Journal of Natural Products
NOTE
(
2.9%) of EtOAc-soluble fraction and 2.24 g (0.6%) of BuOH-soluble
material. The EtOAc-soluble fraction (9.56 g) was chromatographed on
a column of SiO (100 g) using a hexanes-EtOAc step gradient system
Acid Hydrolysis and Enantioselective HPLC Analysis.
Compounds 2 and 3 (0.1 mg each) were suspended in 6 N HCl (0.3
mL) and heated at 115 °C for 18 h in two sealed tubes. The hydrolysates
were concentrated to dryness. The residues were reconstituted in 0.3 mL
2
followed by an EtOAc-MeOH step gradient to give 13 subfractions.
The combined subfractions 7 to 9 (2.0 g), eluting with hexanes-75%
EtOAc, was further chromatographed on a Si-column (100 g) using a
hexanes-EtOAc step gradient system to give eight subfractions. Sub-
fraction 6 (36 mg), eluting with hexanes-25% EtOAc, was further
purified by reversed-phase HPLC (semiprep 250 ꢀ 10 mm, 5 μm, RP-
of H O and analyzed by enantioselective HPLC, comparing the reten-
2
tion times with those of authentic standards [Phenomenex Chirex (D)
penicillamine, 4.6 ꢀ 250 mm, 5 μm]; solvent mixtures of 2.0 mM
CuSO
CuSO
4
4
-CH
-CH
3
CN (85:15 or 90:10); detection at 254 nm. Using 2.0 mM
CN (90:10) with a flow rate of 0.8 mL/min, the retention
3
times (t
19.6), and with a flow rate of 1.0 mL/min the retention times (t
for authentic standards were L-Val (17.0) and D-Val (22.9). Using 2.0
mM CuSO -CH CN (85:15) with a flow rate of 1.0 mL/min, the
R
, min) for authentic standards were L-Pro (10.0) and D-Pro
1
8, flow 3.0 mL/min) using 15% H O-MeOH to give 3 mg of impure
2
(
R
, min)
cocosamide B, 20 mg of majusculamides A and B, 4.0 mg of malynga-
mide 3 (1, yield, 0.001% dry wt), and 1.4 mg of cocosamide A (2, yield,
0
.0004% dry wt). The impure cocosamide B fraction was further
separated by reversed-phase HPLC using 30% H O-MeOH to give
.0 mg of cocosamide B (3, yield 0.0006% dry wt).
Malyngamide 3 (1): colorless, amorphous powder; [R]
4
3
retention times (t
R
, min) for authentic standards were N-Me-L-Phe
2
(34.2) and N-Me-D-Phe (36.6). The retention times (and respective
2
2
5
D
HPLC conditions) of the amino acids in the hydrolysates of 2 and 3
were (min) 10.0 (90:10, 0.8 mL/min), 17.0 (90:10, 1.0 mL/min), and
-10.1
(
c 0.36, MeOH); UV (MeOH) λmax (log ε) 205 (4.19), 274 (3.40) nm;
IR (film) νmax 3320, 2932, 1725, 1636 cm- ; H and C NMR data, see
Table 1, assignments were made by interpretation of 2D DQF COSY,
edited-HSQC, HMBC, and NOESY data; HRESI/TOFMS m/z
1 1
13
34.2 (85:15, 1.0 mL/min), indicating the presence of L-Pro, L-Val, and
N-Me-L-Phe.
Cell Viability Assays. Cells were propagated and maintained in
DMEM (Invitrogen) supplemented with 10% FBS (Hyclone) at 37 °C
þ
35
5
59.3146 [M þ H] (calcd for C H ClN O , 559.3145).
28
48
2 7
25
in humidified air and 5% CO
2
. Cells were seeded in 96-well plates
Cocosamide A (2): white solid; [R]
D
-77.7 (c 0.12, MeOH);
(MCF7 10 500 cells/well; HT-29 13 000 cells/well). After 24 h, cells
UV (MeOH) λmax (log ε) 208 (4.36), 260 (3.26) nm; IR (film) ν
3
Table 2, assignments were made by interpretation of 2D DQF COSY,
edited-HSQC, HMBC, and NOESY data; HRESI/TOFMS m/z
max
-
1
1
13
were treated with various concentrations of the test compound or
solvent control (1% EtOH). After 48 h of incubation, cell viability was
measured using MTT according to the manufacturer’s instructions
330, 2920, 1665, 1634, 1527, 1197 cm ; H and C NMR data, see
þ
(Promega). Paclitaxel was used as a positive control; IC50 values were
7
44.4330 [M þ H] (calcd for C H N O , 744.4331).
42
58
5
2
7
5
7
and 6 nM in HT-29 and MCF7 cell lines, respectively. Experiments
Cocosamide B (3): white solid; [R]
D
-103 (c 0.18, MeOH);
were done in duplicate. IC50 values were determined using nonlinear
regression in GraphPad Prism.
UV (MeOH) λmax (log ε) 208 (4.54), 260 (3.35) nm; IR (film) νmax
3
-
1
1
13
416, 2950, 1665, 1634, 1541, 1032 cm ; H and C NMR data, see
Table 2, assignments were made by interpretation of 2D DQF COSY,
edited-HSQC, HMBC, and NOESY data; HRESI/TOFMS m/z
’ ASSOCIATED CONTENT
þ
7
42.4186 [M þ H] (calcd for C H N O , 742.4174).
42 56 5 7
1
13
S
Supporting Information. H, C, and 2D NOESY
Preparation of (R)-MTPA and (S)-MTPA Esters of 1. Com-
pound 1 (1.0 mg) was dissolved in CHCl (50 μL), and pyridine (50
b
3
NMR spectra in CDCl for malyngamide 3 (1) and cocosamide
3
1 13
μL) and a catalytic amount of 4-DMAP were added. The solution was
treated with S(þ)-MTPA chloride (1.0 μL) and stirred at room
temperature for 12 h. The reaction was terminated with the addition
of MeOH (200 μL), and the solvent was evaporated to give the (R)-
MTPA ester of 1. Similarly, the (S)-MTPA ester of 1 was prepared with
R(-)-MTPA chloride using the same procedure. Both esters were
A (2). H, C, COSY, HMBC, and 2D NOESY NMR spectra in
1
CDCl for cocosamide B (3). H and 2D NOESY NMR spectra
3
in CDCl for pitipeptolide A (5). This material is available free of
3
charge via the Internet at http://pubs.acs.org.
’
AUTHOR INFORMATION
subjected to HPLC (semiprep 250 ꢀ 10 mm, 5 μm, SiO
2
, flow 3.0 mL/
min) using EtOAc-3% MeOH to yield the pure (R)-MTPA ester of 1
Corresponding Author
*Tel: (772) 462-0982. Fax: (772) 461-8154. E-mail: paul@si.edu.
(
0.4 mg) and (S)-MTPA esters of 1 (0.3 mg).
R-MTPA ester of 1: H NMR δ (only key resonances are listed)
1
5
.921 (1H, t, J = 5.4 Hz, NH), 5.292 (1H, m, H-9), 3.584 (3H, s, OMe-
12), 3.536 (2H, s, H
2
-6), 2.624 (2H, d, J = 6.9 Hz, H
2
-10); ESIMS m/z
’ ACKNOWLEDGMENT
þ
þ
7
C
77.4 [M þ H] ; HRESI/TOFMS m/z 777.3404 [M þ H] (calcd for
This research was supported by the NIH, NIGMS Grant
P41GM806210. We thank E. Cruz-Rivera for collecting the
sample. We also thank the Harbor Branch Oceanographic
Institute at Florida Atlantic University spectroscopy facility for
35
38 3 2
H57 ClF N O9, 777.3422).
1
S-MTPA ester of 1: H NMR δ (only key resonances are listed)
5
1
.732 (1H, t, J = 5.4 Hz, NH), 5.292 (1H, m, H-9), 3.656 (3H, s, OMe-
2), 3.535 (2H, s, H -6), 2.652 (2H, d, J = 6.9 Hz, H -10); ESIMS m/z
2
2
6
00 MHz NMR spectrometer time and UV measurements and
þ
þ
7
C
77.4 [M þ H] ; HRESI/TOFMS m/z 777.3443 [M þ H] (calcd for
the Florida Atlantic University, Jupiter Campus, for the use of
their polarimeter and infrared spectrometer. The high-resolution
mass spectrometric analysis was performed by the UCR mass
spectrometer facility, Department of Chemistry, University of
California at Riverside. This is contribution number 840 from the
Smithsonian Marine Station at Fort Pierce.
3
5
38
H
57 ClF
3
N
2
O
9, 777.3422).
Base Hydrolysis of Malyngamide 3. Compound 1 (1.9 mg)
was dissolved in a 0.5 mL solution of 10% KOH in 80% aqueous EtOH
and refluxed for 12 h. The hydrolysate was concentrated in vacuo and
partitioned between H O and CH Cl . The H O layer was separated,
2
2
2
2
acidified, and extracted with CH
2
Cl
2
to yield lyngbic acid (5, 0.4 mg):
25
16
colorless oil; [R] -12 (c 0.04, CHCl ) [lit. -12.6 (c 0.8, MeOH) ];
D
3
1
H NMR (600 MHz, CDCl
3
) δ 5.48 (2H, m), 3.31 (3H, s, OMe), 3.15
’ REFERENCES
(
1H, quin, J = 5.5 Hz), 2.42 (2H, t, J = 7.5 Hz), 2.34 (2H, m), 2.18 (2H,
(
1) Tidgewell, K.; Clark, B. T.; Gerwick, W. H. In Comprehensive
m), 1.43 (2H, m), 1.27 (10H, m), 0.87 (3H, t, J = 7.0 Hz); HRESI/
TOFMS m/z 257.2113 [M þ H] (calcd for C H O , 257.2114).
Natural Products Chemistry, 2nd ed.; Moore, B., Crews, P., Eds.; Elsevier
Limited: Oxford, 2010, in press.
þ
15 29 3
8
75
dx.doi.org/10.1021/np1008015 |J. Nat. Prod. 2011, 74, 871–876