Lamellarins as inhibitors of P-glycoprotein-mediated multidrug resistance in a human colon cancer cell line

Article abstract of DOI:10.1002/asia.201101049

Chemical analysis of a Didemnum sp. (CMB-01656) collected during scientific Scuba operations off Wasp Island, New South Wales, yielded five new lamellarins A1 (1), A2 (2), A3 (3), A4 (4) and A5 (5) and eight known lamellarins C (6), E (7), K (8), M (9), S (10), T (11), X (12) and χ (13). Analysis of a second Didemnum sp. (CMB-02127) collected during scientific trawling operations along the Northern Rottnest Shelf, Western Australia, yielded the new lamellarin A6 (14) and two known lamellarins G (15) and Z (16). Structures were assigned to 1-16 on the basis of detailed spectroscopic analysis with comparison to literature data and authentic samples. Access to this unique library of natural lamellarins (1-16) provided a rare opportunity for structure-activity relationship (SAR) investigations, probing interactions between lamellarins and the ABC transporter efflux pump P-glycoprotein (P-gp) with a view to reversing multidrug resistance in a human colon cancer cell line (SW620 Ad300). These SAR studies, which were expanded to include the permethylated lamellarin derivative (17) and a series of lamellarin-inspired synthetic coumarins (19-24) and isoquinolines (25-26), successfully revealed 17 as a promising new non-cytotoxic P-gp inhibitor pharmacophore. Copyright

Full text of DOI:10.1002/asia.201101049

DOI: 10.1002/asia.201101049
Lamellarins as Inhibitors of P-Glycoprotein-Mediated Multidrug Resistance
in a Human Colon Cancer Cell Line
[a]
Fabien Plisson, Xiao-Cong Huang, Hua Zhang, Zeinab Khalil, and Robert J. Capon*
Abstract: Chemical analysis of a Di-
demnum sp. (CMB-01656) collected
during scientific Scuba operations off
Wasp Island, New South Wales, yielded
five new lamellarins A1 (1), A2 (2), A3
and Z (16). Structures were assigned to
1–16 on the basis of detailed spectro-
scopic analysis with comparison to lit-
erature data and authentic samples.
Access to this unique library of natural
lamellarins (1–16) provided a rare op-
portunity for structure–activity rela-
tionship (SAR) investigations, probing
interactions between lamellarins and
the ABC transporter efflux pump P-
glycoprotein (P-gp) with a view to re-
versing
multidrug
resistance
in
a human colon cancer cell line (SW620
Ad300). These SAR studies, which
were expanded to include the perme-
thylated lamellarin derivative (17) and
a series of lamellarin-inspired synthetic
coumarins (19–24) and isoquinolines
(25–26), successfully revealed 17 as
a promising new non-cytotoxic P-gp in-
hibitor pharmacophore.
(
3), A4 (4) and A5 (5) and eight
known lamellarins C (6), E (7), K (8),
M (9), S (10), T (11), X (12) and
c (13). Analysis of a second Didemnum
sp. (CMB-02127) collected during sci-
entific trawling operations along the
Northern Rottnest Shelf, Western Aus-
tralia, yielded the new lamellarin A6
Keywords: cancer
·
inhibitor
·
lamellarin · multidrug resistance ·
p-glycoprotein
(
14) and two known lamellarins G (15)
Introduction
important anticancer agents, including anthracyclins (doxor-
ubicin and daunorubicin), vinca alkaloids (vinblastine and
vincristine), and taxanes (paclitaxel and docetaxel), render-
ing cancer cell lines overexpressing P-gp multidrug-resist-
Multidrug resistance (MDR) is a major challenge to modern
healthcare, seriously compromising the effectiveness of
a wide variety of therapeutic agents, most notably anticancer
and anti-infective drugs. ATP-binding cassette (ABC) trans-
porters are plasma membrane proteins and an important de-
terminant of MDR, with the human MDR1 gene product P-
glycoprotein (P-gp), also known as ABCB1 and MDR1,
being the first ABC transporter to be characterized by its
ability to confer MDR in cancer cells. P-gp is expressed in
many normal cells, including those in kidney, liver, colon,
pancreas, and adrenal tissues, and in endothelial cells at the
blood–brain barrier, where its broad substrate recognition
and high transport capacity provide cellular protection
against many endogenous and exogenous toxins. Notwith-
standing its important natural role, cancers that acquire the
ability to overexpress P-gp (i.e., acute myeloid leukemia,
lung carcinoma, neuroblastoma, and advanced ovarian and
breast carcinoma) typically correlate with poor patient prog-
[3]
ant. Not surprisingly, pharmaceutical inhibition of P-gp
and the associated drug efflux has been viewed as an attrac-
tive strategy for combating MDR. Unfortunately, and de-
spite much effort, clinically useful P-gp inhibitors have
proved elusive. Key among the hurdles faced in bringing P-
gp inhibitors to the clinic is the need to engineer for high in
vivo P-gp inhibitory potency and selectivity, with low toxici-
ty, while not adversely influencing the normal processes that
regulate the safe and timely distribution and clearance of
highly cytotoxic chemotherapeutic agents. Notwithstanding
these concerns, should a clinically useful P-gp inhibitor be
developed, the implications for cancer treatment and patient
survival could be profound.
While not underestimating the scale of the challenge out-
lined above, we were encouraged to note that a number of
natural product P-gp inhibitors (e.g., cyclosporine A and
quinine) have attracted considerable attention, while also
observing that other natural product-derived inhibitor struc-
ture classes have been reported but have yet to be fully ex-
plored. In the latter category our attention was drawn to the
lamellarin class of marine alkaloids. First isolated in 1985 by
Faulkner et al. from a Pacific Ocean marine prosobranch
mollusk (Lamellaria sp.), several dozen lamellarins have
since been reported from ascidia (Didemnum spp.), sponges
[1]
[
2]
nosis. This correlation is a function of P-gp utilizing ATP
hydrolysis to drive transmembrane efflux of many clinically
[
a] F. Plisson, X.-C. Huang, Dr. H. Zhang, Z. Khalil, Prof. R. J. Capon
Division of Chemistry and Structural Biology
Institute for Molecular Bioscience
The University of Queensland
60 Carmody Road, St Lucia, Brisbane, 4072, Queensland (Australia)
3
Fax : (+61)7-334-62090
(
Dendrilla cactos), and mollusks (Coriocella hibyae) sourced
E-mail: r.capon@uq.edu.au
from many regions (i.e., the Indian, Pacific, and Southern
Oceans, and the Arabian Sea). A number of lamellarins
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201101049.
[4]
Chem. Asian J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
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FULL PAPERS
have been reported to exhibit significant cytotoxicity, with
IC50 values in the submicromolar range, against a range of
Table 1. Chemical structures of lamellarins 1–18.
[4–5]
cancer cell lines,
with a subset of lamellarins being identi-
fied as effective against P-gp–overexpressing MDR cancer
[6]
cell lines. With respect to MDR, Quesada et al. demon-
strated in 1996 that P-gp–mediated efflux of doxorubicin,
daunorubicin, and vinblastine in a murine lymphoid leuke-
mia cell line (P388/Schabel) could be fully reversed by co-
treatment with a subcytotoxic 2 mm dosage of lamellarin I
[
7]
(
18). The same authors alluded to other lamellarins dis-
1
2
3
4
5
6
7
D5,6[a]
#
R
R
R
R
R
R
R
playing P-gp inhibitory properties (although the data were
not shown), and went on to patent the lamellarin class for
the treatment of MDR cancers. While interest in the la-
mellarin structure class has been sustained over the quarter
century since their first discovery, with many valuable con-
tributions from natural products, synthetic, and medicinal
1
2
3
4
5
6
7
8
9
1
1
1
1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
Me
Me
Me
Me
H
Me
Me
Me
H
H
H
H
H
H
H
Me
H
H
Me
Me
Me
H
Me
Me
H
Me
Me
H
H
H
Me
Me
H
Me
Me
Me
H
H
H
OH
H
H
H
OMe
OH
OH
OH
H
OMe
OH
H
–
–
–
–
[7]
Me
Me
H
[8]
5
,6
H
H
D
–
–
–
D
–
–
D
–
-
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
Me
Me
H
H
H
H
Me
Me
[4–5]
chemists,
these reports have not extended to a structure–
5
5
,6
,6
activity relationship (SAR) study aimed at probing and de-
fining molecular interactions between lamellarins and P-gp.
We took the view that such an SAR analysis would advance
our understanding of this unique marine pharmacophore
and potentially promote the future development of lamellar-
in-inspired P-gp inhibitors.
0
1
2
3
H
Me
Me
H
H
Me
H
H
14
15
1
1
1
Me
Me
Me
Me
H
H
H
H
H
H
H
Me
Me
-
-
-
-
6
7
8
H
Me
Me
Me
Me
OMe
Results and Discussion
5
,6
[
a] D , unsaturated bond C-5/C-6.
As part of a marine biodiscovery program we assessed a li-
brary of about 2600 southern Australian marine inverte-
brates and algae for growth inhibitory activity against paren-
tal (SW620) and P-gp–overexpressing (SW620 Ad300) colon
have elected to label the new examples of this structure
class as lamellarins A1–A6.]
cancer cell lines. Chemical and spectroscopic profiling
Structures were assigned to lamellarins 1–16 (Table 1) on
the basis of detailed spectroscopic analysis with comparison
to literature data and authentic samples where available.
The C NMR data for 1–16 are summarized in Table 2,
while comprehensive accounts of 1D and 2D NMR data are
documented in the Supporting Information. The structure
elucidation of the new lamellarins A1–A6 (1–5, 14) and la-
mellarin c (13) are presented below, supported by Fig-
ures 1–7 which illustrate key 2D NMR correlations.
1
(HPLC–DAD–MS and H NMR) of a particularly cytotoxic
extract from a Didemnum sp. (CMB-01656) specimen col-
lected during scientific Scuba operations off Wasp Island,
New South Wales, revealed a complex mixture of aromatic
alkaloids. This mixture was subsequently resolved by solvent
extraction, partitioning and trituration, followed by re-
versed-phase HPLC, to yield thirteen members of the lamel-
larin class of marine alkaloids. These metabolites included
five new examples, lamellarin A1 (1), lamellarin A2 (2), la-
mellarin A3 (3), lamellarin A4 (4), and lamellarin A5 (5),
1
3
High-resolution mass spectrometry (HRMS) data, ac-
quired in ESI+ mode, were obtained for 1–5, 13, and 14.
[9]
+
together with eight known examples, lamellarin C (6), la-
The data for 1 revealed a quasi-molecular ion (M+H) cor-
[
10]
[11]
[11]
mellarin E (7), lamellarin K (8), lamellarin M (9), la-
responding to a molecular formula C H NO , with NMR
2
7
21
8
[12]
[13]
[13]
mellarin S (10), lamellarin T (11), lamellarin X (12),
and lamellarin c (13) (of which the latter has previously
been first described as an acetylated derivative and syn-
[14]
[15]
thesized in 2006 ). To support lamellarin structure–activity
relationship investigations described later in this report, we
examined a second Didemnum sp. (CMB-02127) sample col-
lected during scientific trawling operations along the North-
ern Rottnest Shelf, Western Australia. Although not a priori-
ty hit in our screening, this extract nevertheless analyzed for
a complex mixture of alkaloids that included as minor me-
tabolites the new lamellarin A6 (14) together with the
[10]
[16]
known lamellarin G (15) and lamellarin Z (16). [Note:
in extending the existing lamellarin trivial nomenclature, we
6
Figure 1. Key 2D NMR ([D ]DMSO) correlations for lamellarin A1 (1).
&
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www.chemasianj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 0000, 00, 0 – 0
ÝÝ These are not the final page numbers!

Lamellarins as inhibitors of P-gp-mediated multidrug resistance
1
3
[c]
6
Table 2. C NMR ([D ]DMSO) data for lamellarins 1–16.
[
a]
[a]
#
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(12)
(13)
(14)
(15)
(16)
1
2
3
5
6
6
7
8
9
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
7
8
9
1
1
2
2
114.6
127.6
n.d.
115.7
127.3
112.6
41.7
114.6
127.5
n.d.
42.0
27.8
114.6
127.5
112.1
42.0
27.8
125.7
115.0
146.0
143.5
113.4
118.5
135.9
125.4
117.4
145.8
145.1
116.7
121.2
109.2
144.6
103.2
145.9
141.9
108.8
154.4
–
n.d.
129.0
n.d.
116.0
127.6
n.d.
41.3
21.2
115.0
127.2
112.5
41.6
115.7
127.8
112.9
41.9
115.0
129.1
107.1
121.2
107.7
112.3
148.9
133.5
153.0
97.5
121.1
136.0
125.6
115.2
148.0
146.5
116.6
124.0
108.4
147.0
103.9
145.9
144.8
105.9
154.6
–
114.5
127.3
112.3
41.9
27.6
127.2
115.2
146.9
145.9
109.2
118.2
136.0
125.5
117.7
146.1
145.3
116.6
121.6
109.3
144.7
103.3
146.0
142.0
108.7
154.5
–
114.7
128.3
107.3
120.8
107.2
111.7
145.5
135.7
152.6
97.0
120.7
133.0
127.0
117.8
147.6
148.0
113.2
121.7
108.0
147.6
103.4
146.3
144.5
105.2
n.d.
114.2
127.6
n.d.
114.2
n.d.
n.d.
114.2
126.7
112.4
41.9
114.7
126.9
112.4
41.9
27.5
127.1
115.2
147.0
145.9
109.3
118.0
136.0
125.3
117.8
146.2
145.4
116.7
121.4
110.2
144.6
100.7
147.8
142.8
108.4
154.3
–
42.0
27.6
127.3
115.3
147.0
146.1
109.2
118.2
136.2
125.4
114.6
148.6
146.5
116.4
123.3
109.2
144.7
103.4
145.9
142.4
108.6
n.d.
n.d.
42.0
27.5
127.1
115.3
146.9
145.9
109.2
118.1
135.8
125.4
114.6
148.4
146.5
116.3
123.4
108.8
146.0
103.6
146.6
144.4
105.1
n.d.
41.7
27.3
127.0
115.0
146.7
145.8
108.9
117.0
135.9
125.2
114.2
148.5
146.6
116.1
122.9
110.2
144.4
100.4
147.7
142.7
107.9
n.d.
21.3
112.9
124.2
111.8
147.5
146.6
109.8
118.6
n.d.
125.5
114.5
148.8
147.1
117.2
123.5
108.9
145.4
103.7
147.1
142.3
109.8
n.d.
21.1
21.5
27.4
a
114.3
147.2
136.3
150.7
100.9
122.5
135.4
125.5
114.5
148.5
146.5
116.4
123.1
109.1
144.7
103.3
146.0
142.1
108.6
154.5
–
126.8
111.8
148.8
146.8
108.6
119.2
135.5
125.2
114.5
148.5
146.4
116.3
123.4
108.6
146.2
103.6
146.7
144.6
105.1
n.d.
120.1
150.3
141.8
151.2
104.7
122.4
134.8
125.2
114.1
148.6
146.5
115.9
122.9
108.6
146.8
103.2
145.7
144.5
104.7
n.d.
114.2
147.2
136.3
150.7
100.8
122.2
135.0
127.2
117.7
147.4
147.5
113.3
121.4
108.4
146.7
103.5
145.5
144.3
104.9
154.2
–
114.5
147.6
136.7
151.1
101.3
122.8
135.5
125.9
115.1
148.8
146.8
116.5
123.8
109.0
147.0
103.8
145.9
144.7
105.3
154.3
–
127.0
115.2
146.9
145.8
109.0
117.8
135.9
127.1
117.6
147.3
147.3
113.3
121.3
110.0
144.5
100.6
147.7
142.6
108.2
154.2
–
0
0a
0b
1
2
3
4
5
6
7
8
9
0
1
2
3
-OMe
-OMe
-OMe
3-OMe
4-OMe
0-OMe
1-OMe
b
b
–
–
54.6
55.8
–
–
55.6
54.5
56.0
–
–
55.0
–
–
–
55.9
–
60.5
60.2
54.4
55.7
–
–
54.6
–
60.3
54.6
–
55.8
–
54.6
–
–
54.7
56.0
–
–
–
60.3
54.6
55.9
–
–
–
–
–
–
–
–
–
60.2
54.6
–
56.0
–
55.0
60.5
54.9
56.3
–
–
55.3
60.8
54.9
56.2
–
–
55.2
–
54.6
–
–
–
–
54.5
–
55.6
55.7
–
–
54.7
–
–
55.8
–
54.4
55.7
–
55.6
–
–
–
–
–
–
55.0
–
1
3
n.d., resonances not detected. [a] Values obtained as a mixture. [b] Signals interchangeable. [c] Lack of material precluded measurement of C NMR
data for lamellarin T (11).
(
[D ]DMSO) data suggestive of an O-methyl homologue of
HMBC correlations from 21-OH to C-20 (dC 146.0), C-21
(dC 142.1), and C-22 (dC 108.6), and a ROESY correlation
to H-22 (dH 6.62). These considerations permitted structure
assignment of lamellarin A2 (2) as indicated.
6
[12]
lamellarin S (10). Assignment of NMR resonances to 9-
OMe (dH 2.98) and 13-OMe (dH 3.73) moieties was support-
ed by diagnostic 2D NMR correlations (Figure 1), and per-
mitted structure assignment of lamellarin A1 (1) as indicat-
ed.
HRMS data for
(M+Na) corresponding to a molecular formula C H NO ,
3
revealed
a quasi-molecular ion
+
2
9
25
8
HRMS data for
M+Na) corresponding to a molecular formula C H NO ,
2
revealed
a
quasi-molecular ion
with NMR ([D ]DMSO) data suggestive of a deoxy ana-
logue of lamellarin K (8). Diagnostic 2D NMR correla-
6
+
[11]
(
2
8
23
9
with NMR ([D ]DMSO) data suggestive of an O-demethyl
homologue of lamellarin K (8). Assignment of an NMR
tions (Figure 3), inclusive of an HMBC correlation from 8-
OMe (dH 3.77) to C-8 (dC 148.8), and ROESY correlation
between 8-OMe and H-7 (dH 6.98), permitted structure as-
signment of lamellarin A3 (3) as indicated.
6
[11]
resonance to a 21-OH (d 8.96) moiety was supported by di-
H
agnostic 2D NMR correlations (Figure 2), inclusive of
Figure 2. Key 2D NMR ([D
6
]DMSO) correlations for lamellarin A2 (2).
6
Figure 3. Key 2D NMR ([D ]DMSO) correlations for lamellarin A3 (3).
Chem. Asian J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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HRMS data for
M+H) corresponding to a molecular formula C H NO ,
4
revealed
a quasi-molecular ion
+
(
2
5
17
8
with NMR ([D ]DMSO) data suggestive of a dihydro ana-
6
[10]
3
logue of lamellarin H. The appearance of sp -hybridized
C NMR resonances for C-5 (dC 42.0) and C-6 (dC 27.8),
supported by diagnostic 2D NMR correlations (Figure 4),
13
Figure 6. Key 2D NMR ([D
6
]DMSO) correlations for lamellarin A6 (14).
6
Figure 4. Key 2D NMR ([D ]DMSO) correlations for lamellarin A4 (4).
permitted structure assignment of lamellarin A4 (4) as indi-
cated.
HRMS data for
5
revealed
a
quasi-molecular ion
molecular formula
+
(
(
(
M+H)
corresponding to a
C H NO ) isomeric with lamellarin S (10), with NMR
6
Figure 7. Key 2D NMR ([D ]DMSO) correlations for lamellarin c (13).
26
15
8
[D ]DMSO) data suggestive of an O-methyl homologue of
6
[10]
1
lamellarin H. Assignment of a H NMR resonance for 13-
OMe (d 3.74) was supported by diagnostic 2D NMR corre-
lations (Figure 5) and permitted structure assignment of la-
lamellarin A1 (1). Analysis of the 2D NMR data for 13 re-
vealed diagnostic correlations (Figure 7) inclusive of an
HMBC correlation from 21-OMe (dH 3.35) to C-21 (dC
H
1
44.4), and ROESY correlations between 21-OMe and H-22
(
dH 6.60), that permitted structure assignment of lamellarin
c (13) as indicated.
To assess interactions between the lamellarins 1–16 and P-
gp, we undertook a series of experiments (a summary of the
results is presented in Table 3). In a primary cytotoxicity
(
MTT) assay, the comparative cytotoxicity of 1–16 was as-
sessed against the P-gp–overexpressing human colon adeno-
carcinoma cell line SW620 Ad300 and the corresponding pa-
rental cell line SW620. The significantly reduced cytotoxicity
against SW620 Ad300 compared to SW620 suggested that
lamellarins A1 (1), A2 (2), and S (10) could be P-gp sub-
6
Figure 5. Key 2D NMR ([D ]DMSO) correlations for lamellarin A5 (5).
[17]
strates, whereas comparable levels of cytotoxicity against
both cell lines suggested that lamellarins K (8), E (7), M (9),
A3 (3), c (13), and A6 (14) were not P-gp substrates. The in-
creased cytotoxicity (ꢀ 4-fold) of A1 (1), A2 (2), and S (10)
in the presence of the P-gp inhibitor cyclosporine A in
SW620 Ad300 confirmed these three lamellarins as P-gp
substrates. By contrast, the lack of cytotoxicity to either cell
line exhibited by lamellarins A4 (4), A5 (5), C (6), G (15),
and Z (16) left their P-gp substrate status unresolved. In
a second round of screening (MDR reversal), the P-gp non-
substrates 3, 7–9, and 13–14 were assessed for their ability to
reverse P-gp–mediated doxorubicin drug resistance in the
SW620 Ad300 cell line. Although all these lamellarins ex-
hibited reversal of doxorubicin resistance, with gains in sen-
sitivity (GS) ranging from 2.02 to 4.26, assessment of compa-
mellarin A5 (5) as indicated.
HRMS data for 14 revealed a quasi-molecular ion
+
(
(
M+H)
corresponding to
a
molecular formula
[10]
[11]
C H NO ) isomeric with lamellarins G (15)
and L,
28
23
8
with NMR ([D ]DMSO) data suggestive of an O-methyl ho-
6
[16]
1
mologue of lamellarin Z (16). Assignment of a H NMR
resonance for 13-OMe (dH 3.73) was supported by diagnos-
tic 2D NMR correlations (Figure 6) and permitted structure
assignment of lamellarin A6 (14) as indicated.
HRMS data for 13 revealed a quasi-molecular ion
+
(
(
(
M+Na)
corresponding to
a
molecular formula
with NMR
[D ]DMSO) data suggestive of an O-methyl homologue of
C H NO ) isomeric with lamellarin
L
28
23
8
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Lamellarins as inhibitors of P-gp-mediated multidrug resistance
Table 3. Lamellarin interactions with P-glycoprotein.
ity. In addition, we assembled
selection of lamellarin-in-
spired synthetic coumarins (19–
4) and isoquinolines (25–26) to
test the hypothesis that lamel-
larin P-gp inhibitory activity is
inherent to embedded structure
fragments. Subsequent assays
established that, whereas the la-
mellarin-inspired coumarin and
isoquinoline fragments 19–26
[
a]
#
SW620
IC50 (mM)
SW620 Ad300
MDR reversal
GS
Calcein
FAR
a
[
b]
[d]
[e]
[
b]
[c]
IC50 (mM)
IC50 (mM)+CsA
2
4
5
6
1
1
1
2
1
8
7
9
3
1
1
1
>30
>30
>30
>30
>30
3.59
9.47
8.50
0.50
4.26
1.02
19.7
6.72
23.8
28.2
>30
>30
>30
>30
>30
37.0
55.5
103
1.09
5.35
2.09
28.4
12.3
28.0
23.4
–
–
–
–
–
6.80
10.6
5.42
–
–
–
–
–
–
–
0.96 (20)
1.52 (20)
1.58 (20)
–
2.51 (20)
–
0.63
0.80
0.92
1.15
0.80
1.07
1.14
1.64
39.4
38.6
27.2
18.8
11.7
9.22
27.4
5
6
–
–
0
2.11 (0.1)
4.01 (1)
2.02 (0.5)
4.26 (5)
2.23 (3)
2.91 (10)
7.80 (3)
(
Figure 8) did not exhibit P-gp
inhibitory activity, the hexame-
thylated lamellarin 17, obtained
by K CO /MeI treatment, was
3
4
7
2
3
a potent P-gp inhibitor (fluores-
cence arbitrary ratio FAR of
–
=not tested. [a] Due to a lack of material, 11 and 12 were not tested. [b] Cytotoxicity (MTT) assay for lamel-
2
7.4) and a very strong MDR
larins. [c] Cytotoxicity (MTT) assay for lamellarins in presence of cyclosporine A (CsA 3 mm). [d] MDR rever-
sal (doxorubicin) assay against SW620 Ad300 where GS (gain in sensitivity)=IC50 of doxorubicin without la- reversal agent (GS of 7.80 at
mellarin/IC50 of doxorubicin with lamellarin (test concentration in mM is shown in parentheses). IC50 of doxor- 3 mm). Also of note, 17 was less
ubicin without lamellarin was 4.43 mm; verapamil (2.5 mm) was used as a positive control (GS=6.23). [e] Cell
flow cytometry analysis of intracellular calcein fluorescence: FAR (fluorescence arbitrary ratio)=calcein fluo-
rescence intensity (Geo mean) in the presence of lamellarin at 20 mm/calcein fluorescence intensity (Geo
cytotoxic towards either colon
cancer cell line (Table 3). While
these results support our initial
hypothesis regarding a correla-
mean) in the presence of PBS. Positive control is verapamil at 20 mm which returns a FAR=43.1.
rative potency proved problematic given the need to test la-
mellarins at concentrations below their respective IC50
values (spanning 0.1 to 10 mm). To address this limitation, in
a third round of screening (Calcein AM), the P-gp inhibitory
activities of 3, 7–9, and 13–14 were assessed by brief expo-
sure (0.5 h) of individual lamellarins (20 mm) to SW620
Ad300 cells, followed by immediate quantification of intra-
cellular (calcein) fluorescence by cell flow cytometry. After
compensating for baseline levels of intracellular fluores-
cence, and standardizing to the positive control verapamil
(
7
20 mm), lamellarins could be ranked as either strong (8 and
), moderate (9), or weak (3, 13, and 14) P-gp inhibitors. On
applying the MDR reversal and Calcein AM assays to the
P-gp substrates, lamellarins 1, 2, and 10, and the non-cyto-
toxic lamellarins 4–6 and 15–16, we determined that none of
these metabolites were P-gp inhibitors.
While the lamellarins 1–16 exhibit many common struc-
tural features, SAR analysis of our screening data (Table 3)
highlighted some useful correlations. Lamellarins 1–16 can
be grouped according to the degree of methylation, ranging
across unmethylated 4, monomethylated 5 and 10, dimethy-
lated 1 and 16, trimethylated 2 and 13–15, tetramethylated 3
and 7–9, to pentamethylated 6 and 11–12. Our SAR data
suggest that the P-gp inhibitory activity loosely correlates
with higher levels of methylation on rings A and B (i.e., tet-
ramethylated 7–9 but not the pentamethylated 6). The hex-
amethylated lamellarin I (18), the only lamellarin with
published data supportive of P-gp inhibitory activity, is con-
sistent with this methylation hypothesis. Building on this
emerging SAR, we permethylated lamellarin S to lamellarin
derivative 17 to test the hypothesis that further increasing
lamellarin methylation would enhance P-gp inhibitory activ-
Figure 8. Lamellarin-inspired synthetic coumarins (19–24) and isoquino-
lines scaffolds (25–26).
tion between methylation and P-gp inhibition, they also sug-
gest that the molecular basis behind lamellarin P-gp inhibi-
tory activity requires the intact molecular architecture of the
natural product scaffold.
In an effort to document their broader biological profile,
particularly against indications and biological targets already
associated with lamellarins in the literature, the Didemnum
lamellarins 1–16 were screened for antibiotic and kinase in-
hibitory activities. Lamellarins 1–16 displayed no significant
growth inhibitory activity (MIC>30 mm) against the fungus
Candida albicans (ATCC 90028), the Gram-negative bacte-
[7]
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Robert J. Capon et al.
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ria Escherichia coli (ATCC 11775) and Pseudomonas aerugi-
nosa (ATCC 10145), or the Gram-positive bacteria Staphy-
lococcus aureus (ATCC 9144 and ATCC 25923) and Bacillus
subtilis (ATCC 6051). When 1–16 were tested against Bacil-
lus subtilis (ATCC 6633), only lamellarins E (7), K (8), and
M (9) displayed antibacterial activity (MIC=7.5, 15, and
tiating concept is a salutatory lesson to those who value
and/or devalue natural products merely on the basis of cyto-
toxicity. Our investigation into Didemnum lamellarins reaf-
firms the view that non-cytotoxic natural products can ex-
hibit valuable biological properties that allude to both an
ecological advantage and a pharmacological potential.
7.5 mm respectively). When assessed against the neurodege-
nerative disease targets casein kinase 1 (CK1d) and cyclin-
dependent kinase 5 (CDK5), 1–16 exhibited limited inhibi-
tory activity against CK1d, with lamellarins A4 (4) and S
Experimental Section
General Experimentals
(
10) being the most active (IC =3 mm). On the other hand,
50
1
–16 displayed generally higher levels of inhibition against
General procedures, instrumentation, HPLC chromatograms, characteri-
zation data, and screening and assay results are presented in the Support-
ing Information.
CDK5, with lamellarins A1 (1), A6 (14), and Z (16) all dis-
playing submicromolar inhibition (0.1, 0.3, and 0.4 mm, re-
spectively).
Collection and Taxonomy
Sample CMB-01656 was collected by Scuba diving in July 1994 off Wasp
Island Durras NSW (35840.1’S, 150818.5’E) at a depth of 15–20 m.
Sample CMB-02127 was collected in January 1996 by using an epibenthic
sled off the northern Rottnest Shelf, Western Australia. Freshly collected
samples were frozen (À48C) for shipping to the laboratory, where they
were thawed, catalogued, subjected to taxonomic classification as Didem-
num sp., diced, and steeped in aqueous EtOH at À308C for prolonged
storage.
Conclusions
This study reports the isolation and identification of a selec-
tion of new (1–5, 14) and known (6–13, 15, 16) lamellarins
from two southern Australian Didemnum sp., supported by
comprehensive spectroscopic analysis. Access to a privileged
library of natural lamellarins enabled SAR studies aimed at
understanding interactions between this unique family of
marine alkaloids and the ABC transporter efflux pump P-
glycoprotein, a key determinant in MDR cancers. These
studies determined that 1) the non-cytotoxic lamellarins 4–6
and 15–16 had no demonstrable interaction with P-gp, 2) the
cytotoxic lamellarins 1–2 and 10 were P-gp substrates, and
Extraction and Isolation
A portion of the aqueous EtOH extract of sample CMB-01656 was deca-
nted and concentrated in vacuo, and the residue (737.9 mg) partitioned
between H
ed in vacuo (108.9 mg) and the residue further triturated into light petro-
leum (21.6 mg), CH Cl (27.5 mg), MeOH (55.8 mg), and H O (665.5 mg)
solubles. The CH Cl
2
O and nBuOH. The nBuOH-soluble fraction was concentrat-
2
2
2
2
2
solubles were subjected to HPLC fractionation
Agilent Zorbax Eclipse 5 mm, 250ꢁ9.4 mm column, 4 mLmin gradient
À1
(
2
elution, 90% H O/MeCN to 100% MeCN with a constant 0.01% TFA/
3
) the cytotoxic lamellarins 3, 7–9, and 13–14 were P-gp in-
MeCN modifier over 20 min) to yield eight fractions. Fractions 2–5 yield-
ed lamellarins S (1) (1.4 mg, 0.051%), A1 (1) (1.3 mg, 0.012%), A2 (2)
(1.5 mg, 0.014%), and c (13) (1.5 mg, 0.014%), respectively. Fraction 6
was a mixture (32:35:22:11) of lamellarins E (7) (1.6 mg, 0.015%), K (8)
hibitors capable of reversing MDR. An SAR analysis of
these data suggested that P-gp inhibitory activity correlated
with higher levels of lamellarin methylation, a hypothesis
that was tested and supported by synthesis and evaluation
of the permethylated lamellarin 17. Significantly, not only
did 17 exhibit strong MDR reversal and potent inhibition of
P-gp but these enhancements also coincided with a signifi-
cant reduction in cytotoxicity. By contrast, efforts to attri-
bute the lamellarin P-gp inhibitory activity to the simpler
synthetic structure fragments 19–26 failed, reinforcing the
importance of discovery through the screening of highly
evolved intact natural product scaffolds.
(
2.0 mg, 0.018%), M (9) (0.6 mg, 0.005%), and X (12) (0.3 mg, 0.003%).
Fraction 7 was pure lamellarin A3 (3) (0.9 mg, 0.008%). HPLC resolu-
tion of fraction 8 (1.3 mg) (Agilent Zorbax SB C
column, first with a 1 mLmin gradient elution, 75% H
3
5 mm, 150ꢁ4.6 mm
O/MeOH to
À1
2
100% MeOH with a constant 0.01% TFA/MeOH modifier over 15 min,
À1
followed by a 1 mLmin isocratic elution 100% MeOH with a constant
0
.01% TFA/MeOH modifier over 3 min), afforded lamellarins T (11)
(
0.2 mg, 0.002%) and C (6) (0.3 mg, 0.003%). The MeOH solubles were
subjected to HPLC fractionation (Agilent Zorbax Eclipse C18 5 mm, 250ꢁ
À1
9.4 mm column, 4 mLmin gradient elution, 90% H O/MeOH to 100%
2
MeOH with a constant 0.01% TFA/MeOH modifier over 20 min) to
yield nine fractions. Fractions 3 and 5–8 yielded lamellarins A4 (4)
The observations made above draw attention to the possi-
ble ecological value of maintaining a combinatorial cohort
of differentially methylated lamellarin co-metabolites, which
at first glance may seem a biosynthetic extravagance on
behalf of the Didemnum sp. In an ecological setting the
suite of lamellarin co-metabolites could be viewed as poten-
tially far more valuable to the Didemnum sp. than any indi-
vidual lamellarin, irrespective of relative cytotoxicity. For
example, by a modest biosynthetic investment (methyl trans-
ferase) the Didemnum sp. can deploy more highly methylat-
ed lamellarins (as P-gp inhibitors) to potentiate the ecologi-
cal impact of less methylated lamellarins (cytotoxins) when
encountering competing or threatening organisms/cells
equipped with P-gp–mediated cellular defences. This poten-
(
(
1.3 mg, 0.012%), S (10) (17.9 mg, 0.164%), A5 (5) (1.1 mg, 0.010%), A1
1) (1.7 mg, 0.016%), and A2 (2) (3.2 mg, 0.029%), respectively. HPLC
resolution of fraction 9 (2.2 mg) (Agilent Zorbax SB C18 5 mm, 150ꢁ
4.6 mm column, first with a 1 mLmin gradient elution, 90% H
À1
2
O/
MeCN to 40% H
over 18 min, followed by a 1 mLmin gradient elution 40% H
to 100% MeCN with a constant 0.01% TFA/MeCN modifier over
min), afforded lamellarins K (8) (0.5 mg, 0.005%) and E (7) (0.5 mg,
2
O/MeCN with a constant 0.01% TFA/MeCN modifier
À1
2
O/MeCN
2
0.005%).
A portion of the aqueous EtOH extract of sample CMB-02127 was deca-
nted and concentrated in vacuo, and the residue (869.8 mg) partitioned
between H
ed in vacuo (153.8 mg) and the residue further triturated into light petro-
leum (5.7 mg), CH Cl (9.7 mg), MeOH (129.2 mg), and water (649.1 mg)
2
O and nBuOH. The nBuOH-soluble fraction was concentrat-
2
2
solubles. The MeOH solubles were subjected to HPLC fractionation
(Agilent Zorbax Eclipse 5 mm, 250ꢁ9.4 mm column, 4 mLmin gradient
À1
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Lamellarins as inhibitors of P-gp-mediated multidrug resistance
elution, 90% H
2
O/MeCN to 100% MeCN with a constant 0.01% TFA/
Samples were then analyzed on a BD FACSCantoTM II flow cytometer
(Becton Dickinson, San Jose, CA). Calcein fluorescence was detected
with a 488 nm argon laser and a 530 nm band pass filter. The data were
analyzed by using FCSexpress 3 (De Novo Software, Los Angeles, CA).
10000 events were collected for each sample. Inhibition was evaluated
using the following equation: FAR (fluorescence arbitrary ratio)=calcein
fluorescence intensity (Geo mean) in the presence of lamellarin at 20 mm/
calcein fluorescence intensity (Geo mean) in the presence of PBS. Vera-
pamil at 20 mm was used as a positive control (FAR=43.1).
MeCN modifier over 22 min) to yield fourteen fractions. Fraction 13 was
pure lamellarin Z (16) (0.3 mg, 0.002%). HPLC resolution of fraction 14
(
4.2 mg) (Agilent Zorbax SB C
3
5 mm, 150ꢁ4.6 mm column, first with
O/MeCN to 30% H O/MeCN
À1
a 1 mLmin gradient elution, 90% H
2
2
with a constant 0.01% TFA/MeCN modifier over 18 min, followed by
À1
a 1 mLmin gradient elution 30% H
2
O/MeOH to 100% MeOH with
a constant 0.01% TFA/MeOH modifier over 7 min), afforded lamellarin
G (15) (0.3 mg, 0.002%) and A6 (14) (0.5 mg, 0.003%).
[
Note:% yields are expressed as a mass-to-mass% compared to the
Antibacterial Assay
weight of the nBuOH solubles].
The bacterium to be tested was streaked onto a tryptic soy agar plate
Cell Lines and Cell Culture
and was incubated at 378C for 24 h. One colony was then transferred to
4
fresh tryptic soy broth (15 mL) and the cell density was adjusted to 10 –
The parental cell line SW620 (American Type Culture Collection, Mana-
ssasm VA, CCL-227), is a human colon cell line that originated from
a lymph node metastasis in a patient with primary adenocarcinoma of
the colon. The multidrug-resistant (MDR) cell line SW620 Ad300, which
overexpresses P-gp, was selected from SW620 by growth in the presence
of increasing concentrations of doxorubicin. SW620 and SW620 Ad300
cells were grown in flasks as adherent monolayers in RPMI medium sup-
plemented with 10% fetal bovine serum, 2 mml-glutamine, 100
5
À1
1
0 cfumL . Test compounds were dissolved in DMSO and diluted with
O to give a 300 mm stock solution (10% DMSO). The stock solution
was then serially diluted with 10% DMSO to give final concentrations of
0 mm to 0.01 mm in 1% DMSO. An aliquot (20 mL) of each dilution was
H
2
3
transferred to a 96-well microtiter plate and freshly prepared microbial
broth (180 mL) was added to each well. The plates were incubated at
3
78C for 24 h and the optical density of each well was measured spectro-
À1
À1
photometrically at 600 nm. Each test compound was screened against the
Gram-negative bacterium Escherichia coli (ATCC 11775) and the Gram-
positive bacteria Staphylococcus aureus (ATCC 9144 and ATCC 25923)
and Bacillus subtilis (ATCC 6633 and ATCC 6051).
unitsmL penicillin and 100 mgmL streptomycin in a humidified incu-
bator under 5% CO
2
at 378C. After SW620 Ad300 exhibited stable phe-
À1
notype of P-gp, the cells were maintained in 300 ngmL doxorubicin.
Cytotoxicity (MTT) Assay
Antifungal Assay
The cytotoxicity MTT assay was used to evaluate the cytotoxicity of com-
pounds against cancer cells. This assay was modified slightly from that
The fungus to be tested was streaked onto a Sabouraud agar plate and
was incubated at 26.58C for 48 h. One colony was then transferred to
[
18]
previously described. Briefly, cells (2000 per well in 180 mL of RPMI
640 supplemented with 10% FBS) were seeded evenly in a 96-well mi-
croplate, and the plate was incubated for 18 h at 378C under 5% CO to
4
1
fresh Sabouraud broth (15 mL) and the cell density was adjusted to 10 –
5
À1
10 cfumL . Test compounds were dissolved in DMSO and diluted with
2
H O to give a 300 mm stock solution (10% DMSO). The stock solution
2
allow cells to attach. Compounds to be tested were dissolved in 5%
DMSO (v/v) and diluted in a range from 300 mm to300 nm. Aliquots
was then serially diluted with 10% DMSO to give final concentrations of
30 mm to 0.01 mm in 1% DMSO. An aliquot (20 mL) of each dilution was
transferred to a 96-well microtiter plate and freshly prepared microbial
broth (180 mL) was added to each well. The plates were incubated at
26.58C for 48 h and the optical density of each well was measured spec-
trophotometrically at 600 nm. Each test compound was screened against
the fungus Candida albicans (ATCC 90028).
(
20 mL) of each dilution (or of 5% aqueous DMSO for control wells)
were added to each well in duplicate. To evaluate the cytotoxicity of 1, 2,
or 10 in the presence of cyclosporine A in SW620 Ad300, 2000 cells in
1
1
60 mL medium were plated onto each well. Following incubation for
8 h, increasing doses of these three lamellarins were added in the pres-
ence of 3 mm cyclosporine A. After 68 h incubation (378C; 5% CO
2
), a so-
lution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Kinase (CDK5) Inhibitor Assay
(
MTT; Sigma, USA) in PBS was added to each well to a final concentra-
À1
tion of 0.4 mgmL , and the plate was incubated for a further 4 h (378C;
Test compounds (1% DMSO) were added in duplicate to a 384-well
plate at desired concentrations. CDK5/p25 (0.8 ngmL ) was dispensed
5
% CO
2
). After that the medium was carefully aspirated and precipitat-
À1
ed formazan crystals were dissolved in DMSO (100 mL per well). Finally,
the absorbance of each well at 580 nm was measured with a PowerWave
XS Microplate Reader from Bio-Tek Instruments Inc. (Vinooski, VT).
The IC50 value was calculated as the concentration of the compound re-
quired for 50% inhibition of the cancer cells using Prism 5.0 from
GraphPad Software Inc. (La Jolla, CA).
into wells and the plate incubated for 10 min at rt. Following addition of
ATP (4 mm) and histone (300 mgmL ) to a final assay volume of 25 mL,
À1
the plate was covered with parafilm and incubated at 278C for 60 min.
After equilibrating to rt, 20 mL of Kinase-Glo reagent was added to each
well and the plate incubated for a further 10 min before luminescence
was measured using a Polarstar plate reader. Concentrations given are
for final assay conditions. Controls included a ꢂno kinaseꢃ control (ATP,
histone only), ꢂkinaseꢃ control (CDK5/p25, ATP and histone), and a ꢂvehi-
cleꢃ control (CDK5/p25, ATP, histone and DMSO). Percent inhibition=
((test sample-kinase control)/(no kinase control-kinase control))ꢁ100.
IC50 values were calculated using Prism. The assay buffer consisted of
MDR Reversal (Doxorubicin) Assay
This assay is similar to the above cytotoxicity (MTT) assay. Instead of
measuring the cytotoxicity of lamellarins, this assay was applied to mea-
sure the cytotoxicity of doxorubicin against SW620 Ad300, in the pres-
ence or absence of lamellarin at concentrations that were non-cytotoxic
to SW620 Ad300. Gain in sensitivity (GS) was calculated according to
GS=IC50 of doxorubicin without lamellarin/IC50 of doxorubicin with la-
mellarin (test concentration in mM is shown in parentheses). The IC50
value of doxorubicin without lamellarin was 4.43 mm, 2.5 mm verapamil
was used as a positive control (GS=6.23).
2
6.25 mm MOPS (pH 7.2), 6.25 mm MgCl , 1.25 mm EGTA, 1.25 mm
EDTA, and 0.25% glycerol. Assay components were obtained as follows:
CDK5/p25 (Sigma Aldrich, C0745), ATP (Sigma Aldrich, A7699), his-
tone (Sigma Aldrich, H4524), Kinase-Glo reagent (Promega, V6712),
384-well plates (Perkin–Elmer, 6007290).
Kinase (CK1d) Inhibitor Assay
Flow Cytometry (Calcein AM Assay)
Test compounds (1% DMSO) were added in duplicate to a 384-well
plate at desired concentrations. CK1d (0.4 ngmL ) was dispensed into
[
19]
À1
The flow cytometry assay was based on that described previously. Cells
which overexpress P-gp (SW620 Ad300) were harvested with trypsin, re-
suspended in completed medium to give a final concentration of 50ꢁ
wells and the plate incubated for 10 min at rt. ATP (6 mm) and CK1tide
(125 mm) were added to a final assay volume of 25 mL. The method then
proceeded as described above for the kinase (CDK5) inhibitor assay.
Concentrations given are for final assay conditions. Controls included
a ꢂno kinaseꢃ control (ATP, CK1tide only), ꢂkinaseꢃ control (CK1d, ATP
4
À1
1
3
2
0 cellsmL , and pre-incubated with 20 mm lamellarin or verapamil for
0 min at 378C under 5% CO . Subsequently, cells were incubated with
.5 mm calcein AM for 1 h followed by washing twice with cold PBS.
2
Chem. Asian J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Robert J. Capon et al.
FULL PAPERS
and CK1tide), and
a
ꢂvehicleꢃ control (CK1d, ATP, CK1tide, and
Bates and R. Robey (NCI) for providing the SW620 and SW620 Ad300
cell lines, and A. M. Piggott (IMB, UQ) for the acquisition of HRESIMS
data. X.-C. Huang and Z. Khalil acknowledge the provision of Interna-
tional Postgraduate Scholarships from The University of Queensland. F.
Plisson acknowledges the provision of an IMB Postgraduate Award. This
work was partially funded by the Institute for Molecular Bioscience, The
University of Queensland, the Australian Research Council (ARC
LP0775547), and Noscira (Madrid, Spain).
DMSO). Percent inhibition=((test sampleÀkinase control)/(no kinase
controlÀkinase control))ꢁ100. IC50 values were calculated using Prism.
The assay buffer consisted of 7.5 mm MOPS (pH 7.0), 0.25 mm EDTA,
À1
0
.003% Brij-35, 1% glycerol, 0.03% BME, 0.5 mgmL BSA, and
1
2
2.5 mm Mg ACHTUNGTRENNUNG( OAc) . Assay components were obtained as described above
or as follows: CK1d (Millipore, 14–520) and Ck1tide (Millipore, 12–529).
Characterization Data for Lamellarins A1–A6 and c
Lamellarin A1 (1). A pale brown oil. UV (EtOH) lmax (log e) 207 (4.64),
2
77 (4.34), 315 (4.28), 340 (4.27), 392 (3.54) nm; NMR ([D
6
]DMSO,
6
00 MHz) data, see Table 2 and the Supporting Information; ESI(+)MS
[
[
1] H. Glavinas, P. Krajcsi, J. Cserepes, B. Sarkadi, Curr. Drug Delivery
004, 1, 27–42.
2] C. Barthomeuf, M. L. Bourguet-Kondracki, J. M. Kornprobst, Anti-
cancer Agents Med. Chem. 2008, 8, 886–903.
+
À
m/z 488 [M+H] ; ESI(À)MS m/z 486 [MÀH] ; HRESI(+)MS m/z calcd
for C27 : 488.1340 [M+H] ; found, 488.1340 [M+H] .
Lamellarin A2 (2). A brown oil. UV (EtOH) lmax (log e) 207 (4.62), 278
4.33), 315 (4.26), 340 (4.24) nm; NMR ([D ]DMSO, 600 MHz) data, see
Table 2 and the Supporting Information; ESI(+)MS m/z 540 [M+Na] ;
2
+
+
H
22NO
8
(
6
[3] N. Aouali, L. Eddabra, J. Macadre, H. Morjani, Crit. Rev. Oncol.
Hematol. 2005, 56, 61–70.
[4] H. Fan, J. Peng, M. T. Hamann, J.-F. Hu, Chem. Rev. 2008, 108, 264–
287.
+
+
À
ESI(+)MS m/z 540 [M+Na] ; ESI(À)MS m/z 516 [MÀH] ; HRE-
+
SI(+)MS m/z calcd for
C
28
H
23NO
9
Na [M+Na] : 540.1265; found:
5
40.1257.
Lamellarin A3 (3). A pale yellow oil. UV (EtOH) lmax (log e) 207 (4.60),
77 (4.32), 315 (4.24), 340 (4.20) nm; NMR ([D ]DMSO, 600 MHz) data,
see Table 2 and the Supporting Information; ESI(+)MS m/z 538
[5] J. Kluza, P. Marchetti, C. Bailly, in Modern Alkaloids: Structure, Iso-
lation, Synthesis and Biology (Eds.: E. Fattorusso, O. Taglialatela-
Scafati), Wiley-VCH, Weinheim, 2008, pp. 171–187.
2
6
[
[
[
6] M. Vanhuyse, J. Kluza, C. Tardy, G. Otero, C. Cuevas, C. Bailly, A.
Lansiaux, Cancer Lett. 2005, 221, 165–175.
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J. Cancer 1996, 74, 677–682.
+
À
[
C
M+Na] ; ESI(À)MS m/z 514 [MÀH] ; HRESI(+)MS m/z calcd for
+
29
H
25NO
8
Na [M+Na] : 538.1472; found: 538.1469.
Lamellarin A4 (4). A pale green oil. UV (EtOH) lmax (log e) 208 (4.64),
2
6
8] J. L. F. Puentes, D. Garcia Gravalos, A. R. Quesada, 1997, WO
76 (4.37), 315 (4.20), 337 (4.16), 392 (3.76) nm; NMR ([D
6
]DMSO,
9
701336 (A1).
00 MHz) data, see Table 2 and the Supporting Information; ESI(+)MS
+
+
À
[9] R. J. Andersen, D. J. Faulkner, C.-H. He, G. D. V. Duyne, J. Clardy,
J. Am. Chem. Soc. 1985, 107, 5492–5495.
10] N. Lindquist, W. Fenical, G. D. V. Duyne, J. Clardy, J. Org. Chem.
m/z 460 [M+H] and m/z 482 [M+Na] ; ESI(À)MS m/z 458 [MÀH] ;
+
HRESI(+)MS m/z calcd for
C
25
H
18NO
8
[M+H] : 460.1027; found:
[
4
60.1024.
1
988, 53, 4570–4574.
11] A. R. Carroll, B. F. Bowden, J. C. Coll, Aust. J. Chem. 1993, 46, 489–
01.
Lamellarin A5 (5). A pale yellow oil. UV (EtOH) lmax (log e) 207 (4.67),
[
2
81 (4.38), 308 (4.22), 368 (3.90), 391 (3.97) nm; NMR ([D
6
]DMSO,
5
6
00 MHz) data, see Table 2 and the Supporting Information; ESI(+)MS
[
[
12] S. Urban, R. J. Capon, Aust. J. Chem. 1996, 49, 711–713.
13] M. V. R. Reddy, D. J. Faulkner, Y. Venkateswarlu, M. R. Rao, Tetra-
hedron 1997, 53, 3457–3466.
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Venkateswarlu, Tetrahedron 2005, 61, 9242–9247.
15] P. Ploypradith, T. Petchmanee, P. Sahakitpichan, N. D. Litvinas, S.
Ruchirawat, J. Org. Chem. 2006, 71, 9440–9448.
16] R. A. Davis, A. R. Carroll, G. K. Pierens, R. J. Quinn, J. Nat. Prod.
+
À
À
m/z 472 [M+H] ; ESI(À)MS m/z 470 [MÀH] and m/z 941 [2MÀH] ;
À
HRESI(À)MS m/z calcd for
C
26
H
16NO
8
[MÀH] : 470.0890; found:
4
70.0881.
Lamellarin A6 (14). A pale yellow oil. UV (MeOH) lmax (log e) 203
4.73), 276 (4.27), 315 (4.17), 340 (4.13) nm; NMR ([D ]DMSO,
00 MHz) data, see Table 2 and the Supporting Information; ESI(+)MS
[
[
[
(
6
6
+
À
m/z 502 [M+H] ; ESI(À)MS m/z 500 [MÀH] ; HRESI(+)MS m/z calcd
+
for C28
H
24NO
8
[M+H] : 502.1496; found: 502.1503.
1
999, 62, 419–424.
Lamellarin c (13). A colorless oil. UV (EtOH) lmax (log e) 207 (4.64),
78 (4.35), 313 (4.28), 335 (4.24) nm; NMR ([D ]DMSO, 600 MHz) data,
see Table 2 and the Supporting Information; ESI(+)MS m/z 524
[17] S. Scala, N. Akhmed, U. S. Rao, K. Paull, L. B. Lan, B. Dickstein,
J. S. Lee, G. H. Elgemeie, W. D. Stein, S. E. Bates, Mol. Pharmacol.
1997, 51, 1024–1033.
[18] C. J. Henrich, H. R. Bokesch, M. Dean, S. E. Bates, R. W. Robey,
E. I. Goncharova, J. A. Wilson, J. B. McMahon, J. Biomol. Screening
2
6
+
À
[
M+Na] ; ESI(À)MS m/z 500 [MÀH] ; HRESI(+)MS m/z calcd for
+
C
28
H
23NO
8
Na [M+Na] : 524.1316; found: 524.1319.
2
006, 11, 176–183.
[
19] C. J. Henrich, R. W. Robey, K. Takada, H. R. Bokesch, S. E. Bates,
S. Shukla, S. V. Ambudkar, J. B. McMahon, K. R. Gustafson, ACS
Chem. Biol. 2009, 4, 637–647.
Acknowledgements
Received: December 24, 2011
The authors thank the CSIRO Division of Oceanography and crew of
Revised: February 14, 2012
the RV Franklin for the collection of Didemnum sp. (CMB-02127), S.
Published online: && &&, 0000
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www.chemasianj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Asian J. 0000, 00, 0 – 0
ÝÝ These are not the final page numbers!

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Chem. Asian J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
9
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These are not the final page numbers! ÞÞ