Isolation and structures of pipecolidepsins A and B, cytotoxic cyclic depsipeptides from the madagascan sponge Homophymia lamellosa

Article abstract of DOI:10.1021/np400888e

Two new cyclic depsipeptides, pipecolidepsins A and B (1 and 2), have been isolated from the sponge Homophymia lamellosa collected off the coast of Madagascar. Their structures were determined by a combination of NMR experiments and by LC-MS analysis of the amino acid fragments obtained by hydrolysis and derivatization using Marfey's reagent. In addition to several common amino acids, these peptides contain unusual residues, including 2-amino-3-hydroxy-4,5- dimethylhexanoic acid, 3-ethoxyasparagine, 3,4-dimethylglutamine, 4,7-diamino-2,3-dihydroxy-7-oxoheptanoic acid, and 3-hydroxyaspartic acid as well as a terminal 3-hydroxy-2,4,6-trimethylheptanoic acid residue. Pipecolidepsins A and B displayed cytotoxic activity against a panel of different human tumor cell lines.

Full text of DOI:10.1021/np400888e

Article
pubs.acs.org/jnp
Isolation and Structures of Pipecolidepsins A and B, Cytotoxic Cyclic
Depsipeptides from the Madagascan Sponge Homophymia lamellosa
Laura Coello, Fernando Reyes,† María Jesus Martín, Carmen Cuevas,* and Rogelio Fernandez*
́
́
Medicinal Chemistry Department, PharmaMar S. A., Pol. Ind. La Mina Norte, Avenida de los Reyes 1, 28770, Colmenar Viejo
(Madrid), Spain
S
* Supporting Information
ABSTRACT: Two new cyclic depsipeptides, pipecolidepsins
A and B (1 and 2), have been isolated from the sponge
Homophymia lamellosa collected off the coast of Madagascar.
Their structures were determined by a combination of NMR
experiments and by LC-MS analysis of the amino acid
fragments obtained by hydrolysis and derivatization using
Marfey’s reagent. In addition to several common amino acids,
these peptides contain unusual residues, including 2-amino-3-
hydroxy-4,5-dimethylhexanoic acid, 3-ethoxyasparagine, 3,4-
dimethylglutamine, 4,7-diamino-2,3-dihydroxy-7-oxoheptanoic acid, and 3-hydroxyaspartic acid as well as a terminal 3-hydroxy-
2,4,6-trimethylheptanoic acid residue. Pipecolidepsins A and B displayed cytotoxic activity against a panel of different human
tumor cell lines.
arine sponges continue to be an abundant source of new
molecules with interesting biological properties. Among
compounds incorporate some of the unusual amino acids
present in the depsipeptides cited above, as well as a 3-
ethoxyasparagine residue, which is unprecedented in natural
peptides.
M
them, cyclic depsipeptides constitute a structurally complex
class, with many such compounds incorporating novel amino
acid residues. Particularly, a number of complex sponge
peptides including homophymines A−E and A1−E1 from
Homophymia sp.,1 callipeltins A−M from Callipelta sp. and
Latrunculia sp.,2 mirabamides A−H from Siliquariaspongia
mirabilis,3 papuamides A−F and theopapuamide from Theonella
mirabilis and T. swinhoei,4,5 and neamphamides A and B from
Neamphius huxleyi6 all share one or more of the unusual
residues (2S,3S,4R)-3,4-dimethylglutamine (3,4-diMeGln),
(2S,3R)-β-methoxytyrosine (βOMeTyr), (2R,3R,4S)-4-amino-
7-guanidino-2,3-dihydroxyheptanoic acid (AGDHA),
(2R,3R,4S)-4-amino-2,3-dihydroxy-1,7-heptandioic acid,
(2R,3R,4R)-2-amino-3-hydroxy-4,5-dimethylhexanoic acid,
(2R,3R,4R)-3-hydroxy-2,4,6-trimethylheptanoic acid
(HTMHA), and (2R,3R,4R)-3-hydroxy-2,4,6-trimethyloctanoic
acid (HTMOA). Interestingly, many of these molecules have
been reported to display strong anti-HIV, antifungal, or
antitumor properties.
During the course of our ongoing screening program to find
new antitumor agents from marine organisms, extracts of the
Madagascan sponge Homophymia lamellosa were found to
display cytotoxic activity against the human tumor cell lines A-
549 (lung), HT-29 (colon), and MDA-MB-231 (breast).
Bioassay-guided fractionation of the 2-propanol extract of a
specimen of this organism yielded pipecolidepsins A and B as
the bioactive components. The structures of both compounds,
their cytotoxic properties, and a total solid-phase synthesis of
pipecolidepsin A have been previously disclosed.7,8 Herein, we
report the isolation and the rationale followed in their structural
characterization. The chemical structures of these new
The molecular formula of pipecolidepsin A (1) was
established as C74H126N16O26 by HRMALDIMS of the [M +
H]+ molecular ion at m/z 1655.90918. The peptidic nature of
pipecolidepsin A was evident from the abundance of signals in
the amide NH proton (δ 9.00−6.49 ppm) and α-amino proton
1
(δ 5.42−3.82 ppm) regions of its H NMR spectrum and the
presence of numerous carbonyl carbons (δ 180.2−169.7 ppm)
in the 13C NMR spectrum obtained in CD3OH. Additionally,
1
the H NMR spectrum showed signals corresponding to an N-
methyl group at δ 2.96 ppm (3H, s) and several other methyl
Received: October 21, 2013
Published: January 24, 2014
© 2014 American Chemical Society and
American Society of Pharmacognosy
298
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Journal of Natural Products
Article
Table 1. NMR Data of Pipecolidepsins A (1) and B (2) in CD3OH
1
2
amino acid
Pip
δC, mult
δH, m, J (Hz)
5.27, m
amino acid
Pip
δC, mult
δH, m, J (Hz)
5.26, m
c
1
2
3
170.4, C
53.9, CH
27.6, CH2
1
2
3
170.6, C
53.9, CH
27.6, CH2
2.19, m
1.64, m
1.74, m
1.25, m
1.63, m
1.53, m
3.70, m
3.12, m
2.20, m
1.63, m
1.75, m
1.23, m
1.62, m
1.56, m
3.69, m
3.12, m
4
5
6
22.6, CH2
26.3, CH2
44.6, CH2
4
5
6
22.6, CH2
28.0, CH2
44.6, CH2
c
Asp1
1
2
3
170.5, C
47.2, CH
36.5, CH2
Asp
1
2
3
170.4, C
5.34, m
47.3, CH
36.6, CH2
5.32, m
2.85, m
2.46, m
2.90, dd (17.0, 8.0)
2.46, dd (16.5, 4.0)
h
4
173.8, C
4
NH
1
8.39, d (9.0)
NH
1
8.43, d (7.6)
EtOAsn
169.7, C
56.7, CH
78.5, CH
174.1, C
68.0, CH2
EtOAsn
169.8, C
56.8, CH
g
2
4.92, m
2
4.91
3
4.62, br s
3
78.5, CH
h
4.60, br s
4
4
5
3.67, m
5
68.0, CH2
15.9, CH3
3.67, m
3.48, m
3.48, m
6
16.0, CH3
1.19, t (7.0)
6.49, d (9.0)
7.53, br s
7.31, br s
6
1.18, t (7.0)
NH
NH2
NH
NH2
6.48, d (8.5)
i
N-MeGln
1
2
3
172.2, C
58.2, CH
23.5, CH2
N-MeGln
1
2
3
172.3, C
58.2, CH
23.3, CH2
5.42, d (8.0)
2.46, m
5.43, m
2.48, m
1.78, m
2.22, m
2.12, m
1.81, m
4
31.9, CH2
2.19, m
4
31.7, CH2
2.08, m
5
177.8, C
5
177.7, C
NMe
NH2
31.4, CH3
2.96, s
NMe
NH2
31.4, CH3
2.95, s
i
7.21, br s
6.75, br s
Leu
1
2
3
177.0, C
51.3, CH
38.9, CH2
Leu
1
2
3
177.0, C
51.2, CH
38.9, CH2
4.58, m
4.58, m
2.39, m
2.38, m
1.55, m
1.55, m
4
26.1, CH
20.8, CH3
24.1, CH3
2.03, m
4
26.1, CH
20.8, CH3
24.0, CH3
2.04, m
4-Me
1.03, d (6.5)
4-Me
1.01, d (6.5)
1.09, d (6.9)
7.57, d (5.5)
a
d
5
1.09, d (6.5)
5
NH
1
7.60, d (6.0)
NH
1
b
h
Lys
174.4, C
Lys
2
53.1, CH
30.4, CH2
4.49, m
2.02, m
1.55, m
1.48, m
1.36, m
1.66, m
2
53.4, CH
30.5, CH2
4.44, m
2.02, m
1.58, m
1.50, m
1.35, m
1.63, m
1.59, m
2.95, m
7.88, br s
n.o.
3
3
4
5
23.5, CH2
26.3, CH2
41.0, CH2
4
5
23.6, CH2
27.9, CH2
41.1, CH2
6
2.95, m
6
NH
NH2
1
7.85, br d (8.5)
NH
NH2
1
j
n.o.
Thr
171.8, C
64.3, CH
67.6, CH
20.1, CH3
Thr
171.9, C
64.2, CH
67.6, CH
20.2, CH3
2
3.82, br s
4.39, m
2
3.82, br s
4.38, m
3
3
4
1.34, d (6.0)
4
1.33, d (6.5)
299
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Table 1. continued
1
2
amino acid
NH
δC, mult
δH, m, J (Hz)
8.33, br s
amino acid
AHDMHA
δC, mult
δH, m, J (Hz)
8.39, br s
NH
b
h
AHDMHA
1
174.6, C
55.6, CH
77.2, CH
39.1, CH
8.8, CH3
27.8, CH
21.3, CH3
15.3, CH3
1
2
5.29, m
2
55.6, CH
77.2, CH
39.1, CH
8.7, CH3
27.9, CH
21.3, CH3
15.2, CH3
5.29, m
3
5.62, dd (11.0, 2.0)
1.91, m
3
5.57, br d (11.0)
1.93, m
4
4
4-Me
5
0.74, d (7.5)
1.93, m
4-Me
5
0.73, d (7.0)
1.92, m
5-Me
6
0.94, d (6.5)
0.73, d (7.5)
8.96, d (10.0)
5-Me
6
0.94, d (6.5)
0.71, d (7.5)
8.91, br d
NH
1
NH
1
h
DiMeGln
174.6, C
59.0, CH
37.2, CH
14.3, CH3
42.5, CH
14.4, CH3
180.2, C
DiMeGln
2
4.33, dd (11.0, 5.5)
2.30, m
2
59.0, CH
36.9, CH
14.1, CH3
42.7, CH
14.4, CH3
180.6, C
4.28, dd (10.5, 5.5)
2.31, m
3
3
a
e
d
3-Me
4
1.09, d (7,1)
3-Me
4
1.05, d (6.5)
2.75, qd (7.0, 2.5)
1.24, d (7.0)
2.76, m
4-Me
5
4-Me
5
1.23, d (7.0)
NH
NH2
9.00, d (4.5)
7.45, br s
NH
NH2
9.19, br d
i
6.94, br s
DADHOHA
1
2
3
4
5
176.4, C
72.9, CH
75.7, CH
51.1, CH
28.5, CH2
DADHOHA
1
2
3
4
5
176.3, C
72.8, CH
75.6, CH
51.0, CH
28.5, CH2
3.84, m
3.59, m
4.10, m
1.92, m
1.78, m
2.23, m
3.92, m
3.57, m
4.10, m
1.92, m
1.75, m
2.20, m
6
32.6, CH2
178.6, C
6
32.7, CH2
178.7, C
7
7
f
NH
NH2
7.66, d (9.0)
7.53, br s
NH
NH2
7.53
i
6.83, br s
h
Asp2
1
2
3
174.8, C
51.7, CH
36.6, CH2
HOAsp
1
2
3
g
4.69, dd (12.0, 6.5)
2.92, m
58.2, CH
72.2, CH
4.86
4.38, m
2.84, dd (17.0, 5.0)
h
4
174.7, C
4
NH
8.35, d (8.0)
NH
8.28, d (7.0)
HTMHA
1
179.1, C
HTMHA
1
179.3, C
2
44.9, CH
14.4, CH3
79.6, CH
33.5, CH
17.4, CH3
39.3, CH2
26.2, CH
21.5, CH3
24.7, CH3
2.62, qd (9.5, 7.0)
2
44.9, CH
14.3, CH3
79.9, CH
33.4, CH
17.4, CH
39.1, CH2
26.2, CH
21.5, CH3
24.7, CH3
2.69, qd (9.0, 7.0)
a
e
d
2-Me
1.07, d (6.9)
2-Me
1.06, d (6.5)
3
3.57, m
3
3.55, m
4
1.74, m
4
1.75, m
4-Me
0.99, d (6.5)
1.16, m
4-Me
0.98, d (7.0)
1.16, m
5
5
6
1.66, m
6
1.63, m
6-Me
7
0.87, d (6.5)
6-Me
7
0.86, d (6.5)
0.93, d (6.5)
0.94, d (6.5)
a−f
g
h
Assignment for these signals may be interchanged. Under solvent. Carbonyl signals at these positions (δC 174.7, 174.4, 174.2, 173.8 ppm) were
i
not assigned due to the failure of obtaining 1H−13C long-range connectivities. Three carbonyl signals were overlapped/not detected. Assignments of
j
NH2 at these positions (δH 7.54f/6.81, 7.50/6.96, 7.53f/7.30 and 7.16/6.79 ppm) are interchangeable. Not observed.
groups between 1.34 and 0.73 ppm. Extensive analysis of the
1H and 13C NMR data of 1, including COSY, TOCSY, HMQC-
TOCSY, HSQC, and HMBC spectra (Table 1), established the
presence of the common amino acids threonine, lysine, leucine,
and two aspartic acids, together with six unusual amino acid
moieties: pipecolic acid (Pip), 3-ethoxyasparagine (EtOAsn),
N-methylglutamine (N-MeGln), 2-amino-3-hydroxy-4,5-dime-
thylhexanoic acid (AHDMHA), 3,4-dimethylglutamine (3,4-
diMeGln), and 4,7-diamino-2,3-dihydroxy-7-oxoheptanoic acid
(DADHOHA). The presence of the amino-linked 3-hydroxy-
2,4,6-trimethylheptanoic acid (HTMHA), also present as the
end group in callipeltin B, neamphamide A, and homophy-
mines B and B1,1,2,6 was also inferred from the analysis of the
NMR spectra. The previously unreported amino acid 3-
ethoxyasparagine was identified as one amino acid component
by long-range correlations from the 3-oxymethine proton at
300
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Journal of Natural Products
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Figure 1. Selected NOE (dashed) and HMBC (plain) correlations for 1.
4.62 ppm to a methylene at 3.67 and 3.48 ppm corresponding
to an ethoxy group, coupled in turn in the COSY spectrum to a
triplet methyl group at 1.19 ppm.
In a similar manner, the absolute configurations of DiMeGln,
AHDMHA, and HTMHA were assumed to be the same as
those previously reported for other similar peptides due to the
close resemblance of the 1H and 13C NMR chemical shifts and
use of synthetic standards for Marfey’s analysis.11 Definitive
confirmation of these configurations and the proposed structure
of pipecolidepsin A came from the solid-phase synthesis of the
compound.8 Comparison of the data obtained for the synthetic
compound and the natural pipecolidepsin A revealed a perfect
overlap of the NMR spectra, identical analytical HPLC
behavior, and similar in vitro activity.
Pipecolidepsin B (2) had a molecular formula of
C74H126N16O27, according to the (+)-HRMALDIMS (m/z
1671.89954 [M + H]+). This molecular formula and a
comparison of 1H and 13C NMR spectra with those of
pipecolidepsin A revealed that the only difference between the
compounds was the absence of the methylene group present in
the Asp2 of 1 and its substitution for an oxymethine (δH 4.38
and δC 72.2 ppm) to give a 3-hydroxyaspartic acid (HOAsp) in
2. Further support for this proposal was provided by an
extensive analysis of the 1D and 2D NMR data. Finally,
hydrolysis of 2 with 6 N HCl, derivatization with Marfey’s
reagent, HPLC-MS analysis, and comparison with the four
synthetic stereoisomers12 of 3-hydroxyaspartic acid confirmed
the presence of (2R,3S)-3-hydroxyaspartic acid in pipecolidep-
sin B.
Finally, a second test of the cytotoxic activity of the
pipecolidepsin A against three human tumor cell lines, lung
(A-549), colon (HT-29), and breast (MDA-MB-231),
produced similar results to those previously reported8 with
GI50 values of 0.6, 1.12, and 0.7 μM, respectively. Values of
0.04, 0.01, and 0.02 μM, respectively, were obtained when 2
was assayed against the same cell lines. Interestingly, the
replacement of the Asp2 residue in pipecolidepsin A with an
HOAsp amino acid in pipecolidepsin B gives rise to a 10-fold
(100-fold for the HT-29 cell line) increase in the bioactivity of
the latter compound, revealing perhaps a key role for the
substitution at C-3 and the hydrophilic nature of this amino
acid residue in the mode of action of the compound.
The sequencing of these units was carried out using a
combination of HMBC and ROESY data. Long-range
correlations from α-protons to carbonyl carbons of adjacent
amino acids plus ROESY correlations between α-protons and
NH protons of adjacent amino acids (Figure 1) allowed us to
establish the sequence as Pip-Asp1-EtOAsn-N-MeGln-Leu-Lys-
Thr-AHDMHA-DiMeGln-DADHOHA. The presence of an
ester bond between the carbonyl group of pipecolic acid and
the hydroxy group of AHDMHA was suggested by the
downfield chemical shift of the H-3 oxymethine proton (δH
5.62 ppm) of this last residue5 and confirmed by the presence
of an HMBC correlation between this proton and the Pip
carbonyl carbon at δC 170.4 ppm. Additionally, ROESY cross-
peaks between the DADHOHA-NH (δH 7.66 ppm) and the
Asp2 H-2 proton (δH 4.69 ppm) established an amide linkage
between these two amino acids, and acylation of Asp2 by
HTMHA was evidenced by HMBC correlations from the NH
and H-2 signals of Asp2 to the carbonyl group of HTMHA at
179.1 ppm and a ROESY cross-peak between the Asp2 NH (δH
8.35 ppm) and proton H-2 of HTMHA at δH 2.62 ppm.
The absolute configurations of S-Pip, R-Asp, (2S,3S)-
EtOAsn, S-N-Me-Gln, S-Leu, R-Lys, R-allo-Thr, and R-Asp
were determined by comparing the hydrolysis products of 1 (6
N HCl, 110 °C, 15 h), after derivatization with Marfey’s reagent
(N-(3-fluoro-4.6-dinitrophenyl)-L-alaninamide, L-FDAA), with
appropriate amino acid standards using HPLC-MS chromatog-
raphy.9 Commercially available samples of Pip, Asp, N-Me-Glu,
Leu, and Thr and synthetic samples of 3-EtOAsp10 were used
in these analyses.
The S configuration at C-4 of the 4,7-diamino-2,3-dihydroxy-
7-oxoheptanoic acid (DADHOHA) was determined by
cleavage of the 2,3-diol in 1 with sodium periodate, followed
by oxidative workup, hydrolysis with 6 N HCl, derivatization
with Marfey’s reagent, HPLC-MS analysis of the resulting Glu
residue, and comparison with the corresponding S- and R-Glu
Marfey’s derivatives. The configurations at C-2 and C-3 of the
diol were assumed to be identical to those of the similar amino
acid found in homophymine, callipeltins, and the theopapua-
EXPERIMENTAL SECTION
■
1
mides and were confirmed by analysis of H and 13C NMR
General Experimental Procedures. Optical rotations were
determined using a Jasco P-1020 polarimeter. UV spectra were
performed using an Agilent 8453 UV−vis spectrometer. IR spectra
chemical shifts, their coupling constants, and solid-phase
synthesis of pipecolidepsin A.8
301
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Journal of Natural Products
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were obtained with a Perkin-Elmer Spectrum 100 FT-IR spectrometer
with ATR sampling. NMR spectra were recorded on a Varian “Unity
500” spectrometer at 500/125 MHz (1H/13C). Chemical shifts were
AUTHOR INFORMATION
Corresponding Author
*Tel: +34 91 823 4664. Fax: +34 91 846 6001. E-mail:
rfernandez@pharmamar.com.
■
1
reported in ppm using residual CD3OH (δ 3.30 ppm for H and 49.0
ppm for 13C) as an internal reference. (+)-HRMALDIMS was
performed on an Applied Biosystems 4700 Proteomics Analyzer
spectrometer employing an α-cyano-4-hydroxycinnamic acid matrix.
(+)-ESIMS were recorded using an Agilent 1100 Series LC/MSD
spectrometer.
Present Address
†Fundacion
́
MEDINA, Avenida del Conocimiento 3, 18016,
Granada, Spain.
Notes
Animal Material. The sponge Homophymia lamellosa (order
Lithistida, family Neopeltidae) was collected by hand using scuba
diving near Saint Marie Island, Madagascar (17°07.436′ S/49°47.525′
E) at depths ranging between 3 and 7 m in May 2004 and frozen
immediately after collection. A voucher specimen (ORMA028881) is
deposited at PharmaMar.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We gratefully acknowledge the help of our PharmaMar
colleagues, C. de Eguilior and S. Bueno, for collecting the
■
Extraction and Isolation. A group of specimens of H. lamellosa
(382.5 g) was triturated and exhaustively extracted with 2-propanol (4
× 400 mL, 2 × 300 mL). The combined extracts were concentrated to
yield a crude mass of 13.19 g, which was dissolved in 300 mL of H2O
and extracted with hexane (3 × 300 mL), EtOAc (3 × 300 mL), and n-
butanol (3 × 100 mL).
́
marine samples, L. F. Garcıa for the design of the biological
assays, S. Munt for revision of the manuscript, and S. Gonzalez
́
for performance of the NMR experiments. We also thank J. L.
Carballo for the sponge taxonomy determination.
REFERENCES
■
The n-butanol extract was evaporated to yield 5.86 g of a crude
product that was subjected to VLC on Lichroprep RP-18 with a
stepped gradient from H2O to MeOH and then MeOH/CH2Cl2
(50:50). Fractions eluted with MeOH/H2O (75:25) and MeOH/H2O
(85:15) were pooled to give a fraction of 592.5 mg, which was
subjected to RP-18 column chromatography with a stepped gradient
from H2O/MeOH (35:65) to MeOH. Fractions eluted with H2O/
MeOH (30:70, 223.4 mg) were subjected to preparative HPLC
(Symmetry C18, 7 μm, 19 × 150 mm, gradient H2O + 0.1% TFA/
CH3CN + 0.1% TFA from 22% to 42% CH3CN + 0.1% TFA in 25
min and then from 42% to 100% in 7 min, flow: 15 mL/min, UV
detection) to yield a fraction (95.9 mg) containing a mixture of
pipecolidepsins A and B (retention time from 24.2 to 26.2 min). This
fraction was further purified by semipreparative HPLC (X-Bridge Prep
C18, 5 μm, 10 × 150 mm, isocratic H2O + 0.1% TFA/CH3CN + 0.1%
TFA (65:35), flow 2.3 mL/min, UV detection) to obtain impure
pipecolidepsin A (39.9 mg, retention time 26.49 min) and pure
pipecolidepsin B (13.6 mg, 0.0035% w/w, retention time 24.99 min).
Final purification of pipecolidepsin A (14.9 mg, 0.038% w/w) was
achieved by semipreparative HPLC (Kromasil 100 C8, 10 μm, 10 ×
150 mm, gradient H2O/CH3CN from 30% to 45% in 30 min, flow 2.5
mL/min, UV detection, retention time 20.70 min).
(1) (a) Zampella, A.; Sepe, V.; Luciano, P.; Bellota, F.; Monti, M. C.;
D’Auria, M. V.; Jepsen, T.; Petek, S.; Adeline, M.-T.; Laprev
́
o
̂
te, O.;
Aubertin, A.-M.; Debitus, C.; Poupat, C.; Ahond, A. J. Org. Chem.
2008, 73, 5319−5327. (b) Zampella, A.; Sepe, V.; Bellota, F.; Luciano,
P.; D’Auria, M. V.; Cresteil, T.; Debitus, C.; Petek, S.; Poupat, C.;
Ahond, A. Org. Biomol. Chem. 2009, 7, 4037−4044.
(2) (a) Zampella, A.; D’Auria, M. V.; Gomez-Paloma, L.; Casapullo,
A.; Minale, L.; Debitus, C.; Henin, Y. J. Am. Chem. Soc. 1996, 118,
6202−6209. (b) D’Auria, M. V.; Zampella, A.; Gomez-Paloma, L.;
Minale, L.; Debitus, C.; Roussakis, C.; Le Bert, V. Tetrahedron 1996,
52, 9589−9596. (c) Zampella, A.; Randazzo, A.; Borbone, N.; Luciani,
S.; Trevisi, L.; Debitus, C.; D’Auria, M. V. Tetrahedron Lett. 2002, 43,
6163−6166. (d) Sepe, V.; D’Orsi, R.; Borbone, N.; D’Auria, M. V.;
Bifulco, G.; Monti, M. C.; Catania, A.; Zampella, A. Tetrahedron 2006,
62, 833−840. (e) D’Auria, M. V.; Sepe, V.; D’Orsi, R.; Bellota, F.;
Debitus, C.; Zampella, A. Tetrahedron 2007, 63, 131−140.
(3) (a) Plaza, A.; Gustchina, E.; Baker, H. L.; Kelly, M.; Bewley, C. A.
J. Nat. Prod. 2007, 70, 1753−1760. (b) Lu, Z. Y.; Van-Wagoner, R. M.;
Harper, M. K.; Baker, H. L.; Jooper, J. N. A.; Bewley, C. A.; Ireland, C.
M. J. Nat. Prod. 2011, 74, 185−193.
(4) (a) Ford, P. W.; Gustafson, K. R.; McKee, T. C.; Shigematsu, N.;
Maurizi, L. K.; Panneel, L. K.; Williams, D. E.; de Silva, E. D.; Lassota,
P.; Allen, T. M.; Van Soest, R.; Andersen, R. J.; Boyd, M. R. J. Am.
Chem. Soc. 1999, 121, 5899−5909. (b) Prasad, P.; Aalbersberg, W.;
Feussner, K. D.; Wagoner, R. M. V. Tetrahedron 2011, 67, 8529−8531.
(5) (a) Ratnayake, A. S.; Bugni, T. S.; Feng, X. D.; Harper, M. K.;
Skalicky, J. J.; Mohammed, K. A.; Andjelic, C. D.; Barrows, L. R.;
Ireland, C. M. J. Nat. Prod. 2006, 69, 1582−1586. (b) Plaza, A.;
Bifulco, G.; Keffer, J. L.; Lloyd, J. R.; Beker, H. L.; Bewley, C. A. J. Org.
Chem. 2009, 74, 504−512.
Pipecolidepsin A (1): amorphous, white solid; [α]25 +14.4 (c 0.1,
D
MeOH); UV (MeOH) λmax 196 nm; IR (KBr) νmax 3345, 2961, 2875,
1
1664, 1525, 1460, 1203, 1141, 1013, 827, 801, 722 cm−1; H NMR
(500 MHz) and 13C NMR (125 MHz) see Table 1; (+)HRMALDIMS
m/z 1655.90918 [M + H]+ (calcd for C74H127N16O26, 1655.91020).
Pipecolidepsin B (2): amorphous, white solid; [α]25 +6.1 (c 0.2,
D
MeOH); UV (MeOH) λmax 195 nm; IR (KBr) νmax 3326, 2961, 2931,
1
1663, 1525, 1449, 1204, 1138, 1013, 841, 801, 723 cm−1; H NMR
(6) (a) Oku, N.; Gustafson, K. R.; Cartner, L. K.; Wilson, J. A.;
Shigematsu, N.; Hess, S.; Panell, L. K.; Boyd, M. R.; McMahon, J. B. J.
Nat. Prod. 2004, 67, 1407−1411. (b) Yamano, Y.; Arai, M.; Kobayashi,
M. Bioorg. Med. Chem. Lett. 2012, 22, 4877−4881.
(7) Coello, L.; Fernandez, R.; Reyes, J. F.; Francesh, A.; Cuevas, M.
́
D. C. Anticancer Pipecolidepsins from marine organisms. Int. Appl.
Pat. WO2010/070078 A1, June 24, 2010.
(500 MHz) and 13C NMR (125 MHz) see Table 1; (+)HRMALDIMS
m/z 1671.89954 [M + H]+ (calcd for C74H127N16O27, 1671.90511).
Evaluation of Cytotoxic Activity. The cytotoxic activity of
pipecolidepsins was evaluated against a panel of three human tumor
cell lines as previously described.13
(8) Pelay-Gimeno, M.; García-Ramos, Y.; Martín, M. J.; Spengler, J.;
Molina-Guijarro, J. M.; Munt, S.; Francesh, A. M.; Cuevas, C.; Tulla-
Puche, J.; Albericio, F. Nat. Commun. 2013, 4, 2352.
ASSOCIATED CONTENT
■
S
* Supporting Information
(9) Marfey, P. Calsberg Res. Commun. 1985, 49, 591−596.
(10) (a) Rao, A. V. R.; Chakraborty, T. K.; Reddy, K. L.; Rao, A. S
Tetrahedron Lett. 1994, 35, 5043−5046. (b) Deng, J.; Hamada, Y.;
Shioiri, T. J. Am. Chem. Soc. 1995, 117, 7824−7825.
Tabulated NMR data, 1D and 2D NMR spectra, details of the
biological activity and determination of the absolute config-
uration of amino acids, and LC-MS chromatogram charts of L-
FDAA derivatives of pipecolidepsins A (1) and B (2). This
material is available free of charge via the Internet at http://
pubs.acs.org.
(11) (a) Acebedo, C. M.; Kogut, E. F.; Lipton, M. A. Tetrahedron
2001, 57, 6353−6359. (b) C
̧
alimsiz, S.; Lipton, M. A. J. Org. Chem.
2005, 70, 6218−6221.
302
dx.doi.org/10.1021/np400888e | J. Nat. Prod. 2014, 77, 298−303

Journal of Natural Products
Article
(12) (a) Sendai, M.; Hashigushi, S.; Tomimoto, M.; Kishimoto, S.;
Matsuo, T.; Ochiai, M. Chem. Pharm. Bull. 1985, 33, 3798−3810.
(b) Guzman
1513−1516.
(13) Reyes, F.; Rodríguez-Acebes, R.; Fernan
Francesh, A.; Cuevas, C. J. Nat. Prod. 2010, 73, 83−85.
́
-Martínez, M.; VanNieuwenhze, M. S. Synlett 2007, 10,
́
dez, R.; Bueno, S.;
303
dx.doi.org/10.1021/np400888e | J. Nat. Prod. 2014, 77, 298−303