Lehualides e - K, cytotoxic metabolites from the tongan marine sponge plakortis sp.

Article abstract of DOI:10.1021/np100868t

Spectroscopy-guided chemical analysis of a marine sponge from the genus Plakortis, collected in Tonga, yielded seven new metabolites of polyketide origin, lehualides E-K (5-11), four of which incorporate various sulfur functionalities. The structures of compounds 5-11 were elucidated by interpretation of spectroscopic data and spectral comparison with model compounds. The biological activities of compounds 6-9 were investigated against human promyeloid leukemic HL-60 cells and two yeast strains, wild-type and a drug-sensitive mutant.

Full text of DOI:10.1021/np100868t

ARTICLE
pubs.acs.org/jnp
Lehualides E-K, Cytotoxic Metabolites from the Tongan Marine
Sponge Plakortis sp.
Jacqueline M. Barber,† Natelle C. H. Quek,‡ Dora C. Leahy,‡ John H. Miller,‡ David S. Bellows,‡ and
Peter T. Northcote*,†
†
Centre for Biodiscovery and Schools of Chemical and Physical Sciences and ‡Biological Sciences, Victoria University of Wellington,
PO Box 600, Wellington, New Zealand
S Supporting Information
b
ABSTRACT: Spectroscopy-guided chemical analysis of a mar-
ine sponge from the genus Plakortis, collected in Tonga, yielded
seven new metabolites of polyketide origin, lehualides E-K
(5-11), four of which incorporate various sulfur functional-
ities. The structures of compounds 5-11 were elucidated by interpretation of spectroscopic data and spectral comparison with
model compounds. The biological activities of compounds 6-9 were investigated against human promyeloid leukemic HL-60 cells
and two yeast strains, wild-type and a drug-sensitive mutant.
arine sponges of the genus Plakortis (family Plakinidae) are
known to be rich sources of structurally unique and
biologically active secondary metabolites. Frequently isolated
from Plakortis extracts, the antibacterial plakortin1 and other
polyketides containing cycloperoxide rings2 exhibit wide-ranging
biological effects including antiparasitic activity3 and promotion
of Ca2þ uptake in the cardiac sarcoplasmic reticulum.4
M
The common plakortin carbon skeleton is frequently augmen-
ted by further functionalities, including γ-lactone moieties,2b,4,5
tetrahydrofuran rings,6 and furanoester groups.7 Another common
motif of Plakortis isolates is a long alkyl chain with varying degrees
of saturation. This feature is exemplified by the epiplakinic acids,
isolated from Palauan collections of Plakortis nigra,2b and is
characteristic of lehualides A-D (1-4).8 Reported in 2005 from
a Hawai’ian Plakortis specimen, these cytotoxic polyketides in-
corporate R- or γ-pyrone moieties, coupled with thioacetate or
thiol functionalities.8 Seven additional members of this class,
lehualides E-K (5-11), are reported herein.
requiring seven degrees of unsaturation. Of a possible 24, the
13C NMR spectrum contained 22 distinct resonances, including
11 sp2 centers, indicating some molecular symmetry, while the
HSQC spectrum accounted for all of the 32 protons.
’ RESULTS AND DISCUSSION
A Plakortis sponge specimen, collected from a cave off the
coast of ‘Eua Island, Tonga, was extracted in MeOH and
partitioned over reversed-phase polystyrene divinyl benzene
resin with mixtures of acetone and water. Subsequent iterative,
NMR-guided chromatography on dihydroxypropoxypropyl-
derivatized silica gel (DIOL, CH2Cl2/EtOAc) and reversed-
phase HPLC (C-18, MeCN/H2O) of the 100% and 75% acetone
fractions provided lehualides E-K (5-11) as colorless oils or
glassy solids. The structure of lehualide K (11) was elucidated
from fractions containing persistent impurities (ca. 15%) of
compounds 7 and 8.
Immediately apparent in the CDCl3 1H NMR spectrum were
the resonances of a mono-alkyl-substituted benzene ring (δH
7.27-7.16), an olefinic methine (δH 5.18), two methoxy singlets
(δH 4.19, 3.81), a deshielded methylene doublet and triplet (δH
3.17, 2.57), two olefinic methyl singlets (δH 1.85, 1.66), and an
aliphatic methylene pocket integrating for 12 protons. Three spin
systems were identified from the COSY spectrum: a mono-alkyl-
substituted phenyl ring, an isolated methyl-substituted alkene,
and an extended aliphatic chain.
The positive-ion mode HRESIMS data of lehualide E (5)
showed a [M þ Na]þ pseudomolecular ion peak at m/z
385.2399, consistent with the molecular formula C24H32O4,
Received: November 29, 2010
Published: February 25, 2011
r
Copyright
2011 American Chemical Society and
809
dx.doi.org/10.1021/np100868t J. Nat. Prod. 2011, 74, 809–815
American Society of Pharmacognosy
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COSY and HMBC correlations allowed the facile assembly of
the mono-alkyl-substituted benzene ring C-16-C-21 [C-16 (δC
143.1); CH-17, CH-21 (δC 128.3, δH 7.27 2H); CH-18, CH-20
(δC 128.5, δH 7.17 2H); CH-19 (δC 125.7, δH 7.16 1H)]. The
ring accounted for four of the 11 observed sp2 resonances, four of
the degrees of unsaturation required by the molecular formula,
and the element of molecular symmetry indicated by the 13C
spectrum. HMBC correlations were observed between the pro-
tons of the methylene triplet CH2-15 (δC 36.1, δH 2.57) and
C-16, while reciprocal correlations between the methines CH-
17/-21 and CH2-15 established the attachment point of the
aromatic ring.
The protons of the olefinic methyl singlet CH3-23 (δC 16.4,
δH 1.66) showed HMBC correlations to two other sp2 carbons,
CH-8 (δC 118.2, δH 5.18) and C-9 (δC 138.7). A further HMBC
correlation from H3-23 to the methylene carbon CH2-10 (δC
39.7, δH 1.96) and a COSY correlation from H-8 to the protons
of another methylene, CH2-7 (δC 30.2, δH 3.17), established this
olefinic system as an isolated double bond. The E-geometry of
the double bond was established from NOE enhancements of
H-8/H2-7, H3-23/H2-11, H3-23/H2-7, and H-8/H2-10 follow-
ing selective irradiation of the resonances.
Figure 1. Structural elucidation of lehualide E (5). COSY couplings are
shown in red, and key NOE and HMBC correlations are shown in green
and blue, respectively. Double-headedarrows depict reciprocal correlations.
established a C6 methylene chain linking the mono-alkyl-
substituted benzene ring and the C-2-CH2-10 segment. Selec-
tive irradiation of both methylene centers in a series of 1D
TOCSY experiments confirmed the connection, completing the
structure of 5.
Lehualide F (6) was isolated as a white solid. Positive-ion
mode HRESIMS analysis of 6 (m/z 393.2042 [M þ Na]þ)
indicated the molecular formula C23H30O4, differing from that of
5 by 14 mass units. The multiplicity-edited HSQC spectrum
accounted for 32 of the 33 protons, indicating the presence of
one exchangeable proton, while the 1H spectrum of 6 appeared
essentially identical to that of 5 except for the presence of only
one methoxy.
The remaining five nonprotonated sp2 carbons, C-2 (δC
162.7), C-3 (δC 128.3), C-4 (δC 159.0), and C-5 (δC 108.3),
could be accounted for only by two fully substituted carbon-
carbon double bonds and a carbonyl. HMBC correlations were
observed from H2-7 to C-5 and C-6, indicating the double-allylic
character of H2-7, consistent with its chemical shift. An HMBC
correlation from H-8 to C-6 established the C-7-C-6 bond.
Further HMBC correlations were observed from the protons of
another olefinic methyl, CH3-22 (δC 10.1, δH 1.85), to C-4, C-5,
and C-6, which together with a homoallylic COSY correlation
between H2-7 and H3-22 established the methyl attachment at
C-5 and the C-5-C-4 and C-5-C-6 connections. The sub-
structure was extended by an HMBC correlation from the
protons of the methoxy OCH3-4 (δC 60.5, δH 4.19) to C-4,
while another methoxy, OCH3-3 (δC 60.4, δH 3.81), correlated
with C-3, revealing a final carbon-carbon double bond. Reci-
procal NOE correlations between OCH3-3 and OCH3-4 estab-
lished the C-3-C-4 connectivity, while further NOE correlations
between OCH3-4 and H3-22 and between H2-7 and H3-22
confirmed the tetrasubstituted diene C-3-C-6, with CH2-7
and CH3-22 on the same side of the C-5-C-6 double bond.
The remaining sp2 center, C-2 (δC 162.7), which must be a
carbonyl, was therefore assigned as an R,β-unsaturated ester on
the basis of its shielded chemical shift. The shielded chemical
shift of the oxygenated C-3 (δC 126.2) is consistent with the
attachment of the carbonyl.8,9 All but one degree of unsaturation
associated with the molecular formula were accounted for by the
phenyl ring, the isolated double bond C-8-C-9, and the C-2-C-6
dienenoate. Furthermore the four oxygen atoms required by the
molecular formula are accounted for by the two oxymethyls and
the ester functionality. The deshielded chemical shift of C-6 (δC
154.8) is consistent with oxygen substitution, and therefore an R-
pyrone moiety, C-2-C-6, similar to that of lehualide A (1), is
established. Absorption maxima observed in the UV spectrum
(λmax 291 nm) and stretching frequencies in the IR spectrum
(νmax 1686, 1649 cm-1) support this assignment,10 while the
chemical shifts of the sp2 carbon resonances are consistent with
those reported for similar systems.8
An R-pyrone (λmax 298 nm) similar to that of 5 was quickly
identified. The oxy-substituted double bond between C-5 and
C-6 was established on the basis of the strong HMBC correla-
tions from the protons of the olefinic methyl H3-21 and
methylene H2-7 to both C-5 and C-6, and weak homoallylic
COSY coupling between H3-21 and H2-7. A further HMBC
correlation from H3-22 to C-4 (δC 162.1) was observed; how-
ever the methoxy observed in 5 was absent, suggesting hydroxy
substitution of C-4. This accounted for the exchangeable proton
(IR: νmax 3250 cm-1, δH 6.50 ppm). HMBC correlations from
protons of the lone methoxy OCH3-3 (δC 60.0, δH 3.86) to C-3
(δC 125.0) were observed as in 5. The R-pyrone system was
completed by the R,β-unsaturated ester carbonyl C-2 (δC
160.4). All other chemical shifts and correlations observed in
the NMR spectra were essentially identical to those of 5, thereby
establishing 6 as the C-4 des-methoxy congener of 5.
A pseudomolecular ion peak observed in the positive-ion
mode HRESIMS spectrum for lehualide G (7) (m/z 421.2355
[M þ Na]þ) indicated a molecular formula of C25H34O4,
differing from 6 by two methylene units. Detailed analysis of
the 1D and 2D NMR spectra of 6 and 7 determined that the two
were chain-length congeners. The only perceivable differences
between the spectra of the two compounds were the methylene
regions of the 1H and 13C spectra. The 1D and 2D NMR data of 7
were consistent with the R-pyrone moiety present in 6, and UV
and IR spectroscopic data supported the assignment. The
extended methylene chain of 7, C-10-C-17, was established
by selective 1D TOCSY irradiation of H2-17 (C-17: δC 36.1, δH
2.61). As the mixing time was increased from 0 to 120 ms, the
methylene resonances H2-16-H2-10 (C-10: δC 40.5, δH 2.01)
were sequentially revealed. Analogous 1D TOCSY irradiation of
H2-10 coupled with COSY and HMBC correlations within the
chain provided confirmation of this assignment and the connec-
tion between the methylenes. HSQC-TOCSY analysis of 7 in
CD3OD over a selected bandwidth (δC: 10-50 ppm) allowed
elucidation of the aliphatic chain: [C-12-C-15 (δC 32.8, 30.6,
30.5, 30.3, δH 1.25-1.29) and C-16 (δC 30.1, δH 1.22)]. Using
this method the methylene resonances C-12-C-16 were clearly
A series of sequential COSY and HMBC correlations begin-
ning at the H2-15 methylene triplet and terminating with H2-10
810
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resolved, and the TOCSY correlations aided concise ordering of
the resonances.
Positive-ion mode HRESIMS analysis of lehualide H (8)
generated a pseudomolecular ion peak at m/z 407.1864 [M þ
Na]þ suitable for the formula C20H32O5S, requiring five degrees
of unsaturation, and incorporation of a sulfur atom. Analysis of
the 1H NMR spectrum of compound 8 revealed, in contrast with
the lehualides E-G, the absence of the phenyl, olefinic proton,
and doubly allylic methylene signals. Similar 2D NMR, IR, and
UV spectroscopic data to those of 5 and the presence of two
methoxy signals indicated retention of the dimethoxy R-pyrone,
therefore accounting for four of the five oxygen atoms, and four
degrees of unsaturation. As with 5, the nonprotonated R-pyrone
ring was assigned on the basis of HMBC and NOE correlations of
H2-7, H3-21, and the methoxy groups OCH3-4 and OCH3-3. The
doubly allylic methylene doublet of 5, CH2-7 (δC 30.2, δH 3.17),
is replaced in 8 by a singly allylic methylene triplet, CH2-7 (δC
30.9, δH 2.44), as evidenced by homoallylic COSY correlation
with H3-21 and HMBC correlations with C-5 and C-6. This
confirmed the absences of the isolated double bond and asso-
ciated olefinic methyl of the previously described structures. The
long alkyl chain characteristic of the lehualides extended between
CH2-7 and CH2-16 (δC 29.2, δH 2.86) and selective 1D TOCSY
irradiations of both methylenes H2-7 and H2-16, with increasing
mixing times (0-200 ms), established the connection between
the two terminating methylenes. The final substructure began at
the deshielded methyl singlet terminus (δC 30.8, δH 2.35).
Strong HMBC correlations from the protons of both the methyl
and H2-16 to an ester carbonyl (δC 196.3) were observed. The
large 1JCH coupling constant and low 1H and 13C chemical shifts
Figure 2. Lehualides H-J (8-10). COSY couplings are shown in red,
and key NOE and HMBC correlations are shown in green and blue,
respectively. Double-headed arrows depict reciprocal correlations.
395.1868 [M þ Na]þ). As with compounds 8 and 9 the C-6
alkyl-substituted R-pyrone system was identified through de-
tailed analysis of both 1D and 2D NMR data and corroborated by
UV and IR spectroscopic data. The extended aliphatic chain,
typical of the latter lehualides, terminates in a deshielded
diastereotopic methylene, CH2-16 (δC 54.9, δH a 2.73, b
2.66). A deshielded methyl singlet (δC 38.7, δH 2.56) was
observed at significantly higher chemical shift than that of the
methyl sulfide 8, indicating a change of functionality. Reciprocal
HMBC correlations between these two centers established their
linkage through a heteroatom bridge. Again the large 1JCH values
of CH2-16 (1JCH Ha 137, Hb 135) and the methyl terminus
(1JCH 137) indicated a sulfur bridge.11a An oxygen-bearing
stereogenic sulfoxide center is consistent with the diastereotopic
nature of CH2-16 and the remaining oxygen suggested by the
pseudomolecular ion. The IR stretch of the functionality was
1
of the C-16 methylene (δC 29.2, δH 2.86, JCH 141) indicated
sulfur attachment.11 The chemical shift of the acetate carbonyl
(δC 196.3) is consistent with a thioacetate moiety, as previously
observed in lehualide C (3) and Dysidea sp.12 Reciprocal weak
HMBC correlations observed from the protons of methylene H2-
16 and the methyl terminus to the respective carbons confirmed
the thioester linkage. The elucidation of this substructure ac-
counted for the sulfur atom, the fifth oxygen atom, and the final
degree of unsaturation required by the molecular formula.
Positive-ion mode HRESIMS analysis of lehualide I (9) (m/z
379.1920 [M þ Na]þ) established the molecular formula as
C19H32O4S. Analysis of the 1D and 2D NMR spectroscopic data,
in conjunction with IR and UV spectra, confirmed the same C-6
alkyl-substituted R-pyrone system present in 8, accounting for
the four degrees of unsaturation and the four oxygen atoms
required by the molecular formula. The alkyl chain was again
elucidated by irradiation of methylenes H2-16 and H2-7 using a
1D TOCSY pulse with various mixing times. Similar to 8 a
methyl singlet was observed (δC 15.6, δH 2.09), and inspection
of 2D NMR spectroscopic data revealed weak HMBC and COSY
cross-peaks with the CH2-16 methylene (δC 34.3, δH 2.49),
indicating the presence of a heteroatom between them. The high
1JCH values and shielded chemical shifts of the aliphatic centers
CH2-16 (1JCH 137) and the methyl terminus (1JCH 137)
established the linkage as a sulfur atom.11 This was corroborated
present at 1027 cm-1 13
apparent lack of optical activity ([R]19.1D 0.00 (c 3.69 g mL-1 E-5
.
It is unclear, however, whether the
,
CHCl3)) is due to the natural occurrence of 10 as a racemic mixture
or small sample size. Stoichiometric oxidation of methyloctyl sulfide
with both H2O2 and KIO4 provided the sulfoxide product,14 which
proved to be a spectroscopic match for the aliphatic portion of the
natural product in both chemical shift and 1JCH coupling. For further
spectroscopic comparison with both 9 and 10, methyloctyl sulfone
was prepared via oxidation of the sulfide with activated MnO2 and
KMnO4115 and displayed significant spectroscopic differences.
The H and 13C NMR spectra of lehualide K (11) are
remarkably similar to those of 8, except for the absence of the
acetyl group. Detailed analysis of both the 1D and 2D NMR
spectra suggested that 11 is the thiol analogue of 8, although the
signal attributed to the S-H proton diminished during the
purification process. The positive-ion mode HRESIMS spectrum
showed a [M þ Na]þ pseudomolecular ion peak at m/z
705.3477. This was consistent with the molecular formula
C36H58O8S2 and indicated that the species had formed a disulfide
dimer. In light of the isolation of the thiol lehualide D (4),
dimerization of 11 during the isolation process is suspected. As is
the case with compounds 8-10, the C-16 methylene has a large
1JCH value (δC 39.3, δH 2.67, 1JCH 139) and shielded chemical
shift. Compound 11 was not isolated in purity exceeding ∼85%,
as persistent impurities (compounds 6 and 8) remained despite
multiple normal-phase column chromatographic purifications
by a weak C-S stretch present in the IR spectrum at 698 cm-1
.
Methyloctyl sulfide was prepared from octyl bromide10a and was
found to have identical spectroscopic characteristics to the
aliphatic portion of the natural product, confirming the structure
of 9 as a methyl sulfide.
The molecular formula of lehualide J (10) was established by
positive-ion mode HRESIMS analysis as C19H32O5S (m/z
811
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Figure 3. Dose-dependent growth inhibition of drug-sensitive yeast to lehualides F, G, H, and I.
and attempted isolation by C-18 and DIOL HPLC under various
solvent conditions.
marine organisms, although there have been numerous examples
reported from terrestrial plants including methionine and cysteine
sulfoxides from brassica and alliaceous vegetables13,18 and trisul-
fides from the roots of some angiosperms.19 Examples from
sponge literature include sulfoxides psammaplin N,20 eudistomin
K,21 and didemnolines C and D11b and the sulfide benzylthio-
crellidone from Crella spinulata.22 The moderate biological activity
displayed by compounds of the lehualide class has prompted
synthetic investigations, and the succinct synthesis of compound 2
was recently reported.23 The isolation of lehualides E-K (5-11),
particularly in light of the S-functionalities present in compounds
8-11, adds to the diversity of chemical functionality obtained
from Plakortis sponges.
A cell-based dose-response assay was used to assess the
susceptibility of yeast to lehualides F (6), G (7), H (8), and I
(9). The compounds were tested against wild-type yeast or a
drug-sensitive (DS) mutant strain that is deleted for the genes
encoding PDR1 and PDR3, transcription factors that act as
master regulators of the pleiotropic drug resistance (PDR)
network in yeast.16 The DS strain is crippled in its ability to
upregulate the PDR response and is up to 200 times more
sensitive to xenobiotics17 than wild-type yeast. Compounds 6, 7,
and 9 inhibited growth of the DS strain by 60%, 68%, and 67%,
respectively, at the highest concentration tested (Figure 3).
Interestingly, lehualide H (8) did not inhibit yeast growth at
any concentration, suggesting that this compound either is
inactive in yeast, has a potency profile that is not captured in
the tested concentration range, or is highly unstable. In contrast,
none of the compounds inhibited the growth of wild-type yeast at
any concentration tested (data not shown). The increased
sensitivity of the DS mutant suggests the lehualides are substrates
for one of the PDR efflux pumps. To more accurately gauge the
potency of the lehualides, the dose-response experiment was
repeated with 6 using a higher concentration range. Lehualide F
(6) exhibited an IC50 of 44 μΜ. Furthermore, a 48% growth
inhibition of the wild-type strain at the highest concentration was
observed, indicating a half log decrease in sensitivity compared to
the DS strain and supporting the hypothesis that the lehualides
are substrates for the PDR efflux pumps.
’ EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were ob-
tained on a Autopol II automatic polarimeter, UV spectra were acquired
on an Agilent 8453 UV/visible spectrometer, while IR spectra were
recorded using a Perkin-Elmer Spectrum One FT-IR spectrometer. NMR
spectra were obtained using a 600 MHz Varian DirectDrive spectrometer
equipped with a triple resonance HCN cryogenic probe, operating at 600,
150, and 60 MHz for 1H, 13C, and 15N nuclei, respectively. Spectra were
recorded in CD3OD (Cambridge Isotopes) and CDCl3 (Merck), with
chemical shifts δ (ppm) referenced to the residual solvent peak
[(CD3OD: δC 49.00, δH 3.31) and (CDCl3: δC 77.16, δH 7.26)].24
HREIMS measurements were obtained using a Waters Q-TOF Premier
Tandem mass spectrometer. Silica gel plates were used for analytical thin-
layer chromatography. 2,3-Dihydroxypropoxypropyl-derivatized silica gel
was used for all normal-phase column chromatography, while Supelco
HP20 and HP20SS polystyrene(divinylbenzene) resins were used for
reversed-phase column chromatography. HPLC-grade CH2Cl2 (Merck)
and MeCN (Fluka) were used generally and for HPLC purification; all
other solvents were distilled from glass immediately prior to use. All
chemical reagents were purchased from Sigma-Aldrich and Penta Inter-
national Corporation and used without further purification.
Lehualides F-I (6-9) also displayed cytotoxicity against the
human promyelocytic leukemia (HL-60) cell line. Using a
standard 2-day cell proliferation assay, compounds 6 and 7
exhibited IC50 values for growth inhibition of 6.2 and 5.4 μΜ,
respectively, while interestingly the thioacetate and sulfide
compounds 8 and 9 showed weaker inhibition of the same cell
line with respective IC50 values of 14.6 and 10.8 μΜ.
While the sulfur functionalities of lehualides H (8) and K (11)
have been previously encountered in compounds 3 and 4, the
methyl sulfide and sulfoxide functionalities present in 9 and 10
are thought to be unprecedented in metabolites from sponges of
this genus. Sulfoxides and sulfides have rarely been reported from
Animal Material. The sponge was collected from the horizontal
ceiling of a large cave at a depth of 12-15 m, on ‘Eua Island, Kingdom of
Tonga. It was identified as a species of Plakortis (order Homosclero-
phorida, family Plakinidae), similar to P. ceylonica (Dendy, 1905). The
sponge formed small oval pendant encrustations about 3 cm thick; the
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Table 1. NMR Spectroscopic Data of Lehualide E (5)
(CDCl3, 1H 600 MHz; 13C 150 MHz)
Table 2. NMR Spectroscopic Data of Lehualides F and G
(6, 7) (CDCl3, 1H 600 MHz; 13C 150 MHz)
HMBC
6
7
position δC, mult.
δH (J, Hz) COSY
(HfC)
NOEa
position
δC, mult.
δH (J, Hz)
δC
δH (J, Hz)
2
3
162.7, C
128.3, C
2
160.3, C
125.0, C
59.8, CH3
157.9, C
105.6, C
157.3, C
30.2, CH2
117.2, CH
139.0, C
39.7, CH2
27.8, CH2
160.4, C
3
125.0, C
OCH3 3 60.5, CH3 3.81, s
159.0, C
OCH3 4 60.6, CH3 4.19, s
3
4
OCH3 4
OCH3 3
4
3.90, s
60.0, CH3
3.86, s
4
158.0, C
OCH3 3; 22
5
105.7, C
5
108.3, C
6
157.3, C
6
154.8, C
7
30.3, CH2 3.17, d (6.9)
117.5, CH 5.17, t (7.2)
138.7, C
7; 22b 8; 6; 23; 5b
6; 23 7; 23; 6b
7
3.20, d (7.2)
5.17, t (7.0)
30.3, CH2
117.1, CH
3.21, d (7.0)
5.16, t (7.0)
139.1, C
8
8
10
9
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
OH
1.97, t (8.4)
39.7, CH2
27.8, CH2
29.4, CH2
1.97, t (7.4)
1.36, quin (7.1)
1.20 - 1.33, m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
39.7, CH2 1.96, t (7.5)
27.8, CH2
10; 23 11; 23; 9; 8
11; 9
8
1.38, quin (7.6)
10; 12; 9
29.29, CH2 1.32, quin (8.0)
29.9, CH2
29.26, CH2 1.24 - 1.29, m
29.55, CH2 1.20 - 1.33, m
29.58, CH2 1.20 - 1.33, m
29.45, CH2 1.20 - 1.33, m
29.3, CH2 1.33, quin (7.3) 13; 11
31.5, CH2
36.1, CH2
143.0, C
1.59, q (8.0)
2.57, t (7.2)
31.6, CH2 1.59, quin (8.1) 14; 12 15; 16; 13
36.1, CH2 2.57, t (7.7)
143.0, C
13
14; 16; 17; 21
31.7, CH2
36.1, CH2
143.1, C
1.59, quin (6.9)
2.61, t (7.8)
128.5, CH
128.4, CH
125.6, CH
128.4, CH
128.5, CH
9.4, CH3
7.17, d (8.2)
7.22, t (7.7)
7.16, t (6.0)
7.22, t (7.7)
7.17, d (8.2)
1.96, s
128.5, CH 7.19, d (7.4)
128.4, CH 7.29, t (7.0)
125.7, CH 7.19, t (7.8)
128.4, CH 7.29, t (7.0)
128.5, CH 7.19, d (7.4)
10.3, CH3 1.85, s
18
19; 21
17; 19 16; 20
18; 20 17; 21
19; 21 16; 18
128.5, CH
128.3, CH
125.7, CH
128.3, CH
128.5, CH
7.17, d (7.4)
7.27, t (7.3)
7.16, t (6.3)
7.27, t (7.3)
7.17, d (7.4)
20
7b
17; 19
5; 4; 6; 7b
OCH3 3; 7
16.3, CH3
1.68, s
16.4, CH3 1.66, s
9; 8; 10
11; 7
a Selected correlations. b Weak correlation.
9.5, CH3 1.96, s
16.5, CH3
1.68, s
texture is cork-like and rubbery and covered in wide shallow nodules
about 3 mm high. The sponge was dark chocolate-purplish brown, with a
dense interior. Diod spicules of approximately 180-250 μm long were
found. A voucher specimen (PTN3_20A) has been deposited in the
collection of Dr. James Bell at the School of Biological Sciences, VUW,
Wellington.
6.50, bs
6.50, bs
that regulate the expression of pleiotropic drug resistance efflux
pumps.16 For the assay, yeast cultures were grown to stationary phase
in synthetic complete (SC) media26 and diluted to 5 ꢀ 105 cells/mL in
fresh SC, and 100 μL aliquots were added to 96-well tissue culture plates.
Seven point, half-log serial dilutions of compounds 6-9 (10 to 0.1 mM)
were prepared from 10 mM working stocks dissolved in dimethyl
sulfoxide (DMSO). A 1 μL volume of diluent was added per well, giving
a final concentration range of 100 to 0.01 μΜ. DMSO (1% final
concentration) was added to one well as a negative control. To achieve
the highest concentration for compound 6 in the dose-response experi-
ment, 3 μL of working stock was added to give a final concentration of
300 μM L-1 along with a control well containing 3% DMSO. Plates were
mixed by vortexing and incubated at 30 °C for 18 h. Cell growth was
quantified by measuring optical density (OD) at 590 nm using a Wallac
EnVision 2102 multilabel plate reader (Perkin-Elmer). Residual growth
Extraction of Plakortis and Isolation of Lehualides E-K
(5-11). The frozen sample (36.0 g) was extracted twice for 14 h in 100
mL of MeOH at room temperature. The second extract, followed by the
first, were passed through a column of 40 mL of HP20 resin, and the
eluents were combined, diluted with 200 mL of H2O, passed through
the column again, further diluted to 25% MeOH, and passed through the
column once more. The column was washed with H2O, then eluted,
generating three 100 mL fractions of 30%, 75%, and 100% Me2CO/
H2O. The 75% and 100% Me2CO/H2O fractions were analyzed by 1D
and 2D NMR, then subjected to further reversed-phase purification
using HP20SS resin, generating fractions of 30%, 40%, 50%, 60%, 70%,
80%, 90%, and 100% Me2CO/H2O. Further purification of the 70-
100% Me2CO/H2O fractions by DIOL oil chromatography (hexanes/
CH2Cl2/EtOAc) and reversed-phase HPLC (C-18, 85% MeCN/H2O)
yielded lehualides E-I (5-9) in varying ratios (see compound data)
and lehualide K (11) with persistent impurities of compounds 7 and 8.
DIOL oil chromatography (hexanes/CH2Cl2/EtOAc) and HPLC
(C-18, 95% MeCN/H2O) purification of the 60% Me2CO/H2O
HP20SS fraction afforded lehualide J (10).
(%) was calculated for each concentration using the formula (ODexp
/
ODDMSO) ꢀ 100. Results are reported as the mean ( standard error for
two independent experiments performed in triplicate. The IC50 values
were determined using Prism (Graphpad Software).
Tests for inhibition of growth of human HL-60 promyeloid leukemic
cells were carried out using the MTT cell proliferation assay as
previously described.27
Biological Testing. A dose-response assay was performed using
two Saccharomyces cerevisiae strains: wild-type YCG117 (MATR
ura3Δ0::NAT derived from strain Y709225) and YCG198 (MATR
PDR1 Δ::NAT PDR3 Δ::URA3, derived from strain Y820525), a drug-
sensitive mutant strain that lacks the two main transcription factor genes
Lehualide E (5): clear oil, 540 μg; UV (MeOH) λmax (log ε), 235
(3.80), 295 (3.73); IR (film) νmax 3250, 2928, 2853, 1687, 1647, 1561,
1440, 1221, 1094 cm-1; 1H and 13C NMR data, see Table 1; HRESIMS
m/z 385.2399 [M þ Na]þ (calcd for C24H32O4Na, 385.2398; Δ 0.2
ppm).
813
dx.doi.org/10.1021/np100868t |J. Nat. Prod. 2011, 74, 809–815

Journal of Natural Products
ARTICLE
Lehualide F (6): white solid, 2.79 mg; UV (MeOH) λmax (log ε),
235 (3.81), 282 (3.70) nm; IR (film) νmax 3250, 2929, 2854, 1686, 1648,
Lehualide G (7): white solid, 18.32 mg; UV (MeOH) λmax (log ε),
235 (3.44), 291 (3.55) nm; IR (film) νmax 3250, 2926, 2854, 1686, 1649,
1
1
1560, 1439, 1220, 1094 cm-1; H and 13C NMR data, see Table 2;
1563, 1439, 1276, 1094 cm-1; H and 13C NMR data, see Table 2;
HRESIMS m/z 393.2042 [M þ Na]þ (calcd for C23H30O4Na,
393.2042; Δ 0.0 ppm).
HRESIMS m/z 421.2355 [M þ Na]þ (calcd for C25H34O4Na,
421.2355; Δ 0.0 ppm).
Lehualide H (8): white solid, 1.40 mg; UV (MeOH) λmax (log ε),
255 (3.68), 297 (4.29) nm; IR (film) νmax 2925, 2853, 1709, 1690, 1651,
1
Table 3. H and 13C NMR Spectroscopic Data of Lehualides
1
1569, 1458, 1227, 670 cm-1; H and 13C NMR data, see Table 3;
H and K (8 and 11) (CDCl3, 1H 600 MHz; 13C 150 MHz)
HRESIMS m/z 407.1864 [M þ Na]þ (calcd for C20H32O5SNa,
8
11
δH (J, Hz)
407.1868; Δ -1.0 ppm).
Lehualide I (9): clear oil, 646 μg; UV (MeOH) λmax (log ε), 249
(3.12), 297 (3.96) nm; IR (film) νmax 2926, 2854, 1712, 1652, 1570,
position
δC , mult.
δH (J, Hz)
δC
1
1460, 1370, 1229, 699 cm-1; H and 13C NMR data, see Table 4;
2
3
162.7, C
127.9, C
162.7, C
128.4, C
HRESIMS m/z 379.1920 [M þ Na]þ (calcd for C19H32O4SNa,
379.1919; Δ 0.3 ppm).
OCH3 3
60.4, CH3 3.81, s
158.9, C
60.5, CH3 3.81, s
158.9, C
Lehualide J (10): clear oil, 1.95 mg; [R]19.1D 0.00 (c 3.69 g mL-1
E-5, CHCl3); UV (MeOH) λmax (log ε), 249 (2.88), 288 (3.52) nm; IR
(film) νmax 2925, 2855, 1713, 1652, 1569, 1458, 1371, 1228, 1098, 699
4
OCH3 4
60.6, CH3 4.16, s
108.5, C
60.6, CH3 4.17, s
108.5, C
1
cm-1; H and 13C NMR data, see Table 4; HRESIMS m/z 395.1868
5
[M þ Na]þ (calcd for C19H32O5SNa, 395.1868; Δ 0.0 ppm).
Lehualide K (11): clear oil, 2.12 mg (impure); UV (MeOH) λmax
(log ε), 240 (3.41), 296 (3.60) nm; IR (film) νmax 2926, 2852, 1690,
1651, 1569, 1458, 1227, 672 cm-1; 1H and 13C NMR data, see Table 3;
HRESIMS [M þ Na]þ m/z 705.3477 (calcd for C36H58O8S2Na,
705.3471; Δ 0.9 ppm).
Preparation of Methyloctyl Sulfide. Methyloctylthiol (0.5 mL,
mmol) was stirred under ambient conditions in 20 mL of MeOH. One
equivalent of MeI was added, and the reaction was heated at reflux for 20
min, affording the monomethylation product in a 40% yield.
Methyloctyl sulfide: colorless liquid; IR (film) νmax 698 cm-1; 1H
NMR (CDCl3, 600 MHz) δ 2.48 (2H, t, J = 7.2, H-2), 2.09 (3H, s, H-1),
1.58 (2H, quin, J = 7.2, H-3), 1.37 (2H, quin, J = 7.2, H-4), 1.23-1.30
(8H, m, H-5-H-8), 0.88 (3H, t, J = 7.2, H-9); 13C NMR (CDCl3, 150
MHz) δ 34.2 (C-2), 31.8 (C-8), 29.15 (C-5), 29.13 (C-7), 29.1 (C-3),
28.8 (C-4), 22.6 (C-6), 15.5 (C-1), 14.0 (C-9).
6
155.9, C
155.9, C
7
30.9, CH2 2.44, t (7.7)
30.9, CH2 2.44, t (7.7)
8
27.5, CH2 1.58, quin (7.5) 27.5, CH2 1.59, quin (7.5)
9
29.3, CH2 1.28, m
29.4, CH2 1.27, m
29.54, CH2 1.25, m
29.55, CH2 1.25, m
29.48, CH2 1.23, m
28.9, CH2 1.35, m
29.48, CH2 1.55, t (7.2)
29.2, CH2 2.86, t (7.3)
29.33, CH2 1.27, m
10
11
12
13
14
15
16
29.62, CH2 1.28, m
29.59, CH2 1.28, m
29.55, CH2 1.28, m
29.44, CH2 1.28, m
28.6, CH2 1.37, quin (7.6)
29.3, CH2 1.66, quin (7.4)
39.2, CH2 2.67, t (7.4)
SCdOCH3 196.3, C
SCdOCH3 30.8, CH3 2.35, s
17
10.5, CH3 1.84, s
10.4, CH3 1.84, s
1
Table 4. H and 13C NMR Spectroscopic Data of Lehualides I and J (9, 10) (CDCl3, 1H 600 MHz; 13C 150 MHz)
9
10
position
δC , mult.
δH (J, Hz)
δC
δH (J, Hz)
162.7, C
2
3
162.6, C
127.7, C
127.8, C
OCH3 3
60.4, CH3
158.8, C
3.85, s
60.5, CH3
60.6, CH3
3.81, s
4
158.9, C
OCH3 4
60.5, CH3
108.3, C
4.21, s
4.17, s
5
108.5, C
6
155.8, C
155.9, C
7
30.8, CH2
27.4, CH2
29.5, CH2
29.4, CH2
29.3, CH2
29.2, CH2
29.2, CH2
28.8, CH2
29.2, CH2
34.3, CH2
2.45, t (7.7)
1.59, quin (7.2)
1.28, m
30.9, CH2
27.5, CH2
29.36, CH2
29.28, CH2
28.9, CH2
29.44, CH2
29.39, CH2
29.24, CH2
22.7, CH2
54.9, CH2
2.44, t (7.7)
1.59, quin (7.7)
1.28, m
8
9
10
11
12
13
14
15
16
1.28, m
1.30, m
1.28, m
1.44, m
1.28, m
1.24, m
1.28, m
1.27, m
1.37, quin (8.0)
1.58, quin (7.3)
2.49, bt (7.7)
1.32, m
1.76, quin (7.0)
a 2.73, ddd (13.0, 9.0, 6.1)
b 2.66, ddd (12.7, 9.2, 6.6)
2.56, s
SMe; OdSMe
15.6, CH3
10.3, CH3
2.09, bs
1.85, s
38.7, CH3
10.4, CH3
17
1.85, s
814
dx.doi.org/10.1021/np100868t |J. Nat. Prod. 2011, 74, 809–815

Journal of Natural Products
ARTICLE
Preparation of Methyloctyl Sulfoxide A. Methyloctyl sulfide
(0.5 mL, 2.6 mmol) was stirred in 30 mL of MeOH, after which 1.5 equiv
(4.0 mmol) of KIO4 in 3 mL of H2O was added, and the cloudy solution
stirred at room temperature for 12 h. Methyloctyl sulfoxide was
produced in 100% yield.
Preparation of Methyloctyl Sulfoxide B. Methyloctyl sulfide
(0.5 mL, 2.6 mmol) was stirred in 30 mL of MeOH and treated with 1.5
equiv (4.0 mmol) of 30% H2O2 aqueous solution. The reaction was
stirred for 12 h under ambient conditions, affording the sulfoxide
product in 100% yield.
Methyloctyl sulfoxide: white, crystalline solid; IR (film) νmax
1027, 748 cm-1; 1H NMR (CDCl3, 600 MHz) δ 2.73 (1H, ddd, J = 14.9,
9.8, 5.8, H-2a), 2.64 (1H, ddd, J = 16.2, 9.3, 5.8, H-2b), 2.56 (3H, s, H-1),
1.75 (2H, quin, J = 7.0, H-3), 1.44 (2H, sep, J = 8.4, H-4), 1.22-1.34
(8H, m, H-5-H-8), 0.88 (3H, t, J = 7.1 Hz, H-9); 13C NMR (CDCl3,
150 MHz) δ 54.9 (C-2), 38.7 (C-1), 31.9 (C-8), 29.3 (C-5), 29.2 (C-7),
28.9 (C-4), 22.8 (C-6), 22.7 (C-3), 14.2 (C-9); HRESIMS [M þ Na]þ
m/z 199.1133 (calcd for C9H20OS, Δ -2.0 ppm); HRESIMS [M þ
H]þ m/z 159.1207 (calcd for C9H20S, Δ 1.3 ppm).
Preparation of Methyloctyl Sulfone. Methyloctyl sulfide (0.5
mL, 2.6 mmol) was stirred in 25 mL of CH2Cl2 with excess 3:1 activated
MnO2/KMnO4. The suspension was stirred for 96 h, then filtered, and
the residue was washed with a 1:1 mixture of MeOH and CH2Cl2. The
filtrate was evaporated, affording a white crystalline solid in 100% yield.
Methyloctyl sulfone: white, crystalline solid; IR (film) νmax 1273,
1141, 1128, 1117, 765, 749 cm-1; 1H NMR (CDCl3, 600 MHz) δ 3.00
(2H, t, J = 8.4 Hz, H-2), 2.89 (3H, s, H-1), 1.85 (2H, quin, J = 7.8 Hz,
H-3), 1.44 (2H, quin, J = 7.2 Hz, H-4), 1.24-1.34 (8H, m, H-5 - H-8),
0.88 (3H, t, J = 7.2 Hz, H-9); 13C NMR (CDCl3, 150 MHz) δ 55.0 (C-2),
40.5 (C-1), 31.8 (C-8), 29.14 (C-5), 29.05 (C-6), 28.5 (C-4), 22.7 (C-7),
22.6 (C-3), 14.2 (C-9).
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’ ASSOCIATED CONTENT
Supporting Information. 1H, 13C, gCOSY, HSQCad,
S
b
and gHMBC NMR spectra for compounds 5-11 and the
bsHSQC-TOCSYad spectrum for compound 7. Above-water
photograph of Plakortis specimen. This information is available
free of charge via the Internet at http://pubs.acs.org.
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’ AUTHOR INFORMATION
Corresponding Author
*E-mail: Peter.Northcote@vuw.ac.nz. Tel: þ64 4 463 5960. Fax:
þ64 4 463 5247.
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132, 11422–11424.
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’ ACKNOWLEDGMENT
(25) Tong, A. H. Y.; Boone, C.; Ian, S.; Michael, J. R. S., In Methods in
Microbiology; Academic Press: New York, 2007; Vol. 36, pp 369-386.
(26) Amberg, D. C.; Burke, D.; Strathern, J. N. Methods in Yeast
Genetics: a Cold Spring Harbor Laboratory Course Manual, 2005 ed.; Cold
Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2005; p xvii.
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Miller, J. H. Apoptosis 2001, 6, 207–219.
The authors thank Dr. Y. Lu (Industrial Research Limited) for
HRESIMS measurements. We gratefully acknowledge Dr. J. Bell
in the School of Biological Sciences, Victoria University of
Wellington, for confirmation of sponge identification. We re-
cognize the award of a Victoria University of Wellington Ph.D.
scholarship and a Curtis-Gordon research grant (J.M.B.), and
wish to thank the Tongan Ministry of Fisheries for collection
permission.
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