O-acetylheteronemin
(2)19 (Scheme 1), while oxidation with pyridinium chlorochromate
afforded 12-oxoheteronemin (3) as the major product (Scheme 2).
12-Episcalarin (4a)20 was available from the sponge extract. The
triol 5 was obtained from heteronemin (1) via NaBH4 reduction.
Oxidation of 5 with an excess of barium permanganate (10-20
equiv) at room temperature afforded 12-O-deacetyl-25-deoxy-12-
episcalarin (4b) (Scheme 1). The ease of oxidation of the allylic
C-24 alcohol into the carboxylic acid functionality resulted in rapid
lactone ring formation. The product 4b is identical to the reported
naturally occurring material.20
Owing to their diverse biological properties, interest in
scalarane sesterterpenes and their synthesis has increased notice-
ably in recent years. This family of compounds have been
reported to have anti-inflammatory,7 antimicrobial,8 platelet-
aggregation inhibitory,9 and cytotoxic10 activities. In addition,
the role of these compounds in the defensive mechanism of
sponges has been demonstrated, e.g., their antifeedant,11 anti-
fouling,12 and ichthyotoxic13 activities. Despite the large number
of scalarane sesterterpenes exhibiting moderate to significant
cytotoxic activities, there are no systematic reports on their
structure-cytotoxicity relationship. Additionally, further biologi-
cal studies were prohibited due to the nonselective cytotoxicities
associated with some analogues. Herein, a series of semisynthetic
and natural scalarane sesterterpenes were evaluated for their in
vitro antimicrobial and cytotoxic properties. Structural features
for their cytotoxicity were defined and antimicrobial activities
for a number of scalaranes at nontoxic concentrations were
found.
* To whom correspondence should be addressed. Tel: 662-915-1706.
Fax: 662-915-6975. E-mail: hnkamel@olemiss.edu. Tel: 662-915-1053.
E-mail: slattery@olemiss.edu.
† Department of Pharmacognosy, University of Mississippi.
‡ Department of Medicinal Chemistry, University of Mississippi.
§ Department of Chemistry, Louisiana State University.
Next, we investigated the conversion of heteronemin (1) into
scalarane-based furanosesterterpenes. Treatment of heteronemin (1)
10.1021/np900326a CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/06/2009

Scalarane Sesterterpenoids
Journal of Natural Products, 2009, Vol. 72, No. 8 1493
Scheme 1a
a Reagents and conditions: (i) Ac2O, pyr; (ii) NaBH4, MeOH, rt, 1 h; (iii) p-TsOH, CH2Cl2, reflux, 3 h; (iv) KOH, MeOH, rt, 24 h; (v) Ba(MnO4)2, CH2Cl2, rt, 24 h.
Scheme 2a
Figure 1. ORTEP drawing of compound 6c.
is a new analogue, formed presumably via solvolysis of the allylic
O-acetyl group in a predominantly SN2 fashion.
Compound 6b has a molecular formula of C25H36O2 ([M + H]+
369.2782). Its 13C NMR spectrum lacked the signals pertaining to
a Reagents and conditions: (i) H2/Pd-C, anhydrous MeOH, rt, 24 h; (ii) PCC/
silica gel, CH2Cl2, rt, 2 h; (iii) NaHCO3, MeOH, rt, 5 min; (iv) Ac2O, pyr; (v)
MeOH, ion exchange resin, rt; (vi) K2CO3, MeOH, rt, 2 h.
the acetoxy functionality as well as the methine signal at δ 68.1
(C-16) in 6a and, instead, displayed two olefinic methine signals
at δ 119.2 and 127.6. Its 1H NMR spectrum displayed two olefinic
proton signals at δ 5.79 and 6.50. Analysis of the HMBC
correlations confirmed the structure as 16-deacetoxy-15,16-dehy-
droscalarafuran. This compound was claimed to be formed via
pyrolysis of heteronemin.17 However, no NMR data to support the
structure were presented. Compound 6c has a molecular formula
of C26H40O3 [(M)+ 400.2924]. The structure was elucidated by
comparison of 1H and 13C NMR data with those of sesterstatin 521
(6d). It displayed an O-methyl signal in its 13C NMR spectrum at
δ 56.5, which correlated in the HMQC spectrum with a singlet at
δ 3.35 (3H), suggesting the presence of an O-methyl group at C-16.
The structure and configuration were confirmed by X-ray crystal-
lography (Figure 1).
with 1 equiv of p-toluenesulfonic acid at room temperature in
CH2Cl2 for 30 min yielded the furan analogue scalarafuran (6a).
However, if an excess of p-TsOH is used (10-20 equiv) or if the
reaction is extended for longer periods of time (3 h), the C-15-C-
16 vinylic furan, 16-deacetoxy-15,16-dehydroscalarafuran (6b), is
obtained as the major product. It is notable that under these time-
extended conditions the C-12 equatorial hydroxy group is not
involved in a similar dehydration reaction. The conformational
rigidity of the C ring would not permit a conformation in which
the C-12 hydroxy group and a proton at C-11 would attain the
requisite trans-diaxial relationship for a dehydration reaction.
Scalarafuran (6a) was also obtained from heteronemin (1) via
pyrolysis following reported procedures,11 albeit in a low yield.
Hydrolysis of scalarafuran (6a) in base/methanol afforded 16-O-
methylsesterstatin 4 (6c) and sesterstatin 521 (6d). Compound 6c
Heteronemin (1) undergoes reduction with H2 in the presence
of Pd/C in MeOH to give a mixture of the sesterterpenes 7a-7c.
These 17,24-dihydroheteronemin derivatives are reported for the
first time. Compound 7a exhibited a molecular ion peak at m/z

1494 Journal of Natural Products, 2009, Vol. 72, No. 8
Kamel et al.
Table 1. Cytotoxicity and Antimicrobial Dataa
compound
SKOV3 SKMEL BT549 Vero MRSA M. int.
1
2
3
3.4
15.3
16.7
NA
NA
NA
NA
>37.3
NA
26.4
18.6
>42.7
14.6
14.3
NA
11.2
18.8
>37.6 >37.6
NA
NA
NA
15.9
NA
19.4
12.9
21.0
15.6
15.6
NA
8.2
9.0
NA
NA
NA
NA
NA
NA
NA
NA
22
3.8
6.3
27.8
30.1
NA
0.14
2.04
18.8
5.1
18.8
>37.6
NA
NA
NA
18.3
NA
31
19.4
23.3
41.6
31.2
NA
4a-4b
5
6a-6d
7a
7b-7c
8a
8b
8c
NA
NA
NA
36.7
NA
19.4
20.7
22.2
18.2
15.6
NA
NA
NA
NA
NA
NA
38.8
NA
NA
NA
NA
NA
0.47
8d
8e
9a-9b
Ciprofloxacin
Doxorubicin
0.8
1.2
1.2
6.3
a MRSA ) methicillin-resistant Staphylococcus aureus, M. int. )
Mycobacterium intracellulare, NA ) not active. Data shown are the
IC50 values in µM.
Figure 2. ORTEP drawing of compound 8b.
431.3236 ([M - OAc]+), suggesting a molecular formula of
C29H46O6 with seven degrees of unsaturation. The 13C NMR
spectrum lacked the olefinic carbons at δ 114.0 and 134.9 in
heteronemin (1) and displayed an oxymethylene carbon at δ 70.5
(C-24) and a methine carbon at δ 37.4 (C-17). On the basis of
these results, 7a was defined as 17,24-dihydroheteronemin. Com-
pound 7b had a molecular formula of C25H42O2 (m/z 375.3257 [M
+ H]+). The 13C NMR spectrum lacked the olefinic (δ 114.0, 134.9)
and ester carbonyl carbons (δ 170.3, 172.1) but displayed two
oxymethylene carbons at δ 71.1 and 72.0 and an additional
methylene carbon at δ 16.4, indicating hydrogenolysis of the allylic
C-16 O-acetyl group. Detailed analysis of the 2D NMR data
indicated the structure deduced for 7b (16,25-deacetoxy-17,24-
dihydroheteronemin) as shown. The molecular formula of 7c was
defined as C26H44O3 from a molecular ion peak at m/z 427.3067
products, although the authors have often considered them artifacts
due to the use of MeOH during extraction and/or isolation.19,25
The natural and synthesized scalaranes were evaluated for their
cytotoxicity against SK-MEL melanoma, SKOV3 ovarian, and
BT549 breast cancer cell lines, as well as their antimicrobial activity
against Mycobacterium intracellulare and methicillin-resistant
Staphylococcus aureus (MRSA). The IC50 values are presented in
Table 1. It was originally suggested by Crews and Bescana that
the oxygenation pattern at C-25 of the scalarane sesterterpene exerts
a strong influence on their biological activities.19 Such an influence
was evident among the scalaranes tested, in which the furanoses-
terterpenes 6a-6d, lacking an oxygen functionality at C-25, were
not cytotoxic at doses up to 45 µM. Our data also suggest that the
oxygenation pattern at C-16 is similarly influential on the activity
since the 16-deoxy sesterterpenes 4a and 4b lack cytotoxicity at
doses up to 45 µM. Alternatively, the C-17-C-24 double bond
slightly affects the cytotoxicity of the scalaranes, as evident in
compound 7a. The dialdehydes 8a-8d exhibited cytoxicity against
all the cancer cell lines tested. Natural products with an 1,4-
unsaturated dialdehyde functionality have been proposed to exert
their cytotoxicity by reacting with proteins in the cell membrane
via the formation of adducts between the unsaturated dialdehyde
moieties and the amino groups of amino acid residues.26 Heter-
onemin (1) and pentacylic sesterterpenes, lacking the dialdehyde
moiety, exhibit their cytotoxicity via a different mechanism.
However, heteronemin (1) may act as a prodrug of the dialdehyde
that may be unmasked via hydrolysis of the C-25 ester functionality.
Evaluation of the antimicrobial activities of the scalaranes showed
that sesterterpene 8b is a potent inhibitor of MRSA with an IC50
value of 3.8 µM, while its epimer (8a) was less potent at 6.3 µM.
This is in agreement with the suggestion that the extent of
antimicrobial activity of bicyclic sesquiterpenes with dialdehyde
groups such as warburganal and polygodial depends on the
stereochemical arrangement of B-ring-oxygenated substituents.27
It has been suggested that the unsaturated dialdehyde functionality
and its reactivity toward biological nucleophiles is responsible for
the general antibiotic activity of these compounds.25 Unsaturated
dialdehydes are sensitive; for example, 8a and 8b interconvert via
epimerization at alkaline pH, which may affect their reactivity and
bioactivity.
1
([M + Na]+) in the HRESIMS spectrum. The H NMR spectrum
exhibited an O-methyl resonance at δ 3.37, which correlated with
the carbon at δ 55.2 in the HMQC spectrum and the carbon at δ
108.5 in the HMBC spectrum, indicating the attachment of the
O-methyl group at C-25 of the scalarane skeleton. The structure
was then confirmed by 2D NMR analysis. Compound 7c originated
via trans-acetalization at C-25 and hydrogenolysis of the allylic
16-O-acetyl group of heteronemin (1) in methanolic solution.
We then turned our attention to methods of synthesizing scalarane
sesterterpenes possessing R,ꢀ-unsaturated dialdehyde groups to
provide additional insights into structure-cytotoxicity relationships.
Hydrolysis of heteronemin (1) under basic conditions gave 12-O-
deacetyl-12-episcalaradial (8a) (Scheme 2) via acetal hydrolysis
and subsequent dehydration involving the C-16 hydroxy group.
Compound 8a undergoes facile epimerization at C-18 to give 12-
O-deacetyl-12,18-diepiscalaradial23 (8b), whose structure was
confirmed via X-ray crystallography (Figure 2). The vicinal
dialdehyde functionality in 8a is analogous to that of the antifeedant
drimane sesquiterpene polygodial.24 12-Episcalaradial23 (8c) was
available from 8a via direct acetylation.19 12-O-deacetyl-12-
oxoscalaradial (8d) and 12-O-deacetyl-18-epi-12-oxoscalardial (8e)
were prepared by treating 12-oxoheteronemin (3) with potassium
carbonate in methanol. The physical data of the above compounds
are in accordance with those reported.24
The dimethylacetals 12-O-deacetyl-24,25-dimethoxyscalarins 9a
and 9b were readily obtained by treatment of 12-O-deacetyl-12-
episcalaradial (8a) with an acidic ion-exchange resin in MeOH with
the major product (9a) having both methoxy groups R-oriented.
The 1H NMR chemical shifts of H-24 in 9a and 9b were similar to
those reported,19 confirming syn- and anti-1,3-bismethoxy groups
for 9a (H-24 ) 5.17) and 9b (H-24 ) 5.47), respectively.
Dimethylacetals of dialdehydes have been reported as natural
Scalarane sesterterpenes were reported to possess nonselective
moderate to significant cytotoxic activities, which, in many cases,
inhibited further biological studies. Here we report the preparation
and biological testing of 20 natural and semiynsthetic sesterterpene
analogues. Our data indicate that oxygenation at both C-16 and
C-25 of the scalarane skeleton exerts a strong influence on their
cytotoxicities. Recent studies reported scalaranes with an oxygen

Scalarane Sesterterpenoids
Journal of Natural Products, 2009, Vol. 72, No. 8 1495
(C-16), 78.9 (C-12), 58.4 (C-9), 57.0 (C-14), 56.9 (C-5), 42.2 (C-3),
40.7 (C-7), 40.5 (C-13), 39.8 (C-1), 37.2 (C-10), 37.0 (C-8), 33.5 (C-
4, C-19), 28.1 (C-11), 21.5 (C-20), 18.8 (C-6), 18.7 (C-21), 18.1 (C-
2), 16.3 (C-22), 16.2 (C-23); HRESIMS m/z 369.2782 [M + H]+ (calcd
for C25H37O2, 369.2793).
functionality at C-3 of the A ring from the sponge Hyrtios with
selective activity on the P-388 murine lymphatic leukemia cell line
at nanomolar concentrations.28 This data suggests that the structure
of ring A may play a significant but less obvious role in their
mechanism of action. Our results also identified 12-O-deacetyl-
12,18-diepiscalaradial (8b) and 12-episcalaradial (8c) as potent
inhibitors of MRSA at concentrations that are not significantly
cytotoxic to mammalian cells.
16-O-Methylsesterstatin 4 (6c). A solution of scalarafuran (6a) in
MeOH (3 mg in 1 mL) was stirred with KOH (7 mg) at rt for 24 h.
The reaction was diluted with water (5 mL) and extracted with CH2Cl2
(3 × 5 mL). The organic phase was dried over anhydrous Na2SO4,
filtered, and evaporated to afford a crude residue, which after column
chromatography (hexanes/EtOAc, 8:2) gave 6c (1 mg, 35%) and 6d
Experimental Section
(1.2 mg, 45%). Compound 6c: [R]25 -6.3 (c 0.2, CHCl3); IR (film)
General Experiment Procedures. Optical rotations were measured
with a JASCO DIP-370 digital polarimeter. UV spectra were recorded
on an Agilent Chemstation with 8453 spectrophotometer. NMR spectra
D
1
νmax 3439, 2932, 1461, 1387, 1087, 756 cm-1; H NMR (CDCl3, 400
MHz) δ 7.57 (1H, d, J ) 1.2 Hz, H-25), 7.36 (1H, d, J ) 1.2 Hz,
H-24), 4.36 (1H, d, J ) 1.6 Hz, H-16), 3.70 (1H, bd, J ) 10 Hz, H-12),
3.35 (3H, s, OMe), 1.86 (1H, m, HR -15), 1.55 (1H, m, Hꢀ-15); 13C
NMR (CDCl3, 100 MHz) δ 138.9 (C-24), 137.6 (C-25), 133.4 (C-18),
120.5 (C-17), 79.4 (C-12), 70.6 (C-16), 58.5 (C-5), 56.5 (OMe), 55.5
(C-14), 49.3 (C-13), 42.0 (C-3), 41.5 (C-7), 40.5 (C-8), 39.8 (C-1),
37.4 (C-9), 36.9 (C-10), 33.3 (C-4), 33.2 (C-19), 28.0 (C-11), 24.2
(C-15), 21.2 (C-20), 18.7 (C-6), 18.5 (C-21), 18.1 (C-2), 16.4 (C-22),
16.2 (C-23); HRESIMS m/z 400.2924 [M]+ (calcd for C26H40O3,
400.2977).
1
were recorded on a Bruker Avance DRX-400 spectrometer. H and
13C NMR spectra were measured and reported in ppm by using the
CDCl3 and DMSO-d6 solvent peaks (δH 7.24 and δC 77.2 and δH 2.50
and δC 39.5, respectively) as internal standards. ESIFTMS were
recorded on a Bruker-Magnex BioAPEX 30es ion cyclotron HR HPLC-
FT spectrometer by direct injection into an electrospray interface.
Chromatographic separations were carried out by conventional column
chromatography on Sorbent silica gel 60 (230 × 450 mesh).
Animal Material. The sponge Hyrtios sp. was collected from
American Samoa in February 2004 and identified by one of the authors
(M.S.). A voucher specimen (AS40204070) was deposited at the NOAA
Ocean Biotechnology Center and Repository, Oxford, MS.
17,24-Dihydroheteronemin (7a), 16,25-Deacetoxy-17,24-dihydro-
heteronemin (7b), and 16-Deacetoxy-25-methoxy-17,24-dihydro-
heteronemin (7c). A solution of heteronemin (1) in anhydrous MeOH
(19 mg 2 mL) containing 10% palladium on charcoal catalyst was stirred
under an atmosphere of H2 for 24 h. The catalyst was removed by
filtration and the solvent evaporated to obtain a mixture, which after
column chromatography (hexanes/EtOAc, 9:1) afforded 7a (2.8 mg,
16%), 7b (1.8 mg, 13%), and 7c (6.4 mg, 45%) as white solids.
Compound 7a: [R]25D -2.4 (c 0.1, CHCl3); IR (film) νmax 3404, 2938,
12-Oxoheteronemin (3). Pyridinium chlorochromate (8.6 mg) was
ground to a fine powder with 10 mg of silica gel, and the light orange
mixture was suspended in 2 mL of CH2Cl2. Heteronemin 1 (10 mg in
1 mL of CH2Cl2) was added in one portion to the orange suspension,
and the mixture was stirred for 2 h at rt. The reaction mixture was
filtered through Celite and the solvent removed under vacuum. The
residue was dissolved in CHCl3 (10 mL), washed with H2O (2 × 10
mL), dried over anhydrous Na2SO4, filtered, and evaporated to give a
crude residue, which after column chromatography (hexanes/EtOAc,
8:2) afforded 319 as a white solid (6 mg, 65%).
12-O-Deacetyl-25-deoxy-12-episcalarin (4b). To a solution of the
triol 5 (8 mg) in CH2Cl2 (2 mL) was added Ba(MnO4)2 (40 mg), and
the mixture was stirred for 24 h at rt. Filtration through a Celite pad
and removal of the solvent gave a residue, which after column
chromatography (hexanes/EtOAc, 8:2) afforded 4b20 as a white solid
(4 mg, 50%).
Triol 5. To a solution of heteronemin (1) (25 mg) in MeOH (5 mL)
was added an excess of NaBH4. The mixture was stirred at rt for 1 h.
After acidification with HOAc (1 mL) and addition of H2O (20 mL),
the aqueous mixture was extracted with CH2Cl2 (2 × 10 mL) and dried
over anhydrous Na2SO4, and the solvent was evaporated under vacuum.
The resultant residue was chromatographed over silica gel, eluting with
5% MeOH in CHCl3 to afford 519 as a white solid (16 mg, 87%).
Scalarafuran (6a). Heteronemin (1) (10 mg) was stirred with
p-TsOH (4 mg, 1 equiv) in CH2Cl2 (5 mL) for 30 min at rt. The reaction
mixture was diluted with H2O (20 mL) and extracted with CHCl3 (3 ×
10 mL). The combined organic phases were dried over anhydrous
Na2SO4, filtered, and evaporated to afford a crude residue, which after
column chromatography (hexanes/EtOAc, 9:1) gave 6a22 as a white
solid (5.2 mg, 60%).
1
1676, 1440, 1387, 1205, 1142 cm-1; H NMR (CDCl3, 400 MHz) δ
5.39 (1H, s, H-25), 5.07 (1H, t, J ) 6 Hz, H-16), 3.92 (1H, dd, J ) 11,
8.8 Hz, HR-24), 3.39 (1H, bd, J ) 11 Hz, Hꢀ-24), 3.10 (1H, bd, J ) 9
Hz, H-12), 2.07 (3H, s, AcO-16), 2.04 (3H, s, AcO-25); 13C NMR
(CDCl3, 100 MHz) δ 172.1 (Ac-25), 170.3 (Ac-16), 104.9 (C-25), 83.1
(C-12), 72.9 (C-16), 70.5 (C-24), 59.3 (C-18), 58.9 (C-9), 56.7 (C-5),
54.6 (C-15), 42.1 (C-3), 41.8 (C-1), 40.8 (C-8), 39.9 (C-7), 37.8 (C-
10), 37.6 (C-13), 37.4 (C-17), 33.3 (C-4), 33.2 (C-19), 26.0 (C-11),
21.4 (Ac-25), 21.2 (Ac-16), 21.0 (C-20), 18.6 (C-6), 18.2 (C-2), 18.0
(C-21), 16.2 (C-22), 11.5 (C-23); HRESIMS m/z 431.3236 [M - OAc]+
(calcd for C27H43O4, 431.3161). Compound 7b: [R]25 -8.6 (c 0.2,
D
CHCl3); IR (film) νmax 2924, 1683, 1463, 1206 cm-1; 1H NMR (CDCl3,
400 MHz) δ 4.37 (1H, dd, J ) 9, 2 Hz, HR-24) 3.82 (2H, brd, J ) 9
Hz, Hꢀ-24, HR-25), 3.43 (1H, dd, J ) 12, 9 Hz, Hꢀ-25), 3.33 (1H, bd,
J ) 9 Hz, H-12); 13C NMR (CDCl3, 100 MHz) δ 84.8 (C-12), 72.0
(C-25), 71.1 (C-24), 58.7 (C-9), 56.6 (C-5), 56.3 (C-18), 53.0 (C-14),
42.1 (C-3), 41.6 (C-1), 41.1 (C-8), 39.9 (C-7), 37.9 (C-10), 37.6 (C-
17), 37.4 (C-13), 33.2 (C-4, C-19), 27.8 (C-11), 24.2 (C-15), 21.2 (C-
20), 18.6 (C-6), 18.2 (C-2), 17.4 (C-21), 16.4 (C-16), 16.2 (C-22), 11.7
(C-23); HRESIMS m/z 375.3257 [M + H]+ (calcd for C25H43O2,
375.3263). Compound 7c: [R]25 -12 (c 0.5, CHCl3); IR (film) νmax
D
3421, 2927, 2874, 1466, 1390, 1203, 1093, 1039 cm-1
;
1H NMR
(CDCl3, 400 MHz) δ 5.32 (1H, s, H-25), 4.00 (1H, t, J ) 8.4 Hz,
HR-24), 3.55 (1H, t, J ) 8.4 Hz, Hꢀ-24), 3.43 (1H, bd, J ) 9 Hz, H-12),
3.37 (3H, s, OMe); 13C NMR (CDCl3, 100 MHz) δ 108.5 (C-25), 84.6
(C-12), 71.7 (C-24), 58.7 (C-9), 58.6 (C-18), 56.6 (C-5), 55.2 (OMe),
54.9 (C-13), 42.1 (C-3), 41.6 (C-1), 40.3 (C-8), 39.9 (C-7), 37.8 (C-
14), 37.4 (C-10), 34.8 (C-17), 33.3 (C-19), 33.2 (C-4), 27.2 (C-15),
24.0 (C-16), 21.3 (C-20), 18.6 (C-6), 18.2 (C-2), 17.5 (C-21), 16.4
(C-22), 12.2 (C-23); HRESIMS m/z 427.3067 [M + Na]+ (calcd for
C26H44O3Na, 427.3188).
12-O-Deacetyl-12-oxoscalaradial (8d) and 12-O-deacetyl-12-oxo-
18-episcalaradial (8e). To a solution of 3 (10 mg) in MeOH (2 mL)
was added K2CO3 (4 mg), and the reaction mixture was stirred at room
temperature for 2 h. The reaction was then quenched with H2O,
extracted with CH2Cl2, dried over anhydrous Na2SO4, and evaporated
to give a crude residue, which was chromatographed over silica gel
eluting with 20% EtOAc in hexanes to afford 8d (1.5 mg, 20%) and
8e (1 mg, 13%) as white solids.
16-Deacetoxy-15,16-dehydroscalarafuran (6b). Heteronemin (1)
(20 mg) was stirred with p-TsOH (38 mg) in CH2Cl2 (5 mL), and the
mixture was heated to reflux for 30 min. The reaction mixture was
diluted with H2O (20 mL) and extracted with CHCl3 (3 × 10 mL).
The combined organic phases were dried over anhydrous Na2SO4,
filtered, and evaporated to afford a crude residue, which after column
chromatography (hexanes/EtOAc, 9:1) gave 6a (1 mg, 10%) and 6b
(6.2 mg, 45%) as white solids. Compound 6b: [R]25 -9.2 (c 0.5,
D
1
CHCl3); IR (film) νmax 3429, 2931, 1735, 1383, 1213, 763 cm-1; H
NMR (CDCl3, 400 MHz) δ 7.42 (1H, s, H-25), 7.24 (1H, s, H-24),
6.50 (1H, dd, J ) 9.8, 3 Hz, H-16), 5.79 (1H, dd, J ) 9.8, 2.4 Hz,
H-15), 3.88 (1H, bd, J ) 10 Hz, H-12), 1.94 (1H, bs, H-14), 1.91 (1H,
m, H-7), 1.84 (1H, dd, J ) 10, 2 Hz, H-11), 1.71 (1H, m, H-1), 1.65
(1H, m, H-6), 1.59 (1H, m, H-2), 1.55 (1H, m, H-11), 1.45 (1H, m,
H-6), 1.43 (1H, m, H-2), 1.37 (1H, m, H-3), 1.15 (1H, m, H-3), 1.04
(3H, s, H-23), 1.00 (3H, s, H-21), 0.96 (1H, m, H-9), 0.92 (1H, m,
H-7), 0.88 (3H, s, H-22), 0.85 (3H, s, H-19), 0.84 (1H, m, H-1), 0.83
(3H, s, H-20) 0.79 (1H, m, H-5); 13C NMR (CDCl3, 100 MHz) δ 136.3
(C-25), 135.6 (C-24), 132.6 (C-18), 127.6 (C-15), 121.3 (C-17), 119.2
12-O-Deacetyl-24,25-dimethoxyscalarins (9a and 9b). A solution
of 12-O-deacetyl-12-episcalaradial (8a) (5 mg) in MeOH (1 mL) was
run through a column (5 cm × 2 cm) of Dowex 50w × 8 (100-200

1496 Journal of Natural Products, 2009, Vol. 72, No. 8
Kamel et al.
mesh, H+) ion-exchange resin (prewashed with MeOH) to give a
mixture of dimethylacetals. The mixture was dried under vacuum, and
the residue was chromatographed over silica gel eluting with 15%
EtOAc in hexanes to give the pure dimethylacetals 9a19 (3 mg, 53%)
and 9b19 (1.2 mg, 20%) as white solids.
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Single-Crystal X-ray Diffraction Analysis of Compounds 6c and
8b. Diffraction data for 6c and 8b were collected at low temperature
on a Nonius KappaCCD diffractometer equipped with Mo KR radiation
and an Oxford Cryostream sample chiller. Crystal data: for 6c (crystals
from CHCl3), C26H40O3, monoclinic space group P21, a ) 11.061(4)
Å, b ) 6.048(2) Å, c ) 16.603(7) Å, ꢀ ) 101.298(17)°, V ) 1089.2(7)
Å3, T ) 100.0(5) K, Z ) 2, R ) 0.049 (F2 > 2σ), Rw ) 0.110 (all F2)
for 2067 unique data having 2θ < 50.0° and 270 refined parameters;
for 8b (crystals from CHCl3), C25H38O3, orthorhombic space group
P212121, a ) 8.4330(8) Å, b ) 11.9818(14) Å, c ) 20.782(3) Å, V )
2099.9(4) Å3, T ) 90.0(5) K, Z ) 4, R ) 0.036 (F2 > 2σ), Rw ) 0.096
(all F2) for 4355 unique data having 2θ < 65.8° and 261 refined
parameters.
Cytotoxicity Assay. The in vitro cytotoxic activity was determined
against three human cancer cell lines, SK-MEL, BT-549, and SKOV-
3, as well as Vero noncancerous cell lines. Vero cells were obtained
from the American Type Culture Collection (ATCC, Rockville, MS).
The assay was performed in 96-well tissue culture-treated microplates.
Cells (25 000 cells/well) were seeded in the wells of the plate and
incubated for 24 h. Samples were added, and plates were again
incubated for 48 h. The number of viable cells was determined using
Neutral Red according to a modification of the procedure of Boren-
freund et al.29 IC50 values were determined from logarithmic graphs
of growth inhibition versus concentration. Doxorubicin was used as a
positive control, while DMSO was used as the negative (vehicle)
control.
Antimicrobial Assay. Organisms (methicillin-resistant Staphylo-
coccus aureus ATCC 43300-MRSA and Mycobacterium intracellulare
ATCC 23068) were obtained from the American Type Culture
Collection (Manassas, VA). MRSA was tested using a modified version
of the CLSI methods.30 M. intracellulare was tested using a modified
method of Franzblau et al.31 Samples were serially diluted in 20%
DMSO/saline and transferred in duplicate to 96-well flat bottom
microplates. Microbial inocula were prepared by correcting the OD630
of microbe suspensions in incubation broth to afford final target inocula.
Ciprofloxacin (ICN Biomedicals, Aurora, OH) was included in each
assay. MRSA was read at 630 nm using the Biotek Powerwave XS
plate reader (Bio-Tek Instruments, Winooski, VT) and M. intracellulare
at 544ex/590em using the Polarstar Galaxy plate reader (BMG
LabTechnologies, Germany) prior to and after incubation.
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Acknowledgment. We are grateful to M. Wright, D. Kutrzeba, and
S. Khan for performing the antimicrobial and cytotoxic assays at the
National Center for Natural Products Research. This work was funded
by the National Oceanic and Atmospheric Association’s National
Undersea Research program (NOAA NIUST NA16RU) and the USDA
Agricultural Research Service Specific Cooperative Agreement No. 58-
6408-2-0009.
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Supporting Information Available: CCDC 732431 and 732432
contain the supplementary crystallographic data for this paper. These
data can be obtained free of charge at www.ccdc.cam.ac.uk/conts/
retrieving.html [or from the Cambridge Crystallographic Data Centre
(CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223-
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1
336033; e-mail: deposit@ccdc.cam.ac.uk]. H and 13C NMR spectra of
Biol. 1990, 26, 1030–1034.
compounds 6b, 6c, and 7a-c are available free of charge via the Internet
(30) NCCLS, NCCLS Document M7-A7; 2006; Vol. 26 (2), pp 1-64.
(31) Franzblau, S. G.; Witzig, R. S.; McLaughlin, J. C.; Torres, P.; Madico,
G.; Hernandez, A.; Degnan, M. T.; Cook, M. B.; Quenzer, V. K.;
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at http://pubs.acs.org.
References and Notes
(1) Pettit, G. R.; Cichacz, Z. A.; Hoard, M. S.; Melody, N.; Pettit, R. K.
J. Nat. Prod. 1998, 61, 13–16.
NP900326A