Dudawalamides A-D, Antiparasitic Cyclic Depsipeptides from the Marine Cyanobacterium Moorea producens

Article abstract of DOI:10.1021/acs.jnatprod.7b00034

A family of 2,2-dimethyl-3-hydroxy-7-octynoic acid (Dhoya)-containing cyclic depsipeptides, named dudawalamides A-D (1-4), was isolated from a Papua New Guinean field collection of the cyanobacterium Moorea producens using bioassay-guided and spectroscopic approaches. The planar structures of dudawalamides A-D were determined by a combination of 1D and 2D NMR experiments and MS analysis, whereas the absolute configurations were determined by X-ray crystallography, modified Marfey's analysis, chiral-phase GCMS, and chiral-phase HPLC. Dudawalamides A-D possess a broad spectrum of antiparasitic activity with minimal mammalian cell cytotoxicity. Comparative analysis of the Dhoya-containing class of lipopeptides reveals intriguing structure-activity relationship features of these NRPS-PKS-derived metabolites and their derivatives.

Full text of DOI:10.1021/acs.jnatprod.7b00034

Article
pubs.acs.org/jnp
Dudawalamides A−D, Antiparasitic Cyclic Depsipeptides from the
Marine Cyanobacterium Moorea producens
Jehad Almaliti,†,‡,# Karla L. Malloy, Evgenia Glukhov, Carmenza Spadafora, Marcelino Gutierrez,†,⊥
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 92093, United States
‡
Department Pharmaceutical Sciences, College of Pharmacy, The University of Jordan, Amman, 11942, Jordan
§
⊥
Centro de Biología Celular y Molecular de Enfermedades and Centro de Biodiversidad y Descubrimiento de Drogas, Instituto de
Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT-AIP), City of Knowledge, Panama 0843-01103, Republic of
Panama
*
S Supporting Information
ABSTRACT: A family of 2,2-dimethyl-3-hydroxy-7-octynoic acid (Dhoya)-containing cyclic depsipeptides, named
dudawalamides A−D (1−4), was isolated from a Papua New Guinean field collection of the cyanobacterium Moorea producens
using bioassay-guided and spectroscopic approaches. The planar structures of dudawalamides A−D were determined by a
combination of 1D and 2D NMR experiments and MS analysis, whereas the absolute configurations were determined by X-ray
crystallography, modified Marfey’s analysis, chiral-phase GCMS, and chiral-phase HPLC. Dudawalamides A−D possess a broad
spectrum of antiparasitic activity with minimal mammalian cell cytotoxicity. Comparative analysis of the Dhoya-containing class
of lipopeptides reveals intriguing structure−activity relationship features of these NRPS−PKS-derived metabolites and their
derivatives.
arine cyanobacteria are one of the most prolific sources
kulolide in 1993, a number of structurally similar cyclic
depsipeptides have been isolated from marine cyanobacteria
that contain either the Dhoya or the 3-hydroxy-2-methyl-7-
M
of biologically active secondary metabolites, often of
1
,2
mixed peptide and polyketide biosynthetic origin. Inves-
tigation of these marine bacteria has resulted in the discovery of
a wealth of biologically active compounds, and the majority of
these are peptides and peptide-derived compounds. Of special
note, a number of cyanobacterial peptides and related hybrid
metabolites have been pursued as therapeutic lead com-
8
,14−19
octynoic acid (Hmoya) moiety.
These molecules are
likely produced by hybrid biosynthetic pathways that are
composed of nonribosomal peptide synthetases (NRPS) and
20,21
polyketide synthetases (PKS).
Depsipeptides containing
Dhoya and Hmoya possess an array of biological activities
including cytotoxicity to P388 murine leukemia cells, LoVo
human colon cancer cells, H-460 human lung cancer cells, and
2
−4
pounds.
Recent examples include dolastatin 10, curacin A,
5
−7
largazole, and the carmaphycins.
Expanding on the known
8
,14−19
brine shrimp, as well as in various antiparasitic assays.
assembly of cyanobacterial peptides, we have reported a
number of cyclic depsipeptides including viequeamides A and
B, yanucamides A and B, mantillamide, antanapeptins A−D,
In our continuing search for biologically active compounds
from marine organisms, the extract of the marine cyanobacte-
rium Moorea producens collected from Dudawali Bay, Papua
New Guinea, was found to contain several related molecules
8
−12
apratoxins D, F, and G, and wewakpeptins A−D.
Kulolide, isolated from the cephalaspidean mollusk Phi-
linopsis speciosa by Scheuer and co-workers, was the first cyclic
depsipeptide reported to contain the Dhoya (2,2-dimethy-3-
Received: January 12, 2017
13
hydroxy-7-octynoic acid) moiety. Since the isolation of
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00034
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 1. Structures of dudawalamides A−D (1−4).
that shared similar fragmentation patterns by MS/MS
2
2
analysis. In this study, we present the isolation, structure
elucidation, and biological evaluation of four structurally similar
cyclic Dhoya-containing depsipepeptides, the dudawalamides
A−D, from this Papua New Guinea collection. The planar
structures of these compounds were determined by 2D NMR
and mass spectrometric methods whereas the absolute
configurations were determined by X-ray crystallography,
chiral-phase GCMS, chiral-phase HPLC, or modified Marfey’s
analysis. Biological evaluation of these four new compounds
revealed selective antiparasitic activity with low host cell
cytotoxicity as well as some preliminary structure−activity
relationships (SAR).
RESULTS AND DISCUSSION
A sample of the cyanobacterium M. producens was collected in
006 from shallow marine habitats in Dudawali Bay, Papua
■
2
New Guinea. The sample was preserved in 1:1 2-propanol−
seawater and then subjected to CH Cl −MeOH (2:1)
2
2
extraction, yielding an extract mass of 1.99 g. The extract was
fractionated on normal-phase silica vacuum liquid chromatog-
raphy in a gradient from 100% hexanes to 100% EtOAc to
1
00% MeOH, providing nine subfractions (A−I). Two
relatively polar fractions (F, G), eluting with 80% to 100%
EtOAc, were further purified by reversed-phase HPLC and
analyzed by LCMS to afford a new family of metabolites called
dudawalamides A−D (1−4). This collection also yielded the
known cyanobacterial natural products apratoxin A and
Figure 2. Key 2D NMR correlations of dudawalamide A−D (1−4).
of seven carbonyls at δ 168−175 and a phenyl ring, accounting
C
23,24
majusculamides A and B,
identified by comparison with
for 11 degrees of unsaturation and leaving four additional
double bonds or rings unassigned. The H NMR spectrum was
well dispersed and showed several proton resonances indicative
1
authentic standards and published data, respectively. The
structures of the new dudawalamides A−D (1−4) were
determined by 1D and 2D NMR, mass spectroscopy (MS),
and X-ray crystallography (Figures 1 and 2).
Positive ion LC-LRESI-MS analysis of the extract revealed
four new compounds with protonated molecules at m/z 752.40,
of amide bonds (e.g., NH and NCH groups) and an aromatic
3
ring structure. The presence of four shielded methyl groups
composed of three doublets and one triplet was suggestive of
1
isoleucine- and alanine-type moieties. Integrated H NMR,
7
86.47, 794.87, and 808.93 (LC-HRMS gave m/z 752.4220,
COSY, HSQC, and HMBC data were used to assign six partial
structures consisting of the amino acids glycine, N-methyl-
phenylalanine, N-methylisoleucine, proline (accounting for a
786.4429, 794.4694, and 808.4849 for molecular formulas of
C H N O , C H N O , C H N O , and C H N O ,
40
57
5
9
44 59
5
8
43 63
5
9
44 65
5
9
th
respectively). Their closely aligned molecular formulas
suggested that they were related to one another and, hence,
were given the names dudawalamides A−D (1−4).
12 degree of unsaturation), alanine, and lactic acid.
A final partial structure was shown by these same NMR
methods to be 2,2-dimethyl-3-hydroxy-7-octynoic acid. An
HSQC experiment revealed that a doublet methine proton at
δ 5.58 (J = 10.8 Hz) was connected to a carbon at δ 77.1,
Dudawalamide A (1, C H N O for 15 degrees of
4
0
57
5
9
unsaturation) was found as the major component in the
mixture (0.7% of extract). C NMR data showed the presence
H
C
1
3
indicating that it was also attached to an oxygen atom. COSY
B
DOI: 10.1021/acs.jnatprod.7b00034
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Table 1. NMR Data (CDCl ) for Dudawalamide A (1) ( H 500 MHz, 13C 125 MHz)
Article
1
3
residue
Dhoya
position
δC, type
174.6, C
δH (J in Hz)
HMBC
COSY
1
2
3
47.6, C
77.1, CH
28.0, CH2
5.58, d (10.8)
1.87, m
1, 2, 4, 9, 10, 38
4
4
4
5
6
6
7
8
9
1
a
5
3, 4b, 5
3, 4a
4, 6
b
1.64, m
3, 6, 7
6, 7
24.4, CH2
17.9, CH2
1.63, m
a
2.26, m
4, 5, 7
4, 5, 7
5, 6b
5, 6a
b
2.39, m
83.7, C
69.3, CH
23.9, CH3
17.6, CH3
169.8, C
70.9, CH
18.4, CH3
174.9, C
46.2, CH
15.3, CH3
NH
1.99, t (2.7)
1.25, s
3, 7, 10
3, 7, 9
0
1.33, s
Lac
Ala
11
1
1
2
3
5.40, q (6.7)
1.40, d (6.8)
1, 11, 13
11, 12
13
12
14
15
16
17
4.64, m
14, 16
15
16
15
1.51, d (7.2)
6.78, d (5.6)
11, 15, 16
N-Me-Ile
18
168.5, C
56.6, CH
33.9, CH
23.9, CH2
10.9, CH3
1
2
2
2
2
2
9
0
1
2
3
4
4.85, d (11.0)
2.03, m
18, 24
20
19
19, 23
20, 22
21
1.43, m
19, 20, 22, 23
20, 21
0.83, t (7.0)
0.77, d (6.5)
3.26, s
15.0, CH , CH3
19, 20, 21
14, 18, 19
20
3
30.3, CH3
175.0, C
Pro
25
2
2
2
2
2
2
6
58.4, CH, CH2
46.3, CH2
21.3, CH2
4.64, m
3.33−3.46, m
0.89 m
25, 27, 28, 29
28, 29
29b
7
28a, 28b
27
8a
8b
9a
9b
27, 29
1.43 m
27
30.8, CH2
0.88 m
28
28
26, 28
26, 28
1.84 m
N-Me-Phe
30
171.4, C
57.1, CH
34.3, CH2
3
3
3
3
3
3
3
3
1
5.81, dd (12.2, 4.8)
2.86, m
30, 33
32a, 32b
32b
2a
2b
3
31, 33, 34
31, 33, 34
3.58, m
32a
137.1, C
4
129.1, CH
128.3, CH
126.4, CH
30.3, CH3
172.6, C
7.18−7.22, m
7.24, d (7.4)
7.15−7.19, m
3.26, s
32, 33, 35, 36
33, 34
35
5
34, 36
35
6
35
7
25, 31
Gly
38
3
3
4
9a
40.9, CH2
4.50, dd (18.0, 8.1)
3.57, m
30, 38
30, 38
30
39b
9b
0
39a
NH
9.00, dd (8.2, 4.3)
39a, 39b
and HMBC correlations were used to consecutively assign the
(1), and thus the final degree of unsaturation was an overall
macro-ring structure.
adjacent C-4, C-5, and C-6 methylenes (δ 28.0, 24.4, and
C
1
7.9). The distal methylene protons (δ 2.26−2.39; δ 17.9)
HMBC correlations were used to elucidate the sequence of
residues in dudawalamide A (1). Both the α-proton and N-
methyl protons of the N-Me-isoleucine residue showed HMBC
H
C
showed HMBC correlations to a terminal alkyne group with a
quaternary carbon at δ 83.7 and a methine carbon at δ 69.3
C
C
(
δH 1.99), thereby accounting for two more degrees of
correlations to the alanyl carbonyl carbon at δ 174.9, revealing
C
unsaturation. Additionally, two- and three-bond HMBC
correlations were observed between two singlet methyl groups
that an amide bond bridged these two residues. An HMBC
correlation was also observed between the NH proton of the
alanyl residue to the carbonyl carbon of the lactic acid (δC
169.8), indicating a second amide linkage. The α-methine
proton at δH 5.40 of the lactic acid showed an HMBC
correlation to the carbonyl carbon of the Dhoya moiety (δC
174.6), revealing this ester linkage in dudawalamide A (1). In
to a quaternary carbon (δ 47.6), an ester carbonyl carbon (δC
C
1
74.6), and the above-described C-3 oxygen-bearing carbon
atom (δ 77.1), thus completing this partial structure as 2,2-
C
dimethyl-3-hydroxy-7-octynoic acid. This final residue ac-
counted for all of the remaining atoms in dudawalamide A
C
DOI: 10.1021/acs.jnatprod.7b00034
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 3. Isotropic X-ray crystallographic structure and line drawing of dudawalamide A (1) with solvent water molecules deleted.
turn, the deshielded C-3 proton of the Dhoya group showed an
osine. With interplay of TOCSY, COSY, HSQC, and HMBC
HMBC correlation to the glycyl carbonyl at δ 172.6, indicating
experiments, the residues of dudawalamide B (2) were
elucidated as Dhoya, proline, alanine, phenylalanine, and two
residues not observed in dudalwalmide A, valine and N,O-
dimethyltyrosine. The sequence of the residues of dudawala-
mide B (2) was deduced from the HMBC correlations as cyclo-
[Dhoya−Val−N-Me-Phe−Pro−N,O-diMe-Tyr−Ala] (Figure 2,
Table 2).
C
the presence of a second ester bond. HMBC correlations were
also observed from the glycyl α-methine proton to the N-
methylphenylalanine carbonyl and from the latter residue’s N-
methyl group to the carbonyl of the proline, establishing the
two additional amide linkages between these three amino acids.
However, the α-methine proton of proline (δ 4.64) showed
H
The 1H NMR spectra of two additional analogues,
dudawalamide C (3) and dudawalamide D (4), were well
dispersed and contained several resonances indicative of two N-
methyl groups, alkyl methylene and two singlet methyl signals
indicative of a Dhoya residue, and aromatic protons suggesting
a phenylalanine residue, similar to the structure of dudawala-
mide A (1). Two shielded methyl doublets were suggestive of
valine residues in dudawalamides C (3) and D (4). A
combination of H and C NMR as well as COSY and
HSQC spectra were used to deduce partial structures for these
two additional dudawalamides; both possessed Dhoya, valine,
proline, N-methylphenylalanine, and glycine residues. The
difference between them was the presence of a hydroxyisova-
leric acid (Hiva) in dudawalamide C versus a 2-hydroxy-3-
methylpentanoic acid (Hmpa) unit in dudawalamide D (4).
HMBC correlations delineated the sequence of residues in
these analogues, establishing the sequence for dudawalamide C
(3) as cyclo-[Dhoya−Val−N-Me-Val−Hiva−Pro−N-Me-Phe−
Gly] and for dudawalamide D (4) as cyclo-[Dhoya−Val−N-
Me-Val−Hmpa−Pro−N-Me-Phe−Gly] (Table 3 and Support-
ing Information, Figure 2).
only a very weak HMBC correlation through its N atom to the
next residue in the sequence (N-Me-isoleucine); we have
previously observed a similar small or absent heteronuclear
8
,14
coupling in other proline-containing metabolites.
However,
to account for the last degree of unsaturation based on HRMS
data, the dudawalamide A (1) structure must have an overall
macrocyclic structure. Summarizing, HMBC and MS data
identified the residue sequence of dudawalamide A (1) as cyclo-
1
13
[
Pro-N-Me-Phe-Gly-Dhoya-Lac-Ala-N-Me-Ile] (Figure 2,
Table 1).
Confirmation of the structure of dudawalamide A (1) and
determination of its absolute configuration were achieved by X-
ray crystallography of crystals obtained from a mixture of
CH Cl and hexanes containing residual acetone. This provided
2
2
unambiguous confirmation of the structure of dudawalamide A
1) and revealed that the Lac, Ala, N-Me-Ile, and Pro residues
all have an L-configuration, while the Dhoya residue was R
Figure 3). Remarkably, the N-Me-Phe was shown to be of D-
(
(
configuration. This is the first occurrence of N-Me-D-Phe in a
8
metabolite of the “kulolide superfamily”.
Three analogues of dudawalamide A (1), designated as
dudawalamides B−D (2−4), were also isolated from the same
M. producens extract. Their planar structures were determined
by 2D NMR, and the absolute configurations of dudawalamides
B (2) and D (4) were elucidated by a combination of chiral-
phase HPLC, chiral-phase GCMS, and modified Marfey’s
analysis. However, due to the limited quantities of isolated
dudawalamide C (3), its configurations were only partially
determined.
To determine the absolute configurations of the residues for
dudawalamides B−D, a combination of chiral-phase GCMS,
chiral-phase HPLC, and modified Marfey’s analysis was used
8
(Figure 4). The configuration of the Dhoya residue was
determined for each compound by hydrogenation (H /Pd)
2
followed by acid hydrolysis and then reacting the hydrolysate
with CH N to give the methyl ester of the 2,2-dimethyl-3-
2
2
hydroxyoctanoic acid (Dhoaa) unit. The retention time of this
dudawalamide-derived fragment was compared by chiral-phase
Comparison of the 1H and 13C NMR spectra for
dudawalamide B (2) with those of dudawalamide A (1)
indicated that these metabolites were closely related; both
possessed two N-methyl amide peaks, doublet methyl groups
indicative of alanine, aryl protons indicative of phenylalanine,
and the alkyl methylene protons and two singlet methyl signals
indicative of a Dhoya residue. The most noticeable differences
were the presence in dudawalamide B (2) of doublet methyl
groups indicative of a valine residue, and the aryl protons and
deshielded singlet methyl group suggestive of O-methyltyr-
25
GCMS to previously prepared synthetic standards. Interest-
ingly, while the Dhoya moiety configuration was determined to
be R in dudawalamide A (1) by X-ray diffraction analysis and
confirmed via this chiral-phase GCMS investigation, the
opposite configuration (S) was observed for all of the other
analogues, dudawalamides B−D (2−4). Furthermore, the
configurations of all of the amino acids in dudawalamides B−
D were determined to be L by hydrolysis and either direct
analysis by chiral-phase HPLC or modified Marfey’s analysis
D
DOI: 10.1021/acs.jnatprod.7b00034
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Journal of Natural Products
Table 2. NMR Data (in CDCl ) for Dudawalamide B (2) ( H 500 MHz, 13C 125 MHz)
Article
1
3
residue
Dhoya
position
δC, type
δH (J in Hz)
HMBC
COSY
1
2
3
176.6, C
46.1, C
77.5, CH
27.8, CH2
5.23, dd (11.2, 2.3)
1.84−1.73, m
1.62, m
1, 2, 4, 5, 6, 39
4a, 4b
4
4
5
6
7
8
9
1
a
6
4b, 5
4a, 5
4a, 4b, 6
5
b
6
24.7, CH2
18.3, CH2
83.7, C
1.45−1.53, m
2.24, td (6.8, 2.6)
3, 6, 7
4, 5, 7, 8
69.3, CH
17.6, CH3
23.5, CH3
172.3, C
55.7, CH
31, CH
1.97, t (2.6)
1.30, s
6
2, 10
9
0
1.22, s
Val
11
12
13
14
15
16
4.31, dd (9.5, 7.2)
1.95−1.85, m
1.01, d (6.6)
1, 11, 13, 15
14, 15
13, 16
12, 14, 15
19.5, CH3
18.8, CH3
NH
12, 13, 15
12, 13, 14
1, 12, 13
13
13
12
0.94, d (6.6)
5.73, d (7.2)
N-Me-Phe
17
169.1, C
54.3, CH
38, CH2
1
1
1
2
2
2
2
2
8
5.11, dd (12.2, 3.4)
3.00, dd (11.7, 3.4)
3.20, m
11, 17, 19, 20, 24
18, 20, 21
17a, 17b
18, 19b
18, 19a
9a
9b
0
18, 20, 21
137.8, C
1
130.1, CH
128.7, CH
127.1, CH
32.4, CH3
171.7, C
7.50−7.41, m
7.26, m
19, 20
22
2
20, 21, 23
21, 22
21, 23
22
3
7.24, m
4
3.62, s
11, 18
Pro
25
2
2
2
2
2
2
2
6
56.5, CH
30.2, CH2
3.19, m
25, 27, 28
27a, 27b
7a
7b
8a
8b
9a
9b
0.13, ddt (12.9, 6.5)
0.63, tt (10.2, 7.8)
1.27, m
26, 27b, 28a
26, 27a, 28b
28b, 29a, 29b
28a, 29a, 29b
28a, 28b, 29b
28a, 28b, 29a
26
27
22.3, CH2
46.4, CH2
1.38, m
3.25, m
26, 27, 28
26, 27, 28
3.41, ddd (12.0, 8.3, 3.8)
N,O-diMe-Tyr
30
169, C
3
3
3
3
3
3
3
3
3
1
63.4, CH
34.4, CH2
3.95, dd (8.9, 4.5)
3.60, m
25, 30, 32, 37
30, 31, 33
32a, 32b
31, 32b
31, 32a
2a
2b
3
2.69, dd (14.5, 8.9)
30, 31, 33
130.2, C
130.7, CH
114.4, CH
158.9, C
31.1, CH3
55.6, CH3
169.9, C
48.7, CH
18.4, CH3
NH
4
7.00, d (8.2)
6.80, d (8.3)
36
36
35
34
5
6
7
2.80, s
3.76, s
25, 31
36
8
Ala
39
40
41
42
5.01, dd (8.7, 6.6)
1.57, d (6.9)
30, 41
39, 40
30
42, 41
40
9.05, d (9.0)
40
along with authentic standards. By chiral-phase HPLC, the
configuration of the Hiva residue in dudawalamide C (3) was
established to be L, whereas the Hmpa residue in dudawalamide
D (4) was determined to be D-allo. Unfortunately, due to
limited quantity of isolated dudawalamide C (3), the
configurations of only three residues were determined (S-
Dhoya, L-Hiva, and L-Val).
mides are structurally quite similar to one other, they show
quite different biological activities. In general, they exhibit
broad antiparasitic activity but little to no cytotoxicity against
H-460 cells relative to the control at the maximum
concentration of 30 μM. Dudawalamides A (1) and D (4)
showed the most potent activities against P. falciparum with
IC ’s of 3.6 and 3.5 μM, respectively. However, these two were
50
The dudawalamides were evaluated for their cytotoxic
properties using the H-460 human lung cancer cell line and
for antiparasitic properties using malaria, leishmaniasis, and
distinguished in their activity to the other two parasites, with 1
possessing weaker activity against both Trypanasoma cruzi and
Leishmania donovani, whereas 4 was relatively potent against
L. donovani (2.6 μM) (Table 4). Dudawalamides B (2) and C
26−30
Chagas disease assays.
Intriguingly, while the dudawala-
E
DOI: 10.1021/acs.jnatprod.7b00034
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Journal of Natural Products
Article
Table 3. NMR Data (in CDCl ) for Dudawalamides C (3) and D (4) ( H 500 MHz, 13C 125 MHz except Where Noted)
1
3
dudawalamide C (3)
δC, type
dudawalamide D (4)
δC, type
a
residue
Dhoya
position
δH, mult (J in Hz)
residue
Dhoya
position
δH, mult (J in Hz)
1
2
3
175, C
1
175, C
46.8, C
2
46.8, C
77.6, CH
28.5, CH2
5.00, dd (10.0, 2.4)
1.59, m
3
77.8, CH
28.6, CH2
4.97, m
1.58, m
1.80, m
1.52, m
2.23, m
4
4
5
6
7
8
9
1
a
4a
4b
5
b
1.80, m
25.1, CH2
18.4, CH2
84.1, C
1.52, m
25.1, CH2
18.4, CH2
84, C
2.22, m
6
7
69.2, CH
18.7, CH3
24.2, CH3
172, C
1.96, t (2.5)
1.38, s
8
69, CH
1.97, s
1.37, s
1.21, s
9
18.7, CH3
24.1, CH3
172, C
0
1.22, s
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31a
31b
32a
32b
33a
33b
34
35
36a
36b
37
38
39
40
41
42
43a
43b
Val
11
Val
1
1
1
1
1
2
3
4
5
6
53.2, CH
32, CH
5.18, dd (8.5, 2.6)
2.08, m
53.3, CH
32, CH
5.17, d (8.6)
2.08, m
15.8, CH3
20.2, CH3
NH
0.77, d (6.9)
0.95, d (6.8)
6.62, d (8.6)
15.8, CH3
20.3, CH3
NH
0.78, d (6.5)
0.95, m
6.65, d (8.6)
N-Me-Val
17
168.8, C
63.9, CH
26.8, CH
17.9. CH3
20, CH3
28.8, CH3
167.5, C
77.2, CH
29.7, CH
17.3, CH3
19.2, CH3
172.1, C
55.4, CH
28.1, CH2
N-Me-Val
Hmpa
Pro
168.7, C
64, CH
18
19
20
21
22
3.87, d (11.0)
2.41, m
3.87, d (11.0)
26.9, CH
17.9, CH3
19.9, CH3
28.7, CH3
167.7, C
75, CH
2.41, dq (13.6, 6.7)
0.90, d (6.8)
0.95, m
0.89, d (6.8)
0.97, d (6.8)
2.65, s
2.66, s
Hiva
Pro
23
24
25
26
27
4.78, d (5.2)
2.03, m
4.97, m
1.71, m
0.95, m
1.40, m
0.92, m
36, CH
0.99, d (6.9)
1.00, d (6.9)
14.1, CH3
26.5, CH2
12, CH3
171.9, C
55.6, CH
28, CH2
28
2
3
3
3
3
3
3
9
4.31, dd (8.1, 5.2)
0.88, m
0a
0b
1a
1b
2a
2b
4.34, dd (8.1, 5.2)
0.88, m
0.94, m
25.5, CH2
47.5, CH2
2.03, m
0.96, m
1.65, m
25.5, CH2
47.4, CH2
1.61, m
3.37, m
2.03, dt (13.4, 6.9)
3.36, q (8.1)
3.76, q (7.5)
3.80, dt (9.7, 7.1)
N-Me-Phe
33
169.7, C
63.8, CH
34, CH2
3
3
3
3
3
3
3
4
4
4.77, m
N-Me-Phe
169.8, C
63.9, CH
33.9, CH2
5a
5b
6
2.95, dd (14.5, 11.5)
3.38, m
4.74, dd (11.6, 2.9)
2.94, dd (14.6, 11.5)
3.42, dd (14.7, 2.8)
138.6, C
127, CH
129, CH
129.4, CH
29.3, CH3
169.7, C
40.2, CH2
7
7.22, m
138.7, C
127, CH
129, CH
129.4, CH
29.4, CH3
169.7, C
40.2, CH2
8
7.28, d (1.5)
7.12, m
7.22, t (7.4)
7.29, m
9
0
2.79, s
7.12, d (7.6)
2.80, s
Gly
41
4
4
4
2a
4.02, dd (18.3, 1.9)
4.67, ddd (18.2, 9.8, 1.9)
8.93, d (9.6)
Gly
2b
3
4.03, d (17.9)
4.67, d (18.0, 9.7)
8.91, d (9.7)
NH
4
4
NH
a
Data collected at 800 MHz.
(
3) were significantly less potent than dudawalamides A and D
Val, L-Ala to L-N-Me-Val, and L-N-Me-Ile to D-allo-Hmpa,
results in at least a 5-fold difference for Leishmania inhibition
(Table 4). Because residues 1 and 2 in dudawalamide D (4) are
rather similar in dudawalamide B (2), and the latter has weak
activity against Leishmania, it is possible that stereochemical
inversion at residue 3 is responsible for this change in activity
(Tables 4 and 5). In the case of wewakpeptins A and C,
against P. falciparum, and 2 also showed decreased potency to
the other two parasites.
Intriguing SAR features emerge from comparison of the
Dhoya class of depsipeptides in several measures of bioactivity.
The constellation of changes between residues 1−3 of
dudawalamide A (1) and dudawalamide D (4), L-Lac to L-
F
DOI: 10.1021/acs.jnatprod.7b00034
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 4. Stereostructures of dudawalamides B−D (2−4).
3
4
Table 4. Biological Activities of Dudawalamides A−D (1−4)
sp.), and the recent viequeamides (isolated from Rivularia
8
sp.). These natural products are members of the “kulolide
Plasmodium
falciparum
(IC , μM)
Trypanosoma
Leishmania
donovani
superfamily”, which is characterized by a β-hydroxyoctanoic
acid derivative in addition to a sequence of amino acids or
hydroxy acid residues in an overall cyclic structure (Table 5).
The dudawalamides A−D (1−4), like other members of the
cruzi (% GI at 10
a
compound
μg/mL)
(IC , μM)
5
0
50
dudawalamide A (1)
dudawalamide B (2)
dudawalamide C (3)
dudawalamide D (4)
3.6
8.0
10
3.5
12
>10
7
>10
“
kulolide superfamily”, are believed to derive from a mixed
not tested
60
not tested
2.6
NRPS−PKS biosynthetic pathway. Specifically, the biosynthesis
of the distinctive Dhoya moiety likely arises via PKS acetate
extensions (possibly to a hexanoic or hexenoic acid starter unit)
and terminal desaturation by oxidative dehydrogenation, and S-
adenosylmethionine is likely responsible for methylation at C-2
a
Percentage growth inhibition.
modification of residue 6 from L-Hiva to D-Pla results in a 20-
12
fold decrease in neuro-2a cytotoxic activity (Table 5).
35,36
of this residue.
Although the configuration of dudawalamide C is only partially
defined, it is noteworthy that dudawalamides C (3) and D (4)
are only different by one methyl group at residue 3, L-Hiva to D-
allo-Hmpa, but this single methyl group and stereochemical
inversion result in a more than 3-fold difference in their activity
for P. falciparum. Thus, it appears that minor changes in
configuration and sequence of residues strongly impact the
bioactivity of these Dhoya-containing natural products. Never-
theless, it will be important to more fully explore these SAR
relationships in the future using a synthetic medicinal chemistry
approach.
In summary, we report the isolation of four new Dhoya-
containing natural products, dudawalamides A−D (1−4), from
the marine cyanobacterium M. producens. This newly identified
group of natural products further demonstrates that the Dhoya
residue can be considered a molecular fingerprint of
cyanobacterial secondary metabolism. The dudawalamides
exhibit variable antiparasitic activity against malaria-, leishma-
niasis-, and Chagas disease-causing organisms, with many of the
IC50 values in the low micromolar range. This set of natural
products further expands on SAR features for Dhoya-containing
cyanobacterial compounds, generally known as the “kulolide
superfamily”.
The dudawalamides A−D (1−4) join several other Dhoya-
containing cyclic lipopeptides such as the wewakpeptins
1
2,13
(
isolated from Lyngbya semiplena),
kulolide (isolated from
pitipeptolides A and B (isolated from Moorea
19
1
3,31
EXPERIMENTAL SECTION
a mollusk),
sp.),
■
3
1−33
antanapeptin A (isolated from Moorea sp.),
mantillamide A, cocosamides A and B (isolated from Moorea
General Experimental Procedures. A Jasco P-2000 polarimeter
1
0
was used to measure optical rotations. IR spectra were measured on a
8
,35
Table 5. Selected Dhoya-Containing Secondary Metabolites from the “Kulolide Superfamily”
residue
compound
1
2
3
4
5
6
7
8
9
dudawalamide A
dudawalamide B
dudawalamide C
dudawalamide D
viequeamide A
viequeamide B
kulolide
R-Dhoya
S-Dhoya
S-Dhoya
S-Dhoya
S-Dhoya
S-Dhoya
R-Dhoya
S-Dhoya
R-Dhoya
R-Dhoya
L-Lac
L-Val
L-Val
L-Val
L-Val
L-Val
L-Val
L-Val
L-Ile
L-Ala
N-Me-L-Ile
L-Pro
L-Pro
N-Me- L-Phe
L-Ala
Gly
N-Me-L-Phe
N-Me-Val
N-Me-O-Me-L-Tyr
Pro
Hiva
N-Me-Phe
N-Me- L-Phe
N-Me- L-Val
N-Me- L-Ala
L-Val
Gly
N-Me-L-Val
N-Me-L-Val
N-Me-L-Val
N-Me-D-Val
N-Me-L-Phe
N-Me-L-Ala
N-Me-L-Ala
D-allo-Hmpa
D-allo-Hmpa
L-Pro
Gly
L-Pro
L-Thr
L-Val
L-Ala
Gly
a
L-Pla
L-Pro
a
L-Pla
L-Pro
pitipeptolide A
wewakpeptin A
wewakpeptin C
L-Hmpa
L-Pro
L-Ile
N-Me- L-Val
N-Me- L-Ala
N-Me- L-Val
N-Me- L-Val
L-Hiva
L-Pro
L-Pro
L-Pro
L-Pro
L-Val
L-Val
L-Ile
D-Pla
a
Pla = Phenyl Lactic Acid.
G
DOI: 10.1021/acs.jnatprod.7b00034
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Thermo Electron Corporation Nicolet IR 100 FT-IR. NMR spectra
were recorded on a Varian 500 MHz spectrometer (500 and 125 MHz
for the 1H and C nuclei, respectively) and Bruker 800 MHz
Supporting Information; (+)-HRESIMS m/z 794.4694 [M + H]+
(calcd for C H N O , 794.4704).
Dudawalamide D (4): colorless oil; [α]
43
63
5
9
13
25
−50.1 (c 0.16, CHCl );
3
D
1
13
spectrometer (800 and 200 MHz for the H and C nuclei,
respectively) using CDCl3 as solvent from Cambridge Isotope
Laboratories, Inc. (99.8% D). Spectra were referenced to residual
CDCl solvent as internal standard (δ 7.26 and δ 77.1). LCMS data
IR (neat) νmax 3281, 2964, 2927, 2361, 1747, 1660, 1500, 1447, 1406,
−1
1
13
1307, 1193, 1123 cm ; H NMR (CDCl
(CDCl , 125 MHz) data, Table 3 and Supporting Information;
, 800 MHz) and C NMR
3
3
+
(+)-HRESIMS m/z 808.4849 [M + H] (calcd for C44H N O ,
3
H
C
65 5 9
for stereochemical analysis of the hydrolysates of 1−4 were obtained
with a Thermo Finnigan Surveyor Autosampler-Plus/LC-Pump-Plus/
PDA-Plus system and a Thermo Finnigan LCQ Advantage Max mass
spectrometer (monitoring 200−600 nm and m/z 250−2000 in
positive ion mode). LC-HRMS data for analysis of compounds 1−4
were obtained on an Agilent 6239 HR-ESI-TOFMS equipped with a
Phenomenex Luna 5 μm C18 100 Å column (4.6 × 250 mm). GCMS
was conducted with a Thermo Electron Corp. DSQ/TRACE-GC-
Ultra GCMS system with a Cyclosil-B column (Agilent Technologies
J&W Scientific, 30 m × 0.25 mm). Semipreparative HPLC purification
was carried out using a Waters 515 with a Waters 996 photodiode
array detector using Empower Pro software. All solvents were HPLC
grade.
Organism Collection and Identification. The Moorea producens
sample (PNG-04/22/06-2) yielding compounds 1−4 was collected in
April 2006 at a depth of 3−9 m by snorkel near Dudawali Bay in
Papua New Guinea (10°17.274′ S and 151°00.390′ E). The specimen,
measuring 8 L in total biomass, was stored in 70% EtOH at −20 °C
until extraction. The 16S rRNA gene sequences of algal biomass for
this sample are available in the DDBJ/EMBL/GenBank databases
under acc. no. EU492878.1.
808.4861).
X-ray Crystallographic Data for Compound 1. The single-
crystal X-ray diffraction studies were carried out on a Bruker Kappa
APEX-II CCD diffractometer equipped with Cu Ka radiation (λ =
1
.5478). A 0.33 × 0.30 × 0.08 mm colorless plate was mounted on a
Cryoloop with Paratone oil. Data were collected in a nitrogen gas
stream at 100(2) K using ϕ and ϖ scans. The crystal-to-detector
distance was 50 mm, and the exposure time was 1 s per frame using a
scan width of 1.0°. Data collection was 99.7% complete to 68.00° in θ.
A total of 42 468 reflections were collected covering the indices −17 ≤
h ≤ 17, −12 ≤ k ≤ 11, and −18 ≤ l ≤ 18. A total of 8830 reflections
were found to be symmetry independent, with an R of 0.0395.
int
Indexing and unit cell refinement indicated a primitive, monoclinic
lattice. The space group was found to be P2 . The data were integrated
1
using the Bruker SAINT software program and scaled using the
SADABS software program. Solution by direct methods (SHELXT)
produced a complete phasing model consistent with the proposed
structure.
All non-hydrogen atoms were refined anisotropically by full-matrix
least-squares (SHELXL-2014). All hydrogen atoms were placed using
a riding model. Their positions were constrained relative to their
parent atom using the appropriate HFIX command in SHELXL-2014.
The absolute configurations of the stereocenters in dudawalamide A
were established by anomalous dispersion using the Parson’s method
with a Flack parameter of 0.023(47). Crystallographic data are
summarized in Table S1 in the Supporting Information; CCDC
Extraction and Isolation. Approximately 1 L of the preserved
M. producens biomass was filtered using cheesecloth and extracted six
times with 500 mL of CH Cl −MeOH (2:1) with heating at gentle
2
2
reflux. The combined organic layers were dried under vacuum to afford
.99 g of dark green solids. A small portion was preserved for future
1
1
549804.
analysis, and the rest was purified on silica gel (type H, 10−40 μm,
Sigma-Aldrich) using vacuum liquid chromatography with a hexanes−
EtOAC−MeOH gradient to generate nine subfractions, A−I (100%
hexanes, 10% EtOAc−90% hexanes, 20% EtOAc−80% hexanes, then
Hydrogenations. A 0.2−0.4 mg aliquot of compounds 2−4 was
dissolved in 300 μL of EtOH, and a catalytic amount of 10% Pd/C was
added under argon. The argon was replaced with hydrogen gas
followed by stirring for 3−4 h at room temperature. The crude
reaction product was filtered with a Nalgene syringe filter (2 μm, 13
mm) and dried under reduced pressure. The crude product was used
without purification for acid hydrolysis reactions.
2
0% increments to 100% EtOAc, 25% MeOH−75% EtOAc, and 100%
MeOH, named A−I, respectively). The most cytotoxic fractions in the
H-460 lung carcinoma cell line were F (90% EtOAc−hexanes; 17.6
mg) and G (100% EtOAc, 29.2 mg). Fraction F exhibited 33% and
Acid Hydrolysis and Chiral HPLC. Hydrogenated compounds
99% inhibition at 1 and 10 μg/mL, respectively, while fraction G
2
−4 were dissolved in 500 μL of 6 N HCl in sealed tubes, flushed with
exhibited 7% and 89% inhibition at 1 and 10 μg/mL, respectively,
against the H-460 cell lines. These fractions (combined weight 46.8
mg) were filtered using a Waters RP C18 SPE cartridge with 100%
MeCN followed by 100% MeOH. The MeCN filtrate from both
fractions was further purified by RP-HPLC using a Phenomenex
Kinetex C-18, 100 Å, 150 × 10 mm column using linear gradient
N , and heated at 105 °C for 16 h. The reaction mixture was dried
2
using a stream of N and resuspended in MeOH, and an aliquot was
2
taken for further analysis. The configuration of the proline residue in
2
−4 was determined by chiral-phase HPLC (Chirex 3126, 4.6 × 50
mm, D-penicillamine) using isocratic conditions [2 mM CuSO −
MeCN (95:5), 0.8 mL/min] and matched the retention time (t , min)
4
R
elution with MeCN−H O (50:50) for 5 min to MeCN−H O (90:10)
2
2
of L-Pro (8.1 min; D-Pro, 11.8 min). The Hiva residue in 3 and Hmpa
residue in 4 were analyzed using the same chiral-phase HPLC system
with slightly varying conditions. The Hiva residue in 3 was analyzed
in 30 min at 3 mL/min. The purification of both fractions yielded a
combined yield of dudawalamide A (1, 1.8 mg), dudawalamide B (2,
1.1 mg), dudawalamide C (3, 0.3 mg), dudawalamide D (4, 0.8 mg),
using 2 mM CuSO −MeCN (85:15), 0.8 mL/min, while the Hmpa
4
and the known compounds apratoxin A (1.0 mg) and majusculamides
residue in 4 was analyzed using 2 mM CuSO −MeCN (87.5:12.5), 0.8
4
A and B (2.0 mg combined).
Dudawalamide A (1): white, crystalline solid; mp 96 °C, [α]
mL/min. Dudawalamide C (3) was thus shown to possess L-Hiva (t
R
25
D
for L-Hiva, 16.6 min; D-Hiva, 24.2 min). The retention times for the
authentic Hmpa standards were as follows: L-Hmpa (20.4 min), D-
Hmpa (31.0 min), L-allo-Hmpa (18.5 min), D-allo-Hmpa (26.6 min).
For dudawalamide D (4), the results were consistent with D-allo-Hmpa
(26.7 min).
−
62.5 (c 0.17, CHCl ); IR (neat) ν 3385, 3270, 3050, 2987, 2945,
3 max
−1
1
2
876, 1733, 1512, 1488, 1376, 1285, 1195, 1094 cm ; H NMR
13
(
(
7
CDCl , 500 MHz) and C NMR (CDCl , 125 MHz) data, Table 1;
+)-HRESIMS m/z 752.4220 [M + H] (calcd for C H N O ,
52.4235).
3
3
+
40
57
5
9
Modified Marfey’s Analysis. A second portion of the acid
Dudawalamide B (2): colorless oil; [α]25 −79.4 (c 0.17, CHCl );
D
3
hydrolysis product of compounds 2−4 was reacted with D-FDVA
IR (neat) νmax 3286, 2920, 2852, 1742, 1660, 1633, 1512, 1458, 1305,
(0.1% in acetone and 0.1 M NaHCO ) and heated at 90 °C in a sealed
3
−1
1
13
1249, 1142 cm ; H NMR (CDCl , 500 MHz) and C NMR
tube for 5 min. The reaction was quenched with 50 μL of 1 N HCl(aq)
and diluted with 100 μL of MeCN, and 10 μL of this mixture was
analyzed by HPLC-ESIMS in comparison to authentic amino acid
standards that had been derivatized with D-FDVA. The N-Me-Phe,
Pro, Val, N-Me-Val, and N,O-diMe-Tyr residues in 2−4 were analyzed
by RP-HPLC [HP LiChrospher 100 RP-18 (5 μm, 4 × 125 mm); 0.8
mL/min, UV detection at 340 nm] using a linear gradient (30/70 to
3
(
CDCl , 125 MHz) data, Table 2; (+)-HRESIMS m/z 786.4429 [M +
3
+
H] (calcd for C H N O , 786.4442).
4
4
59
5
8
25
D
Dudawalamide C (3): colorless oil; [α] −10.8 (c 0.11, CHCl );
3
IR (neat) νmax 3287, 2963, 2923, 2853, 1744, 1645, 1499, 1407, 1308,
1
MHz) and C NMR (CDCl , 125 MHz) data, Table 3 and
−
1
1
258, 1194, 1124, 1090, 1021, 801 cm ; H NMR (CDCl , 500
3
13
3
H
DOI: 10.1021/acs.jnatprod.7b00034
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
7
0/30 MeCN−H O in 0.1% HCOOH for 60 min). The retention
2
(CIF)
times for the authentic amino acid standards were as follows: N-Me-L-
Phe (18.5 min), N-Me-D-Phe (20.1 min), L-Pro (8.1 min), D-Pro (10.7
min), L-Val (34.4 min), D-Val (51.7 min), N-Me-L-Val (15.7 min), N-
Me-D-Val (21.0 min), L-Pro (8.1 min), D-Pro (10.7 min), N,O-diMe-L-
Tyr (17.2 min), N,O-diMe-D-Tyr (18.9 min), L-Ala (13.9 min), and D-
Ala (13.0 min). The hydrolysate peaks with the expected masses for
dudawalamide B (2) were found at 34.4, 18.5, 8.1, 17.2, and 13.9 min,
which correspond to L-Val, N-Me-L-Phe, L-Pro, N,O-diMe-L-Tyr, and
L-Ala. For dudawalamide D (4), the retention time matched those for
L-Val (34.4 min), N-Me-L-Val (15.7 min), L-Pro (8.1 min), and N-Me-
L-Phe (18.5 min).
AUTHOR INFORMATION
Corresponding Author
Tel: (858)-534-0578. E-mail: wgerwick@ucsd.edu.
■
*
ORCID
William H. Gerwick: 0000-0003-1403-4458
Author Contributions
J. Almaliti and K. L. Malloy contributed equally to the
manuscript.
#
Dhoya Configuration Analysis. The hydrolysates of compounds
1
−4 were used for the Dhoya configuration analysis. A small portion
Notes
of each was dissolved in MeOH, and CH N (in ether) was added.
The reaction mixture was stirred for 10 min, dried under N , and
2
2
The authors declare no competing financial interest.
2
extracted several times with CH Cl −H O and a few drops of 1 N
2
2
2
ACKNOWLEDGMENTS
■
HCl. The combined organic layer was dried under N , redissolved in
2
The authors thank J. Lee for running the H-460 cytotoxicity
assay on the extract, A. Mrse for assistance with NMR
experiments, E. Theodorakis (UCSD Chemistry) for use of the
microwave reactor, the Walter Reed Army Institute of Research
for providing the drug-resistant P. falciparum strain W2 and the
L. donovani strain WR2810, and the UCSD Chemistry and
Biochemistry Mass Spectrometry Facility for LC-HR-ESI-
TOFMS experiments and analysis. We thank T. Matainaho
for facilitating collections of marine cyanobacteria in Papua
New Guinea. We also acknowledge A. L. Rheingold and C.
Moore of the UC San Diego X-ray Crystallography Facility for
determining the X-ray structure of dudawalamide A. We
acknowledge funding for this work by the NIH under grants
TW006634 and CA100851, as well as partial funding to C.S.
and M.G. from the SNI-Panama.
CH Cl , and analyzed by chiral-phase GCMS (J &W Scientific Cyclosil
2
2
C, 0.25 μm, 30 mm × 0.250 mm) using a stepwise temperature
gradient [40 to 100 °C (20 °C/min), hold for 5 min, then 100 to 110
°
C (5 °C/min)]. The Dhoaa synthetic standards (3.0 mg) were
subjected to the same reaction conditions as above to give the methyl-
25
ester derivatives. The Dhoaa residue deriving from compound 1
matched the tR of R-Dhoaa while those from compounds 2−4
matched the t of S-Dhoaa (t for S-Dhoaa, 51.5 min; R-Dhoaa, 52.8
R
R
min, Supporting Information).
Cytotoxicity Assay. H-460 human lung cancer cells were added to
6-well plates at 3.33 × 10 cells/mL in Roswell Park Memorial
Institute (RPMI) 1640 media with fetal bovine serum (FBS) and 1%
4
9
penicillin−streptomycin. The cells were incubated overnight (37 °C,
5
% CO ) in a volume of 180 μL per well to permit recovery before
2
treatment with test compounds. The test substances were dissolved in
DMSO to a stock concentration of 1 mg/mL. Working solutions were
prepared through serial dilution in RPMI 1640 media without FBS,
with 20 μL added to each well to give final compound concentrations
of 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, and 0.001 μg/mL. Negative
control wells were prepared by adding an equal volume of RPMI 1640
media lacking FBS for each plate. Plates were incubated for
approximately 48 h prior to MTT staining. Plates were read with a
ThermoElectron Multiskan Ascent plate reader at 570 and 630 nm to
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ASSOCIATED CONTENT
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■
*
S
Supporting Information
(
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ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00034.
2
005, 70, 3133−3139.
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