Palmyrolide A, an unusually stabilized neuroactive macrolide from palmyra atoll cyanobacteria

Article abstract of DOI:10.1021/ol101752n

Palmyrolide A (1) is a new neuroactive macrolide isolated from a marine cyanobacterial assemblage composed of Leptolyngbya cf. and Oscillatoria spp. collected from Palmyra Atoll. It features a rare N-methyl enamide and an intriguing t-butyl branch; the latter renders the adjacent lactone ester bond resistant to hydrolysis. Consistent with its significant suppression of calcium influx in cerebrocortical neurons (IC₅₀ = 3.70 μM), palmyrolide A (1) showed a relatively potent sodium channel blocking activity in neuro-2a cells (IC₅₀ = 5.2 μM), without appreciable cytotoxicity.

Full text of DOI:10.1021/ol101752n

ORGANIC
LETTERS
2010
Vol. 12, No. 20
4490-4493
Palmyrolide A, an Unusually Stabilized
Neuroactive Macrolide from Palmyra
Atoll Cyanobacteria
Alban R. Pereira,† Zhengyu Cao,‡ Niclas Engene,† Irma E. Soria-Mercado,†,§
Thomas F. Murray,‡ and William H. Gerwick*,†
Center for Marine Biotechnology and Biomedicine, Scripps Institution of
Oceanography and Skaggs School of Pharmacy and Pharmaceutical Sciences,
UniVersity of California San Diego, La Jolla, California 92037, Department of
Pharmacology, Creighton UniVersity School of Medicine, Omaha, Nebraska 68178,
and Facultad de Ciencias Marinas, UniVersidad Autonoma de Baja California,
Ensenada, BC, 22830, Mexico
wgerwick@ucsd.edu
Received July 27, 2010
ABSTRACT
Palmyrolide A (1) is a new neuroactive macrolide isolated from a marine cyanobacterial assemblage composed of Leptolyngbya cf. and
Oscillatoria spp. collected from Palmyra Atoll. It features a rare N-methyl enamide and an intriguing t-butyl branch; the latter renders the
adjacent lactone ester bond resistant to hydrolysis. Consistent with its significant suppression of calcium influx in cerebrocortical neurons
(IC50 ) 3.70 µM), palmyrolide A (1) showed a relatively potent sodium channel blocking activity in neuro-2a cells (IC50 ) 5.2 µM), without
appreciable cytotoxicity.
Suppression and/or activation of spontaneous Ca2+ oscilla-
tions of murine cerebrocortical neurons1 has proven to be
an extremely sensitive screening method for the discovery
of new neurotoxins, including the recently reported cyano-
bacterial metabolites hoiamide A,2a alotamide A,2b and
palmyramide A.2c In the case of hoiamide A, further
pharmacological characterization found it to be a partial
agonist at neurotoxin site 2 of voltage-gated sodium channels
(VGSCs).2a
the Ca2+-sensitive fluorescent dye, fluo-3.1 First, those
metabolites that trigger Ca2+ influx can be easily revealed
by monitoring for this ion using a Fluorometric Imaging Plate
Reader (FLIPR). Second, because these cultures display
spontaneous Ca2+ oscillations, they provide a robust screen-
ing system for the discovery of small molecule ion channel
modulators. Synchronized Ca2+ oscillations in cultured
neurons are considered to be a neuronal network phenomenon
dependent on VGSC-mediated action potentials.1,3 Thus, the
latter measurement allows detection of VGSC antagonists
Primary cultures of cerebrocortical neurons allow the
detection of two distinct actions when cells are loaded with
(2) (a) Pereira, A.; Cao, Z.; Murray, T. F.; Gerwick, W. H. Chem. Biol.
2009, 16, 893–906. (b) Soria-Mercado, I. E.; Pereira, A.; Cao, Z.; Murray,
T. F.; Gerwick, W. H. Org. Lett. 2009, 11, 4704–4707. (c) Taniguchi, M.;
Nunnery, J. K.; Engene, N.; Esquenazi, E.; Byrum, T.; Dorrestein, P. C.;
† University of California San Diego.
‡ Creighton University School of Medicine.
§ Universidad Autonoma de Baja California.
(1) Dravid, S. M.; Murray, T. F. Brain Res. 2004, 1006, 8–17.
Gerwick, W. H. J. Nat. Prod. 2010, 73, 393–398
.
10.1021/ol101752n  2010 American Chemical Society
Published on Web 09/16/2010

Table 1. NMR Spectroscopic Data (600 MHz, CDCl3) of Palmyrolide A (1)
a
carbon
δC
type
δH, multiplicity (J in Hz)b
HMBCc
COSY
NOESY
1
2
172.9
34.4
24.3
CH2
2.38 m
1, 3, 4
3a, 3b
4a, 4b, 18
3a
3b
4a
4b
5
6a
6b
7
CH2
1.57 m
1.78 m
1.06 m
1.33 m
1, 2, 4, 5
1, 2, 4, 5
2, 3, 5, 6, 12
2, 3, 5, 12
6, 7
4, 5, 12
4, 5, 7, 12
5, 8, 9/10/11, 13
2, 3b, 4a, 4b
2, 3a, 4a, 4b
3a, 3b, 4b, 5
3a, 3b, 4a
4a, 6a, 12
5, 6b, 7
18
7, 18, 19
2, 6a, 7
2, 6b, 7, 18, 19
7, 18, 19
4a, 9/10/11, 12
4b, 9/10/11, 12
3b, 4a, 4b, 5, 9/10/11, 12
34.6
CH2
29.3
35.6
CH
CH2
1.48 m
1.38 ddd (1.5, 8.5, 14.5)
1.66 ddd (3.0, 11.0, 14.5)
4.88 dd (1.5, 11.0)
6a, 7
6a, 6b
76.9
35.2
26.1
20.6
175.3
38.8
32.8
27.0
117.3
130.6
16.8
31.7
CH
8
9/10/11
12
13
14
15
16
17
18
19
20
3×CH3
0.86 s
0.90 d (6.5)
6, 7, 8
4, 5, 6
6a, 6b, 7, 19
6a, 6b, 7
CH3
5
CH
2.47 m
1.78 m
2.29 m
5.27 dt (7.5, 14.0)
6.47 d (14.0)
1.21 d (7.0)
3.04 s
13, 15, 16, 19
13, 14, 16, 17, 19
14, 15, 17, 18
15, 16, 18
1, 16, 17, 20
13, 14, 15
15, 19
14, 16
15, 17, 18
16, 18
16, 17
14
CH2
CH2
CH
CH
CH3
17
19
15, 20
2, 3a, 3b, 4b, 5
3b, 4b, 5, 9/10/11, 16
17
NCH3
1, 17, 18
a Recorded at 150 MHz. b Recorded at 600 MHz. c From proton to the indicated carbon.
(as opposed to activators), glutamate (AMPA and mGluR)
receptor antagonists, adenosine A1 receptor agonists, and
other compounds that disrupt neuronal action potentials or
glutamate neurotransmission.1,3 Glutamate-mediated intra-
cellular Ca2+ overload is known to contribute to neuronal
death in several human pathological conditions (hypoxia-
ischemia, hypoglycemia, trauma, epilepsy)4,5 and possibly
to neurodegenerative disorders such as Alzheimer’s, Hun-
tington’s, and motor neuron disease.6,7
lecular formula C20H35O3N and four degrees of unsaturation.
The most intriguing resonance in its H NMR spectrum
1
(Table 1 and SI) was an intense singlet at δ0.86 (nine
protons), which could be attributed to three isochronous
methyl groups comprising a t-butyl moiety.8 Also present
were the methyl doublets at δ0.90 and δ1.21, as well as the
N-methyl singlet at δ3.04. The 1H NMR of 1 was completed
by a deshielded methine proton at δ4.88 and a terminal 1,2-
disubstituted vinylic system represented by protons at δ5.27
(dt) and δ6.47 (d). The presence of a t-butyl moiety was
supported in the 13C NMR spectrum of 1 by a very intense
resonance at δ26.1, as well as a quaternary carbon at δ35.2.
Additionally, the deshielded carbons at δ117.3 and δ130.6
were in agreement with a 1,2-disubstituted double bond,
whereas the carbonyls at δ172.9 and δ175.3 indicated a total
of two ester or/and amide functionalities. As detailed below,
extensive analysis of these 1H and 13C NMR resonances using
HSQC, HMBC, COSY, and NOESY led us to deduce the
planar structure of 1.
The intense singlet at δ0.86 (H9, H10, H11) showed
HMBC correlations with the quaternary carbon C8 (δ35.2),
oxymethine C7 (δ76.9), and methylene carbon C6 (δ35.6)
(Figure 1A). This last carbon was found by HSQC to bear
the diastereotopic proton resonances H6a (δ1.38) and H6b
(δ1.66), which according to the COSY spectrum participated
in a spin system (I in Figure 1) that included the flanking
methines H7 (δ4.88) and H5 (δ1.48), both diastereotopic
methylene pairs H4a (δ1.06)/H4b (δ1.33) and H3a (δ1.57)/
H3b (δ1.78), and a final third methylene H22 (δ2.38). The
As part of our drug discovery screening efforts, we
determined that the extract of a cyanobacterial assemblage,
collected from Palmyra Atoll in the Northern Pacific Ocean,
exhibited significant suppression of Ca2+ oscillations in
cultured mouse cerebrocortical neurons. A two-pronged
taxonomic approach, consisting of both detailed morphologi-
cal characterization and phylogenetic analysis based on the
SSU (16S) rRNA genes (see Supporting Information, SI),
revealed two genera of filamentous cyanobacteria, Leptolyn-
gbya cf. sp. and Oscillatoria sp. A portion of the cyanobac-
1
terial extract (0.2768 g) was subjected to bioassay and H
NMR-guided fractionation to yield 10.2 mg (3.7%) of pure
palmyrolide A (1) {[R]23D -29 (c 0.9, CHCl3)}, as the only
neuroactive component. Unfortunately, efforts directed to
identify the actual producer of 1 in the assemblage via
MALDI imaging techniques2c were not successful, due in
part to the lack of live cultures of these organisms.
HRESIMS of 1 yielded an [M + H]+ peak at m/z 338.2687
(calcd for C20H36O3N, 338.2690), consistent with the mo-
(3) Dravid, S. M.; Baden, D. G.; Murray, T. F. J. Neurochem. 2004,
89, 739–749
.
(8) Identification of this substructure facilitated dereplication efforts
because only a handful of cyanobacterial-derived secondary metabolites
possess such a structural feature, according to MarinLit, Marine Natural
Products Bibliography Software (update March 2010), University of
Canterbury: Christchurch, New Zealand, 2010.
(4) Berman, F. W.; Murray, T. F. J. Neurochem. 2000, 74, 1443–1451
(5) Choi, D. W. Neuron 1998, 1, 623–634
(6) Choi, D. W. Ann. N.Y. Acad. Sci. 1994, 747, 162–171
.
.
.
(7) Choi, D. W. J. Neurobiol. 1992, 23, 1261–1276
.
Org. Lett., Vol. 12, No. 20, 2010
4491

The relative configuration for the 1,3-methine system
C5-C7 was determined via J-based configuration analysis.9
First, the diastereotopic methylene protons H26 (δa1.38,
δb1.66) were stereospecifically assigned with respect to H7
(δ4.88), according to the magnitude of the coupling constants
10
provided by the H NMR, HETLOC, and HSQMBC11
spectra (Figure 1B). Thus, H7 was found to be gauche to
H6a (δ1.38) and anti to H6b (δ1.66), and the rotamer was
assigned as C-1 (S, L, S, S, S, S, L). In a second step, we
evaluated the relationship between methine H5 (δ1.48) and
these stereospecifically defined H6-methylene protons. In this
1
3
case, intermediate values for the homonuclear JH5,H6a (8.5
Hz) and four out of the five heteronuclear coupling constants
led us to consider two major rotamers around the C5-C6
carbon bond, with H5/H6a exhibiting both anti and gauche
orientations. Fortunately, all six of the possible rotamer pairs
with the different prochiral assignments for methylene
protons can be unequivocally distinguished using their full
arrangement of J values.9 The only combination in agreement
with our data corresponded to the rotamer pair D-1/D-2 (M,
S, M, S, M, M, M). Finally, the proposed syn configuration
between H5-H26-H7 (Figure 1A) was in agreement with
the NOE interactions observed among these protons.
Figure 1. (A) Representative 2D NMR correlations and (B) J-based
configuration analysis for palmyrolide A (1) (S, small; M, medium;
L, large; H6a, proton at high field; H6b, proton at low field).
aRotamer designation according to Murata.9
HMBC data obtained for all of these protons correlated with
the COSY-derived sequence above and enabled us to position
both the carbonyl C1 (δ172.9) as the terminating element
of the carbon chain (via correlations from H3a, H3b, and
H2) and the methyl branch H12 (δ0.90, d, J ) 6.5 Hz) at
C5 [correlations from H12 to δ34.6 (C4), δ29.3 (C5), and
δ35.6 (C6)]. Finally, the oxymethine proton H7 (δ4.88)
displayed, among others, a strong HMBC correlation with
carbonyl C13 (δ175.3), confirming the presence of an ester
Determination of the absolute configuration of 1 was
challenging due to a remarkable resistance of the ester group
at C13 to hydrolysis (Figure 2, route A). Our initial strategy
involved selective hydrolysis of this moiety, followed by
1
linkage as suggested by its deshielded H NMR chemical
shift value.
This ester functionality accounted for the second of four
unsaturations present in metabolite 1 and comprised the start
of a second carbon chain further elongated by the methyl-
substituted methine H14 (δ2.47), according to HMBC correla-
tions from methyl H19 (δ1.21) and H14 itself to C13. COSY
data indicated that both of these proton resonances belonged
to a second spin system (II) that sequentially included the two
methylene pairs H15 (δ1.78) and H16 (δ2.29), as well as vinylic
protons H17 (δ5.27, dt, J ) 7.5, 14.0 Hz) and H18 (δ6.47, d,
3
J ) 14.0 Hz). A vicinal JH17,H18 of 14.0 Hz and the doublet
multiplicity displayed by H18 confirmed this as a terminal (E)-
1,2-disubstituted double bond.
1
Furthermore, both H (δ6.47) and 13C (δ130.6) chemical
shifts at C18 were consistent with the presence of an attached
nitrogen atom. Conclusive proof of an N-methyl amide
functionality at this position was provided by HMBC
correlations from the methyl singlet H20 (δ3.04) to carbon
resonances C17 (δ117.3), C18 (δ130.6), and C1 (δ172.9).
The latter key HMBC correlation, along with H18 to C1,
assembled a 15-membered macrocyclic ring structure for
palmyrolide A (1) which satisfied all of the required degrees
of unsaturation and possessed a molecular formula consistent
with the HRESIMS data.
Figure 2. Derivatization of palmyrolide A (1). (A) All reactions
approximately 7 mM of 1 (2 mL). (B) Representative section of
the H NMR spectrum (500 MHz, CDCl3, see SI).
1
(9) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.; Tachibana,
K. J. Org. Chem. 1999, 64, 866–876.
4492
Org. Lett., Vol. 12, No. 20, 2010

sequential derivatization of the resulting C7-secondary hy-
droxy and the C13-carboxy groups with Mosher’s acid
chloride and PGME, respectively.12,13 However, both acid
and basic hydrolysis conditions afforded compound 2, which
undergoes further addition reactions to the aldehyde func-
tionality, as indicated by the presence of several minor
analogues in the crude reaction products (see SI for a
mechanistic proposal on the formation of 2). Furthermore,
the expected C1-C12 t-butyl hydroxy acid derived from 1
was not isolated even under more strenuous reaction condi-
tions, suggesting the possibility of dehydration at C7 (the
donated from S-adenosyl-L-methionine (SAM).16 Both the
C19 and C20 methyl groups likely also come from SAM,
whereas the C12 methyl resides at a predicted carbonyl site
in the nascent polyketide and, thus, should be produced by
a ꢀ-branch mechanism involving an HMGCoA synthase
cassette.17 A predicted NRPS module next adds glycine that
is ketide extended, reduced at the ꢀ-carbonyl, dehydrated,
and double bond isomerized from C16-C17 to form the
C17-C18 enamide. Cyclization to 1 likely occurs coincident
with offloading from this final NRPS module but must
overcome the extreme steric congestion that is present about
this ester functionality.18
1
δ4.88 oxymethine disappears in the H NMR spectra of
reaction crudes).
Pure palmyrolide A (1) showed significant inhibition of
Ca2+ oscillations in murine cerebrocortical neurons, with an
IC50 value of 3.70 µM (2.29-5.98 µM, 95% CI) (see SI).
Additionally, in an assay designed to detect sodium channel
blocking activity using mouse neuroblastoma (neuro2a) cells,
compound 1 suppressed the veratridine- and ouabain-induced
sodium overload that leads to cytotoxicity, with an IC50 of
5.2 µM. Finally, palmyrolide A was not cytotoxic when
tested against H-460 human lung adenocarcinoma cells up
to 20 µM. These results suggest that palmyrolide A (1) may
function as a VGSC antagonist to suppress spontaneous Ca2+
oscillations and to protect against veratridine-induced sodium
influx, making it an intriguing candidate for further phar-
macological exploration.
The unexpected stability of this ester, clearly due to a
combination of steric and electronic effects provided by the
contiguous t-butyl substituent at C7, and perhaps assisted
by the C19 methyl group, leads us to speculate that
cyanobacteria might produce secondary metabolites exhibit-
ing this architecture to prevent cleavage of the lactone ester
bond and preserve the bioactive macrocycle under a wide
variety of conceivable environmental conditions. Our specu-
lation that the t-butyl group in palmyrolide A protects against
lactone hydrolysis is consistent with known principles in
medicinal chemistry (e.g., pivalic acid esters) and represents
an interesting chemical biology concept that warrants further
exploration.
The configuration of the stereocenter at C14 was deter-
mined by ozonolysis and oxidative workup of palmyrolide
A (1), followed by acid hydrolysis of the resulting crude
reaction product to afford 2-methylglutaric acid (3), which
is commercially available as its pure (R)-(-)- and (S)-(+)-
enantiomers (route B). Further derivatization with (S)-(+)-
2-octanol and acetyl chloride afforded the amiable diester 4
which was more easily detected and purified than its diacid
precursor. Comparison of NMR data and specific rotation
values measured for 4 and the similarly derivatized standards
(see SI) led to assignment of the C14 chiral center as R.
Unfortunately, this procedure failed to produce a suitable
C1-C12 derivative, hindering our efforts to assign the
absolute configuration at C5 and C7 (relative configuration
shown in Figure 1A).
Palmyrolide A (1) is structurally related to the laingolides,
initially reported from Papua New Guinea specimens of
Lyngbya bouillonii.14 However, given the morphological
similarities between Lyngbya and Oscillatoria cyanobacteria,
and the fact that the laingolides producer was described solely
from morphological characters rather than with the inclusion
of phylogenetic analysis, we speculate that the laingolides
may have been actually isolated from an Oscillatoria sp.
Biogenetically,15 1 appears to derive from a combination of
PKS and NRPS pathways, with its t-butyl appendage likely
assembled from malonyl-CoA and three methyl groups
Acknowledgment. We thank A. Jansma (UCSD) and Y.
Su (UCSD) for assistance with NMR and HRMS data
acquisition, respectively. Support was provided by NIH
NS053398 (W.H.G and T.F.M), NSF CHE-0741968 (NMR,
UCSD), and Consejo Nacional de Ciencia y Tecnologia of
Mexico (I.S.M).
Supporting Information Available: Experimental, full
NMR data of 1, 2, and 4 (all stereoisomers), bioassay data,
and taxonomic characterization. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL101752N
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