Two cytotoxic stereoisomers of malyngamide C, 8-epi-malyngamide C and 8-O-acetyl-8-epi-malyngamide C, from the marine cyanobacterium Lyngbya majuscula

Article abstract of DOI:10.1016/j.phytochem.2010.07.001

Two epimers of malyngamide C, 8-O-acetyl-8-epi-malyngamide C (1) and 8-epi-malyngamide C (3) have been isolated along with known compounds 6-O-acetylmalyngamide F (5), H (6), J (7) K (8), and characterized from a Grenada field collection of the marine cya

Full text of DOI:10.1016/j.phytochem.2010.07.001

Phytochemistry 71 (2010) 1729–1735
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Phytochemistry
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Two cytotoxic stereoisomers of malyngamide C, 8-epi-malyngamide C
and 8-O-acetyl-8-epi-malyngamide C, from the marine cyanobacterium
Lyngbya majuscula
Harald Gross a, Kerry L. McPhail b, Douglas E. Goeger b, Frederick A. Valeriote c, William H. Gerwick b,d,
*
a Institute for Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn, Germany
b College of Pharmacy, Oregon State University, Corvallis, OR 97331, USA
c Henry Ford Health System, Detroit, MI 48202, USA
d Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography and Skaggs School of Pharmacy and Pharmaceutical Sciences,
University of California at San Diego, La Jolla, CA 92093-0212, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 April 2010
Received in revised form 9 June 2010
Available online 9 August 2010
Two epimers of malyngamide C, 8-O-acetyl-8-epi-malyngamide C (1) and 8-epi-malyngamide C (3) have
been isolated along with known compounds 6-O-acetylmalyngamide F (5), H (6), J (7) K (8), and charac-
terized from a Grenada field collection of the marine cyanobacterium Lyngbya majuscula. The structures
of these compounds were deduced by 1D and 2D NMR spectroscopic and mass spectral data interpreta-
tion. Absolute configurations were determined by a combination of CD-spectroscopy, chemical degrada-
tion and the variable temperature Mosher’s method. Compounds 1–5, 7 and 8 displayed moderate
cytotoxicity to NCI-H460 human lung tumor and neuro-2a cancer cell lines, with IC50 values ranging
Keywords:
Lyngbya majuscula
Malyngamide
Variable temperature Mosher ester method
between 0.5 and 20 lg/mL.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
oxygen-containing functional groups such as ketones, alcohols,
epoxides and lactones. In addition to the structural variations,
differences in absolute configuration have also been observed
(Kan et al., 2000; Milligan et al., 2000; Suntornchashwej et al., 2007).
In the present study, a collection of L. majuscula from Grenada
was chemically analyzed in order to identify the compounds
responsible for the cytotoxicity observed in the crude extract
(Fig. 1). Subsequent bioassay-guided fractionation led to the isola-
tion of two new stereoisomers of malyngamide C (1, 3), along with
a suite of malyngamides previously reported, including 6-O-acetyl-
malyngamide F (4) and malyngamides H (5), J (6), and K (7). This
paper describes the isolation, structure elucidation, and biological
activity of the two new malyngamides 1 and 3.
Malyngamides are a class of secondary metabolites frequently
encountered during chemical investigations of filamentous cyano-
bacteria, particularly Lyngbya majuscula or sea hares of the genus
Bursatella and Stylocheilus (Gerwick et al., 2001; Tan, 2007). In the
latter case, however, it has been shown that molluscs incorporate
these compounds as a consequence of their diet of cyanobacteria
(Pennings and Paul, 1993). Thus, malyngamides are considered
metabolites of cyanobacterial origin. To date, more than 30 mem-
bers belonging to this compound class have been isolated. Structur-
ally, malyngamides consist of a methoxylated fatty acid tail, known
as ‘lyngbic acid’, and a presumed amino acid derived head which are
joined through an amide linkage. Whereas for the lipid part of
malyngamides only five variations are known, namely lyngbic acid
and its 12-, 16- and 20-carbon analog (Gerwick et al., 2001) and
one 7R-epimer (Suntornchashwej et al., 2007), the amine portion
of the molecule shows a remarkable structural diversity in carbon
skeletons and bears a multitude of functional groups. These include
unsaturations, covalently-attached chlorine atoms, and various
2. Results and discussion
Compound 1 showed an [M+H]+ peak at m/z 498.2607, consis-
tent with the molecular formula C26H41ClNO6 by HRESITOFMS.
The 1H NMR spectroscopic data exhibited several resonances typi-
cal of a malyngamide-type metabolite. For example, a methoxy
singlet signal (d 3.31) and its corresponding
a-methoxy methine
multiplet (d 3.14), two olefinic resonances (d 5.46), three methyl-
enes between d 2.17 and 2.31, a methylene envelope (d 1.26–
1.42) and a terminal methyl (d 0.87) were indicative of the amide
of a lyngbic acid moiety in 1. In addition, the 1H and 13C NMR spec-
tra contained resonances consistent with the presence of an
* Corresponding author. Address: Scripps Institution of Oceanography, University
of California San Diego, 9500 Gilman Drive, MC 0212, La Jolla, CA 92093, USA. Tel.:
+1 (858) 534 0578; fax: +1 (858) 534 0529.
E-mail address: wgerwick@ucsd.edu (W.H. Gerwick).
0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.07.001

1730
H. Gross et al. / Phytochemistry 71 (2010) 1729–1735
Fig. 1. Structures of lyngbic acid and all malyngamide analogs obtained during this study.
Table 1
1H and 13C NMR spectroscopic data for 8-O-acetyl-8-epi-malyngamide C (1) and malyngamide C acetate in CDCl3.
Position
1
8-O-acetyl-8-epi-malyngamide C (1)a
Malyngamide C acetatea,b
dH
dC
dH
dC
D(dC)
3.82 ddd (14.8, 4.8, 0.7)
3.99 ddd (14.7, 6.0, 1.2)
40.6 CH2
3.85 dd (4.8, 14.7)
3.97 dd (4.8, 14.7)
40.3 CH2
0.3
2
3
4
5
6
132.8 qC
122.7 CH
60.7 qC
201.7 qC
32.2 CH2
133.1 qC
122.8 CH
61.5 qC
200.9 qC
35.0 CH2
0.3
0.1
0.8
0.8
2.8
6.39 s
6.39 s
2.49 m
2.35 m
2.60 dt (4.0, 17.8)
2.01 m
7
1.95 m
22.4 CH2
21.5 CH2
0.9
2.17 m
8
5.49 ddd (3.7, 2.9)
3.67 d (2.9)
66.5 CH
61.6 CH
5.44 m
3.64 s
68.4 CH
62.6 CH
1.9
1.0
0.2
0.0
0.0
0.0
0.0
0.1
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.1
0.2
9
10
172.2 qC
36.3 CH2
28.4 CH2
130.7 CH
127.6 CH
36.4 CH2
80.7 CH
33.3 CH2
31.8 CH2
25.3 CH2
29.3 CH2
29.7 CH2
22.6 CH2
14.1 CH3
56.5 CH3
170.2 qC
172.4 qC
36.3 CH2
28.4 CH2
130.7 CH
127.6 CH
36.3 CH2
80.7 CH
33.3 CH2
31.8 CH2
25.3 CH2
29.3 CH2
29.7 CH2
22.6 CH2
14.1 CH3
56.4 CH3
170.4 qC
20
2.23 m
2.31 m
5.46 m
5.46 m
2.17 m
3.14 m
1.42 m
1.26 m
1.26 m
1.26 m
1.26 m
1.26 m
0.87 t (6.7)
3.31 s
2.17 m
2.29 m
5.46 m
5.46 m
2.25 m
3.12 m
1.42 m
1.25 m
1.25 m
1.25 m
1.25 m
1.25 m
0.86 m
3.30 s
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
NH
2.10 s
6.02 dd (4.8, 4.8)
20.8 CH3
2.14 s
6.10 br t (4.8)
20.9 CH3
0.1
a
The coupling constants (J) are in parentheses and reported in Hz; chemical shifts are given in ppm.
NMR values taken from Wright and Coll (1990).
b

H. Gross et al. / Phytochemistry 71 (2010) 1729–1735
1731
exomethylene functionality possessing a vinyl chloride (dH 6.39/dC
122.7) and an exchangeable amide signal (d 6.02), revealing further
typical structural features of this compound class. Based on its
mass and overall NMR features, isolate 1 was initially dereplicated
as malyngamide C acetate (Ainslie et al., 1985; Wright and Coll,
1990) using the MarinLit database (Blunt and Munro, 2005). How-
ever, when the spectroscopic data were carefully compared with
those reported in the literature, deviations in the specific rotation
in sign and value as well as in the 13C NMR spectra between 1
and malyngamide C acetate became apparent (Table 1). Analysis
of the 2D NMR data confirmed that 1 had the same structure as
malyngamide C acetate, and hence, the two molecules must differ
in configuration of either the stereogenic centers C-4, C-8, C-9 and
of malyngamide C acetate, obtained from a Lyngbya species, col-
lected in Madagascar (see Supplementary data).
In order to investigate the absolute configuration at C-8, 1 was
deacetylated and the hydrolysis product was subsequently analyzed
by single derivatization with methoxyphenylacetic acid (MPA) fol-
lowed by variable temperature 1H NMR spectroscopy analysis (Laty-
pov et al., 1998). Complete deacetylation of 1 without ring opening
of the epoxide function was accomplished by use of porcine liver
esterase. Calculation of the shift differences
D
dT1,T2 gave negative
values for H2-6 and H2-7, and a positive value for H-9 (Fig. 2), hence
establishing the 8-R absolute configuration (Seco et al., 2001). Com-
pound 1 was thus proven to be the C-8 epimer of the known com-
pound malyngamide C acetate, and for which we propose the
trivial name 8-O-acetyl-8-epi-malyngamide C.
C-70 and/or the geometry of the D2 and D4 double bonds.
0
Base hydrolysis of 1 yielded the acid portion, which was identical
in all respects to that previously reported for 7(S)-methoxytetra-
dec-4-(E)-enoic acid (2), thus establishing the 70S-configuration for
Compound 3 was analyzed for C24H38ClNO5 by HRMS. Inspection
of the 1H and 13C NMR data of 3 showed these data to be extremely
similar to those of 8-O-acetyl-8-epi-malyngamide C (1), the major
differences being the missing proton and carbon NMR resonances
accounting for the acetyl function (Table 2). Indeed, compound 3
was identical in all respects to the hydrolysis product of 8-O-acet-
yl-8-epi-malyngamide C (1). Thus, compound 3 was shown to be
the C-8 epimer of malyngamide C (4) and given the name 8-epi-mal-
yngamide C. Late in the preparation of this manuscript, we became
aware that Kwan et al. (2010) also recently isolated and defined the
structure of compound 3. In this latter work, the absolute configura-
tion was determined using a selective Mitsunobu inversion of C-8.
The reported structural data for 3 are in good agreement with our
findings and hence, corroborate the assignment of the absolute con-
figuration. The identities of the known compounds 4–7 were readily
established by direct comparison of HR-MS, 1H NMR and 13C NMR
spectroscopic data with published data (Gerwick et al., 1987; Orjala
et al., 1995; Wu et al., 1997).
1. The Z-configuration of the
D
2-double bond was evident from
the observation of ROE correlations from H-1 and H-3 and NOE
a
correlations from NH to H-3. The geometry of the D40-double bond
was inferred from 1H decoupling experiments. The olefinic proton
signals of C-40 and C-50 overlapped in CDCl3 (Table 1), but resolved
to a distinct pair of multiplets in C6D6. Decoupling of the adjacent
methylene protons H2-30 and H2-60 allowed measurement of the
0
3
JH4 -H5 as 15.4 Hz, confirming an E-geometry for D4 in 1.
0
0
These results focused our efforts on elucidation of the configu-
ration of the cyclohexyl amino moiety of 1. Moreover, this was the
part of the molecule where the majority of 13C NMR chemical shift
differences were observed between 1 and malyngamide C acetate
(Table 1). Because the empirical reversed octant rule can be applied
to determine the absolute configuration of an epoxy-ketone ring
system (Djerassi et al., 1965; Kan et al., 1998), the configuration
of carbons C-4 and C-9 was clarified on the basis of a circular
dichroism (CD) spectrum. The CD spectrum of 1 showed a negative
Cotton effect by the C-5 carbonyl group at 308 nm, indicating that
the absolute configuration of the epoxide was 4S and 9S, as ob-
served in malyngamide C acetate. This result was also supported
by comparison of the CD spectra of 1 with an authentic sample
All malyngamide compounds obtained during this study were
tested for cytotoxicity and for modulatory activity in the mamma-
lian Voltage-Gated Sodium Channel. Cytotoxicity (VGSC) was eval-
uated by testing the metabolites towards NCI-460 human lung
tumor, neuro-2a mouse neuroblastoma and HCT-116 cells and
in the disk-diffusion assay (Table 3). All compounds except
O-MPA
+ 0.024
- 0.078
H
H
-0.034
H
H
7
6
9
O
1
R
-0.057
N
H
H
O
3
Cl
Fig. 2. Results of the variable temperature NMR study on the R-methoxyphenylacetate (MPA) derivative of 8-epi-malyngamide C. Significant shifts in the 1H NMR spectrum of
the protons of interest are shown on the left panel, while the corresponding
dT1T2 values (ppm) are detailed on the right (R = acyl portion of lyngbic acid).
D

1732
H. Gross et al. / Phytochemistry 71 (2010) 1729–1735
Table 2
1H and 13C NMR spectroscopic data for 8-epi-malyngamide C (3) and malyngamide C in CDCl3.
Position
1
8-Epi-malyngamide C (3)a
Malyngamide Ca,b
dH
dC
dH
dC
D(dC)
3.84 m
3.99 m
41.6 CH2
3.81 ddd (14.1, 6.2, 1.3)
4.03 ddd (14.1, 4.7, 0.8)
40.2 CH2
1.4
2
3
4
5
6
132.9 qC
123.6 CH
61.0 qC
202.6 qC
31.8 CH2
133.3 qC
122.3 CH
61.8 qC
202.0 qC
35.4 CH2
0.4
0.7
0.8
0.6
3.6
6.42 s
6.38 dd (1.3, 0.8)
2.48 m
2.24 ddd (16.9, 12.5, 6.1)
2.57 ddd (16.9, 4.4, 3.9)
1.93 dddd (13.1, 12.5, 9.5, 4.4)
2.01 dddd (13.1, 6.1, 5.5, 3.9, 1.1)
4.39 ddd (9.5, 5.5, 1.1)
3.61 d (1.1)
7
1.89 m
2.11 m
4.48 s
3.59 d (2.6)
26.1 CH2
24.8 CH2
1.3
8
64.1 CH
64.3 CH
66.2 CH
65.3 CH
2.1
1.0
0.4
0.0
0.1
0.2
0.3
0.1
0.0
0.0
0.0
0.0
0.1
0.0
0.1
0.0
0.0
9
10
173.0 qC
36.3 CH2
28.3 CH2
130.7 CH
127.8 CH
36.2 CH2
80.7 CH
33.3 CH2
31.8 CH2
25.3 CH2
29.3 CH2
29.7 CH2
22.7 CH2
14.1 CH3
56.4 CH3
172.6 qC
36.3 CH2
28.4 CH2
130.5 CH
127.5 CH
36.3 CH2
80.7 CH
33.3 CH2
31.8 CH2
25.3 CH2
29.2 CH2
29.7 CH2
22.6 CH2
14.1 CH3
56.4 CH3
20
2.29 m
2.30 m
5.46 m
5.46 m
2.18 m
3.15 m
1.42 m
1.27 m
1.27 m
1.27 m
1.27 m
1.27 m
0.88 t (6.7)
3.32 s
2.29 m
2.30 m
5.46 m
5.46 m
2.20 m
3.15 m
1.42 m
1.25 m
1.25 m
1.25 m
1.25 m
1.25 m
0.86 t
30
40
50
60
70
80
90
100
110
120
130
140
150
NH
3.31 s
6.13 dd (6.2, 4.7)
6.07 br s
a
The coupling constants (J) are in parentheses and reported in Hz; chemical shifts are given in ppm.
NMR values taken from Ainslie et al. (1985).
b
Table 3
Cytotoxic activities of malyngamide analogs towards selected cancer cell lines in conventional cell line assays and in the disk-diffusion assay.
Compound
Cell line assay LC50
NCI-H460d
(l
g/mL)a,b
Neuro-2ad
Disk-diffusion assay (zu)a,c
HCT-116e
C38DL1210
e
8-O-acetyl-8-epi-malyngamide C (1)
Malyngamide C acetate
8-Epi-malyngamide C (3)
Malyngamide C
4.2
0.98
4.5
1.4
7.6
–
5.3
0.91
10.9
3.1
3.1
–
n.d.
0.8
n.d.
0.2
–
<250
<250
500
<250
250
6-O-acetylmalyngamide F (4)
Malyngamide H (5)f
–
–
Malyngamide J (6)
10.8
1.1
4.0
0.49
–
–
<250
<250
Malyngamide K (7)f
a
b
c
d
e
f
Dash = not active.
n.d. = not determined.
zu = zone units.
Four replicates at each test concentration were averaged to construct dose–response curves; LC50 values were determined graphically.
A minimum of two measurements were made at each concentration tested.
Since in this study, malyngamide H (5) and K (7) were obtained as an inseparable mixture, for testing, pure reference compounds were taken from an in-house compound
library.
Table 4
Mammalian Voltage-Gated Sodium Channel (VGSC) modulatory activity of malyngamide analogs.
Compound/conc. (
lg/mL)
Activation (%)a,b
3
Blocking (%)a,b,c
1
0.3
10
3
1
0.3
8-O-acetyl-8-epi-malyngamide C (1)
Malyngamide C acetate
Malyngamide C
–
–
–
–
–
–
–
–
–
n.d.
n.d.
n.d.
n.d.
82
71 16
–
–
28
69
10 15
–
–
15
33
–
–
–
–
9
–
–
Toxic
45
19
–
–
–
67
19
–
–
–
2
1
8
6-O-acetylmalyngamide F (4)
Malyngamide H (5)d
Malyngamide J (6)
n.d.
n.d.
44
–
5
19
–
4
Malyngamide K (7)d
–
a
b
c
Values are averages of four replicates.
Dash = not active.
n.d. = not determined.
d
Since in this study, malyngamide H (5) and K (7) were obtained as an inseparable mixture, for testing, pure reference compounds were taken from an in-house compound
library.

H. Gross et al. / Phytochemistry 71 (2010) 1729–1735
1733
malyngamide H (5) exhibited moderate activity with IC50 values
ranging from 0.5 to 20 g/mL. Of these, only metabolite 3 showed
lected from shallow waters (1–3 m) in True Blue Bay, Grenada,
on July 26, 1995 and stored in 2-propanol at ꢀ20 °C until workup.
Taxonomy was assigned by microscopic comparison with the
description given by Desikachary (1959).
l
selectivity toward solid-tumor cell lines. 8-O-acetyl-8-epi-mal-
yngamide C (1) and its free alcohol (3) were proven to be less ac-
tive than their respective epimers malyngamide C acetate and
malyngamide C, indicating the importance of the absolute config-
uration at this position for the interaction of malyngamides with
their corresponding target structure. In the sodium channel assay
(Table 4) compounds 1 and 5 were shown to have moderate block-
ing activity and malyngamide C and C acetate moderate activating
properties.
4.3. Extraction and isolation
A total of 19.1 g (dry wt.) of the cyanobacterium L. majuscula
was extracted five times with CH2Cl2–MeOH (2:1, v/v) to produce
a cytotoxic crude organic extract (662 mg), solid tumor selective
at 15 lg/disk: U251DCEM = 400 units. The extract was fractionated
by vacuum liquid chromatography (VLC) over silica gel using a
stepwise gradient of hexanes-EtOAc and EtOAc–MeOH to give nine
fractions. Fractions 5, 6 and 7, eluted with 60% and 80% EtOAc in
hexanes and EtOAc, respectively, showed cytotoxic effects (solid
3. Conclusions
In conclusion, the present work reports on two new natural
products (1, 3) in the malyngamide structure class which are C-8
epimers of the reported compounds malyngamide C and malynga-
mide C acetate. Biosynthetically, the NH, C-1 and C-2 carbons of
these malyngamide C derivatives are likely incorporated from gly-
cine, C-3 from an HMGCoA synthase-like reaction as determined
for jamaicamide A (Edwards et al., 2004), and this is followed by
three rounds of acetate extension. This is interesting in that the
C-8 carbon is predicted to derive from C-2 of acetate, and thus,
the C-8 hydroxyl functionality is likely introduced as a post-assem-
bly P450 oxidation. That both stereoisomers at C-8 in the malynga-
mide C structure class are produced in different populations is
intriguing, and appears to be differentiated by the ocean of origin.
Literature reports of the 8-S isomer (=malyngamide C) all derive
from Pacific collections [Fanning Island by Ainslie et al. (1985)
and Palmyra Atoll by Taniguchi et al. (2010)], whereas the 8-R iso-
mer has now been reported from two Caribbean collections [Gre-
nada in this work and Florida in the recent publication by Kwan
et al. (2010)]. Thus, cytochrome P450 enzymes with opposite chiral
preferences may have evolved in these different populations of L.
majuscula. It is additionally intriguing that this modest structural
change in the malyngamide C class (e.g. C-8 epimerization) results
in an approximately 5-fold reduction in biological activity, and
therefore identifies this as an important stereogenic site to be con-
sidered in any future medicinal chemistry efforts.
tumor selective at 15
ther investigated. Fraction 5 was further separated by RP-HPLC
m; MeOH–H2O
lg/disk: C38DL1210 = 300 units) and were fur-
(column: YMC ODS AQ-323, 250 ꢁ 10.0 mm, 5
l
(85:15), 2 mL/min) giving two distinct fractions. A second chroma-
tography of the first fraction by RP-HPLC on a Phenomenex Synergi
Fusion-RP 80 column (250 ꢁ 10.0 mm, 4
lm; MeOH–H2O (79:21),
2 mL/min) yielded 24.3 mg of 8-O-acetyl-8-epi-malyngamide C
(1). Further separation of VLC fraction 6 by RP-HPLC (column:
YMC ODS AQ-323, 250 ꢁ 10.0 mm, 5
lm; MeOH–H2O (85:15),
2 mL/min) resulted in additional quantities of compound
1
(9.4 mg) and an inseparable mixture of malyngamide H (6) and K
(8) (1.9 mg). Reversed-phase HPLC (column: YMC ODS AQ-323,
250 ꢁ 10.0 mm, 5
lm; MeOH–H2O (85:15), 2 mL/min) of VLC frac-
tion 7 yielded compounds 3 (0.9 mg), 6-O-acetyl malyngamide F
(5) (1.7 mg) and additional quantities of the inseparable mixture
of malyngamide H (6) and K (8) (1.0 mg). 1H NMR profiling of all
VLC fractions indicated fraction 8 (eluted with MeOH–EtOAc, 1:3,
v/v) also contained a malyngamide-type metabolite. Therefore,
VLC fraction 8 was first subjected to C18 HPLC (YMC ODS AQ-323,
250 ꢁ 10.0 mm, 5
lm; MeOH–H2O (90:10), 2 mL/min), and those
materials eluting at 12–14 min were then further purified with
RP-HPLC (Phenomenex Synergi Fusion-RP 80, 250 ꢁ 10.0 mm,
4 lm; MeOH–H2O (80:20), 2 mL/min) to give malyngamide J (7).
4.4. Basic hydrolysis of 8-O-acetyl-8-epi-malyngamide C (1)
4. Experimental
Compound 1 (6.9 mg) was dissolved in 2 mL solution of 10%
KOH in EtOH–H2O (4:1) and the stirred mixture was heated until
reflux began, this being maintained for 15 h. The hydrolysate was
concentrated in vacuo and partitioned between H2O and CH2Cl2.
The H2O layer was isolated, acidified, and extracted with CH2Cl2
to yield 1.6 mg (45.1%) of lyngbic acid (2).
4.1. General experimental procedures
Optical rotation measurements were recorded on
a Jasco
P-1010 polarimeter. CD spectra were measured using a model J-
720 Jasco spectropolarimeter. UV and FT-IR spectra were obtained
employing Hewlett Packard 8452A and Nicolet 510 instruments,
respectively. All NMR spectra were recorded on Bruker Avance
DRX300, DPX400 and DRX600 spectrometers. Spectra were refer-
enced to residual solvent signal with resonances at dH/C 7.26/77.1
(CDCl3) and dH/C 7.15/128.0 (C6D6). Low-resolution ESI-MS spectra
were obtained on a Thermo Finnigan LCQ Advantage mass spec-
trometer. High-resolution ESI-TOF and CI mass spectra were re-
corded on Waters Micromass LCT Classic and JEOL MSRoute mass
spectrometers, respectively. HPLC was carried out using a Waters
system consisting of a Rheodyne 7725i injector, two 515 pumps,
a pump control module and a 996 photodiode array detector. TLC
4.5. Enzymatic hydrolysis of 8-O-acetyl-8-epi-malyngamide C (1)
Compound 1 (4 mg, 8.0 lmol) was dissolved in MeOH (0.2 mL)
and the resulting solution was added to 0.3 M K2HPO4 buffer
(6 mL) (pH 8.0) containing 100 units porcine liver esterase (Sig-
ma–Aldrich). The solution was stirred for 12 h at room tempera-
ture and then repeatedly extracted with Et2O (3 ꢁ 10 mL). The
extracts were dried (MgSO4) and the solvent removed in vacuo to
yield 8-epi-malyngamide C (3, 3.6 mg, 98.3% yield).
grade (10–40 lm) Si gel was used for vacuum liquid chromatogra-
phy. All solvents were purchased as HPLC grade.
4.6. Preparation of the (R)-MPA-Cl
Oxalyl chloride (51.9
lL, 0.6 mmol) was added to a mixture of
4.2. Collection
R-MPA (10 mg, 0.06 mmol) and DMF (0.47
lL, 0.006 mmol) in hex-
anes at room temperature. After 48 h, the solvent was evaporated
to dryness at reduced pressure to afford 0.06 mmol of R-MPA-Cl
(11.1 mg, 100%).
The marine cyanobacterium L. majuscula (voucher specimen
available from WHG as collection no. GBI-26Jul95-07) was col-

1734
H. Gross et al. / Phytochemistry 71 (2010) 1729–1735
4.7. Preparation of the R-MPA ester derivative of 8-epi-malyngamide C
(3)
antiproliferation zone of 300 zu or greater. The differential cytotox-
icity of a pure compound was expressed by observing a zone differ-
ential of 250 units or greater between any solid-tumor cell (murine
colon C38, human colon HCT-116, human lung H125) and either
leukemia cells (murine L1210 or human CEM) or normal cells
(CFU-GM).
To a solution of 8-epi-malyngamide C (3) (3.6 mg) in CH2Cl2
(800 lL) were added DMAP (3 mg) and R-MPA-Cl (7.3 mg). The
reaction mixture was reacted for 24 h and then partitioned be-
tween EtOAc and 0.1 M NaHCO3, with the EtOAc layers then
washed with 0.1 M HCl. The EtOAc layer was evaporated and then
separated by RP-HPLC (column: Phenomenex Synergi Fusion-RP
4.11. Voltage-gated sodium channel modulation assays
80, 250 ꢁ 10.0 mm, 4
l
m; MeOH–H2O (80:20), 2 mL/min) to yield
The malyngamides were evaluated for their capacity to either
activate or block sodium channels using the following modifica-
tions to the cell-based bioassay of Manger et al. (1995). Twenty-
four hours prior to chemical testing, cells were seeded in 96-well
lL. Test chemicals
were dissolved in DMSO, serially diluted with medium and added
the R-MPA ester of 3 (1.8 mg, 37.7% yield).
4.8. Variable temperature NMR experiments
plates at 8 ꢁ 104 cells/well in a volume of 200
1H NMR and 1H–1H-COSY spectra (300 MHz) of the R-MPA ester
of 3 dissolved in CS2/CD2Cl2 (4:1) were recorded at T1 = 25o
(298 K), T1a = ꢀ50o (223 K) and T2 = ꢀ70o (203 K). Chemical shifts
(ppm) were internally referenced to the TMS signal (0 ppm). For
low-temperature NMR spectroscopy, the probe temperature was
cooled by a constant stream of liquid nitrogen, controlled by a
standard unit (see Supplementary data) calibrated with a methanol
reference. The sample was allowed to equilibrate for 15 min at
at 10
1.0 and 0.3
Plates to evaluate sodium channel activating activity received
20 L/well of either a mixture of 3 mM ouabain and 0.3 mM verat-
lL/well in quadruplicate, providing concentrations of 10, 3.0,
lg/mL. DMSO was less than 1% final concentration.
l
ridine (Sigma Chemical Co.) in 5 mM HCl, or 5 mM HCl alone in
addition to the test chemical. Plates were incubated for 18 h and
results compared to similarly treated solvent controls with 10 lL
medium added in lieu of the test chemical. The sodium channel
activator brevetoxin PbTx-1 (Calbiochem) was added at 10 ng/well
in 10 lL of medium and used as the positive control. Sodium chan-
nel blocking activity was assessed in a similar manner except that
the ouabain and veratridine stock solution was 5.0 and 0.5 mM,
respectively, and the sodium channel blocker saxitoxin (Calbio-
chem) was used as the positive control. Plates were incubated for
approximately 22 h.
each temperature before recording the spectra.
D
dT1T2 values
(dT1 ꢀ dT2) of all protons surrounding the chiral secondary alcohol
were calculated and applied to the corresponding model for the
assignment of the absolute configuration of secondary alcohols
by single derivatization, established by the Riguera group (Latypov
et al., 1998; Seco et al., 2001).
4.9. Cancer cell line assays (HCT-116, NCI-460, neuro-2a)
Human colon carcinoma cells HCT-116 (Brattain et al., 1981)
were grown in 5 mL culture medium (RPMI-1640 + 15% fetal bo-
vine serum containing 1% penicillin–streptomycin, and 1% gluta-
mine) (Moore and Woods, 1977) at 37 °C and 5% CO2 from a
starting cell density of 5 ꢁ 104 cells/T25 flask. On day 3, duplicate
wells containing HCT-116 cells were exposed to various concentra-
tions of the malyngamide analogs. Flasks were incubated for 120 h
(5 days) at 5% CO2 and 37 °C, and the cells harvested with trypsin,
washed once with Hanks’ balanced salt solution (HBSS), re-sus-
pended in HBSS, and counted using a hemocytometer. The results
were normalized to an untreated control. The IC50 value was deter-
mined using Prism 4.0 software (GraphPad, San Diego, CA). Cyto-
toxicity to NCI-H460 lung tumor cells and neuro-2a cells were
run in quadruplicate using the method of Alley et al. (1988) with
cell viability being determined by MTT reduction (Manger et al.,
1995). Cells were seeded in 96-well plates at 5000 and 8000
4.12. 8-O-acetyl-8-epi-malyngamide C (1)
Clear oil; [
a]26D +6 (c 0.35, EtOH); UV (EtOH) kmax 216 nm (log e
3.99), 278 nm (log
e
3.64); IR (KBr) mmax 3305, 2925, 2854, 1726,
1718, 1652, 1541, 1435, 1373, 1233, 1093, 1043 cmꢀ1; for 1H and
13C NMR spectroscopic data, see Table 1; HRESITOFMS m/z
498.2607 [M+H]+ (calcd. for C26H4135ClNO6, 498.2622).
4.13. Lyngbic acid (2)
Brownish oil; [
a
]
26D ꢀ6 (c 0.5, CHCl3) [lit. ꢀ5 (c 0.22, CHCl3) Kan
et al., 2000; ꢀ10o (c 0.5, CHCl3), Cardellina et al., 1978); HRCIMS
m/z 257.2110 [M+H]+ (calcd. for C15H29O3, 257.2117); 1H NMR
(300 MHz, CDCl3): d 0.88 (3H, t, J = 6.9 Hz, H-140), 1.27 m (10H,
m, H-90, H-100, H-110, H-120 and H-130), 1.42 (2H, m, H-80), 2.19
(2H, m, H-60), 2.36 (2H, m, H-20), 2.42 (2H, d, J = 5.9 Hz, H-30),
3.15 (1H, t, J = 5.8 Hz, H-70), 3.32 (3H, s, H-150), 5.49 (2H, m, H-
40and H-50).
cells/well in 180
Twenty-four hours later, the test malyngamide was dissolved in
DMSO and diluted into medium, and then added at 20 L/well pro-
ducing concentrations between 15 and 0.5 g. DMSO was less than
lL for H460 and neuro-2a cells, respectively.
l
l
4.14. 8-Epi-malyngamide C (3)
1% final concentration. After 48 h, the medium was removed and
cell viability determined.
Clear oil; [
3.59), 282 nm (log
a
]
26D ꢀ8 (c 0.5, EtOH); UV (EtOH) kmax 208 nm (log
e
e
1.46); IR (KBr) mmax 3322, 2927, 2855, 1714,
4.10. Disk diffusion soft agar colony formation assay
1651, 1537, 1432, 1265, 1092, 1071, 971, 915 cmꢀ1; for 1H and
13C NMR spectroscopic data, see Table 2; HRCIMS m/z 456.2506
[M+H]+ (calcd for C24H3935ClNO5, 456.2517).
An in vitro cell-based assay using murine L1210 (leukemia), C38
(colon), and CFU-GM (normal) cells and human HCT116 (colon),
H125 (lung), and leukemia (CEM) cells, assessed general and differ-
ential cytotoxicity of pure compounds (Valeriote et al., 2002). Sam-
4.15. R-MPA ester of 8-epi-malyngamide C
ples were dissolved in 250
l
L of DMSO, and duplicate 15
l
L
Yellow oil; 1H NMR (300 MHz, CDCl3, see Supplementary data):
d 0.88 (3H, t, J = 6.9 Hz, H-140), 1.26 m (10H, m, H-90, H-100, H-110,
H-120 and H-130), 1.42 (2H, br t, H-80), 1.93–2.50 (10H, m, H-6, H-7,
H-20, H-30, H-60), 3.13 (1H, t, J = 5.4 Hz, H-70), 3.31 (3H, s, H-150),
3.40 (3H, s, MPA-OCH3), 3.47 (1H, d, J = 2.6 Hz, H-9), 3.77 (1H, dd,
J = 15.4, 4.7, H-1a), 3.93 (1H, dd, J = 15.4, 5.7, H-1b), 4.80 (1H, s,
aliquots were applied to cellulose disks in agar plates containing
cells. After a period of incubation, a zone of cell colony inhibition
(z) was measured from the edge of each disk to the edge of colony
growth, and expressed as zone units (zu), where 200 zu = 6 mm.
General cytotoxic activity for a given sample was defined as an

H. Gross et al. / Phytochemistry 71 (2010) 1729–1735
1735
MPA-CH), 5.46 (2H, m, H-40and H-50), 5.54 br s (1H, br s, H-8), 5.96
(1H, br t, NH), 6.30 (1H, br s, H-3), 7.32–7.44 (5H, m, aromatic pro-
tons of MPA); LRESIMS m/z [M + Na]+ 626.3.
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Acknowledgments
Kan, Y., Sakamoto, B., Fujita, T., Nagai, H., 2000. New malyngamides from the
Hawaiian cyanobacterium Lyngbya majuscula. J. Nat. Prod. 63, 1599–1602.
Kwan, J.C., Teplitski, M., Gunasekera, S.P., Paul, V.J., Luesch, H., 2010. Isolation and
We gratefully acknowledge M.I. Schimerlik and M. Harder, nu-
cleic acid and protein core, EIHS Center at OSU (supported by
NIH/NIEHS 00210) and J. Morré, mass spectrometry facility, EIHS
Center at OSU for obtaining CD spectra and for all MS measure-
ments, respectively. We also thank T.L. Simmons and E.H. Andri-
anasolo for providing authentic samples of malyngamides C and
C acetate, respectively, and T. Byrum for help with data analysis.
This research was supported by NIH grants CA CA100851 and
NS053398.
biological evaluation of 8-epi-malyngamide
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