Homophymines B-E and A1-E1, a family of bioactive cyclodepsipeptides from the sponge Homophymia sp.

Article abstract of DOI:10.1039/b910015f

Nine new cyclodepsipeptides, homophymines B-E (2-5) and A1-E1 (1a-5a), were isolated from the polar extracts of the sponge Homophymia sp. The new structures, featuring new polyketide-derived end groups, were determined by interpretation of NMR and MS data

Full text of DOI:10.1039/b910015f

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Homophymines B–E and A1–E1, a family of bioactive cyclodepsipeptides
from the sponge Homophymia sp.†
Angela Zampella,a Valentina Sepe,a Filomena Bellotta,a Paolo Luciano,a Maria Valeria D’Auria,*a
Thierry Cresteil,b Ce´cile Debitus,c Sylvain Petek,d Christiane Poupatb and Alain Ahondb
Received 21st May 2009, Accepted 23rd June 2009
First published as an Advance Article on the web 22nd July 2009
DOI: 10.1039/b910015f
Nine new cyclodepsipeptides, homophymines B–E (2–5) and A1–E1 (1a–5a), were isolated from the
polar extracts of the sponge Homophymia sp. The new structures, featuring new polyketide-derived end
groups, were determined by interpretation of NMR and MS data. The configurations of the new end
groups was secured by the application of J-based configurational analysis. Homophymines displayed
very potent antiproliferative activity (IC50 in the nM range) against a panel of human cancer cell lines.
A careful examination and fractionation of the crude polar
extracts of Homophymia sp. led to the isolation of nine new
homophymines, which we named homophymines B–E (2–5) and
A1–E1 (1a–5a).
In this paper, we describe the isolation and the structure
determination of the new compounds, together with the analysis
of their biological activity.
Introduction
Marine sponges have proven to be a significant source of
biologically active cyclic peptides and depsipeptides.1 Among
these sponge depsipeptides, callipeltin A (from a New Caledonian
sponge Callipelta sp.),2 neamphamide A (from a Papua New
Guinea sponge Neamphius huxleyi),3 papuamides A–D4 and
theopapuamide5 (from Papua New Guinea sponges Theonella
mirabilis and Theonella swinhoei), mirabamides, (from the
Results and discussion
Micronesian sponge Siliquariaspongia mirabilis)6 are well known
for their potent HIV-inhibitory activity and their structurally
unique features incorporating several non proteinogenic amino
acid residues. The rarity of these atypical amino acid residues has
inspired their chemical synthesis.7
The lyophilized sponge (300 g) was extracted with MeOH, and the
combined extracts were fractionated according to the Kupchan
partitioning procedure.9 The bioactive chloroformic extract was
In the frame of our long-standing interest towards bioactive
marine compounds we recently reported the isolation from the
sponge Homophymia sp. (order Lithistida) of a related cyclodep-
sipeptide homophymine A (1).8 This undecapeptide shares some
residues with the abovementioned cyclodepsipeptides, such as
the 2,3-dimethylglutamine residue common to all compounds;
the pipecolic acid, the N-methylglutamine and leucine also
found in neamphamide and theopapuamide; the 3-hydroxy-2,4,6-
trimethyloctanoic acid (HTMOA) also found in theopapuamide.
Homophymine A (1) also contains two unprecedented residues
in natural sources, i.e. the 4-amino-2,3-dihydroxy-1,7-heptandioic
acid (ADHA) and 2-amino-3-hydroxy-4,5-dimethylhexanoic acid
(AHDMHA). As the related cyclodepsipeptides, homophymine A
exhibited cytoprotective activity against the HIV-1 infection with
an IC50 of 75 nM.
aDipartimento di Chimica delle Sostanze Naturali, Universita` di Napoli
“Federico II”, via D. Montesano 49, 80131, Napoli, Italy. E-mail:
madauria@unina.it; Fax: +39-081676552; Tel: +39-081678527
bInstitut de Chimie des Substances Naturelles, CNRS-91198, Gif-sur-Yvette
cedex, France
cUMR7138, Institut de Recherche pour le De´veloppement (IRD), BP529,
98713 Papeete, Tahiti, French Polynesia
dIRD Pharmacochimie des Substances Naturelles et Pharmacophores
Redox UMR152 IRD-Universite´ Paul Sabatier, Toulouse III BPA5, 98848,
NOUMEA cedex Nouvelle Cale´donie
† Electronic supplementary information (ESI) available: Tabulated NMR
data and NMR spectra for homophymines B–E (2–5). Murata’s method
for homophymines B and D. See DOI: 10.1039/b910015f
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Fig. 1 New acyl units in homophymine B–E with COSY/TOCSY connectivities (bold bonds) and HMBC correlations (arrows).
purified by DCCC followed by reverse-phase HPLC (MeOH
aqueous) to give homophymines B–E (2–5) and A1–E1 (1a–5a).
Homophymine B (2) was isolated as a colourless amorphous
solid and showed the pseudomolecular ion peak at m/z 1584.9055
(M + H)+ in the HRESIMS spectrum, corresponding to the molec-
uration was secured by synthesis.7i,j Further confirmation of the
relative configuration arose from the results of J-based NMR
configurational analysis12,13 performed on intact homophymine B
(see ESI†).
HRESIMS data indicated that homophymine B1 (2a) (m/z
1583.9304 (M + H)+ corresponding to the molecular formula
C72H126N16O23) differs from 2 for the presence of a NH2 group
in the place of a OH group, therefore one of the two carboxy-
bearing amino acid residues (Asp or ADHA) in homophymine
B is in amide form in homophymine B1. This hypothesis was
confirmed by the 15N HSQC (CD3OH) experiment that evidenced
the presence of three primary amide groups (dN 108.6, dH 6.90 s
and 7.59 s; dN 109.0, dH 7.01 s and 7.69 s; dN 109.2, dH 6.83 s
and 7.19 s). The amide proton pair at dH 6.90 and 7.59 showed
HMBC correlation with an acyl group at dC 177.8 and with
an allylic methylene carbon at dC 32.4, that on the basis of
COSY and HMBC data were assigned as C6 and C7 of a
5-acyl-4-amino-2,3-dihydroxyheptanoic acid. Therefore the new
4-amino-6-carbamoyl-2,3-dihydroxyhexanoic acid was assigned
and the structure of homophymine B1 as 2a was secured by
NMR data, which was almost superimposable with those of
homophymine B (2).
The MS/MS fragmentation pattern of 2a was consistent with
the replacement of 4-amino-2,3-dihydroxy-1,7-heptandioic acid
(ADHA) with the 4-amino-6-carbamoyl-2,3-dihydroxyhexanoic
acid residue.
Homophymine A1 (1a) was analyzed for molecular formula
C73H128N16O23 by HRESIMS data m/z [M + H]+ 1597.9456.
NMR data indicated that homophymine A1 differs from ho-
mophymine A for the replacement of 4-amino-2,3-dihydroxy-
1,7-heptandioic acid (ADHA) with the 4-amino-6-carbamoyl-2,3-
dihydroxyhexanoic acid residue.
1
ular formula C72H125N15O24. The H NMR spectrum indicated a
close structural analogy with homophymine A (1).
Extensive analysis of 1D and 2D NMR spectra by comparison
with those of homophymine A evidenced the same amino acid
spin systems found in the parent compound whereas a different
b-hydroxy acyl end group was observed. A 3-hydroxy-2,4,6-
trimethylheptanoic acid (HTMHA) residue, also found as the end
group in callipeltin A2 and neamphamide A4 was inferred from
the analysis of COSY and HMBC data (Fig. 1). The connectivity
between amino acid residues was established on the basis of careful
analysis of HMBC and ROESY data acquired in CD3OH (Table 1
in ESI†) and was proved to be the same found in homophymine A.
Further confirmation of the proposed sequence arose from
MS/MS analysis performed on the intact peptide. In addition
to a pseudomolecular ion corresponding to a linear peptide
derived from ester cleavage with concomitant proton transfer
from AHDMHA to a proline residue, the ESI Q/TOF spectrum
provided several fragmentation ion peaks (b series) which were
consistent with the amino acid sequence as shown in 2 (see ESI†).
The close resemblance of the 1H and 13C NMR chemical
shifts of all the amino acid residues with the corresponding
values of homophymine A suggest that they have the same
configuration. To confirm this hypothesis, homophymine B was
subjected to the same procedures described for the stereo-
chemical characterization of the amino acid residues in homo-
phymine A. Therefore the presence of L-Leu, two D-Orn, D-Asp,
L-Pip, (3S,4R)-3,4-diMe-L-Gln, L-ThrOMe, (2R,3R,4S)-4-amino-
2,3-dihydroxy-1,7-heptandioic acid (ADHA) and (2R,3R,4R)-
2-amino-3-hydroxy-4,5-dimethylhexanoic acid (AHDMHA) was
established by Marfey’s analysis.10 The D configuration of Asp
and the L configuration of N-MeGln were determined by LC-
MS analysis of GITC derivatives.11 The D configuration of Ser
residue was determined by chiral HPLC analysis of the hydrolysate
mixture (see Experimental).
The molecular formula C74H129N15O24 of homophymine C (3),
deduced by HRESIMS, indicated the presence of one additional
carbon atom with respect to homophymine A. A careful analysis
of the NMR data, including COSY, HSQC, TOCSY, 15N HSQC
indicated the presence of the same amino acid residues found
in homophymine A and a variation of the end group. Even if
1
from H NMR and HMQC data an additional methylene group
The 1H and 13C NMR chemical shifts and the homonuclear cou-
pling constant pattern of the 3-hydroxy-2,4,6-trimethylheptanoic
acid (HTMHA) residue match with those of the corresponding
residue in callipeltin A for which the 2R,3R,4R absolute config-
with respect to homophymine A was easily detected (dC 21.3, dH
1.27, 1.42), the analysis of the proton spin system of the end
group subunit was not straightforward due to the heavy overlap
1
in the H NMR high field region and to the absence of some
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scalar coupling in the COSY spectrum (Fig. 1). The 1H and 13
C
triplet appeared in the high field region of the 1H NMR spectrum.
A spin system ascribable to a sec-butyl subunit was evidenced from
the COSY spectrum. Even if no scalar coupling was observed
between H2-7 and H-8, the new spin system as 3-hydroxy-
2,4,6,8-tetramethyldecanoic acid (HTMDA) was easily inferred
from analysis of the COSY and HMBC correlation depicted in
Fig. 1.
NMR resonances of C-1/C-6 nuclei relative to this portion were
almost superimposable with the corresponding values found for
the 3-hydroxy-2,4,6-trimethyloctanoic acid (HTMOA) residue in
homophymine A. Additionally, a n-propyl group was inferred by
COSY data that indicated a sequence CH2-CH2-CH3 (C7-C9).
Even if any COSY correlation was observed between H6 and H2-
7 diagnostic HMBC correlation H312/C7 and H28/C6 secured
the linkage of the n-propyl chain to C6. Therefore the new 3-
hydroxy-2,4,6-trimethylnonanoic acid (HTMNA) end group was
established (Fig. 1). The sequence of the amino acids, the location
of nonanoic acid derivative and the macrocyclic structure of
homophymine C (3) was established on the basis of careful analysis
of HMBC and ROESY data acquired in CD3OH (Table 2 in
ESI†) and was proved to be the same as found in homophymine
A. The stereochemistry of amino acid residues was established
as previously described for homophymine B whereas similarity
in 1H and 13C NMR shifts observed for HTMOA residue in
1 and HTMNA in 3 implied that the stereogenic centres in the
b-hydroxyacid fragment had the same configurations.
The new end group has an additional stereogenic centre with
respect with the other end groups found in homophymines. To
assign the relative configuration of the five stereogenic centres,
three contiguous and two methylene-spaced, we used Murata’s
method.12,13 The pattern of homonuclear and heteronuclear cou-
pling constants was almost superimposable to the corresponding
values found for the 3-hydroxy-2,4,6-trimethyloctanoic acid (HT-
MOA) residue in homophymine A, indicating the same 2,3-anti-
3,4-anti-4,6-syn relative configuration (Fig. 2). Stereochemistry
at positions C6 and C8 were related through the intervening C7
diastereotopic methylene protons. The low-field proton at C7
3
(H7a) showed a large (9.9 Hz, anti) JHH to H6, whereas the
3
high-field proton (H7b) showed a small (3.0 Hz, gauche) JHH
As deduced from HRESIMS data and NMR data homo-
phymine C1 (3a) (HRESIMS m/z 1611.9538 [M + H]+) differs
from the homophymine C for the replacement of 4-amino-2,3-
dihydroxy-1,7-heptandioic acid (ADHA) with the 4-amino-6-
carbamoyl-2,3-dihydroxyhexanoic acid residue.
to H6. Small 3JHH were observed between H7a/H8 and H7b/H8.
3
Additionally, small (~1 Hz) JCH were observed from H7a to C5
3
and from H6 to C8, and a large JCH (6.2 Hz) from H7b to Me
at C8 (C14) supported the relative orientation depicted (6,8-syn).
Analysis of the NOESY spectra revealed a series of correlations
that corroborate the proposed stereochemistry.
Absolute stereochemistry of amino acid residues was deter-
mined by Marfey, GITC and chiral HPLC analysis as described
for homophymine B.
As deduced from HRESIMS and NMR data homophymine
E1 (5a) (HRESIMS m/z 1639.9832 [M + H]+) differs from the
homophymine E (5) by the replacement of 4-amino-2,3-dihydroxy-
1,7-heptandioic acid (ADHA) with the 4-amino-6-carbamoyl-2,3-
dihydroxyhexanoic acid residue.
Homophymine D (4) analyzed for C75H131N15O24 by HRES-
IMS. The molecular formula indicated further C1 homologation
with respect to homophymine C, that also in this case was referred
to the end group, as the combined analysis of 2D NMR spectra
indicated the presence of the same amino acid spin systems
1
found in homophymines B and C. The analysis of the H NMR
spectrum indicated the presence of two doublet methyl signals
(0.87, d, J = 6.7 Hz and 0.91, d, J = 6.7 Hz) that replace the
methyl triplet signal at 0.89 observed in the 1H NMR spectrum of
homophymine A, suggesting the presence of an isopropyl group
as the terminal appendage of the end group. The presence of an
isopropyl group was clearly inferred by COSY data that indicated
the scalar coupling of both methyl doublets with a methine signal
at d 1.70, this latter in turn coupled with two diastereotopic
methylene protons at d 1.23 and 0.94. Starting from this methylene,
COSY, HMQC and HMBC indicated a C-1/C-6 spin system
identical to that present in homophymines A–C (Fig. 1). Therefore
the new 3-hydroxy-2,4,6,8-tetramethylnonanoic acid (HTEMNA)
end group was established. The stereochemistry of amino acid
residues was established by Marfey’s methods whereas J-based
NMR configurational analysis allowed us to define the relative
stereochemistry for HTEMEA (see ESI†).
As deduced from HRESIMS data and NMR data homo-
phymine D1 (4a) (HRESIMS m/z 1625.9773 [M + H]+) differs
from the homophymine D for the replacement of 4-amino-2,3-
dihydroxy-1,7-heptandioic acid (ADHA) with the 4-amino-6-
carbamoyl-2,3-dihydroxyhexanoic acid residue.
The HRESIMS data of homophymine E (5) (m/z 1640.9782
[M + H]+) indicated the presence of an additional carbon atom
with respect to homophymine D. Also in this case the difference
with respect to the other homophymines is the b-hydroxy acid that
acylates the N-terminus. After subtracting the signals relative to
the methyls ascribable to amino acid spin systems (Leu, ThrOMe,
DiMe-Gln, AHDMHA) four methyl doublets and one methyl
Biological evaluation
In a first attempt, the antiproliferative activity of homophymines
was determined on a wide panel of cell lines that includes human
and simian cancer and non-cancer cells. Table 1 reports the
observed IC50 values, in the range 2–100 nM, that evidenced a
potent cytotoxic activity. A comparison of the activity against
different tumor cell lines showed a moderate selectivity toward
human prostate (PC3) and ovarian (OV3) carcinoma. When
sensitive and their resistant counterpart cell lines were compared
(MCF7/MCF7R, HCT116/HCT15, HL60/HL60R), no signifi-
cant difference was observed, indicating that overexpression of P-
gp (MDR1, ABCB1) did not affect the intracellular accumulation
of homophymines and therefore evidences that they are not
substrates for the efflux pump.
Concerning the structure–activity relationship it is possible
to point that homophymines A1–E1, featuring the 4-amino-
6-carbamoyl-2,3-dihydroxyhexanoic acid residue, exert stronger
potency, when compared with the corresponding A–E in which
the same residue is in its carboxy form. This assay based on
a 3-day exposure to the compounds is a mixed figure between
antiproliferative and acute toxic activity. To discriminate between
these two biological effects, we decided to use the quiescent EPC
cell line and the IC50 values obtained (Table 1, last line) were in
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Table 1 IC50 (nM) of homophymines A–E (1–5) and A1–E1 (1a–5a) against various cell linesa
Compounds
Cell line
1
1a
7.1
12.4
13.5
6.1
13.5
30.9
5.1
2
2a
16.4
14.2
12.3
11.4
14.1
93.8
6.5
3
3a
6.8
6.3
5.4
4
4a
5
5a
KB
7.3
18.0
16.8
26.3
13.8
22.9
101.9
8.0
8.5
8.8
12.7
19.6
37.7
19.8
43.2
81.3
8.1
10.6
3.5
3.5
6.0
12.5
3.9
7.1
MCF7
MCF7R
HCT116
HCT15
HT29
OVCAR8
OV3
23.6
22.9
6.0
22.5
70.0
5.4
14.2
15.6
5.5
27.2
35.1
4.6
10.8
4.9
19.2
62.8
4.3
2.7
1.8
2.3
17.2
38.2
2.6
2.4
2.6
11.4
32.2
1.6
1.4
1.4
10.1
31.8
4.0
2.7
3.5
7.5
4.2
5.5
3.7
9.9
6.2
8.0
4.7
3.7
3.0
10.6
6.3
4.2
3.9
PC3
Vero
MRC5
HL60
HL60R
K562
PaCa
SF268
A549
MDA231
MDA435
HepG2
EPC
5.0
6.1
7.8
17.3
11.1
12.8
19.2
6.3
6.0
8.4
27.0
91.4
7.8
8.6
6.1
4.2
3.1
8.0
14.6
17.1
11.9
14.4
7.1
10.9
16.9
29.6
24.9
35.3
37.4
17.9
13.8
18.9
49.9
78.7
11.1
1.8
7.0
9.5
23.3
21.4
22.2
18.1
8.1
4.4
11.0
24.1
22.4
24.0
31.4
9.9
17.1
43.1
36.7
26.7
62.0
17.2
19.8
17.0
40.1
99.0
8.0
10.2
18.7
25.8
16.6
22.2
11.7
8.6
18.2
29.5
100.3
6.6
16.8
23.0
23.5
22.5
25.9
13.6
8.3
16.2
35.0
72.1
9.3
10.5
13.1
21.9
12.9
17.6
7.9
12.3
20.5
23.2
17.8
10.6
10.1
11.4
20.0
37.0
62.8
29.0
8.3
6.2
5.0
9.6
10.9
39.0
68.6
5.0
15.8
20.3
58.6
12.2
11.1
23.4
80.4
7.7
13.3
38.3
60.5
9.5
a The values are the mean of duplicate experiments.
Fig. 2 Relative configuration for HTMDA residue in homophymine E (5).
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the same low nanomolar range, indicating that homophymines
display a toxic rather than an antiproliferative activity.
The cell viability/number was measured as MTS reduction and
LDH release assays on HL60 cells.14 After a 24 h exposure to 20
and 50 nM homophymines, the MTS assay indicated that the cells
were only moderately affected by 20 nM homophymines, whereas
a 50 nM dose caused a marked reduction in cell viability/number
(panel A in Fig. 3).
comparable figure was obtained when cells were exposed for 48 h
to 50 or 100 nM homophymines (data not shown). This suggests
that cell death occurred rapidly after the homophymine addition.
Several cell death pathways can be explored with inhibitors:
z-VAD-fmk is a potent inhibitor of caspases3/7 which totally
prevents its activation and activity and thus irreversibly blocks
the apoptotic pathway at a dose of 50 mg/mL (data not shown).
When VAD alone was added to HL60 cells, proliferation and
viability remained unchanged. When given in association with
25 nM homophymine C1 (3a), homophymine D1 (4a) or 100 nM
homophymine B (2), VAD moderately increased the toxicity
of homophymines, suggesting that the cell death pathway is
caspase-independent (Fig. 4). Similarly, 2 mM 3-methyladenine
has been reported to abrogate autophagy but did not reduce the
toxic effect of homophymines. Lastly, necrostatin-1 is a selective
blocker of necroptosis: 50 mM necrostatin-1 had no additive
effect on the reduction of proliferation and viability elicited by
homophymines in treated HL60 cells. To confirm the lack of
effect of homophymines on caspases 3/7 activation, the catalytic
cleavage of Ac-DEVD-AMC was assayed in HL60 and treated
for 48 h with 100 nM, 1 mM and 10 mM homophymine C1,
homophymine D1 and homophymine B. At doses 100 nM and
1 mM these three homophymines had no effect, whereas at 10 mM
a moderate activation could be noticed (from 1.15 to 1.96-fold).
In the same conditions, 1 mM doxorubicine elicited a 12.8-fold
activation of caspases 3/7 activity.
All together the biological data strongly suggest that the toxic
effect of homophymines likely results from an acute direct and non
specific toxicity on various human cell lines.
Experimental
For general experimental procedures and sponge collection see
Zampella et al.8
Extraction and isolation
The lyophilized material (300 g) was extracted with methanol (4 ¥
2.5 L) at room temperature and the crude methanolic extract (70 g)
was subjected to a modified Kupchan’s partitioning procedure
as follows. The methanol extract was dissolved in a mixture
of MeOH/H2O containing 10% H2O and partitioned against
n-hexane. The water content of the MeOH extract was adjusted to
30% (v/v) and partitioned against CHCl3. The aqueous phase
was concentrated to remove MeOH and then extracted with
n-BuOH. Part of the chloroform-soluble material (ca. 2.7 g) was
fractionated by DCCC (droplet counter current chromatography,
CHCl3:MeOH:H2O 7:13:8, ascending mode) and fractions of
4 mL were collected and further purified by reverse phase
chromatography. Fraction 11 was purified by HPLC on a Vydac
C18 column (10 m, 250 ¥ 10 mm, 4 mL/min) eluting with 42%
aqueous acetonitrile in 0.1% TFA to afford homophymine A1 (1a)
(23.5 mg, tR = 7.0 min), B1 (2a) (12.6 mg, tR = 6.0 min), C1 (3a)
(25.5 mg, tR = 9.6 min), D1 (4) (54.5 mg, tR = 13.0 min). Fraction
12 was purified using the same conditions to afford homophymines
A1–D1 together A–D series: A (1) (41.8 mg, tR = 7.4 min), B (2)
(13.3 mg, tR = 6.4 min), C (3) (19.1 mg, tR = 10.6 min), D (4)
(20.1 mg, tR = 14.8 min).
Fig. 3 Effect of homophymines on the proliferation/viability of HL60
cells treated for 24 h with 20 nM (left bars) and 50 nM (right bars)
homophymines. Panel A: MTS reduction assayed in microplates. Results
are expressed as the percentage of absorbance at 490 nm compared
with cells treated with DMSO only. Values are the mean SE for three
experiments. Panel B: release of LDH assayed in the culture medium after
lysis of cells and the fluorescence units measured with em = 560 nm and
ex = 590 nm. Results are expressed in fluorescence units and values are the
mean SE for three experiments. Panel C: release of LDH assayed in the
culture medium of non-lysed cells.
A similar behaviour was observed in the LDH release by lysed
cells that was indicative of the total cells number in each well
(panel B in Fig. 3). However when the LDH release was evaluated
in non-lysed cells, no effect was observed (panel C in Fig. 3). A
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Fig. 4 Effect of VAD, necrostatin-1 and 3-methyladenine on the proliferation/viability of HL60 cells treated with homophymines. HL60 cells were plated
and treated for 72 h with 25 nM homophymine C1 (3a), 25 nM homophymine D1 (4a) or 100 nM homophymine B (2) in the absence or the presence of
50 mM VAD, 50 mM necrostatin-1 or 2 mM 3-methyladenine. Results are expressed as the percentage of absorbance at 490 nm after addition of MTS
reagent compared with cells treated with DMSO only. Values are the mean SE for three experiments.
Fraction 14 was purified by HPLC on a Vydac C18 column (10 m,
250 ¥ 10 mm, 4 mL/min) eluting with 46% aqueous acetonitrile in
0.1% TFA to afford homophymine D (4) (35.1 mg, tR = 6.0 min)
and E (5) (6.2 mg, tR = 10.4 min).
Homophymine A1 (1a). White solid; [a]D +5.21 (c 0.96,
MeOH); tabulated NMR data in ESI†; ESI(+)MS m/z 1597.9
[M + H]+; HRESI(+)MS: 1597.9456 [M + H]+ (C73H129N16O23
requires 1597.9416), accurate mass error of 3.1 ppm.
[M + H]+. HRESI(+)MS: 1625.9773 [M + H]+ (C75H133N16O23:
requires 1625.9729), accurate mass error of 2.70 ppm.
Homophymine E (5). White solid; [a]D = +5.46 (c 0.43,
MeOH); tabulated NMR data in ESI†; ESI(+)MS m/z 1640.8
[M + H]+. HRESI(+)MS: 1640.9782 [M + H]+ (C76H134N15O24:
requires 1640.9726), accurate mass error of 3.40 ppm.
Homophymine E1 (5a). White solid; [a]D = +3.18 (c 0.62,
MeOH); tabulated NMR data in ESI†; ESI(+)MS m/z 1640.0
[M + H]+. HRESI(+)MS: 1639.9832 [M + H]+ (C76H135N16O23
requires 1639.9886), accurate mass error of -3.29 ppm.
Homophymine B (2). White solid; [a]D = -1.28 (c 0.57,
MeOH); tabulated NMR data in ESI†; ESI(+)MS m/z 1584.9
[M + H]+; HRESI(+)MS: 1584.9055 [M + H]+ (C72H126N15O24
requires 1584.9100), accurate mass error of -2.84 ppm.
Determination of the absolute configuration
Homophymine B1 (2a). White solid; [a]D = -1.48 (c 0.50,
MeOH); tabulated NMR data in ESI†; ESI(+)MS m/z 1583.9
[M + H]+. HRESI(+)MS: 1583.9304 [M + H]+ (C72H127N16O23
requires 1583.9260), accurate mass error of 2.77 ppm.
General procedure for peptide hydrolysis. Peptide samples
(200 mg) were dissolved in degassed 6 ◦N HCl (0.5 mL) in an
evacuated glass tube and heated at 160 C for 16 h. The solvent
was removed in vacuo and the resulting material was subjected to
further derivatisation.
Homophymine C (3). White solid; [a]D = +5.66 (c 0.45,
MeOH); tabulated NMR data in ESI†; ESI(+)MS m/z 1612.8
[M + H]+; HRESI(+)MS: 1612.9465 [M + H]+ (C74H130N15O24:
requires 1612.9413), accurate mass error of 3.22 ppm.
General procedures for LC-MS analysis of Marfey’s (FDAA)
derivatives. A portion of the hydrolysate mixture (800 mg) or
the amino acid standard (500 mg) was dissolved in 80 mL of a 2:3
solution of TEA:MeCN and treated with 75 mL of 1% 1-fluoro-2,4-
dinitrophenyl-5-L-alaninamide (FDAA) in 1:2 MeCN:acetone.
The vials were heated at 70 ◦C for 1 h, and the contents were
neutralised with 0.2 N HCl (50 mL) after cooling to room
temperature. An aliquot of the L-FDAA derivative was dried
under vacuum, diluted with MeCN–5% HCOOH in H2O (1:1),
and separated on a Vydac C18 (25 ¥ 1.8 mm i.d.) column by
means of a linear gradient from 10% to 50% aqueous acetonitrile
containing 5% formic acid and 0.05% trifluoracetic acid, over
45 min at 1 mL/min. The RP-HPLC system was connected to
the electrospray ion source by inserting a splitter valve and the
Homophymine C1 (3a). White solid; [a]D = +4.71 (c 0.31,
MeOH); tabulated NMR data in ESI†; ESI(+)MS m/z 1612.0
[M + H]+. HRESI(+)MS: 1611.9538 [M + H]+ (C74H131N16O23:
requires 1611.9573), accurate mass error of -2.17 ppm.
Homophymine D (4). White solid; [a]D = +4.19 (c 0.36,
MeOH); tabulated NMR data in ESI†; ESI(+)MS m/z 1627.0
[M + H]+. HRESI(+)MS: 1626.9521 [M + H]+ (C75H132N15O24:
requires 1626.9570), accurate mass error of -3.01 ppm.
Homophymine D1 (4a). White solid; [a]D = +1.94 (c 0.65,
MeOH); tabulated NMR data in ESI†; ESI(+)MS m/z 1626.0
4042 | Org. Biomol. Chem., 2009, 7, 4037–4044
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flow going into the mass spectrometer source was set at a value of
100 mL/min. Mass spectra were acquired in positive ion detection
mode (m/z interval of 320–900) and the data were analyzed
using the suite of programs Xcalibur (ThermoQuest, San Jose´,
California); all masses were reported as average values. Capillary
temperature was set at 280 ◦C, capillary voltage at 37 V, tube lens
offset at 50 V and ion spray voltage at 5 V.
Retention times (min) of FDAA-amino acids are given in
parentheses: D-Pip (26.3), L-Pip (27.8), D-Leu (36.5), L-Leu (31.3),
D-alloThrOMe (23.1), L-alloThrOMe (18.3), D-ThrOMe (24.8),
L-ThrOMe (19.6), D-Orn (8.6), L-Orn (11.1).
To determine the absolute configuration of 3,4-diMeGln,
4-amino-2,3-dihydroxy-1,7-heptandioic acid (ADHA) or 4-
amino-6-carbamoyl-2,3-dihydroxyhexanoic acid and 2-amino-3-
hydroxy-4,5-dimethylhexanoic acid (AHDMHA), an authentic
sample of homophymine A was used as a standard. The hy-
drolysate of homophymine A contained: (2R,3R,4S)-4-amino-2,3-
dihydroxy-1,7-heptandioic acid (19.5), (2S,3S,4R)-3,4-diMeGlu
(20.6) and (2R,3R,4R)-2-amino-3-hydroxy-4,5-dimethylhexanoic
acid (33.5).
The hydrolysate of homophymines B–E (2–5) and of ho-
mophymines A1–E1 (1a–5a) contained: L-Pip, D-Orn, L-
Leu, L-ThrOMe, (2S,3S,4R)-3,4-diMeGlu, (2R,3R,4S)-4-amino-
2,3-dihydroxy-1,7-heptandioic acid and (2R,3R,4R)-2-amino-3-
hydroxy-4,5-dimethylhexanoic acid as determined by co-elution
with corresponding standards.
grown in D-MEM medium supplemented with 10% fetal calf
serum, in the presence of penicilline, streptomycine and fungizone
in 75 cm2 flask under 5% CO2. HCT116, HCT15, HT29 (colon
adenocarcinoma) and MCF7 (breast adenocarcinoma) were given
by Dr Matthias Kassack (Bonn University, Germany), MDA435
and MDA231 (breast adenocarcinoma), HL60 (promyeocytic
leukaemia), K562 (chronic myelogenous leukaemia) SK-OV3
(ovary adenocarcinoma from NCI), OVCAR-8 (ovary adenocarci-
noma), PC-3 (prostate adenocarcinoma), A549 (lung carcinoma),
MiaPaCa (pancreas carcinoma) and SF268 (glioblastoma from
NCI) were grown in RPMI medium. Resistant MCF7 and
resistant HL60 cells were obtained by prolonged treatment with
doxorubicin. EPC cells (carp epithelium) provided by Dr Bremond
(INRA France) were grown in Stocker-MacPherson MEM at
room temperature.
Cell proliferation assay. Cells were plated in 96-well tissue
culture plates in 200 mL medium and treated 24 h later with com-
pounds dissolved in DMSO; compound concentrations ranged
from 0.025 nM to 6 mM and were prepared by use of a Biomek 3000
(Beckman). Control cells received the same volume of DMSO (1%
final volume). After 72 h exposure to the drug, MTS reagent (Cell
titer Aqueous, Promega, Madison, WI) was added and incubated
for 3 h at 37 ◦C: the absorbance was monitored at 490 nm
and results are expressed as the inhibition of cell proliferation
calculated as the ratio [1 - (OD490 treated/OD490 control)¥100].
For IC50 determinations (50% inhibition of cell proliferation)
experiments were performed in duplicate.
LC-MS analysis of GITC derivatives. Triethylamine (10 mL)
and a GITC (2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl isothio-
cyanate) solution (50 mL, made with 3.9 mg/mL in MeCN) were
added to the acid hydrolysate (100 mg) of homophymines B–E
(2–5) and of homophymines A1–E1 (1a–5a) or homophymine
A, or an authentic amino acid standard (100 mg). The reaction
mixture was kept for 30 min at room temperature, and then the
reaction was quenched by adding 40 mL of MeCN in 5% AcOH in
H2O (1:1). An aliquot was dried under vacuum and then diluted
with the same solvent mixture and subjected to LC-MS analysis as
described above, except for monitoring the absorption at 254 nm.
Retention times (min): L-NMeGlu from homophymine A (25.3),
L-Asp (16.9), D-Asp (17.9). The hydrolysate of 2–5 and 1a–5a gave
peaks for L-NMeGlu and D-Asp.
Caspase activity assay. Caspases3/7 activity was assayed in
HL60 cell after treatment with chemicals for 48 h. HL60 cells
(20 000 cells/well in 180 mL RPMI medium) were plated in black
96-well culture microplates and treated with homophymine D1,
homophymine C1 and homophymine B at final concentrations of
100 nM, 1 mM and 10 mM. Plates were kept under 5% CO2 for
48 h. Lysis buffer (20 mL of a 10¥ stock solution), consisting of
250 mM Hepes (pH7.5), 5 mM EDTA, 0.5% NP40, 0.1% SDS
and 50 mM dithiothreitol, was added with DEVD-AMC at a
final concentration of 50 mM. Plates were incubated at 37 ◦C and
fluorescence was recorded (exc 360 nm, em 435 nm) after 0, 30, 60,
120, and 180 min. Reaction rates were calculated from the slope
of the linear time-dependent reaction and are expressed as the
fold-activation relative to the control (HL60 with DMSO alone).
10-6 M doxorubicin was used as a positive control.
Chiral HPLC analysis. The acid hydrolysate of homo-
phymines (aliquot of 10 mL) was analysed by chiral HPLC on
a Phenomenex D-penicillamine column [Chirex phase 3126 (D)
(150 ¥ 4.6 mm)]. The identity of serine in the acid hydrolysate
was confirmed by comparison of its retention times with those of
authentic standards using HPLC under the following conditions:
mobile phase, 2 mM CuSO4, flow rate 1.0 mL/min; detection UV
254 nm; retention times of the standards (min): L-Ser (5.7), D-
Ser (5.1). The hydrolysate of homophymines B–E (2–5) and of
homophymines A1–E1 (1a–5a) contained D-serine.
LDH release assay. The release of cytoplasmic LDH into the
culture medium was estimated with the Cytotox-ONE reagent
from Promega (Madison, WI). HL60 cells (20 000 cells/well
in 100 mL RPMI) were plated in 96-well culture microplates
and treated with compounds at final concentrations of 20 and
50 nM. Plates were kept under 5% CO2 for 24 and 48 h. One set
of cells was lysed with 2 mL 9% Triton X-100 for 30 min at room
temperature under orbital agitation. Lysed and non-lysed cells
were spun down for 1 min at 500 g and 25 mL supernatant removed
for LDH activity: 25 mL of reagent was added and incubated for
20 min at room temperature in the dark. The fluorescent signal was
monitored with a Paradigm microplate reader (Beckman) with
excitation set at 560 nm and emission at 590 nm.
Biological evaluation
Cell culture. Cell lines were purchased from ECACC (Sal-
isbury, UK) or ATCC (LGC Standards, Molsheim, France)
except when otherwise indicated. The human cell lines KB
(nasopharyngeal epidermoid carcinoma), HepG2 (hepatocarci-
noma), Vero (monkey kidney), MRC5 (fetal human lung) were
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T. M. Allen, R. Van Soest, R. J. Andersen and M. R. Boyd, J. Am.
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Conclusion
5 A. S. Ratnayake, T. S. Bugni, X. Feng, M. K. Harper, J. J. Skalicky,
K. A. Mohammed, C. D. Andjelic, L. R. Barrows and C. M. Ireland,
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6 A. Plaza, E. Gustchina, H. L. Baker, M. Kelly and C. Bewley, J. Nat.
Prod., 2007, 70, 1753–1760.
Homophymines A–E and A1–E1 represent further examples of
bioactive natural cyclodepsipeptides featuring a complex molec-
ular architecture. As found in some lipopeptides of bacterial
origin,15 the main distinction between the members of this family is
the length of the side chain. The PKS involved in the formation of
the homophymine side chain could be an iteratively acting enzyme
with the intrinsic capacity to produce polyketide chains of varying
length.16,17
The rationale of this observed slight structural variability in the
same sponge should be explained with the need of a modulation of
the toxic activity. In particular the replacement of OH with an NH2
group in the A1–E1 series invariably produces an improvement in
cytotoxicity.
7 (a) B. Liang, P. J. Carroll and M. M. Joullie´, Org. Lett., 2000, 2,
4157–4160; (b) N. Okamoto, O. Hara, K. Makino and Y. Hamada,
Tetrahedron: Asymmetry, 2001, 12, 1353–1358; (c) S. Chandrasekhar,
T. Ramachandar and B. Rao Venkateswara, Tetrahedron: Asymmetry,
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Hara, K. Makino and Y. Hamada, J. Org. Chem., 2002, 67, 9210–
9215; (g) A. Ravi Kumar and B. Rao Venkateswara, Tetrahedron
Lett., 2003, 44, 5645–5647; (h) V. Guerlavais, P. J. Carroll and M. M.
Joullie´, Tetrahedron: Asymmetry, 2002, 13, 675–680; (i) A. Zampella,
M. Sorgente and M. V. D’Auria, Tetrahedron: Asymmetry, 2002, 13,
681–685; (j) A. Zampella and M. V. D’Auria, Tetrahedron: Asymmetry,
2002, 13, 1237–1239; (k) J. A. Turk, G. S. Visbal and M. A. Lipton,
J. Org. Chem., 2003, 68, 7841–7844; (l) D. B. Hansen, X. Wan, P. J.
Carrol and M. M. Joullie´, J. Org. Chem., 2005, 70, 3120–3126; (m) J.
Jeon, S.-K. Hong, J. S. Oh and Y. G. Kim, J. Org. Chem., 2006, 71,
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Acknowledgements
This work was supported by grants from MIUR (PRIN 2007)
“Sostanze Naturali ed Analoghi Sintetici con Attivita` Antitu-
morale” Rome, Italy. NMR spectra were provided by the CRIAS
Centro Interdipartimentale di Analisi Strumentale, Faculty of
Pharmacy, University of Naples. We thank the diving team of IRD
in Noume´a (New Caledonia) for the collection of the sponge, Prof.
C. Le´vi (Muse´um National d’Histoire Naturelle, Paris, France) for
the identification of the sponge.
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©