Isolation and structural elucidation of euryjanicins B-D, proline-containing cycloheptapeptides from the caribbean marine sponge Prosuberites laughlini

Article abstract of DOI:10.1021/np9004135

Three new cyclic peptides, euryjanicins B (2), C (3), and D (4), have been isolated from the Puerto Rican marine sponge Prosuberites laughlini, and the structures were elucidated by chemical degradation, ESIMS/MS, and extensive 2D NMR methods. When tested

Full text of DOI:10.1021/np9004135


Copyright 2009 by the American Chemical Society and the American Society of Pharmacognosy
Volume 72, Number 9
September 2009
Full Papers
Isolation and Structural Elucidation of Euryjanicins B-D, Proline-Containing
†
Cycloheptapeptides from the Caribbean Marine Sponge Prosuberites laughlini
Brunilda Vera, Jan Vicente, and Abimael D. Rodr ´ı guez*
Department of Chemistry, UniVersity of Puerto Rico, P.O. Box 23346, U. P. R. Station, San Juan, Puerto Rico 00931-3346
ReceiVed July 9, 2009
Three new cyclic peptides, euryjanicins B (2), C (3), and D (4), have been isolated from the Puerto Rican marine
sponge Prosuberites laughlini, and the structures were elucidated by chemical degradation, ESIMS/MS, and extensive
2
D NMR methods. When tested against the National Cancer Institute 60 tumor cell line panel, all of the purified isolates
displayed weak cytotoxicity.
1
4
Cyclic peptides are a frequently encountered class of natural
products that often exhibits a wide variety of essential biological
functions. A large number of cyclic peptides with unique structures
and diverse pharmacological activities have been reported from
invertebrate animals and microorganisms typically associated with
Suberitidae) collected in the waters off Puerto Rico. As a result,
we recently reported the structure of euryjanicin A (1), a crystalline
cyclopeptide whose basic structural motif consists of a 21-
membered ring constructed from seven amino acid residues, two
1
5
of which are prolines. In this paper we report the isolation,
structure characterization, and bioactivity of three additional
members of the family, named euryjanicins B-D (2-4), from the
above marine sponge. All three compounds are proline-containing
cycloheptapeptides, which, unlike euryjanicin A (1), generate well-
1
marine habitats. Additionally, a smaller number of cyclic peptides
2
have been isolated from higher plants. Because of greater resistance
to in ViVo enzymatic degradation and higher bioavailability than
their noncyclic counterparts, cyclic peptides often possess superior
3
therapeutic potential. A noteworthy class of marine cyclopeptides
3
resolved NMR spectra in CDCl . From these spectra it was obvious
is represented by proline-rich compounds, usually containing seven
or eight amino acid residues. The role of proline in these molecules,
which often occur as complex mixtures of structurally related
derivatives, has been associated with control of conformation of
the molecule in solution because of the restricted dihedral angle of
that for each cyclopeptide one conformation strongly predominated
in this solvent.
Results and Discussion
4
The lyophilized sponge was extracted with MeOH, and the
residue obtained was partitioned according to a modified Kupchan
procedure, affording two extracts of increasing polarity: EtOAc and
proline. Examples of this kind of bioactive natural products are
5
6
7
8
the axinellins, axinastatins, phakellistatins, hymenamides, sty-
9
10
11
12
lopeptides, stylostatin 1, wainunuamide, dominicin, and
16
1
3
nBuOH. Our initial biological screenings showed that the EtOAc
fraction retained most of the original antitumor activity ascribed
to the extract but showed no antituberculosis activity. However,
the nBuOH extract was strongly antitubercular but weakly cytotoxic.
others.
Within the context of a long-term effort to discover new
metabolites with antitumor and anti-infective properties from marine
invertebrates from the Caribbean region, we recently had the
opportunity to chemically scrutinize the bioactive extract of the
marine sponge Prosuberites laughlini (order Hadromerida, family
1
13
Preliminary chromatographic and H and C NMR analyses
revealed that the cytotoxic EtOAc fraction contained substances
of a peptidic nature, whereas the strongly anti-infective nBuOH
extract was mainly comprised of a mixture of the known pyrrole
†
Dedicated to the memory of Dr. Clifford W. J. Chang (1938-2007),
17
18
alkaloids monobromoisophakellin and hymenidin. Therefore,
the EtOAc-soluble material was selected for further isolation work.
This extract was subjected to normal-phase Si gel column chro-
who led the discovery of the marine isocyano series of natural products.
*
To whom correspondence should be addressed. Tel.: +787-764-0000,
ext. 4799. Fax: +787-756-8242. E-mail: abrodriguez@uprrp.edu.
1
0.1021/np9004135 CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/10/2009

1
556 Journal of Natural Products, 2009, Vol. 72, No. 9
Vera et al.
Table 1. H (500 MHz), 13C NMR (125 MHz), TOCSY, HMBC, and NOESY Correlations for Euryjanicin B (2)a
1
b
c
d
e
amino acid
L-alanine1
position
δ
C
(mult)
δ
H
, mult (J in Hz)
TOCSY
HMBC
NOESY
2
CO
R
173.3, qC
R, ꢀ, RThr, NHThr
ꢀ, NH
52.4, CH
18.7, CH
4.28, dd (7.0, 7.5)
1.54, d (7.5)
7.72, d (7.0)
ꢀ, NH
R, NH
R, ꢀ
ꢀ
ꢀ
3
3
R, NH
NH
R, ꢀ, δPro ,
7
NH
2
6
NHThr , RPro
L-threonine2
CO
R
ꢀ
γ
NH
CO
R
ꢀ
γ
169.8, qC
55.3, CH
69.0, CH
20.0, CH
R
3
4.75, dd (3.0, 8.0)
4.02, br s
1.13, d (6.0)
7.54, d (8.0)
ꢀ, NH
γ, NH
γ, NH
R, γ
ꢀ, γ, δPro
3
γ, δPro
R, ꢀ, NH
R, ꢀ, γ
R, ꢀ
3
R, RPro
L-proline3
trans
171.3, qC
62.2, CH
29.5, CH
25.7, CH
R, NHPhe
4
2
4.15, dd (8.5, 8.0)
2.35, m
1.55, m
ꢀ
δ
R, δ
γ, ꢀThr
2
2
R, γ, δ
δ
R
1
.95, m
3.68, m
.42, m
R, ꢀ, δ
γ, R
γ
δ
γ
δ
γ
γ
δ
47.7, CH
2
3
L-phenylalanine4
CO
R
ꢀ
171.0, qC
53.6, CH
37.7, CH
R, ꢀ, NHVal
ꢀ, NH
5
4.80, dd (9.5, 15.5)
3.13, dd (5.5, 14.0)
ꢀ, NH
NH
ꢀ
2
R, ortho, ΝΗ
ortho, NH
3
.07, dd (7.5, 14.0)
γ
136.9, qC
129.7, CH
128.6, CH
127.0, CH
R, ꢀ, meta
ꢀ, meta, para
para
ortho
meta
para
NH
CO
R
7.16, d (7.0)
7.34, m
7.29, m
R, ꢀ, NH
ortho
ortho
R, ꢀ
ortho
ortho
R, ꢀ, ortho, NH
6.14, d (9.5)
L-valine5
169.5, qC
56.5, CH
30.3, CH
19.8, CH
R
R
6
4.36, dd (8.5, 9.0)
2.06, m
0.97, d (6.5)
0.94, d (6.5)
6.60, d (8.5)
ꢀ, NH
γ
ꢀ, γ, NH
R, γ
γ, δPro
R
ꢀ
γ
3
3
R, ꢀ
R
1
7.9, CH
R, NH
R, ꢀ, γ
R, NH
R, ꢀ, γ, NHPhe
4
NH
CO
R
L-proline6
trans
170.7, qC
59.3, CH
28.6, CH
R, ꢀ
δ
R, δ
7
4.59, dd (5.0, 8.5)
2.14, m
ꢀ, γ, δ
δ
δ
RPro
R
ꢀ
2
2
2
1
.75, m
2.11, m
.06, m
3.63, m
.42, m
γ
δ
24.8, CH
47.6, CH
R
2
R, γ
γ
ꢀ, γ
γ
3
L-proline7
cis
CO
R
ꢀ
γ
171.4, qC
60.7, CH
31.8, CH
22.0, CH
R, ꢀ, γ, NHAla
1
6
4.35, d (7.0)
2.44, dd (7.0, 12.3)
1.95, m
ꢀ, δ
R, γ, δ
R
δ
R, γ
γ, δ
R, δ
R, δ
ꢀPro , ꢀ, γ
2
2
1
.75, m
R, δ
γ
δ
46.6, CH
2
3.58, m
R
a
Spectra were recorded in CDCl
3
at 25 °C. Chemical shift values are in ppm relative to the residual CHCl
3
(7.25 ppm) or CDCl
3
(77.0 ppm) signals.
b
13
c
d
C NMR multiplicities were obtained from APT experiments. NMR data recorded in a Bruker (400 Mz) spectrometer. HMBC mixing time ) 50
e
ms. NOESY mixing time ) 200 ms.
matography (CC) using a mixture of CHCl
3
and MeOH that had
spin systems of the type X-CH-CH
2
-CH
2
-CH -X′ were defined
2
been previously saturated with NH
3
(80/20). Similar fractions were
using TOCSY, COSY-GPQF, and HSQC (Table 1), indicating the
presence of three proline units. This contention was further
substantiated by the observation of only four peptide-bond NH
pooled together on the basis of their TLC, NMR, and biological
1
activity profiles. Subsequent evaluation of the first fraction by H
1
3
1
and C NMR indicated that this fraction contained a mixture of
small peptides. Further fractionation was performed by normal-
phase Si gel (100 g) CC with a mixture of hexane and EtOAc (95/
proton signals in the H NMR spectrum. The spin systems
5
1
). Close inspection by NMR indicated that subfractions 10 and
1 appeared to contain most of the peptides observed in the initial
NMR analysis. Purification of subfraction 10 was achieved by C18
1
5
2
Si gel reversed-phase CC; this yielded pure euryjanicin A (1),
1
euryjanicin B (2), and the known cyclic octapeptide dominicin (5).
Subsequent purification of fraction 11 by reversed-phase HPLC
afforded pure euryjanicin C (3) and euryjanicin D (4).
Structure Elucidation of Euryjanicin B. Compound 2 was
obtained as an optically active, colorless oil that showed a
pseudomolecular ion peak at m/z 710 in the positive ion ESIMS
spectrum. The molecular formula of 2 was determined to be
+
C
1
36
H
51
N
7
O
8
by the HRESIMS (m/z 710.3871 [M+H] ), requiring
13
5 sites of unsaturation. The C NMR spectrum revealed reso-
nances consistent with seven amide carbonyls (δ 173.3, 171.4,
1
6
71.3, 171.0, 170.7, 169.8, 169.5), seven R-methine carbons (δ 62.2,
0.7, 59.3, 56.5, 55.3, 53.6, 52.4), a secondary carbinol (δ 69.0),
and a monosubstituted phenyl [δ 136.9 (C), 129.7 (CH), 128.6 (CH),
27.0 (CH)] ring system, suggesting a heptapeptide with pheny-
1
lalanine and threonine units. The seven amino acids were unam-
biguously identified by 2D NMR techniques. Three independent
Figure 1. HMBC (HfC) and NOE correlations (dashed arrows)
for euryjanicin B (2) in CDCl .
3

Cycloheptapeptides from the Sponge Prosuberites laughlini
Journal of Natural Products, 2009, Vol. 72, No. 9 1557
amino acid composition was confirmed by HPLC analyses of the
acid hydrolysate of 2 after derivatization with the Marfey reagent
(
FDAA), allowing the absolute configurations at the R-carbons to
19
be assigned as the L configuration for all residues. Because only
4 of the calculated 15 degrees of unsaturation could be accounted
1
for by the functionalities in the seven individual amino acids, it
became obvious that euryjanicin B was a cyclic peptide. Indeed,
the cyclic nature of 2 was evident also by the high degree of
chemical shift dispersion observed for the peptide-bond amide
proton signals resonating between δ 7.72 and 6.14, its solubility in
organic solvents, and the fact that it was negative to a ninhydrin
test. The sequence of amino acids was assigned on the basis of a
combined approach of 2D-NMR and electrospray tandem mass
spectrometry techniques (ESIMS/MS).
Figure 2. Major fragmentation pathways of 2 following opening
of the protonated cyclic structure during ESIMS/MS .
n
3 2 3 3
X-CH-CH(CH ) , X-CHCH , and X-CH(OH)CH were identi-
fied, suggesting the existence of valine, alanine, and threonine
residues. The remaining independent spin system of the type
The carbonyl carbons within each residue were assigned from
HMBC correlations between the CdO and their respective R-pro-
tons (Table 1), whereas each peptide-bond amide proton was
X-CH-CH -X′ was attributed to phenylalanine by the HSQC
2
4
4
correlations: δ 3.13 and 3.07 [ꢀH (Phe )]/δ 37.7 [ꢀC(Phe )]. The
2
Table 2. H NMR (500 MHz), 13C NMR (125 MHz), TOCSY, HMBC, and NOESY Correlations for Euryjanicin C (3)a
1
b
c
d
amino acid
L-serine1
position
δ
C
(mult)
δ
H
, mult (J in Hz)
TOCSY
HMBC
NOESY
2
CO
R
ꢀ
NH
CO
R
169.5, qC
54.4, CH
R, NHIle
2
2
4.50, m
3.80, d (2.0)
6.54, d (8.5)
ꢀ
ꢀ, γIle , δIle
63.5, CH
2
7
R, ꢀ
NHIle , R
L-isoleucine2
172.9, qC
55.3, CH
37.2, CH
R
ꢀ
R
γ
4.31, dd (9.3, 10.5)
1.79, m
1.63, m
ꢀ, ꢀ
γ, ꢀ
δ, ꢀ
ꢀ, δ, ꢀ
ꢀ
γ
24.6, CH
2
1
.20, m
δ
10.6, CH
14.9, CH
3
0.94, t (7.5)
0.86, d (7.0)
6.87, d (9.0)
ꢀ
3
R
1
1
1
2
NH
CO
R
R
COSer
RSer , ꢀSer , ꢀIle
L-proline3
cis
171.8, qC
61.4, CH
NHLeu , R
4
2
2
4.46, d (7.0)
2.40, dd (7.0, 12.0)
ꢀ, γ, δ
γ
ꢀ, γ, δ
RIle , ꢀIle , ꢀ
ꢀ
31.7, CH
22.1, CH
46.4, CH
2
2
2
R, δ
2
.05, m
1.97, m
.71, m
3.62, m
.49, m
γ
δ
δ
δ
ꢀ, γ
ꢀ, γ
R, δ
R, ꢀ
δ
1
ꢀ, γ
3
L-leucine4
CO
R
ꢀ
171.5, qC
50.5, CH
R, ꢀ, NHPhe
ꢀ, γ
R
5
4.51, m
1.61, m
ꢀ, δ
γ, δ
γ, δ
δ
ꢀ, δ
γ
36.6, CH
2
1
.42, m
γ
δ
24.8, CH
16.0, CH
1.46, m
ꢀ, δ
ꢀ, δ
3
3
0.89, d (3.5)
0.81, d (7.0)
7.60, d (6.0)
2
1.8, CH
3
ΝΗ
CO
R
ꢀ
γ
R
COPro , δ
R, ꢀ
L-phenylalanine5
171.0, qC
54.1, CH
ꢀ
5
6
4.62, dt (3.5, 8.3)
3.05, dd (5.0, 8.3)
ꢀ
γ
ꢀ, NHPhe
ꢀ, RPro
38.0, CH
2
ortho
134.9, qC
129.7, CH
128.9, CH
127.5, CH
ꢀ, meta
ꢀ, para
ortho
ortho
meta
para
NH
CO
R
7.24, d (7.5)
7.31, m
7.28, m
R, ꢀ
ortho
4
4
7.55, d (3.5)
R
R, ꢀ, RLeu , NHLeu
L-proline6
cis
7
171.0, qC
61.1, CH
R, ΝΗIle
ꢀ, γ
R, δ
5
5
3.89, d (7.0)
2.18, dd (7.0, 12.0)
ꢀ, γ
γ, δ
ꢀ, RPhe , ꢀPhe
ꢀ
30.9, CH
21.7, CH
46.5, CH
2
2
2
1
.16, m
1.77, m
.57, m
3.59, m
.45, m
γ
δ
ꢀ
δ
ꢀ, γ
R, ꢀ, δ
ꢀ
R, ꢀ
1
γ
γ
3
L-isoleucine7
1
CO
R
ꢀ
γ
170.8, qC
59.6, CH
36.1, CH
R, ΝΗSer
NHIle , ꢀ, γ
R
R
R, γ
7
4.28, dd (5.5, 9.0)
2.09, m
1.42, m
ꢀ, δ, ꢀ
ꢀ
δ
δ
ꢀ, ꢀ
24.8, CH
2
1
.21, m
δ
ꢀ
NH
11.4, CH
22.9, CH
3
0.86, t (7.5)
0.90, d (4.0)
7.50, d (9.0)
3
δ
R, ꢀ
a
Spectra were recorded in CDCl
3
at 25 °C. Chemical shift values are in ppm relative to the residual CHCl
3
(7.25 ppm) or CDCl
3
(77.0 ppm) signals.
b
13
c
d
C NMR multiplicities were obtained from APT experiments. HMBC mixing time ) 50 ms. NOESY mixing time ) 200 ms.

1
558 Journal of Natural Products, 2009, Vol. 72, No. 9
Vera et al.
1
1
identified from H- H COSY correlation to its adjacent R-proton
and by reference to known values for these residues. The two
7
1
2
3
4
5
fragments Pro -Ala -Thr and Pro -Phe -Val were assigned by two-
1
13
1
7
bond H- C correlation as follows: NH (Ala )/CO (Pro ), NH
Thr )/CO (Ala ) and NH(Phe )/CO (Pro ), NH(Val )/CO (Phe ).
2
1
4
3
5
4
(
The complete amino acid sequence of euryjanicin B (2) was
established as cyclo-(-Ala-Thr-Pro-Phe-Val-Pro-Pro-) by the
6
7
5
following pivotal NOE correlations: RH(Pro )/RH(Pro ); RH(Val )/
6
2
3
δH
2
2
(Pro ); ꢀH(Thr )/δH (Pro ) (Figure 1). Although the complete
sequence was not unequivocally solved using the available NMR
data, it was confirmed on the basis of the results of ESIMS/MS
experiments. The mass spectrum of euryjanicin B displayed [M +
+
n
H] at m/z 710, the fragmentation of which was followed by MS .
The ESIMS/MS spectrum of euryjanicin B showed preferential
5
6
opening of the macrocycle at the Val -Pro amide bond, followed
by a series of major fragmentation pathways. One ion series started
5
6
7
1
with loss of 99 amu due to Val , leaving m/z 611 (Pro -Pro -Ala -
2
3
4
3
4
Thr -Pro -Phe plus H), which then lost 244 amu (Pro -Phe ),
affording m/z 367 (Pro -Pro -Ala -Thr plus H). The latter fragment
lost 101 amu (Thr ), yielding m/z 266 (Pro -Pro -Ala plus H)
6
7
1
2
2
6
7
1
(
[
[
Figure 2). Another pathway left the major fragment m/z 593
5
+
5
M+H-H
M+H-H
2
O-Val ] after loss of 99 amu (Val ) from m/z 664
4
+
2
O] , which then lost sequentially 147 amu (Phe ) and
3
5
4 +
9
7 amu (Pro ), leaving m/z 446 [M+H-H
2
O-Val -Phe ] and 349
Figure 3. HMBC (HfC) and NOE correlations (dashed arrows)
for euryjanicin C (3) in CDCl .
3
5
4
3 +
[
M+H-H
2
O-Val -Phe -Pro ] , respectively. The daughter ion
spectrum also contained additional fragment ions of lower abun-
molecular ion of euryjanicin C at m/z 768 was fragmented by
ESIMS/MS. A complex spectrum was recorded due to simultaneous
fragmentation of two isomeric linear peptides, following the opening
6
+
dance ascribable to the y ion series at m/z 613 [M+H-Pro ] , 516
6
7 +
6
7
1
+
[
M+H-Pro -Pro ] , and 445 [M+H-Pro -Pro -Ala )] .
The structure of euryjanicin B (2) was thus confirmed as cyclo-
-Ala-Thr-Pro-Phe-Val-Pro-Pro-). The geometry of the peptidic
1
of the cyclic structure through preferential fragmentation at the Ser -
(
7
6
7
Ile and Pro -Ile amide bonds. After loss of H
2
O the major linear
13
linkages was assigned on the basis of the differences in C chemical
1
7
peptide arising upon fragmentation at Ser -Ile left a major fragment
shifts of the Cꢀ and Cγ of the proline residues. The 13C NMR
20
+
at m/z 750 [M+H-H
2
O] , which in turn underwent N-terminal
data of euryjanicin B indicated that two proline peptide bonds were
7 +
fragmentation, leaving m/z 637 [M+H-H
2
7
O-Ile ] , 540 [M+H-
13
trans, as shown by the small C NMR chemical shift difference
7
6 +
6
5 +
H
[
2
O-Ile -Pro ] , 393 [M+H-H
2
O-Ile -Pro -Phe ] , and 280
3
6
of Pro ∆δCꢀ-Cγ ) 3.8 and Pro ∆δCꢀ-Cγ ) 3.8, and one cis,
7
6
5
4 +
M+H-H
2
O-Ile -Pro -Phe -Leu ] . On the other hand, fragmenta-
1
3
as indicated by the large C chemical shift difference of
Pro ∆δCꢀ-Cγ ) 9.8 (Table 1).
residues, which have been previously found in cyclic peptides such
as wainunuamide and phakellistatin 8, are known to be powerful
-turn inducers.
tion of the lesser abundant linear peptide generated after cleavage
7
21,22
Adjacent cis and trans proline
6
7
of the Pro -Ile peptide bond left a major fragment at m/z 655,
corresponding to [M+H-Ile ] , which underwent sequential losses
of 87 amu (Ser ), 113 amu (Ile ), and 97 amu (Pro ), leaving m/z
7 +
11
23
1
2
3
4
a
ꢀ
7
1 +
7
1
2 +
5
[
68 [M+H-Ile -Ser ] , 455 [M+H-Ile -Ser -Ile ] , and 358
7 1 2
Structure Elucidation of Euryjanicin C. Euryjanicin C (3),
3 +
M+H-Ile -Ser -Ile -Pro ] , respectively. These related patterns
a white semisolid, showed a high-resolution ESIMS spectral
of b fragments could be clearly distinguished in the daughter ion
mass spectrum, leading to the reconstruction of the sequences
inferred previously from 2D-NMR experiments.
+
quasimolecular ion peak at m/z 768.4662 ([M+H] ) consistent with
62 7 8
the molecular formula C40H N O , requiring 14 degrees of unsat-
-1
uration. The IR absorption bands at 3324 and 1651 cm were
attributed to amino and amide carbonyl groups, respectively. The
heptapeptide nature of 3 was evident from the molecular formula
The solution (CDCl
3
) conformation about the proline peptide
linkages of 3 could be assigned from the chemical shift differences
3
6
between the proline ꢀ- and γ-carbons. Thus, Pro and Pro showed
δCꢀ-Cγ ) 9.6 and 9.2, respectively, indicating that these proline
peptide bonds are both cis. Confirmation for these assignments came
1
3
and C NMR spectra, which showed seven carbonyl signals (δ
∆
1
72.9, 171.8, 171.5, 171.0, 171.0, 170.8, 169.5) and seven R-me-
thine carbons (δ 61.4, 61.1, 59.6, 55.3, 54.4, 54.1, 50.5) (Table 2).
Only five amide NH signals were detected in the H NMR spectrum,
3
2
from the NOESY spectrum. Cross-peaks between RH(Pro )/RH(Ile )
1
3
established the cis geometry for the Pro peptide bond, while cross-
6
5
suggesting a heptapeptide with two proline units. As in 2, the amino
acid residues were identified by extensive NMR analysessCOSY-
GPQF, TOCSY, HSQC, HMBC, and NOESYswhich allowed us
to establish the identity of the seven residues: 2 × Pro, 2 × Ile,
Phe, Ser, and Leu. These residues accounted for 13 degrees of
unsaturation out of the 14, requiring that 3 is a cyclopeptide, too.
The absolute configurations at the R-carbon for these amino acids
were based on Marfey’s method, and we also found in this case
that all residues belong to the L series. The amino acid sequence
analysis was conducted in a similar manner to that for 2, by 2D
NMR analysis, e.g., HMBC and NOESY spectra (Table 2). From
the results of the useful HMBC correlations, as shown in Figure 3,
the structure was established to be cyclo-(-Ser-Ile-Pro-Leu-Phe-
Pro-Ile-), which was also confirmed by the correlations observed
during NOESY NMR experiments (Figure 3).
peaks between RH(Pro )/RH(Phe ) confirmed the cis-peptide link
6
for the Pro peptide bond. In point of fact, stylopeptide 1, isolated
by the Pettit group from the South and Western Pacific Ocean
sponges Stylotella sp. and Phakellia costata and whose structure
was established as cyclo-(-Pro-Leu-Ile-Phe-Ser-Pro-Ile-), is a
9
a
structural isomer of euryjanicin C (3). However, while both
cyclopeptides are comprised of the same amino acid residues, there
is no sequential homology among them.
Structure Elucidation of Euryjanicin D. Euryjanicin D (4)
was obtained as an optically white semisolid that gave a [M +
+
H] ion in the HRESIMS at m/z 802.4514, appropriate for a
molecular formula of C H N O , requiring 18 sites of unsaturation.
4
3
59
7
8
1
13
General features of both the H and C NMR spectra (Table 3)
1
3
suggested 4 was a peptide. Seven C NMR resonances had
chemical shifts appropriate for amide carbonyls, and seven reso-
nances had chemical shifts appropriate for amino acid R-carbons,
Analogously to the preceding cyclopeptide, the protonated

Cycloheptapeptides from the Sponge Prosuberites laughlini
Journal of Natural Products, 2009, Vol. 72, No. 9 1559
Table 3. H NMR (500 MHz), 13C NMR (125 MHz), TOCSY, HMBC, and NOESY Correlations for Euryjanicin D (4)a
1
b
c
d
amino acid
L-phenylalanine1
position
δ
C
(mult)
δ
H
, mult (J in Hz)
TOCSY
HMBC
NOESY
2
CO
R
ꢀ
γ
170.8, qC
53.8, CH
38.6, CH
135.0, qC
R, ꢀ, NHSer
ꢀ
4.51, m
3.05, m
ꢀ
ꢀ, ortho
ortho
2
ortho
ꢀ, meta
ꢀ, para
ortho
meta
para
NH
CO
R
129.2, CH
128.9, CH
127.5, CH
7.22, d (7.0)
7.33, m
7.27, m
ortho
7
7.26, d (5.0)
R, ꢀ
ꢀ
ꢀ
ꢀ
R
R, ꢀIle
L-serine2
170.0, qC
54.9, CH
63.2, CH
7
4.51, m
3.89, br d (14.5)
ꢀ
ꢀ, RPro , ΝΗ
ꢀ
2
1
3
.71, dd (5.0, 11.0)
ꢀPhe
1
ΝΗ
CO
R
7.47, d (6.0)
R, ꢀ
R, COPhe
R
L-proline3
cis
171.7, qC
61.4, CH
30.9, CH
21.6, CH
46.1, CH
171.4, qC
60.4, CH
32.3, CH
R, NHIle
4
3.59, d (7.5)
1.96, m
ꢀ, γ, δ
ꢀ, γ, δ
γ, δ
ꢀ
ꢀ
2
2
2
γ
0
.95, m
1.68, m
.44, m
γ
γ
ꢀ
R, ꢀ, δ
R, ꢀ
1
ꢀ
δ
3.38, d (9.5)
ꢀ, γ
L-isoleucine4
CO
R
R, NHPhe
5
4.15, d (8.8)
1.91, m
1.40, m
ꢀ, ꢀ
R, δ, ꢀ
δ
ꢀ, NH
R, δ, ꢀ
R, δ, ꢀ
δ, ꢀ
γ, δ, ꢀ
δ
ꢀ
γ
24.4, CH
2
0
.95, m
δ
9.89, CH
15.8, CH
3
3
0.77, d (7.0)
0.84, d (6.5)
7.11, d (8.8)
ꢀ
R, ꢀ, γ
ΝΗ
CO
R
ꢀ
γ
R, ꢀ
ꢀ, γ, δ, ꢀ
ꢀ
L-phenylalanine5
171.6, qC
53.2, CH
37.8, CH
135.4, qC
129.6, CH
128.9, CH
127.5, CH
6
4.51, m
3.08, m
ꢀ
ꢀ, ortho, NH
ortho, meta, NH
R, meta
para
ꢀ, RPro , NH
2
ortho, NH
ortho
meta
para
NH
CO
R
7.26, d (5.0)
7.33, m
7.27, m
ortho
7.71, d (7.0)
R, ꢀ
ꢀ, γ, ꢀ
L-proline6
cis
170.6, qC
61.1, CH
30.7, CH
21.7, CH
46.4, CH
R, NHIle
ꢀ, δ
R, γ
7
3.92, d (7.5)
2.18, dd (6.0, 12.0)
ꢀ, γ, δ
ꢀ, orthoPhe5
ꢀ
2
2
2
γ
1
.04, m
1.45, m
.74, m
3.54, dt (8.0, 11.8)
.41, m
γ
γ
ꢀ
γ
δ
ꢀ
R, ꢀ
R, ꢀ
1
ꢀ
ꢀ, γ
3
γ
L-isoleucine7
CO
R
ꢀ
γ
171.6, qC
56.4, CH
36.2, CH
R, ΝΗPhe
1
1
4.12, d (9.3)
2.09, m
1.44, m
ꢀ
ꢀ
δ
γ, δ
ΝΗPhe
γ, δ, ꢀ
R, δ, ꢀ
ꢀ, R, δ
25.0, CH
2
1
.12, m
δ
δ
11.1, CH
15.8, CH
3
3
0.85, d (7.0)
0.90, d (7.0)
7.67, d (9.3)
R, γ
γ
R, γ
ꢀ
NH
R, ꢀ, δ
ꢀ, γ, ꢀ
a
Spectra were recorded in CDCl
3
at 25 °C. Chemical shift values are in ppm relative to the residual CHCl
3
(7.25 ppm) or CDCl
3
(77.0 ppm) signals.
b
13
c
d
C NMR multiplicities were obtained from APT experiments. HMBC mixing time ) 50 ms. NOESY mixing time ) 200 ms.
consistent with a heptapeptide. Detailed analyses of the COSY-
GPQF, TOCSY, HSQC, and HMBC data (Table 3) revealed that
euryjanicin D (4) contained one serine and two each of proline,
isoleucine, and phenylalanine residues. Hydrolysis of peptide 4 with
feature to be elucidated was the geometry of the peptidic linkages
at the Pro residues. In this instance, the NMR data of 4 pointed to
6
N HCl followed by Marfey’s derivatization and HPLC analyses
of the amino acids in the hydrolysate confirmed the presence of
these four amino acids and established that all seven amino acid
residues had the L configuration. The seven amino acid residues
accounted for all of the atoms in the molecular formula of
euryjanicin D (4) and 17 of the sites of unsaturation. Furthermore,
there was no evidence for terminal amino or carboxylic acid
functionalities, and therefore, euryjanicin D (4) was presumed to
be a cyclic peptide.
The sequence analysis was conducted in a similar fashion to those
in 2 and 3 by 2D-NMR analysis, e.g., HMBC and NOESY spectra.
From the HMBC correlations as shown in Figure 4, the structure
was established to be cyclo-(-Phe-Ser-Pro-Ile-Phe-Pro-Ile-), which
was also confirmed by NOE correlations observed in the NOESY
spectrum. Finally, this sequence was confirmed by tandem MS
fragmentation analysis of a linear acylium ion at m/z 784 generated
2
after loss of H O following preferential opening of the macrocycle
1
7
at the Phe -Ile amide bond (Figure 5). Once the amino acid
Figure 4. HMBC (HfC) and NOE correlations (dashed arrows)
sequence of euryjanicin D (4) was determined, the final structural
3
for euryjanicin D (4) in CDCl .

1
560 Journal of Natural Products, 2009, Vol. 72, No. 9
Vera et al.
Table 4. Percent Growth of Selected Human Cancer Cell Lines
a
Treated with Euryjanicins A-D (1-4) and Dominicin (5)
cell line
1
2
3
4
5
b
NCI-H522
72.2
89.0
82.6
73.6
83.9
88.6
91.3
76.3
95.1
88.0
100
83.4
94.6
87.6
100
100
100
100
c
LOX IMVI
d
IGROV1
e
UO-31
82.7
32.8
a
-5
Inhibitory activity tested at a single high dose (10 M) against the
b
c
full NCI 60 cell line panel. Non-small cell lung cancer. Melanoma.
Ovarian cancer. Renal cancer.
d
e
n
Figure 5. N-Terminal and C-terminal MS fragmentations of the
acyclic acylium ion at m/z 784 generated from cyclopeptide 4 after
in-source protonation, ring-opening, and loss of H
2
O.
(1), C (3), and D (4) each contains a serine residue, whereas
euryjanicin B (2) and dominicin (5) possess one threonine unit.
Except for metabolites 1 and 5, one set of resonances was
observed for each of the amino acid residues in compounds 2-4,
indicating that in each instance one rigid solution conformation
2
3
5
6
a cis geometry for both the Ser -Pro and Phe -Pro peptide bonds
∆δCꢀ-Cγ ) 9.3 and 9.0, respectively). Subsequent confirmation
for these assignments followed from the NOESY spectrum. Strong
(
2
3
5
6
cross-peaks between RH(Ser )/RH(Pro ) and RH(Phe )/RH(Pro )
confirmed that indeed the proline peptide bonds were cis.
Our overall chemical investigation of the cell growth inhibitory
sponge extract yielded four new cyclic heptapeptides, euryjanicin
A-D (1-4), and the previously known cyclic octapeptide dominicin
1
13
dominates in CDCl
spectra of 1 and 5 in CDCl
the equilibria of slow exchanging conformers about the peptidic
3
. On the other hand, the H and C NMR
3
gave broad signals resulting from
12,15
ring.
The remarkable analogy in structure and in amino acid
content between cyclic peptides isolated from marine sponges
and those stemming from marine-derived cultured bacteria or
fungi suggests a possible microbial or symbiotic origin for
(
5). These molecules are structurally related to several other bio-
5-13
active proline-rich cyclopeptides reported from marine sponges.
Yet, the isolated peptides 1-5 were found to be only marginally
active to inactive against the National Cancer Institute (NCI) 60
tumor cell line panel (Table 4). The problem of lost activity
associated with proline-rich cyclic peptides has been observed by
25
sponge-derived cyclic peptides such as 1-5.
Experimental Section
General Experimental Procedures. Optical rotations were re-
corded with a Rudolph Autopol IV polarimeter. The UV data were
recorded with a Shimadzu UV-2401PC spectrometer, and the IR
analyses were performed with a Nicolet Magna IR 750 spectrometer.
1D and 2D NMR data were recorded on a Bruker DRX-500 or
9
a,13b
others.
This phenomenon may be due to changes in the
conformation of the cyclic peptide during isolation or to its ability
to bind to a potent antineoplastic substance present in very low
concentration and therefore detectable only during biological
7
,24
1
Bruker AV-500 FT-NMR spectrometer. H NMR chemical shifts
screenings.
(
Peptides 1-5 have been tested for their antimalarial
were recorded with respect to the residual CHCl
3
signal (7.26 ppm),
and C NMR chemical shifts were reported in ppm relative to the
CDCl signal (77.0 ppm). Mass spectrometric measurements were
Plasmodium falciparum W2 clone), anti-Mtb, antiviral [HBV,
1
3
HSV-1, HSV-2, FluA (H5N1), FluB, RSV A, WNV, Rift Valley
fever], and anti-inflammatory activity in rat neonatal microglia and
were inactive in all of the assays.
Cycloheptapeptides 1-4 and cyclooctapeptide 5 are character-
ized by the presence of two or three prolines, an array of apolar
residues such as Leu, Ile, and Val, and (in the case of 1-4) one
or two aromatic residues such as Phe or Trp. Euryjanicins A
3
generated at the Mass Spectrometry Laboratory of the University
of Illinois at Urbana-Champaign. Column chromatography was
performed on Analtech Si gel (35-75 mesh) and monitored with
TLC analyses carried out on Analtech glass precoated Si gel plates
and visualized using UV light and I2 vapors. All solvents were
distilled from glass prior to use.
Chart 1

Cycloheptapeptides from the Sponge Prosuberites laughlini
Journal of Natural Products, 2009, Vol. 72, No. 9 1561
Animal Material. The Caribbean sponge Prosuberites laughlini
Diaz, Alvarez & van Soest, 1987) (phylum: Porifera; class:
(46.85), L-Leu (47.40), L-Thr (26.15), L-Ala (31.74), L-Val (40.70),
L-Phe (39.23), L-Ser (23.2), L-Trp (46.0).
2
6
(
Demospongiae; order: Hadromerida; family: Suberitidae) was col-
lected at a depth of 50 feet by scuba off Aguadilla, Puerto Rico, in
April 2006. A voucher specimen (No. PLAG-01) is stored at the
Chemistry Department of the University of Puerto Rico. The
encrusting (0.3-4.0 cm thick) sponge was dull orange to yellow
externally, lighter internally with surface visually smooth in thin
specimens, rugose on thicker ones. The specimen collected possessed
oscules with transparent membranes (1-6 mm in diameter) with
thin (1-2 mm) canals departing radially and was soft and compress-
ible, easy to tear.
Electrospray Ionization Mass Spectroscopic (ESIMS/MS) Analyses.
ESIMS/MS analyses were performed with a LTQ ion trap mass
spectrometer (Thermo Fisher). Aliquots of the peptide (100 µL) in
3
solution (50% CH CN(aq)/0.1% formic acid) were injected in the
ion source at a flow rate of 1 µL/min, the capillary temperature was
200 °C, and the applied voltage was 1 kV. Fragmentation experi-
ments were carried out using a collision energy of dissociation of
20%. All ESIMS are reported as averaged mass.
Biological Assays. Additional experimental details for our primary
in Vitro antimicrobial assays against Mycobacterium tuberculosis and
27,28
Plasmodium falciparum have been previously described.
All of the
Extraction and Isolation. The freshly collected sponge was freeze-
dried for 5 days, and the dried organism (622.7 g) was repeatedly
extracted with MeOH (16 L). The combined MeOH extracts were
evaporated to dryness, and the resulting brown oil (83.1 g) was
in Vitro antiviral, anti-inflammatory, and cancer cell cytotoxicity assays
2
9-31
for cyclopeptides 1-5 were used as indicated.
Acknowledgment. We thank Dr. I. I. Rodr ´ı guez for helping us
during the collection of P. laughlini and the UPR-RISE and UPR-
MARC Fellowship Programs for financial support to B.V. and J.V.,
respectively. Sponge extracts were screened for antitumor activity
by Dr. S. Nam at the City of Hope Beckman Research Institute and
for anti-TB activity by Dr. Y. Wang at the Institute for Tuberculosis
Research of the University of Illinois. Antitumor, antimycobacterial,
antiviral, antiplasmodial, and anti-inflammatory bioassays of the pure
cyclopeptides were conducted at the National Cancer Institute (NCI),
the Institute for Tuberculosis Research of the UIC, the NIAID’s
Antimicrobial Acquisition and Coordinating Facility (AACF), the
Instituto de Investigaciones Avanzadas y Servicios de Alta Tecno-
log ´ı a (Panama), and the Midwestern University (by Dr. A. M. S.
Mayer), respectively. ESIMS/MS data were kindly provided by Dr.
I. E. Vega (Department of Biology, University of Puerto Rico).
Major financial support was provided by the NIH-MBRS SCORE
program (Grant S06GM08102) of the University of Puerto Rico.
partitioned between EtOAc (3 × 1 L), nBuOH (3 × 1 L), and H
1 × 1.5 L). The combined EtOAc extracts were concentrated in
Vacuo to give 6.1 g of a dark brown oil that was chromatographed
in CHCl as eluent. The
first fraction (2.79 g) was subsequently chromatographed over Si
gel (100 g) with 5% EtOAc in hexane as eluent. Subfractions 10
2
O
(
over Si gel (201 g) with 20% MeOH · NH
3
3
(
182.4 mg) and 11 (155.3 mg) contained all of the peptides. Fraction
0 was purified by reversed-phase C18 column chromatography (5
g) using 15% H O in MeOH, yielding pure euryjanicin A (1) (43.6
mg; 0.007%), euryjanicin B (2) (21 mg; 0.003%), and dominicin
5) (32.3 mg; 0.005%). Purification of fraction 11 via C18 reversed-
phase HPLC using a 10 mm × 25 cm Ultrasphere ODS column, 5
µm, with 30% H O in MeOH yielded pure euryjanicin C (3) (15.3
mg; 0.002%) and euryjanicin D (4) (39.9 mg; 0.006%).
1
2
(
2
2
0
Euryjanicin B (2): colorless oil; [R]
D
-55 (c 0.8, CHCl
3
); IR
-
1
(
film) νmax 3345, 3087, 3064, 2973, 1667, 1518, 1454, 753 cm
;
1
UV (MeOH) λmax (log ꢀ) 204 (4.2) nm; H NMR (500 MHz, CDCl
3
)
1
3
and C NMR (125 MHz, CDCl
H]+ 710.3871 (calcd for C36
Euryjanicin C (3): white semisolid; [R]
IR (film) νmax 3324, 3063, 3028, 2961, 1651, 1530, 1449, 754 cm
3
) (see Table 1); HRESIMS m/z [M
+
H
52
N
7
O
8
, 710.3877).
Supporting Information Available: Representative copies of the
2
0
1
13
D
-60 (c 1.1, CHCl
3
-
);
;
NMR ( H and C), 2D NMR (TOCSY, HSQC, HMBC, and NOESY),
and ESIMS/MS spectra for euryjanicins B-D (2-4). This material is
available free of charge via the Internet at http://pubs.acs.org.
1
1
3
UV (MeOH) λmax (log ꢀ) 204 (3.9) nm; H NMR (500 MHz, CDCl )
and 13C NMR (125 MHz, CDCl
) (see Table 2); HRESIMS m/z [M
, 768.4660).
3
+
+
H] 768.4662 (calcd for C40
H
62
N
7
O
8
2
0
References and Notes
Euryjanicin D (4): white semisolid; [R]
D
3
-123 (c 0.85, CHCl );
IR (film) νmax 3307, 3063, 3029, 2963, 1667, 1516, 1454, 1347,
(1) (a) Piel, J. Curr. Med. Chem. 2006, 13, 39–50. (b) Aneiros, A.;
Garateix, A. J. Chromatogr. B 2004, 803, 41–53. (c) Tincu, J. A.;
Taylor, S. W. Antimicrob. Agents Chemother. 2004, 48, 3645–3654.
(d) Matsunaga, S.; Fusetani, N. Curr. Org. Chem. 2003, 7, 945–
-
1
1
7
52, 702 cm ; UV (MeOH) λmax (log ꢀ) 205 (4.0); H NMR (500
13
3 3
MHz, CDCl ) and C NMR (125 MHz, CDCl ) (see Table 3);
+
HRESIMS m/z [M + H] 802.4514 (calcd for C43
02.4503).
Acid Hydrolysis of Euryjanicins B-D (2-4). Pure euryjanicins
60 7 8
H N O ,
9
1
1
66. (e) Moore, R. E. J. Ind. Microbiol. Biotechnol. 1996, 16, 134–
43. (f) Fusetani, N.; Matsunaga, S. Chem. ReV. 1993, 93, 1793–
806.
8
B-D (0.5 mg) were hydrolyzed in 0.5 mL of 6 N HCl at 110 °C
for 12 h in a 1.0 mL reaction vial. The cooled reaction mixture was
evaporated to dryness, and traces of HCl were removed from the
(
2) (a) Ma, X.; Wu, C.; Wang, W.; Li, X. Asian J. Tradit. Med. 2006,
, 85–90. (b) Tan, N. H.; Zhou, J. Chem. ReV. 2006, 106, 840–
895.
1
residual hydrolysate by repeated evaporation from H
mL) using N gas.
Absolute Configuration of Amino Acids. To a 4 mL vial
containing 1 µmol of pure amino acid standards in 200 µL of H
2
O (3 × 0.5
(3) Wipf, P. Chem. ReV. 1995, 95, 2115–2134.
(4) (a) Herald, D. L.; Cascarano, G. L.; Pettit, G. R.; Srirangam, J. K.
J. Am. Chem. Soc. 1997, 119, 6962–6973. (b) Mechnich, O.; Hessler,
G.; Kessler, H. HelV. Chim. Acta 1997, 80, 1338–1353.
2
2
O
(
5) (a) Randazzo, A.; Piaz, F. D.; Orru, S.; Debitus, C.; Roussakis, C.;
Pucci, P.; Paloma, L. G. Eur. J. Org. Chem. 1998, 2659–2665. (b)
Kong, F.; Burgoyne, D. L.; Andersen, R. J. Tetrahedron Lett. 1992,
was added 1 µmol of N-R-(2,4-dinitro-5-fluorophenyl)-L-alanine
amide (L-FDAA) in 400 µL of acetone followed by 100 µL of 1 N
NaHCO . The mixture was heated for 1 h at 40 °C. After cooling to
3
33, 3269–3272. (c) Tabudravu, J. N.; Morris, L. A.; Van den Bosch,
rt, 100 µL of 2 N HCl was added and the resulting solution was
filtered through a small 4.5 mm filter and stored in the freezer until
HPLC analysis. Half of each peptide hydrolysate mixture was
J. J. K.; Jaspars, M. Tetrahedron 2002, 58, 7863–7868.
(6) (a) Pettit, G. R.; Herald, C. L.; Boyd, M. R.; Leet, J. E.; Dufresne,
C.; Doubek, D. L.; Schmidt, J. M.; Cerny, R. L.; Hooper, J. N.;
Rutzler, K. C. J. Med. Chem. 1991, 34, 3339–3340. (b) Pettit, G. R.;
Gao, F.; Cerny, R. Heterocycles 1993, 35, 711–717. (c) Pettit, G. R.;
Gao, F.; Schmidt, J. M.; Chapuis, J.-C.; Cerny, R. L. Biorg. Med.
Chem. Lett. 1994, 4, 2935–2940. (d) Pettit, G. R.; Gao, F.; Cerny,
R. L.; Doubek, D. L.; Tackett, L. P.; Schmidt, J. M.; Chapuis, J.-
C. J. Med. Chem. 1994, 37, 1165–1168.
dissolved in 200 µL of H
2
O, and to this was added 1.5 µmol of
L-FDAA in 400 µL of acetone followed by 100 µL of 1 N NaHCO
3
.
The derivatization reaction was carried out and worked up as
described above. An 8 µL aliquot of the resulting mixture of L-FDAA
derivatives was analyzed by reversed-phase HPLC. A 5 mm × 250
mm Spheri-5 C18 column, 5 µm, with a linear gradient of (A) 9:1
(7) Pettit, G. R.; Tan, R. J. Nat. Prod 2005, 68, 60–63, and references
therein.
triethylammonium phosphate (50 mM, pH 3.0)/CH
3
CN and (B)
(
8) (a) Kobayashi, J.; Tsuda, M.; Nakamura, T.; Mikami, Y.; Shigemori,
H. Tetrahedron 1993, 49, 2391–2402. (b) Tsuda, M.; Shigemori,
H.; Mikami, Y.; Kobayashi, J. Tetrahedron 1993, 49, 6785–6796.
CH CN with 0% B at start to 40% B over 55 min (flow rate ) 1
3
mL/min) was used to separate the L-FDAA derivatives with UV
detection at 340 nm. Each chromatographic peak was identified by
comparing its retention time with the L-FDAA derivative of the pure
L-amino acid standard and by co-injection. In all cases a peak at
(
c) Tsuda, M.; Sasaki, T.; Kobayashi, J. Tetrahedron 1994, 50, 4667–
4
680. (d) Kobayashi, J.; Nakamura, T.; Tsuda, M. Tetrahedron 1996,
5
2, 6355–6360.
3
9.2 min was observed, which was attributed to excess L-FDAA.
(9) (a) Pettit, G. R.; Srirangam, J. K.; Herald, D. L.; Xu, J.-P.; Boyd,
Retention times (min) are given in parentheses: L-Pro (32.11), L-Ile
M. R.; Cichacz, Z.; Kamano, Y.; Schmidt, J. M.; Erickson, K. L.

1
562 Journal of Natural Products, 2009, Vol. 72, No. 9
Vera et al.
J. Org. Chem. 1995, 60, 8257–8261. (b) Brennan, M. R.; Costello,
C. E.; Maleknia, S. D.; Pettit, G. R.; Erickson, K. L. J. Nat. Prod.
(22) Dorman, D. E.; Borvey, F. A. J. Org. Chem. 1973, 38, 2379–
2383.
(23) Pettit, G. R.; Xu, J.-P.; Dorsaz, A.-C.; Williams, M. D.; Boyd, M. R.;
Cerny, R. L. Biorg. Med. Chem. Lett. 1995, 5, 1339–1344.
(24) Tabudravu, J. N.; Jaspars, M.; Morris, L. A.; Van den Bosch, J. J. K.;
Smith, N. J. Org. Chem. 2002, 67, 8593–8601.
(25) (a) Tan, L. T.; Cheng, X.-C.; Jensen, P. R.; Fenical, W. J. Org. Chem.
2003, 68, 8767–8773. (b) Renner, M. K.; Shen, Y.-C.; Cheng, X.-C.;
Jensen, P. R.; Frankmoelle, W.; Kauffman, C. A.; Fenical, W.;
Labbovsky, E.; Clardy, J. J. Am. Chem. Soc. 1999, 121, 11273–11276.
(26) D ´ı az, M. C. Caribb. J. Sci 2005, 41, 465–475.
(27) Collins, L. A.; Franzblau, S. G. Antimicrob. Agents Chemother. 1997,
41, 1004–1009.
2
008, 71, 453–456.
(
10) (a) Pettit, G. R.; Srirangam, J. K.; Herald, D. L.; Erickson, K. L.;
Doubek, D. L.; Schmidt, J. M.; Tackett, L. P.; Bakus, G. J. J. Org.
Chem. 1992, 57, 7217–7220. (b) Forns, P.; Pir o´ , J.; Cuevas, C.;
Garc ´ı a, M.; Rubiralta, M.; Giralt, E.; Diez, A. J. Med. Chem. 2003,
4
6, 5825–5833.
(
(
(
11) Tabudravu, J.; Morris, L. A.; Van den Bosch, J. J. K.; Jaspars, M.
Tetrahedron Lett. 2001, 42, 9273–9276.
12) Williams, D. E.; Patrick, B. O.; Behrisch, H. W.; Soest, R. V.; Roberge,
M.; Andersen, R. J. J. Nat. Prod. 2005, 68, 327–330.
13) (a) Shin, J.; Seo, Y.; Lee, H.-S.; Rho, J.-R.; Mo, S. J. J. Nat. Prod.
2
003, 66, 883–884. (b) Mohammed, R.; Peng, J.; Kelly, M.; Hamann,
(28) Corbett, Y.; Herrera, L.; Gonz a´ lez, J.; Cubilla, L.; Capson, T.; Colley,
P. D.; Kursar, T. A.; Romero, L. I.; Ortega-Barria, E. J. Trop. Med.
Hyg. 2004, 70, 119–124.
(29) Mayer, A. M. S.; Oh, S.; Ramsey, K. H.; Jacobson, P. B.; Glaser,
K. B.; Romanic, A. M. Shock 1999, 11, 180–186.
(30) Rodr ´ı guez, I. I.; Shi, Y.-P.; Garc ´ı a, O. J.; Rodr ´ı guez, A. D.; Mayer,
A. M. S.; S a´ nchez, J. A.; Ortega-Barria, E.; Gonz a´ lez, J. J. Nat. Prod.
2004, 67, 1672–1680.
(31) Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paul, K.; Vistica,
D.; Hose, C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A. J. Natl. Cancer
Inst. 1991, 83, 757–766.
M. T. J. Nat. Prod. 2006, 69, 1739–1744.
(
(
14) This sponge was formerly described as Eurypon laughlini.
15) Vicente, J.; Vera, B.; Rodr ´ı guez, A. D.; Rodr ´ı guez-Escudero, I.; Raptis,
R. G. Tetrahedron Lett. 2009, 50, 4571–4574.
(
16) Kupchan, S. M.; Britton, R. W.; Ziegler, M. F.; Siegel, C. W. J. Org.
Chem. 1994, 38, 178–179.
17) Assmann, M.; K o¨ ck, M. Z. Naturforsch. 2002, 57, 153–156.
18) Kobayashi, J.; Ohizumi, Y.; Nakamura, H.; Hirata, Y. Experientia
(
(
1
986, 42, 1176–1177.
(
19) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591–596.
(20) Siemion, I. Z.; Wieland, T.; Pook, K.-H. Angew. Chem., Int. Ed. Engl.
1
975, 14, 702–703; Angew. Chem. 1975, 88, 712-714.
21) Dorman, D. E.; Borvey, F. A. J. Org. Chem. 1973, 38, 1719–
722.
(
1
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