Psammaplysin derivatives from the balinese marine sponge Aplysinella strongylata

Article abstract of DOI:10.1021/np300560b

Twenty-one new psammaplysin derivatives (4-24) exhibiting a variety of side chains, as well as six previously known psammaplysins, were identified from the Indonesian marine sponge Aplysinella strongylata. The double bond on the side chain of the fatty acid-containing psammaplysins was located by GC-MS analysis of the fatty acid methyl esters and their pyrrolidide derivatives. HPLC and Mosher ester studies confirmed that the isolated metabolites possessing a 19-OH substituent were mixtures of diastereomers. Selected compounds (4, 5, 7, 8, 12, 18, and 22) were screened for in vitro activity against chloroquine-sensitive (3D7) P. falciparum malaria parasites. Of the new psammaplysins, 19-hydroxypsammaplysin E (4) showed the best antimalarial activity, with an IC₅₀ value of 6.4 μM.

Full text of DOI:10.1021/np300560b

Article
pubs.acs.org/jnp
Psammaplysin Derivatives from the Balinese Marine Sponge
Aplysinella strongylata
I Wayan Mudianta,†,‡ Tina Skinner-Adams,§,⊥ Katherine T. Andrews,§,⊥ Rohan A. Davis,§ Tri A. Hadi,∥
Patricia Y. Hayes,† and Mary J. Garson*,†
†School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia
‡Department of Analytical Chemistry, Faculty of Mathematics and Natural Sciences, Ganesha University of Education, Bali, Indonesia
§Eskitis Institute, Griffith University, Brisbane, QLD 4111, Australia
⊥Queensland Institute of Medical Research, Locked Bag 2000, Herston 4029, Australia
∥Research Center for Oceanography, LIPI; Jl. Pasir Putih I, Ancol Timur, Jakarta 11048, Indonesia
S
* Supporting Information
ABSTRACT: Twenty-one new psammaplysin derivatives (4−24)
exhibiting a variety of side chains, as well as six previously known
psammaplysins, were identified from the Indonesian marine sponge
Aplysinella strongylata. The double bond on the side chain of the fatty
acid-containing psammaplysins was located by GC-MS analysis of
the fatty acid methyl esters and their pyrrolidide derivatives. HPLC
and Mosher ester studies confirmed that the isolated metabolites
possessing a 19-OH substituent were mixtures of diastereomers.
Selected compounds (4, 5, 7, 8, 12, 18, and 22) were screened for in
vitro activity against chloroquine-sensitive (3D7) P. falciparum
malaria parasites. Of the new psammaplysins, 19-hydroxypsamma-
plysin E (4) showed the best antimalarial activity, with an IC50 value
of 6.4 μM.
arine sponges of the order Verongida are well known to
contain alkaloids derived from bromotyrosine.1 One of
including hexanes, EtOAc, and BuOH (see Experimental
Section). The EtOAc fraction was resolved by normal-phase
Si gel chromatography and was subsequently purified by C18
HPLC to afford the known psammaplysins A (1), B (2), D, and
E (3), ceratinamides A and B, and 21 new psammaplysin
derivatives (4−24).
Psammaplysin Derivatives with Modified Aromatic
Ring Substituents. 19-Hydroxypsammaplysin E (4) was
obtained as a yellow oil. The presence of four bromine atoms
in the molecule was defined from an ion cluster at m/z 874/
876/878/880/882 [M + Na]+ in the (+)-LRESIMS. This mass
is 16 Da higher than that of psammaplysin E (3) and suggested
an additional hydroxy group. A molecular formula of
C27H25Br4N3O9 was derived from (+)-HRESIMS, consistent
with 15 double-bond equivalents (DBE).
M
the most interesting and bioactive categories of bromotyrosine-
derived metabolites in these sponges is the small group of
alkaloids possessing a spirooxepinisoxazoline moiety. To date,
there have been 12 such bromotyrosine derivatives, named
psammaplysins A−J and ceratinamides A and B, isolated from
eight different sponge species. Some of these derivatives
contain fatty acyl side chains.2−9 Psammaplysin C was found to
have moderate in vitro cytotoxicity against the human colon
tumor cell line HCT116,3 while psammaplysin D showed
activity against a Haitian strain of HIV-1.4 Psammaplysins G
and H have shown promising activity toward Plasmodium
falciparum malaria parasites.6,7 We report here the isolation of
additional members of the psammaplysin family, along with the
known psammaplysins A (1), B (2), D, and E (3) and
ceratinamides A and B, from an extract of the sponge
Aplysinella strongylata (order Verongida, family Aplysinellidae),
collected at Tulamben, Bali, Indonesia. Five of the new
psammaplysins showed variation in the structural motif
attached to C-16 of the aromatic ring, while the remaining
compounds contained fatty acid side chains.
The presence of the psammaplysin carbon framework in 4
1
could be deduced from H and 13C NMR chemical shifts
(MeOH-d4) compared to those of psammaplysin E (3) (Tables
1 and 2). A signal at δH 7.15 (1H, s) assigned to H-1 had
HMBC correlations to signals at δC 103.5 (C-2), 148.9 (C-3),
and 122.1 (C-6). Two distinctive geminal protons at δH 3.06
and 3.38 could be attributed to H-5a and H-5b, respectively.
This AB system correlated to signals for C-4 (δC 105.7) and C-
RESULTS AND DISCUSSION
■
The combined MeOH−CH2Cl2 extract of the frozen sponge
was partitioned between H2O and various organic solvents
Received: August 17, 2012
Published: December 6, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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secured the connection between the propyloxy side chain and
the substituted aromatic ring.
1
The H NMR spectra of 3 and 4 were consistent except for
the signals corresponding to H-19 and H-20. In psammaplysin
E, H-19 appeared at δH 2.83 (2H) and H-20 at δH 3.55 (2H); in
contrast for 4, H-19 was shifted to δH 4.77 (1H, dd, J = 3.7, 7.2
Hz), while H-20 appeared as an AB system [δH 3.42 (1H, dd, J
= 7.2, 13.4 Hz) and δH 3.59 (1H, dd, J = 3.7, 13.4 Hz)]. Since
the H-20 signals were partially obscured by those of H-5b and
H-10, they were further resolved by a 1D TOCSY experiment
involving irradiation of H-19. The chemical shift of C-19 was
also shifted downfield from δC 36.0 in 3 to δC 71.8 in 4,
consistent with a C-19 hydroxy substituent, as found previously
in both psammaplysins B and D.2,4
Psammaplysin K (5) was obtained as a colorless glass. The
(+)-LRESIMS displayed a 1:4:6:4:1 ion cluster at m/z 737/
739/741/743/745 [M + Na]+, which indicated the presence of
four bromine atoms. The HRESIMS analysis of 5 gave a
quasimolecular ion (M + H+) consistent with a molecular
formula of C20H18Br4N2O7 requiring 10 DBEs. The presence of
1
the psammaplysin scaffold in 5 was evident from the H NMR
spectrum (CDCl3), in addition to the associated HSQC and
HMBC data. There were isolated protons at δH 7.02 and 8.03
(2H), two geminal protons at δH 3.12 and 3.37, an isolated
methine at δH 5.14, three mutually coupled methylenes at δH
3.75, 2.22, and 4.23, and a methoxy group at δH 3.65. The most
notable difference between 5 and 3 was the absence of two
methylene signals attributed to H-19 and H-20 as well as the
disappearance of the signals associated with the cyclo-
pentenedione moiety. Instead, a signal at δH 9.86 linked to a
carbon signal at δC 188.3 indicated the presence of a formyl
moiety. HMBC correlations of the formyl proton to both C-15
and C-17, and from H-15/H-17 to C-19, confirmed the
6 as well as to C-7 at δC 79.3, diagnostic for a hydroxymethine
carbon. The H-7 signal appeared at δH 4.99 as an isolated
singlet and correlated to an amide carbon atom at δC 159.1 (C-
9). These assignments secured the presence of the spiroox-
epinisoxazoline system in 4.4 Furthermore, three mutually
coupled methylenes at δH 3.62, 2.13, and 4.07 suggested the
presence of a −CH2CH2CH2O− moiety attached to the amide
bond of the spiro ring system. A singlet at δH 7.60 was
designated to the symmetric aromatic ring protons H-15 and
H-17. HMBC correlation between H-12 and C-13 (δC 153.2)
1
Table 1. H NMR Assignments for 3−8
a
b
a
a
c
c
position
δH (J in Hz) 3
δH (J in Hz) 4
7.15, s
δH (J in Hz) 5
δH (J in Hz) 6
δH (J in Hz) 7
δH (J in Hz) 8
1
7.02, s
7.02, s
7.02, s
7.18, s
7.18, s
5
3.12, d (16.0)
3.37, d (16.0)
5.13, s
3.06, d (16.0)
3.38, d (16.0)
4.99, s
3.12, d (16.0)
3.37, d (16.0)
5.14, s
3.12, d (16.0)
3.37, d (16.0)
5.13, s
3.12, d (16.0)
3.37, d (16.0)
5.08, s
3.14, d (16.2)
3.45, d (16.2)
5.08, d (7.0)
3.64, m
7
10
11
12
15
17
19
20
3.72, dt (6.4, 6.5)
2.12, m
3.62, t (6.9)
2.13, m
3.75, q (6.5)
2.22, m
3.74, q (6.3)
2.12, m
3.66, m
2.12, m
2.15, m
4.09, t (5.6)
7.31, s
4.07, t (5.9)
7.60, s
4.23, t (5.6)
8.03, s
4.11, t (5.6)
7.61, s
4.15, t (6.0)
7.72, s
4.09, t (6.1)
7.50, s
7.31, s
7.60, s
8.03, s
7.61, s
7.72, s
7.50, s
2.83, t (6.9)
3.55, q (6.9)
4.77, dd (3.7, 7.2)
3.42, dd (7.2, 13.4)
3.59, dd (3.7, 13.4)
7.35, s
5.31, s
5.65, t (8.3)
3.53, m
2.80, t (6.8)
3.37, q (6.8)
d
d
4.05, t (8.3)
21
7.17, s
22
3.55, s
24
6.71, d (6.2)
6.77, d (6.2)
3.89, brs
6.69, d (6.2)
6.76, d (6.2)
25
7-OH
3-OCH3
19-OCH3
C-9NH
C-20NH
C-21NH
CHO
6.01, d (7.1)
3.65, s
6.02, d (7.0)
3.65, s
3.69, s
3.65, s
3.65, s
3.69, s
3.32, s
7.20, t (6.5)
7.10, t (6.5)
7.19, t (6.3)
7.85, brs
8.01, s
7.87, brs
8.01, s
8.38, brt (6.9)
9.86, s
a
b
Chemical shifts (ppm) referenced to CHCl3 (δH 7.26) at 500 MHz. Chemical shifts (ppm) referenced to CH3OH (δH 3.31) at 500 MHz.
c
d
Chemical shifts (ppm) referenced to acetone (δH 2.05) at 500 MHz. Chemical shifts and multiplicity were resolved by 1D TOCSY experiment.
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Table 2. 13C NMR Assignments for 3−8
work. The H-10 signal overlapped with the signal of the
methoxy group at δH 3.66; therefore its chemical shift and
multiplicity were resolved by 1D TOCSY irradiation of the H-
11 signal at δH 2.12. The remaining structural fragment in 7 was
determined to be a 2-oxazolidinone group from 13C NMR and
HMBC data. A triplet signal resonating at δH 5.65 assigned to
H-19 had HMBC correlations to the aromatic carbons C-15/C-
17 and to a carbonyl at δC 159.1 assigned to C-21 (Figure 1).
a
b
a
d
c
c
position
3
4
5
6
7
8
1
145.4
103.4
148.6
105.4
37.1
145.2
103.5
148.9
105.7
37.1
145.2
105.8
149.0
103.5
37.2
145.6
105.8
148.9
103.3
37.1
146.4
103.6
149.4
104.2
37.9
146.4
103.6
149.4
104.2
37.6
2
3
4
5
6
122.0
79.5
122.1
79.3
122.7
79.5
122.3
79.3
120.0
80.2
120.0
80.2
7
8
155.8
159.0
37.1
156.2
159.1
37.1
156.1
159.2
37.2
155.9
159.1
37.1
158.5
159.1
37.5
158.6
159.1
37.6
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
3-OMe
19-OMe
29.2
29.1
29.2
29.3
30.0
30.1
71.1
70.9
71.3
70.8
72.2
72.1
Figure 1. Selected HMBC correlations of the 2-oxazolidinone
fragment of 7.
151.9
118.6
133.1
135.9
133.1
118.6
36.0
153.2
118.5
130.2
142.3e
130.2
118.5
71.8
157.6
119.6
133.9
135.8
133.9
119.6
188.3
152.5
118.0
131.4
134.3
131.4
118.0
101.1
154.0
119.1
131.3
140.0
131.3
119.1
75.9
151.9
118.5
134.1
139.9
134.1
118.5
35.1
An AB system at δH 4.05 and 3.53 attributed to a methylene
group (H-20a and H-20b) showed HMBC correlations to the
quaternary carbon C-16 as well as to C-21. These data for the
oxazolidinone ring matched well with those of the derivative
prepared from xestoaminol A isolated from Xestospongia sp.10
and of oxazolidinone derivatives from halaminols from
Haliclona n. sp.11 The configuration at C-19 was not
determined.
50.6
56.3
48.5
42.2
148.7
99.5
149.3
99.8
159.1
157.6
51.5
197.8
142.3
142.1
194.1
59.1
197.9
142.1
142.2
194.4
59.0
Psammaplysin M (8) was isolated as a colorless oil by RP
HPLC. Accurate mass determination of 8 confirmed a formula
1
of C23H25N3Br4O8Na at m/z 813.8226 [M + Na]+. The H
NMR, HSQC, and HMBC data clearly suggested the presence
of the same spirooxepinisoxazoline carbon framework as in 7.
Again, the chemical shift and the multiplicity of the H-10 signal
were resolved by a 1D TOCSY experiment due to overlap with
the methoxy signal at δH 3.64. The most prominent differences
58.8
58.9
52.6
59.1
59.1
a
b
c
Chemical shifts (ppm) referenced to CDCl3 (δC 77.16) at 400 MHz.
Chemical shifts (ppm) referenced to CD3OD (δH 49.0) at 400 MHz.
Chemical shifts (ppm) referenced to acetone-d6 (δC 29.84) at 400
1
d
in the H NMR spectra of 7 and 8 were the signals associated
MHz. Chemical shifts (ppm) taken from HSQC and HMBC spectra
with H-19 and H-20. In 7, the signal corresponding to H-19
appeared at δH 5.65 (1H, t), while in 8 it was drastically shifted
upfield to δH 2.80 (2H, t), where it overlapped with the residual
water signal.12 The chemical shift and its multiplicity were
resolved by 1D TOCSY irradiation of H-20, a quartet at δH 3.37
in contrast to the two signals observed for H-20 in 7. An
important structural hint was also seen from the 13C NMR data.
The signal corresponding to C-19 in 7 was shifted from δC 75.9
to δC 35.1 in 8. This inferred that C-19 in 8 no longer had an
adjacent oxygen substituent, and as a consequence the AB
system was absent from the spectrum.
referenced to CDCl3 (δC 77.16).
substitution of the formyl group at C-16; thus the structure 5
was assigned to psammaplysin K.
Psammaplysin K dimethoxy acetal (6), obtained as a
colorless glass, had a molecular formula of C22H24Br4N2O8
determined by (+)-HRESIMS. The 1H NMR spectrum
(MeOH-d4) of 6 again displayed signals for a spirooxepinisox-
azoline moiety. Comparison of the 1H and 13C NMR data of 6
with those of 5 revealed a high degree of structural similarity,
but the key difference was that the diagnostic formyl signal in 5
was replaced by a signal at δH 3.32 (6H) that was attributed to
two methoxy groups. Moreover, acetal functionality could be
inferred from signals for H-19 at δH 5.31 and C-19 at δC 101.1.
However, 6 was suspected to be an artifact produced during the
chromatographic separation since the diagnostic signal
The side chain in 8 was proposed to be a glycolamide12
based on the above NMR data and additional HMBC
correlations (Figure 2). The H-20 signal was found to correlate
to the quaternary aromatic carbon at δC 139.9 (C-16) and to
the amide carbonyl at δC 157.6. Furthermore, H-15 showed
HMBC correlations to C-19, and vice versa. A signal
corresponding to H-22 correlated only to the carbonyl at C-21.
Psammaplysin Derivatives Containing Saturated
Fatty Acid Side Chains. Psammaplysin N (9) displayed a
1
corresponding to the dimethoxy group was absent in the H
NMR spectrum of the fraction from which 6 was isolated. This
was also confirmed by the absence of an ion cluster at m/z 783/
785/787/789/791 [M + Na]+ corresponding to 6 in the
LRESIMS data of the fraction prior to RP HPLC separation.
Psammaplysin L (7) was isolated as a colorless oil by RP
HPLC. The (+)-LRESIMS analysis revealed a cluster of ions at
m/z 794/796/798/800/802 [M + Na]+ consistent with a
molecular formula of C22H21N3Br4O8 by HRESIMS measure-
1
ment. Analysis of the H NMR, HSQC, and HMBC data
(acetone-d6) indicated that the molecule also contained the
signals anticipated for a spirooxepinisoxazoline carbon frame-
Figure 2. Selected HMBC correlations of the glycolamide fragment of
psammaplysin M (8).
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Chart 1
1:4:6:4:1 ion cluster at m/z 990/992/994/996/998 [M + Na]+,
and the molecular formula was defined to be C37H53N3Br4O7
from the (+)-HRESIMS. This is 14 Da higher (equivalent to
one methylene) than those of 19-deoxypsammaplysin D and
ceratinamide B.9 The 1H /13C NMR data (CDCl3) of 9
matched those of the known ceratinamide B including the data
corresponding to the spirooxepinisoxazoline carbon framework
as well as to an iso fatty acid chain. The presence of a doublet
signal at δH 0.86 (6H) further confirmed the presence of an iso-
branched fatty acyl substituent. The GC-MS chromatogram of
the corresponding fatty acid methyl ester (FAME) derivative of
9 showed a single peak with m/z 256. The diagnostic fragment
ions at m/z 241 [M+ − 15], 225 [M+ − 31], 213 [M+ − 43],
and 74 (McLafferty) indicated a 13-methyltetradecanoate fatty
acid ester (iso 15:0).
presence of two additional methylenes in the fatty acid
component. GC-MS analysis of the associated FAME derivative
indicated the presence of a C18:0 fatty acid chain attached to
the psammaplysin structure. Metabolite 12 gave an adduct ion
peak at m/z 1034.0771 [M + Na]+, larger than that of 11 by 16
Da, suggesting the presence of an additional oxygen in the
1
molecule. The H and 2D NMR data of 12 were similar to
those in 11 except for the signals attributed to H-19 and H-20.
The signal for H-19 was a doublet of doublets at δH 4.81 (1H, J
= 3.0, 6.5 Hz), and H-20 presented as an AB system at δH 3.31
(dd) and 3.67 (dd). These data indicated that a hydroxy group
was present at C-19; thus 12 was determined to be the 19-
hydroxy derivative of 11. Previous research has identified the iso
C15:0-containing psammaplysin D, which also has a 19-
hydroxy substituent.4 Similarly, 12, with a molecular formula of
C35H49O8N3Br4, was deduced as the 19-hydroxy derivative of
13. Compounds 13 and 14 displayed molecular formulas of
C35H49O7N3Br4 and C35H49O8N3Br4, respectively, which
revealed an additional hydroxy group in 14 compared to 13.
The next group of psammaplysin derivatives identified from
this sponge extract all contained saturated straight-chain fatty
acids (10−14). Psammaplysin O (10) displayed a molecular
1
formula of C37H53O7N3Br4, and its H NMR data were similar
1
to those of 9 except that the terminal methyl signal in 10
appeared as a triplet at δH 0.88 (3H) and so confirmed a linear
fatty acid chain. FAME analysis concluded that 10 contained a
16:0 straight-chain fatty acid residue. Psammaplysin P (11) was
found to return a molecular formula of C39H57O7N3Br4, i.e., a
molecular mass 28 Da larger than in 10, suggesting the
The H and 2D NMR data of 14 matched a 19-hydroxy
derivative of 13. Both compounds yielded a C14:0 straight-
chain fatty acid on GC-MS analysis of their FAME derivatives.
The next group of psammaplysin derivatives (15−19) all
contained an anteiso-branched saturated fatty acid. Psamma-
plysin R (15) was obtained as a colorless oil by RP HPLC with
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Figure 3. Preparation of FAME and pyrrolidide derivatives of 21.
a molecular formula of C37H53O8N3Br4 obtained from
HRESIMS data. The H and 2D NMR data of 15 displayed a
rule formulated by Anderson and Holman,15 the double bond
in 21 was located between C-10 and C-11.
1
The relative configuration of psammaplysin A (1) has been
determined as 6R*,7S* by single-crystal X-ray crystallographic
analysis of psammaplysin acetamide acetate.2 By chemical shift
comparison, and also based on ROESY correlation data and
modeling studies,8 other known psammaplysins have been
shown to possess the same relative configuration. The close
high degree of similarity with those of 14, except that the signal
attributed to the terminal methyl of the linear fatty acyl chain
was absent; instead there were methyl signals at δH 0.88 (t) and
at δH 0.83 (d). HMBC correlations on the fatty acid side chain
3
provided further support for this anteiso assignment. JCH
HMBC correlations were observed between the terminal triplet
methyl at δH 0.88 and the methylene carbon at δC 22.5 (C-34)
and the methine carbon at δC 31.9 (C-33). FAME analysis
confirmed that 15 is a psammaplysin derivative featuring an
anteiso C16:0 fatty acid chain. A pair of psammaplysins, 16 and
17, both shared the same anteiso C17:0 fatty acid chain, but
differed in that 17 had a 19-hydroxy group. These two
1
similarity of H and 13C NMR shifts with those of known
psammaplysins supports a 6R*,7S* configuration for the
spirooxepinisoxazoline ring in psammaplysins 4−24.
The specific rotations of psammaplysin A (1), psammaplysin
B (2), 5, and 6 were calculated as follows: 1 [α]24D = −57.8 (c
0.32, MeOH); 2 [α]24 = −55.6 (c 0.28, MeOH); 5 [α]24
=
D
D
−16 (c 0.09, CHCl3); 6 [α]24 = −15 (c 0.03, CHCl3). The
D
1
compounds showed almost superimposable H NMR signals,
specific rotations of previously isolated psammaplysins are all
negative;2−8 thus it is likely that the various psammaplysins all
share the same absolute configuration. Both psammaplysins and
related spirocyclohexadienyloxazolines may derive biosyntheti-
cally from the same bromotyrosine precursor as shown in
Figure 4. The spirocyclohexadienyloxazolines may derive from
except for those of H-19 and H-20. 19-Hydroxypsammaplysin S
(17) displayed a 1:4:6:4:1 ion cluster at m/z 1020/1022/1024/
1026/1028 [M + Na]+, which is 16 Da larger than that of 16.
The molecular formulas of 16 and 17 were deduced from
(+)-HRESIMS as C38H55O7N3Br4 and C38H55O8N3Br4, re-
spectively. A final pair of compounds, 18 and 19, possessed
1
similar H NMR and 2D NMR data including the signals
corresponding to an anteiso-branched C19:0 fatty acyl side
chain, but differed in the signals of H-19 and H-20. A molecular
formula of C40H59O8N3Br4 was deduced for 19 by HRESIMS.
Psammaplysin Derivatives Containing Monoenoic
Fatty Acid Side Chains. Five new psammaplysin derivatives
containing a monoenoic fatty acid side chain were isolated from
the sponge extract. Two of them (20 and 21) contained an iso-
branched fatty acid, and the rest (22−24) featured unbranched
chain fatty acids. Psammaplysin U (20) showed a molecular
formula of C38H53Br4N3O7, while 21 was the 19-hydroxy
derivative of 20 due to a molecular weight 16 Da larger than
that of 20. The presence of the hydroxy group at C-19 in 21
was again evident from the 1H and 13C NMR chemical shifts of
the signals at C-19 and C-20. In addition, an iso-branched fatty
acid was inferred from a signal at δH 0.86 (d) integrating for six
protons. In the side chain, an olefinic functionality was apparent
Figure 4. Proposed biosynthesis of spirooxepinisoxazoline and
spirocyclohexadienyl-oxazoline ring systems (arrows show proposed
mechanistic steps for path b).
nucleophilic attack of the oxime hydroxy group directly onto
the arene oxide (path a) with C−O bond cleavage and
protonation, giving a hydroxy group at C-1.16 In contrast, the
psammaplysin skeleton may be derived by a two-step process
involving first arene oxide−oxepin rearrangement,2 then a ring
closure that involves protonation of a double bond and
nucleophilic attack by the oxime hydroxy group. Alternatively
in a concerted mechanism, as shown in path b of Figure 4, the
psammaplysin skeleton could be generated by hydroxy attack
on the epoxide, with simultaneous C−C bond cleavage, and
protonation at C-5. Whereas the stereochemical consequences
of path a are well understood, the stereochemical implications
of path b (or an equivalent two-step mechanism) are not as
predictable. The ECD spectrum of psammaplysin A (1) when
run in MeOH showed a positive Cotton effect at 240 nm. The
relationship between ECD data and absolute configuration in
bromotyrosine-derived spirocyclohexadienyloxazolines is well
documented.17−21 Despite the close structural similarities of the
1
from H NMR signals at δH 5.36; a Z geometry was inferred
from the carbon chemical shifts of the adjacent vinylic carbon
atoms, which were less than 30 ppm.13 GC-MS analysis of the
FAME derivative of 21 revealed a molecular ion at m/z 282
accompanied by ions at m/z 250 [M − 32] and m/z 208 [M −
74; McLafferty] as well as a base peak at m/z 74. The location
of the double bond was deduced from the GC-MS pattern of
the corresponding pyrrolidide derivative (Figure 3), which
showed a molecular ion peak at m/z 321 together with the
characteristic base peak of a pyrrolidide derivative [m/z 113]
formed by a McLafferty rearrangement.14 By implementing the
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Journal of Natural Products
Article
Figure 5. Acetylation, hydrolysis, and MPA ester analysis of psammaplysin B (2).
spirocyclohexadienyloxazoline and psammaplysin chromo-
phores, the experimental ECD data do not necessarily secure
the absolute configuration of the psammaplysins due to the
greater conformational flexibility of the spirocycloheptadiene
ring system compared to the spirocyclohexadienes.
concentration. Further biological evaluation of 4 showed an
IC50 value of 6.4 ( 1.4) μM.
The absolute configuration of psammaplysin A was not
pursued in the original X-ray crystallographic work due to
inferior crystal quality.2 In principle, this information is
accessible if the diffraction data are collected using Cu Kα
radiation at low temperature.22 Acetylation of psammaplysin A
(1) or B (2) gave the known psammaplysin A acetamide
acetate and psammaplysin B acetamide diacetate (25),
respectively,2 but neither product produced crystals suitable
for X-ray crystallography. HPLC of 25 using a DAICEL
Chiralpak AD column with UV detection at 254 nm revealed
two components, each possessing a negative optical rotation,
and therefore suggesting the presence of diastereomers differing
in configuration at C-19. Hydrolysis of 25 gave carbamate 26,
the 19-OH analogue of a carbamate product that had been
characterized in the original report of Kashman et al., although
their proposed structure was incorrect.23 Carbamate 26 had a
specific rotation close to zero, in agreement with it being a
racemate; however HPLC as above failed to differentiate the
two enantiomers of 26. Preparation of the (R)-MPA ester
derivative of 26 was undertaken in an attempt to establish the
presence of diastereomers differing in configuration at C-19;
The biological data of 4, in conjunction with the reported
antimalarial data for psammaplysins F (28), G (29), and H
(30), clearly identify the importance of the N-substitution of
the ethylamino moiety to antimalarial activity. Furthermore, the
data indicate that the replacement of an amine, urea, or
enamine derivative with a secondary amide functionality
adversely effects antiparasite activity. However, the higher
lipophilicity (i.e., log P) and larger molecular weights associated
with the new amide psammaplysin analogues (8, 9, 12, 18, 22)
tested in this study would also minimize bioavailability,24 thus
reducing the biological activity. Further analogues of this
structure class are required in order to elucidate more detailed
structure−activity relationships.
1
CONCLUSIONS
however the H NMR spectra of the reaction product did not
■
clearly show the presence of diastereomers. Attempts to purify
individual (R)-MPA ester derivatives were complicated by their
ease of hydrolysis.
In this study, 21 new psammaplysin derivatives were identified
from an extract of the marine sponge A. strongylata collected
from Bali. A group of psammaplysins including the 19-hydroxy
derivative of psammaplysin E (4) together with four derivatives
containing modified side chains (5−8) were identified. Another
group of psammaplysin derivatives containing an unbranched
chain, an iso- or anteiso-branched chain, or monoenoic fatty acid
(9−24) side chains were also isolated. An HPLC study using a
chiral column revealed that psammaplysin B acetamide
diacetate (25) was a mixture of diastereomers differing in
configuration at C-19. Hydrolysis of 25 gave a racemic
carbamate product (26). 19-Hydroxypsammaplysin E (4)
displayed modest in vitro growth inhibition of chloroquine-
sensitive P. falciparum parasites with an IC50 value of 6.4 μM.
Owing to recent literature reports detailing the antimalarial
activity of psammaplysins F (28), G (29), and H (30),6,7 we
tested the more abundant metabolites (>2 mg) from this study
(4, 5, 7−9, 12, 18, 22) in an in vitro growth inhibition assay
using the chloroquine-sensitive P. falciparum 3D7 malaria
parasite line. Psammaplysins F, G, and H had been previously
shown to display IC50 values of 1.92, 5.22, and 0.41 μM,
respectively, against P. falciparum 3D7 parasites.7 In this study,
compounds 4, 5, 7−9, 12, 18, and 22 were initially screened at
10 μM against the same P. falciparum line; however only
compound 4 showed any inhibition of parasite growth at this
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Journal of Natural Products
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a
1
Table 3. H NMR Assignments for 9−16
position
9
10
7.02, s
11
12
13
7.02, s
14
15
16
1
7.02, s
7.02, s
7.02, s
7.02, s
7.02, s
7.02, s
5
3.12, d (16.0)
3.37, d (16.0)
5.13, s
3.12, d (16.0)
3.37, d (16.0)
5.13, s
3.12, d (16.0)
3.37, d (16.0)
5.13, s
3.12, d (16.0)
3.37, d (16.0)
5.13, s
3.12, d (16.0)
3.37, d (16.0)
5.13, s
3.12, d (16.0)
3.37, d (16.0)
5.13, s
3.12, d (16.0)
3.37, d (16.0)
5.13, s
3.12, d (16.0)
3.37, d (16.0)
5.13, s
7
10
11
12
15
17
19
20
3.74, q (6.5)
2.14, m
3.74, q (6.0)
2.14, m
3.74, q (6.2)
2.13, m
3.73, q (6.5)
2.12, m
3.73, q (6.5)
2.14, m
3.73, q (6.2)
2.12, m
3.74, q (6.2)
2.11, m
3.74, q (6.2)
2.12, m
4.09, t (5.5)
7.34, s
4.09, t (5.5)
7.34, s
4.09, t (5.6)
7.34, s
4.10, t (5.6)
7.52, s
4.09, t (5.5)
7.34, s
4.10, t (5.6)
7.52, s
4.10, t (5.6)
7.52, s
4.09, t (5.6)
7.34, s
7.34, s
7.34, s
7.34, s
7.52, s
7.34, s
7.52, s
7.52, s
7.34, s
2.74, t (6.8)
3.46, q (6.8)
2.75, t (6.8)
3.46, q (6.8)
2.74, t (6.8)
3.46, q (6.8)
4.81, dd (3.0, 6.5) 2.74, t (6.8)
4.81, dd (2.5, 6.5) 4.81, dd (2.5, 7.0) 2.74, t (6.9)
3.31, dd (3.0,
13.0)
3.45, q (6.8)
3.31, dd (2.5,
13.0)
3.31, dd (2.5,
13.0)
3.45, q (6.9)
3.65, dd (6.5,
13.0)
3.65, dd (6.5,
13.0)
3.65, dd (7.0,
13.0)
22
2.14, m
2.14, m
2.14, m
2.21, t (6.0)
2.14, m
2.21, t (6.0)
2.21, t (5.6)
2.14, m
bc
,
bc
,
bc
,
bc
,
bc
,
bc
,
bc
,
bc
,
23
1.60, m
1.25−1.28,
1.61, m
1.25−1.28,
1.60, m
1.25−1.28,
1.63, m
1.61, m
1.25−1.28,
1.64, m
1.25−1.28, m
1.62, m
1.60, m
1.25−1.28,
bc
,
bc
,
bc
,
24−31
1.25−1.28, m
1.25−1.28, m
1.25−1.28, m
1.25−1.28, m
1.25−1.28, m
1.25−1.28, m
bc
,
bc
,
bc
,
bc
,
bc
,
m
m
m
m
m
bc
,
b
,
c
bc
,
32
33
34
1.25−1.28,
1.25−1.28,
1.25−1.28,
1.25−1.28,
1.26, m
1.28, m
1.25−1.28,
bc
,
bc
,
bc
,
bc
,
bc
,
m
m
m
m
m
bc
,
bc
,
bc
,
bc
,
bc
,
1.15, m
1.51, m
1.25−1.28,
1.25−1.28,
1.29, m
1.26, m
1.25−1.28,
bc
,
bc
,
bc
,
m
m
m
bc
,
bc
,
bc
,
1.25−1.28,
1.25−1.28,
0.88, t (6.5)
0.88, t (6.7)
0.86, m
1.28, m
1.26, m
bc
,
bc
,
m
m
bc
,
bc
,
35
0.86, d (7.0)
0.86, d (7.0)
1.29, m
1.25−1.28,
bc
1.25−1.28, m
0.88, t (6.8)
0.85, m
1.28, m
,
m
bc
,
bc
,
36
0.88, t (6.5)
1.25, m
1.28, m
1.26, m
1.29, m
0.83, d (6.8)
0.88, t (6.7)
0.83, d (6.7)
bc
,
bc
,
37
38
0.88, t (6.7)
3.69, s
0.88, t (6.8)
3.69, s
OCH3
C-9NH
3.69, s
3.69, s
3.69, s
3.69, s
3.69, s
3.69, s
7.19, t (6.5)
7.19, t (6.0)
5.84, t (6.5)
7.20, t (6.2)
5.43, t (6.4)
7.19, t (6.5)
5.84, t (6.0)
7.19, t (6.5)
5.44, t (6.5)
7.19, t (6.2)
5.83, t (6.0)
7.19, t (6.2)
5.83, t (5.9)
7.20, t (6.2)
5.86, t (6.0)
C-21NH 5.44, t (6.5)
a
b
c
Chemical shifts (ppm) referenced to CHCl3 (δH 7.26) at 500 MHz. Unresolved chemical shifts due to overlapping signals. Signal multiplicity
unresolved due to overlapping signals.
Biological Material. The sponge Aplysinella strongylata was
harvested in Tulamben Bay, Bali, at a depth of approximately 20 m
by hand using scuba in November 2010. The sponge was spherical in
shape, fleshy to the touch, and compressible. The color in life was
milky-colored inside and gray outside, compressible with mucus
secretion, turning deep brown in 70% EtOH preservative. The sponge
identification was undertaken at the Research Center of Ocean-
ography, Indonesian Institute of Science, where a voucher specimen
(TL-20) is deposited.
Extraction and Purification. The freshly collected sponge (147 g
wet wt) was frozen before being extracted with 1:1 DCM−MeOH (3
× 200 mL) at room temperature. The combined extracts were dried
under vacuum to produce 64.5 g (wet wt) crude extract. The crude
extract was sequentially partitioned between hexanes (3 × 200 mL)
and H2O, followed by EtOAc and H2O (3 × 200 mL), and finally
BuOH and H2O (3 × 200 mL). This gave hexanes (1.2 g), EtOAc (3.1
g), and BuOH (0.84 g) fractions, respectively. A portion of the EtOAc
fraction (3.1 g) was resolved by normal-phase VLC (3 × 10 cm in
diameter) with step gradient elution from 100% hexanes to DCM,
EtOAc, and 100% MeOH and gave 11 fractions. Based on TLC
analysis, the fourth and the sixth fractions were combined to give a 433
mg fraction coded as TL-20EV4-6, while the seventh until the 11th
fractions were grouped to produce a 2.1 mg fraction coded as TL-
20EV7-11. Subsequent normal-phase flash column chromatography on
the more polar fraction TL-20EV7-11 employing step gradient elution
from 100% DCM to 100% MeOH gave nine fractions. These fractions
were combined on the basis of their TLC profile to give five fractions,
coded as TL-20EV7-11F2-3 (47.0 mg), TL-20EV7-11F4 (902 mg),
TL-20EV7-11F5 (636 mg), TL-20EV7-11F6 (433 mg), and TL-
EXPERIMENTAL SECTION
■
General Experimental Procedures. All NMR spectra were
referenced to solvent signals as follows: δ 7.26 and 77.16 ppm for
CDCl3; δ 2.05 and 29.84 for acetone-d6; and δ 3.31 and 49.00 ppm for
methanol-d4. 1D and 2D NMR spectra were acquired using a Bruker
Avance 400 or a Bruker Avance 500 spectrometer at 298 K. Optical
rotations were obtained using a Jasco P2000 polarimeter. The
electronic circular dichroism spectra were run on a Jasco J-710
spectrophotometer in MeOH solution. Positive ion electrospray mass
spectra (LRESIMS) were determined using a Bruker Esquire HCT or
(HRESIMS) using a MicroTof Q instrument each with a standard ESI
source. Reversed-phase HPLC was carried out on an Agilent 1100
Series instrument fitted with a variable-wavelength UV and refractive
index detector, an Agilent D1311A quaternary pump, and a
semipreparative Phenomenex C18 Gemini 5 μm 110 Å column (10
mm × 250 mm). Analytical HPLC was performed on an Agilent 1200
Series liquid chromatograph system equipped with both a UV detector
(set at 254 nm) and an ALP detector (Advanced Laser polarimeter,
PDR-Chiral Inc.) using a Chiralpak AD column (4.6 × 250 mm,
i
DAICEL Chemical IND, LDT) and with a gradient of PrOH−
hexanes (5 to 40%iPrOH) at a flow rate of 0.5 mL per min. Normal-
phase flash column chromatography was performed by wet-packing a
glass column, containing a glass frit, to give a column bed height of 18
cm. The column packing used was silica gel 60 (40−63 μm; Scharlau).
Gas chromatography was carried out on a Shimadzu GCMS-QP2010
Plus. Initial temperature was 100 °C, isothermal for 3 min, then
ramped 16 °C/min for 10 min. The final temperature was 270 °C,
injection temperature 250 °C, and flow rate 1.5 mL/min.
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Journal of Natural Products
Article
a
1
Table 4. H NMR Assignments for 17−24
position
17
18
7.02, s
19
20
7.02, s
21
22
7.02, s
23
7.02, s
24
1
7.02, s
7.02, s
7.02, s
7.02, s
5
3.11, d (16.0)
3.37, d (16.0)
5.13, s
3.12, d (16.0) 3.12, d (16.0)
3.37, d (16.0) 3.37, d (16.0)
3.12, d (16.0) 3.12, d (16.0)
3.37, d (16.0) 3.37, d (16.0)
3.12, d (16.0) 3.12, d (16.0) 3.12, d (16.0)
3.37, d (16.0) 3.37, d (16.0) 3.37, d (16.0)
7
5.13, s
5.13, s
5.13, s
5.13, s
5.13, s
5.13, s
5.13, s
10
11
12
15
17
19
20
3.74, q (6.0)
2.12, m
3.74, q (6.2)
2.13, m
3.74, q (6.0)
2.13, m
3.75, q (6.2)
2.13, m
3.74, q (6.0)
2.13, m
3.75, q (6.2)
2.14, m
3.74, q (6.2)
2.12, m
3.75, q (6.1)
2.13, m
4.09, t (5.5)
7.51, s
4.09, t (5.5)
7.34, s
4.09, t (5.5)
7.51, s
4.09, t (5.6)
7.34, s
4.09, t (5.5)
7.51, s
4.09, t (5.5)
7.34, s
4.09, t (5.6)
7.34, s
4.10, t (5.6)
7.34, s
7.51, s
7.34, s
7.51, s
7.34, s
7.51, s
7.34, s
7.34, s
7.34, s
4.80, dd (2.5, 7.0) 2.74, t (7.0)
4.80, dd (3.0, 7.0) 2.74, t (6.9)
4.80, dd (2.5, 7.0) 2.74, t (6.8)
2.74, t (6.8)
3.46, q (6.8)
4.80, dd (2.5, 7.0)
3.31, dd (2.5,
12.5)
3.46, q (7.0)
3.30, dd (3.0,
12.5)
3.45, q (6.9)
3.30, dd (2.5,
12.5)
3.45, q (6.8)
3.30, dd (2.5,
12.7)
3.65, dd (7.0,
12.5)
3.66, dd (7.0,
12.5)
3.66, dd (7.0,
12.5)
3.66, dd (7.0,
12.7)
22
2.20, t (7.0)
2.13, m
2.13, m
2.13, m
2.21, m
2.14, m
2.14, m
2.13, m
1.60, m
bc
,
bc
,
bc
,
23
1.62, m
1.60, m
1.25−1.28,
1.60, m
1.60, m
1.62, m
1.62, m
1.60, m
bc
,
bc
,
bc
,
bc
,
24−28
1.25−1.28, m
1.25−1.28, m
1.25−1.28, m
1.25−1.28, m
1.25−1.28, m
1.25−1.28, m
1.25−1.28, m
1.25−1.28, m
1.25−1.28, m
1.25−1.28, m
1.25−1.28, m
1.25−1.28, m
1.25−1.28,
1.25−1.28, m
1.25−1.28,
1.25−1.28,
1.25−1.28, m
1.25−1.28, m
1.25−1.28, m
1.25−1.28, m
2.00, m
bc
,
bc
,
bc
,
bc
,
m
m
m
m
bc
,
bc
,
bc
,
bc
,
bc
,
29
30
31
32
33
34
35
1.25−1.28,
2.00, m
5.34, m
5.34, m
2.00, m
1.25, m
1.16, m
1.51, m
2.00, m
5.34, m
5.34, m
2.00, m
1.25, m
1.16, m
1.51, m
2.01, m
5.34, m
5.34, m
2.01, m
1.26, m
1.26, m
1.29, m
1.25−1.28,
bc
,
bc
,
m
m
bc
,
bc
,
bc
,
bc
,
bc
,
bc
,
1.25−1.28,
1.25−1.28,
bc
,
bc
,
m
m
bc
,
bc
,
bc
,
bc
,
bc
,
bc
,
1.25−1.28,
1.25−1.28,
bc
,
bc
,
m
m
bc
,
bc
,
b,
c
b,c
1.25−1.28,
2.00, m
5.35, m
5.35, m
2.00, m
bc
,
m
bc
,
bc
,
bc
,
bc
,
bc
,
bc
bc
,
,
1.06, m, 1.25, m 1.25−1.28,
bc
5.35, m
5.35, m
2.00, m
,
m
bc
,
bc
,
bc
,
bc
bc
,
,
1.25, m
1.25−1.28,
bc
,
m
bc
,
bc
,
0.86, m,
1.07, m
1.26, m
1.25, m
0.85, m
1.28, m
,
1.07, m
1.26, m
1.34, m
0.85, m
1.28, m
,
1.28, m
bc
,
bc
bc
,
bc
,
,
36
37
0.87, t (6.5)
0.83, d (6.5)
0.86, d (6.5)
0.86, d (6.5)
0.86, d (6.5)
0.86, d (6.5)
0.88, t (7.0)
1.25, m
1.25, m
1.25, m
1.25, m
bc
,
bc
,
bc
,
bc
,
38
0.88, t (6.7)
0.83, d (6.7)
3.69, s
0.88, t (6.8)
0.83, d (6.8)
3.69, s
1.28, m
1.29, m
39
0.88, t (6.6)
3.69, s
0.88, t (6.6)
3.69, s
OCH3
C-9NH
3.69, s
3.69, s
3.69, s
3.69, s
7.21, t (6.0)
7.20, t (6.2)
5.44, t (6.4)
7.20, t (6.0)
5.44, t (5.9)
7.21, t (6.2)
7.20, t (6.0)
5.86, t (5.5)
7.20, t (6.2)
5.44, t (6.5)
7.20, t (6.2)
5.44, t (5.6)
7.20, t (6.1)
5.44, t (6.0)
C-21NH 5.89, t (6.0)
5.86, t (5.5)
a
b
c
Chemical shifts (ppm) referenced to CHCl3 (δH 7.26) at 500 MHz. Unresolved chemical shifts due to overlapping signals. Signal multiplicity
unresolved due to overlapping signals.
20EV7−-11F7−9 (4128 mg). Fraction TL-20EV7-11F5 was purified
through HPLC (isocratic MeCN−H2O, 85:15% for 40 min) to give
psammaplysins A (1) and B (2).2 The other fraction obtained after the
vacuum liquid chromatography, TL-20EV4-6, was put through NP
flash column chromatography eluting with gradient solvent from 100%
hexanes to hexanes−CHCl3 (3:1, 1:1, 1:3) to MeOH 100% to yield 13
fractions. These fractions were then grouped into four fractions based
on their TLC profile: TL-20EV4-6F4 (20 mg), TL-20EV4-6F5 (48
mg), TL-20EV4-6F6−7 (60 mg), and TL-20EV4-6F8 (209 mg). The
last fraction was then chromatographed using C18 bonded silica HPLC
(15:85% MeOH−H2O for 45 min) to yield 19-hydroxypsammaplysin
E (4) (3.2 mg). The less polar fraction, TL-20EV4-6F6-7, was
subjected to RP HPLC (a gradient of 50% to 100% MeOH−H2O over
20 min) to give psammaplysin D,4 ceratinamide A,9 and seven
psammaplysin derivatives (12, 14, 15, 17, 18, 21, and 24). Fraction
TL-20EV4-6F5 gave psammaplysin E (3)4 and four psammaplysins (9,
13, 22, and 23) after RP HPLC eluting with 85:15% MeCN−H2O for
45 min. The least polar fraction in this group, TL-20EV4-6F4, gave
ceratinamide B9 and seven psammaplysins (5, 6, 10, 11, 16, 19, and
20) by RP HPLC using 95:5% MeOH−H2O for 35 min. Besides
containing mostly lipid components, the hexanes fraction (TL-20H)
also afforded psammaplysin M (3.7 mg) (7) and psammaplysin N (1.5
mg) (8) after successive NP flash column and RP HPLC (90:10
MeOH−H2O for 50 min).
19-Hydroxypsammaplysin E (4): yellowish oil; [α]22 = −79.6 (c
D
0.21, CHCl3); 1H and 13C NMR (methanol-d4, 500 MHz), see Table 1
and Table 2; (+)-LRESIMS m/z (rel int) 874 (23), 876 (66), 878
(100), 880 (70), 882 (24) [M + Na]+; (+)-HRESIMS m/z 877.8148
[M + Na]+ (calcd for C27H25Br4N3O9Na, 877.8176; Δ 3.1 ppm).
Psammaplysin K (5): colorless glass; [α]22D = −16 (c 0.09, CHCl3);
1H and 13C NMR (acetone-d6) see Table 1 and Table 2.
(+)-LRESIMS m/z (rel int) 737 (10), 739 (50), 741 (100), 743
(43), 745 (14) [M + Na]+; (+)-HRESIMS m/z 740.7698 [M + Na]+
(calcd for C20H18Br4N2O7Na, 740.7699; Δ 0.1 ppm).
Psammaplysin K dimethoxy acetal (6): colorless glass; [α]24
=
D
−15 (c 0.03, CHCl3); H and 13C NMR (CDCl3, 500 MHz), see
Table 1 and Table 2; (+)-LRESIMS m/z (rel int) 783 (30), 785 (70),
787 (100), 789 (79), 791 (30) [M + Na]+; (+)-HRESIMS m/z
786.8107 [M + Na]+ (calcd for C22H24Br4N2O8Na, 786.8117; Δ 1.4
ppm).
1
Psammaplysin L (7): colorless glass; [α]22 = −65.6 (c 0.19,
D
1
acetone); H and 13C NMR (acetone-d6, 500 MHz), see Table 1 and
Table 2; (+)-LRESIMS m/z (rel int) 794 (21), 796 (58), 798 (100),
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800 (83), 802 (38) [M + Na]+; (+)-HRESIMS m/z 797.7930 [M +
Na]+ (calcd for C22H21Br4N3O8Na, 797.7913; Δ −2.0 ppm).
19-Hydroxypsammaplysin U (21): colorless glass; [α]22D = −69 (c
0.19, CHCl3); 1H and 13C NMR (CDCl3, 500 MHz), see Table 4 and
Table 5; (+)-LRESIMS m/z (rel int) 1018 (14), 1020 (70), 1022
(100), 1024 (80), 1026 (24) [M + Na]+; (+)-HRESIMS m/z
1022.0463 [M + Na]+ (calcd for C38H53Br4N3O8Na, 1022.0417; Δ
−4.4 ppm).
Psammaplysin M (8): colorless glass; [α]22 = −33 (c 0.05,
D
1
acetone); H and 13C NMR (acetone-d6, 500 MHz), see Table 1 and
Table 2; (+)-LRESIMS m/z (rel int) 810 (17), 812 (67), 814 (100),
816 (63), 818 (18) [M + Na]+; (+)-HRESIMS m/z 813.8226 [M +
Na]+ (calcd for C23H25Br4N3O8Na, 813.8226; Δ 0.1 ppm).
Psammaplysin V (22): colorless glass; [α]22 = −22 (c 0.03,
D
Psammaplysin N (9): colorless glass; [α]22 = −43 (c 0.01,
CHCl3); 1H and 13C NMR (CDCl3, 500 MHz), see Table 4 and Table
5; (+)-LRESIMS m/z (rel int) 988 (14), 990 (72), 992 (100), 994
(82), 996 (18) [M + Na]+; (+)-HRESIMS m/z 1008.0093 [M + K]+
(calcd for C37H51Br4N3O7K, 1008.0051; Δ −4.1 ppm).
D
CHCl3); 1H and 13C NMR (CDCl3, 500 MHz), see Table 3 and Table
5; (+)-LRESIMS m/z (rel int) 990 (29), 992 (78), 994 (100), 996
(76), 998 (30) [M + Na]+; (+)-HRESIMS m/z 994.0495 [M + Na]+
(calcd for C37H53Br4N3O7Na, 994.0468; Δ −2.7 ppm).
Psammaplysin W (23): colorless glass; [α]22 = −43 (c 0.04,
D
Psammaplysin O (10): colorless glass; [α]24D = −74 (c 0.08,
CHCl3); 1H and 13C NMR (CDCl3, 500 MHz), see Table 3 and Table
5; (+)-LRESIMS m/z (rel int) 990 (17), 992 (67), 994 (100), 996
(70), 998 (20) [M + Na]+; (+)-HRESIMS m/z 994.0488 [M + Na]+
(calcd for C37H53Br4N3O7Na, 994.0468; Δ −2.0 ppm).
CHCl3); 1H and 13C NMR (CDCl3, 500 MHz), see Table 4 and Table
5; (+)-LRESIMS m/z (rel int) 1030 (20), 1032 (64), 1034 (100),
1036 (60), 1038 (20) [M + Na]+; (+)-HRESIMS m/z 1034.0767 [M
+ Na]+ (calcd for C40H57Br4N3O7Na, 1034.0781; Δ 2.3 ppm).
19-Hydroxypsammaplysin W (24): colorless glass; [α]22D = −84 (c
0.04, CHCl3); 1H and 13C NMR (CDCl3, 500 MHz), see Table 4 and
Table 5; (+)-LRESIMS m/z (rel int) 1046 (10), 1048 (50), 1050
(100), 1052 (54), 1054 (14) [M + Na]+; (+)-HRESIMS m/z
1046.0773 [M + Na]+ (calcd for C40H57Br4N3O8Na, 1046.0771; Δ
−0.1 ppm).
Psammaplysin P (11): colorless glass; [α]24 = −11 (c 0.09,
D
CHCl3); 1H and 13C NMR (CDCl3, 500 MHz), see Table 3 and Table
5; (+)-LRESIMS m/z (rel int) 1018 (18), 1020 (68), 1022 (100),
1024 (66), 1026 (24) [M + Na]+; (+)-HRESIMS m/z 1022.0799 [M
+ Na]+ (calcd for C39H57Br4N3O7Na, 1022.0799; Δ −1.8 ppm).
19-Hydroxypsammaplysin P (12): colorless glass; [α]24 = −74 (c
Typical Procedure for Preparation of Fatty Acid Methyl Ester
Derivatives of Psammaplysins. A sample of psammaplysin U (0.5
mg) was treated with HCl in MeOH (6.0 M, 2 mL) in a 4 mL round-
bottom flask, equipped with a condenser and drying tube, and refluxed
for 16 h at 70 °C. The mixture was taken to dryness under a stream of
N2 gas before addition of toluene (0.5 mL), which was then also
removed by a stream of N2 gas. The resulting transmethylated fatty
acid ester was dissolved in 1 mL of hexanes before passing through a
short silica column (0.2 g silica, 1 cm height) eluting with hexanes (10
mL). The eluted FAME was evaporated to dryness prior to GC-MS
analysis. A similar procedure was used to prepare FAME esters of
psammaplysins N (9), O (10), Q (13), and R (15) and 19-
hydroxypsammaplysin U (21).
D
0.08, CHCl3); 1H and 13C NMR (CDCl3, 500 MHz), see Table 3 and
Table 5; (+)-LRESIMS m/z (rel int) 1034 (20), 1036 (52), 1038
(100), 1040 (80), 1042 (22) [M + Na]+; (+)-HRESIMS m/z
1034.0748 [M + Na]+ (calcd for C39H57Br4N3O8Na, 1034.0771; Δ 2.3
ppm).
Psammaplysin Q (13): colorless glass; [α]24 = −55 (c 0.01,
D
CHCl3); 1H and 13C NMR (CDCl3, 500 MHz), see Table 3 and Table
5; (+)-LRESIMS m/z (rel int) 962 (38), 964 (70), 966 (100), 968
(78), 970 (40) [M + Na]+; (+)-HRESIMS m/z 966.0137 [M + Na]+
(calcd for C35H49Br4N3O7Na, 966.0155; Δ 1.9 ppm).
19-Hydroxypsammaplysin Q (14): colorless glass; [α]24D = −92 (c
0.03, CHCl3); 1H and 13C NMR (CDCl3, 500 MHz), see Table 3 and
Table 5; (+)-LRESIMS m/z (rel int) 878 (32), 980 (68), 982 (100),
984 (68), 986 (38) [M + Na]+; (+)-HRESIMS m/z 982.0123 [M +
Na]+ (calcd for C35H49Br4N3O8Na, 982.0104; Δ −1.9 ppm).
(Z)-Methyl 15-methylhexadec-10-enoate (iso 17:1n−10).25 GC-
MS m/z 282 [M+] (1), 74 (100), 43 (66), 69 (42), 87 (26), 250 (17),
98 (13), 227 (7), 123 (6), 152 (5), 208 (5), 195 95), 166 (4), 137 (3),
177 (3), 252 (1), 217 (0.9).
Psammaplysin R (15): colorless glass; [α]22 = −88 (c 0.09,
D
CHCl3); 1H and 13C NMR (CDCl3, 500 MHz), see Table 3 and Table
5; (+)-LRESIMS m/z (rel int) 1006 (24), 1008 (64), 1010 (100),
1012 (68), 1014 (30) [M + Na]+; (+)-HRESIMS m/z 1006.0460 [M
+ Na]+ (calcd for C37H53Br4N3O8Na, 1006.0458; Δ −0.2 ppm).
Pyrrolidide Derivative of the FAME Product of 21. The fatty acid
methyl ester resulting from the FAME reaction was dried under N2 gas
in a 4 mL screw-capped vial before adding fresh distilled pyrrolidine
(0.5 mL) and 2 drops of Ac2O. The mixture was saturated with N2 gas,
and a single crystal of BHT was added; then the vial was placed in a
100 °C oil bath for 1 h. The mixture was then cooled to 0 °C, Et2O (3
mL) was added, and the product was transferred into a 20 mL vial.
The 4 mL vial was rinsed with Et2O (3 × 3 mL), and the combined
organic extracts were washed with 5% HCl (3 × 5 mL) followed by
H2O (5 mL). The organic layer was dried over anhydrous MgSO4,
filtered through a 1 cm NP silica column using hexanes as eluent, and
prepared for GC-MS analysis.
Psammaplysin S (16): colorless glass; [α]22 = −98 (c 0.05,
D
CHCl3); 1H and 13C NMR (CDCl3, 500 MHz), see Table 3 and Table
5; (+)-LRESIMS m/z (rel int) 1004 (24), 1006 (58), 1008 (100),
1010 (66), 1012 (20) [M + Na]+; (+)-HRESIMS m/z 1008.0637 [M
+ Na]+ (calcd for C38H55Br4N3O7Na, 1008.0625; Δ −1.2 ppm).
19-Hydroxypsammaplysin S (17): colorless glass; [α]22D = −117 (c
0.18, CHCl3); 1H and 13C NMR (CDCl3, 500 MHz), see Table 4 and
Table 5; (+)-LRESIMS m/z (rel int) 1020 (16), 1022 (54), 1024
(100), 1026 (48), 1028 (16) [M + Na]+; (+)-HRESIMS m/z
1020.0619 [M + Na]+ (calcd for C38H55Br4N3O8Na, 1020.0615; Δ
−0.4 ppm).
(Z)-15-Methyl-1-(pyrrolidin-1-yl)hexadec-10-en-1-one. GC-MS
m/z 321 [M+] (3), 43 (23), 55 (19), 69 (6), 70 (15), 85 (6), 98
(12), 113 (100), 126 (19), 140 (2), 154 (1), 168 (2), 182 (2), 196 (1),
222 (0.7), 236 (0.8), 250 (0.9), 278 (1), 306 (1), 321 (3).
Acetylation, Hydrolysis, and MPA Ester Formation of Psamma-
plysin B (2). Acetylation of Psammaplysin B (2). A sample of
psammaplysin B (19.3 mg) in pyridine (0.5 mL) was cooled in an ice
bath. Ac2O (0.5 mL) was added, and the solution was warmed to room
temperature and stirred for 1.5 h. Toluene (1 mL) was added, and the
resulting mixture was evaporated in vacuo. The reaction mixture was
then filtered through a short NP silica column eluted with hexanes
before purification using RP HPLC (40 → 90% MeOH−H2O over 30
min) to give psammaplysin B acetamide diacetate (25) (26.4 mg). A
similar procedure was used to prepare the acetamide acetate derivative
of psammaplysin A.
Psammaplysin T (18): colorless glass; [α]22 = −90 (c 0.04,
D
CHCl3); 1H and 13C NMR (CDCl3, 500 MHz), see Table 4 and Table
5; (+)-LRESIMS m/z (rel int) 1032 (14), 1034 (50), 1036 (100),
1038 (58), 1040 (14) [M + Na]+; (+)-HRESIMS m/z 1052.0679 [M
+ Na]+ (calcd for C40H59Br4N3O7Na, 1052.0677; Δ −0.2 ppm).
19-Hydroxypsammaplysin T (19): colorless glass; [α]22D = −133 (c
0.13, CHCl3); 1H and 13C NMR (CDCl3, 500 MHz), see Table 4 and
Table 5; (+)-LRESIMS m/z (rel int) 1048 (14), 1050 (74), 1052
(100), 1054 (72), 1056 (22) [M + Na]+; (+)-HRESIMS m/z
1048.0924 [M + Na]+ (calcd for C40H59Br4N3O8Na, 1048.0928; Δ 0.3
ppm).
Psammaplysin U (20): colorless glass; [α]22 = −9.2 (c 0.17,
D
CHCl3); 1H and 13C NMR (CDCl3, 500 MHz), see Table 4 and Table
5; (+)-LRESIMS m/z (rel int) 1002 (18), 1004 (58), 1006 (100),
1008 (60), 1010 (34) [M + Na]+; (+)-HRESIMS m/z 1006.0492 [M
+ Na]+ (calcd for C38H53Br4N3O7Na, 1006.0468; Δ −2.4 ppm).
Psammaplysin B acetamide diacetate (25)23: colorless glass;
1
[α]24 = −58.4 (c 0.35, MeOH); H NMR (methanol-d4, 500 MHz)
D
δH 7.19 (1H, s, H-1), 3.07 (1H, d, J = 16.2 Hz, H-5a), 3.24 (1H, d, J =
16.2 Hz, H-5b), 6.31 (1H, s, H-7), 3.59 (2H, t, J = 7.7 Hz, H-10), 2.12
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(2H, m, H-11), 4.12 (2H, t, J = 6.0, H-12), 7.59 (2H, s, H-15 and H-
17), 5.73 (1H, dd, J = 4.5 Hz, 7.6 Hz, H-19), 3.44 (1H, dd, J = 7.7 Hz,
14.1 Hz, H-20a), 3.54 (1H, dd, J = 7.7 Hz, 14.1 Hz, H-20a), 3.63 (3H,
s, −OCH3); (+)-LR-ESIMS m/z (rel int) 893.8 (1), 895.8 (3), 897.8
(5), 899.8 (3), 901.8 (1) [M + Na]+.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +61-7-3365 3605. Fax: +61-7-3365 4273. E-mail:
m.garson@uq.edu.au.
Hydrolysis of Psammaplysin B Acetamide Diacetate (25).
Psammaplysin B acetamide diacetate (25) (12.1 mg) was dissolved
in dry MeOH (1.0 mL) along with K2CO3 (5.0 mg) and stirred at 40
°C overnight. The resulting cloudy solution was neutralized with 1 M
HCl (1.0 mL) and partitioned between CHCl3 (10 mL) and H2O (3 ×
5 mL). The organic phase was washed with saturated Na2CO3 and
H2O, dried over MgSO4, and evaporated to dryness before being
purified by NP HPLC using a mobile phase of EtOAc−hexanes
(70:30) to return the carbamate 26 (6.4 mg).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Australia Research Council (Discovery Grant to
M.J.G. and a Future Fellowship to K.T.A.), the School of
Chemistry and Molecular Biosciences and The University of
Queensland for financial support, and an International
Postgraduate Research Scholarship (to I.W.M.). The assistance
of Dr. T. Le (NMR) and G. McFarlane (MS) is acknowledged.
Sample collection was carried out by D. Prasetya (Ganesha
University of Education Bali) and Teja (Mimpi Resort
Tulamben) and with the permission of the Governor of Bali
under Permit No. 070/7601/BID II/KBPM. We also acknowl-
edge the Australian Red Cross Blood Service for the provision
of human blood and sera.
N-[3-(4-(2-Acetamido-1-hydroxyethyl)-2,6-dibromophenoxy)-
propyl]-2-methoxyacetamide (26): colorless glass; [α]24 = +0.7 (c
D
1
0.13, MeOH); H NMR (methanol-d4, 500 MHz) δH 7.59 (2H, s, H-
15 and H-17), 4.68 (1H, dd, J = 5.0, 7.5 Hz, H-19), 4.04 (2H, t, J = 6.0
Hz, H-12), 3.63 (s, 3H, OCH3), 3.39 (t, 2H, J = 7.5 Hz, H-10), 3.25
(1H, dd, J = 7.5, 13.5 Hz, H-20a), 3.40 (1H, signal under H-10, H-
20b), 2.04 (p, 2H, J = 7.0 Hz, H-11), 1.93 (s, 3H, NHMe);
(+)-LRESIMS m/z (rel int) 637 (1), 639 (3), 641 (1) [M + Na]+;
(+)-HRESIMS m/z 488.9643 [M
C15H20Br2N2O5Na, 488.9631; Δ −2.5 ppm).
+
Na]+ (calcd for
Preparation of (R)-MPA Ester of 26. N-[3-(4-(2-Acetamido-1-
hydroxyethyl)-2,6-dibromophenoxy)propyl]-2-methoxyacetamide (2.0
mg) was dissolved in dry DCM (0.2 mL), to which (R)-MPA (2 equiv,
0.83 mg) was added, followed by DCC (2 equiv, 1.03 mg) and DMAP
(2 equiv, 0.61 mg). The reaction was stirred at room temperature for
16 h and filtered through a cotton wool plug. The solvent was
removed by rotary evaporation, and the residue passed through a silica
column eluting with DCM−MeOH (8:2) to give an (R)-MPA ester
mixture (0.8 mg).
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ASSOCIATED CONTENT
■
S
* Supporting Information
Figures S1−S30. 1H and selected 2D NMR data for compounds
4−24 and enantioselective HPLC traces for 25 and 26. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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