Perthamides C-F, potent human antipsoriatic cyclopeptides

Article abstract of DOI:10.1016/j.tet.2011.07.077

Two new cyclopeptides, perthamides E and F were isolated from the polar extracts of the sponge Theonella swinhoei. The new structures, featuring an unprecedented β-amino acid unit (AHMOA), were determined by interpretation of NMR and MS data. The absolute

Full text of DOI:10.1016/j.tet.2011.07.077

Tetrahedron 67 (2011) 7780e7786
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Perthamides CeF, potent human antipsoriatic cyclopeptides
a
a
a
a
b
Carmen Festa , Simona De Marino , Valentina Sepe , Maria Valeria D’Auria , Giuseppe Bifulco ,
c
c
c
d
a,
*
Rosa Andr eꢀ s , Maria Carmen Terencio , Miguel Pay aꢀ , C eꢀ cile Debitus , Angela Zampella
Dipartimento di Chimica delle Sostanze Naturali, Universit aꢁ di Napoli “Federico II”, via D. Montesano 49, 80131 Napoli, Italy
Dipartimento di Scienze Farmaceutiche, Universit aꢁ di Salerno, via Ponte don Melillo, 84084 Fisciano (SA), Italy
Department of Pharmacology, University of Valencia and Center of Molecular Recognition and Technological Development (IDM), Av. V. Andr ꢀe s Estelles,
a
b
c
s/n 46100 Burjassot, Valencia, Spain
d
Institut de Recherche pour le Developpement (IRD), Polynesian Research Center on Island Biodiversity, BP529, 98713 Papeete, Tahiti, French Polynesia, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 May 2011
Received in revised form 5 July 2011
Accepted 26 July 2011
Available online 2 August 2011
Two new cyclopeptides, perthamides E and F were isolated from the polar extracts of the sponge The-
onella swinhoei. The new structures, featuring an unprecedented
b-amino acid unit (AHMOA), were
determined by interpretation of NMR and MS data. The absolute configuration of the AHMOA residue
was proposed on the basis of quantum chemical calculation of NMR chemical shifts. Perthamides were
proved to inhibit TNF-
a and IL-8 release in primary human keratinocytes cells and therefore could
represent potentially leads for the treatment of psoriasis.
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Marine compounds
Cyclic peptides
Theonella swinhoei
Anti-Inflammatory activity
1
. Introduction
acid). Perthamide C (1), when tested in a well characterised model
of inflammation in vivo, i.e., mouse paw oedema, significantly re-
duced carrageenan-induced paw oedema, displaying a dose-
dependent anti-inflammatory activity.
Psoriasis is a chronic autoimmune inflammatory skin disorder
affecting approximately 2e3% of the general population in Europe
and North America. Cutaneous and systemic overexpression of
Despite this promising activity, the mechanism of action at the
molecular level of perthamide C is unknown. Although the modular
peptidic nature and the ready chemical access to perthamide C may
open the possibility to investigate new pharmacophores, the iso-
lation of further natural derivatives represents an alternative ap-
proach to investigate the structureeactivity relationships and to
shed light on the biological target of this promise anti-
inflammatory lead. In this respect, the sponge T. swinhoei (order
Lithistida, family Theonellidae), recognized as one of the most
various proinflammatory cytokines (TNF-
a
, IL-8, IFN-
g
, etc.) has
been demonstrated in psoriatic patients. Notably it has been
postulated that TNF- produced locally in psoriatic lesions creates
a TNF- positive feedback loop that amplifies and sustains the in-
flammatory process within plaques. In fact, recently developed
anti-inflammatory therapies based on blocking TNF- signalling
1
,2
a
a
3
a
have been shown to be effective in the treatment of psoriasis and
could become a highly promising option for the treatment of this
skin condition.4
,5
8
prolific sources of bioactive secondary metabolites, could repre-
Recently, we reported the isolation and the chemical charac-
terization of two cyclic octapeptides, which we named perthamides
C (1) and D (2), from the Solomon marine sponge Theonella swin-
sent a source of useful perthamide derivatives. Continuing in-
vestigation of the polar extracts of this sponge afforded a large
amount of perthamide C together with two new derivatives, per-
thamides E (3) and F (4).
6
,7
hoei. Perthamide C has an unprecedented primary structure that
comprises a 25-membered macrocycle with 6 out the 8 residues
In this paper we describe the isolation, the structure elucidation
including the stereochemical characterization and the biological
activity of the new peptides (Fig. 1).
d
constituted by unusual amino acids:
moyl- -OSO asparagine, o-tyrosine,
-amino acid AHMHA (3-amino-2-hydroxy-6-methylheptanoic
g
-methylproline, N -carba-
b
3
D-Abu, O-methylthreonine and
the
b
2. Results and discussion
The lyophilized sponge (400 g) was extracted with MeOH, and
the combined extracts were fractionated according to the Kupchan
*
Corresponding author. Tel.: þ39 081 678525; fax: þ39 081 678552; e-mail
addresses: angela.zampella@unina.it, azampell@unina.it (A. Zampella).
0
040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.07.077

C. Festa et al. / Tetrahedron 67 (2011) 7780e7786
7781
H
2
N
O
O
H N
O
2
O
O
O
O
O
O
N
H
N
H
N
H
N
H
NH
NH
OH
N
NH
NH
OH
N
O
HN
O
O
O
HN
O
O
H
N
O
H
N
OH
O
N
NHCONH2
H
O
N
NHCONH2
O
OSO
3
OSO H
O
3
MeO
MeO
Perthamide C (1)
Perthamide D(2)
O
H
2
N
O
H
2
N
O
O
O
O
O
O
O
N
H
N
H
N
H
N
H
NH
NH
OH
N
O
NH
NH
OH
N
HN
O
HN
O
O
O
O
O
H
N
H
N
OH
O
N
NHCONH2
H
O
N
NHCONH2
O
OSO
3
O
OSO H
3
MeO
MeO
Perthamide E (3)
Perthamide F (4)
Fig. 1.
9
1
partitioning procedure. The n-BuOH extract was purified by DCCC
n-BuOH/Me CO/H O, descending mode) followed by reverse-
(AHMHA in perthamide C). Even if from H NMR and HSQC data an
additional methylene group with respect to perthamide C was
(
2
2
phase HPLC to afford perthamides CeF.
C H
easily detected (d 28.7, d 1.03,1.22), the analysis of the proton spin
Perthamide E (3) was isolated as a colourless amorphous solid
system of this amino acid unit was not straightforward due to the
1
and showed the pseudomolecular ion peak at m/z 1080.4361
heavy overlap in the H NMR high field region and to the absence of
ꢀ
1
13
[
MꢀH] in the HRESIMS spectrum, corresponding to the molecular
some scalar coupling in the COSY spectrum. The H and C NMR
resonances of C-1/C-5 nuclei relative to this portion were almost
superimposable with the corresponding values found for the 3-
amino-2-hydroxy-6-methylheptanoic acid (AHMHA) residue in
67 11
formula C45H N O18S and indicating the presence of one addi-
tional carbon atom with respect to perthamide C (1).
A careful analysis of the NMR data (Table 1), including COSY,
1
HSQC, TOCSY, indicated the presence of the same
a-amino acid
perthamide C. Additionally, inspection of H NMR spectrum in-
residues found in 1 and a variation of the
b
-amino acid unit
dicated the presence of one methyl doublet (0.74, d, J¼6.2 Hz) and
Table 1
NMR data (700 MHz, DMSO-d
6
) of perthamides E (3) and F (4)
Perthamide E (3)
(J in Hz)a
Position
Position
Perthamide F (4)
(J in Hz)a
a
a
d
H
d
C
d
H
d
C
ThrOMe
ThrOMe
1
2
3
4
OMe
NH
d
170.9
55.4
72.9
14.3
54.7
1
2
3
4
OMe
NH
d
170.9
55.4
72.9
14.4
54.5
4.91d (9.5)
4.16m
1.23d (5.9)
3.26s
4.81d (9.6)
4.14e4.22m
1.21d (5.8)
3.25s
9.01d (9.1)
8.90d (8.8)
g
1
2
3
4
5
6
MePro
g
1
2
3
4
5
6
MePro
d
170.7
63.4
36.2
33.2
53.2
15.8
d
170.7
63.3
36.3
33.5
53.2
15.6
3.89dd (11.1, 6.6)
0.71m, 2.05e2.13m
2.20e2.30m
3.39 ovl, 4.11 ovl
1.03d (5.8)
3.98dd (11.0, 6.7)
1.09 ovl, 2.14e2.18m
2.19e2.27m
3.38 ovl, 4.10 ovl
1.01d (5.7)
o-Tyr
Phe
1
2
1
2
3
d
170.6
56.4
30.7
124.3
154.6
114.5
128.0
119.9
130.5
d
170.6
55.8
36.2
137.8
128.0
128.3
126.3
128.3
128.0
4.11 ovl
2.86dd (13.2, 3.2), 2.93t (13.2)
d
4.28e4.36m
2.83dd (13.5, 3.5), 3.14 ovl
d
3
0
0
1
1
2
3
4
0
0
0
0
0
2
3
4
5
d
7.19d (7.4)
7.30t (7.4)
7.22d (7.3)
7.30t (7.4)
7.19d (7.4)
6.91d (8.0)
0
0
0
6.87d (7.8)
7.10t (7.8)
6.80t (7.3)
7.16 ovl
7.20 ovl
5
0
0
6
6
NH
NH
(continued on next page)

7782
C. Festa et al. / Tetrahedron 67 (2011) 7780e7786
Table 1 (continued )
Position
Perthamide E (3)
(J in Hz)a
Position
Perthamide F (4)
(J in Hz)a
a
a
d
H
d
C
d
H
d
C
OH
Asn
1
10.1s
OH
Asn
1
d
d
d
170.4
48.0
170.4
47.9
2
4.67e4.71m
2
4.66 br s
3
4
2.41d (16.3), 3.14dd (16.3, 4.7)
d
36.8
172.6
3
4
2.43d (16.0), 3.15 ovl
d
36.8
172.5
NH
7.19 ovl
NH
7.12 ovl
NH
2
6.52 br s, 7.83 br s
NH
2
6.55 br s, 7.80 br s
AHMOA
AHMOA
1
2
3
4
5
6
7
8
d
169.8
72.8
51.9
23.6
31.8
33.4
28.7
11.0
18.5
1
2
3
4
5
6
7
8
d
169.8
72.9
52.0
23.5
31.6
33.3
28.8
11.1
18.4
4.14 ovl
4.12 ovl
4.01t (9.9)
1.09m, 1.41e1.47m
1.06m
4.01t (9.8)
1.07 ovl, 1.36e148m
1.04m
1.17m
1.19m
1.03m, 1.22 ovl
0.76t (7.0)
0.74d (6.2)
6.37d (9.1)
4.81 br s
1.05m, 1.23m
0.77t (6.9)
0.75d (6.2)
6.43d (9.0)
4.81 br s
9
NH
OH
9
NH
OH
d-Abu
d-Abu
1
2
3
4
d
167.5
132.3
126.5
12.0
1
2
3
4
d
167.4
132.1
126.0
12.0
d
d
5.77q (6.8)
1.54d (6.8)
8.62s
5.75q (6.5)
1.54d (6.5)
8.66s
NH
NH
NMeGly
NMeGly
1
2
NMe
d
167.2
51.5
34.6
1
2
NMe
d
167.2
51.6
34.6
3.31d (17.5), 4.19d (17.5)
2.70s
3.30 ovl, 4.18d (17.0)
2.70s
d
d
N -c-
b
-OSO
3
Asn
N -c-
b
-OSO
3
Asn
1
2
3
4
d
167.5
53.0
74.9
1
2
3
4
d
167.2
52.9
75.0
5.04t (7.3)
4.59d (7.1)
d
5.01t (7.3)
4.58d (6.9)
d
170.5
170.5
b
NH
7.16 ovl
b
NH
7.15 ovl
NH
CO
NH
8.89s
d
NH
CO
NH
8.93s
d
n.o
n.o
2
7.29 br s, 7.51 br s
2
7.33 br s, 7.50 br s
Ovl: overlapped; n.o: not observed.
a
1H and 13C assignments aided by COSY, TOCSY, HSQC and HMBC experiments.
one methyl triplet (0.76, t, J¼7.0 Hz) that replaced the two highly
Therefore the new 3-amino-2-hydroxy-6-methyloctanoic acid
(AHMOA) unit, which is also unprecedented in natural peptides,
was established in perthamide E.
As shown in Fig. 3, the sequence of the amino acids in pertha-
mide E (3) was established on the basis of careful analysis of ROESY
data and was proved to be the same found in 1.
H
overlapped methyl doublet signals at d 0.77 and 0.78 observed in
1
the H NMR spectrum of 1, suggesting the presence of a terminal
appendage for this unit different from the isopropyl group located
on AHMHA.
Two spin systems, a CHeCH
8.5, C-9) and a CH eCH 1.03, 1.22,
C-8) were disclosed from COSY and HSQC data (Fig. 2). Even if no
COSY correlation was observed between the proton at 1.17 (H-6)
3
(d
H
1.17,
d
C
33.4, C-6;
d
H
0.74,
d
C
1
2
3
(d
H
d
C
28.7, C-7; 0.76,
d
H
d
C
11.0,
d
H
and protons ascribable to the methylene at C-7, the connection
between C-6 and C-7 was inferred by HMBC data as shown in Fig. 2,
defining a sec-butyl subunit. The linkage of this latter with the
H N
O
H
2
O
O
O
N
H
N
H
methylene carbon at C-5 (
diagnostic heteronuclear long range correlation between the
methyl protons at 0.74 (C-9) with the carbon resonance at 31.8
C-5). Definitive confirmation to the above unit derived from 2D-
C H
d 31.8, d 1.06) was deduced from the
H
HO
N
H
O
O
NH
H
NH
N
d
H
d
C
(
HN
O
H
TOCSY analysis that showed correlations starting from H-9 to H-3.
O
H
H H H
OH
N
O
NHCONH2
9
OH
O
OSO
3
H
8
MeO
1
6
O
HN
Fig. 3. Selected ROE correlations for perthamide E.
The close resemblance of H and 13C NMR chemical shifts of all
1
a-amino acid residues with the corresponding values of pertha-
Fig. 2. AHMOA unit in perthamides E and F with COSY/TOCSY connectivities (bold
bonds) and HMBC correlations (arrows).
mide C suggested that they have the same configuration. To confirm

C. Festa et al. / Tetrahedron 67 (2011) 7780e7786
7783
this hypothesis, 3 was subjected to the same procedures described
for the stereochemical characterization of the amino acid residues
in perthamide C,6 establishing the presence of
,7
L
-Asn,
L
-o-Tyr, cis-
L-
d
gMePro,
L
-ThrOMe, erythro-
D
-N -c-b-OSO
3
Asn and Z-
D
-Abu.
As concern AHMOA, this unit has an additional stereogenic
centre with respect to AHMHA in perthamide C. Chemical shifts and
the homonuclear coupling constant pattern of C-2/C-3 centres
closely matched with those of the corresponding centres in
AHMHA for which the 2R,3R absolute configuration was established
7
by stereoselective synthesis. The impossibility to relate this stereo-
triad to C-6 through the intervening of two methylene carbon that
showed highly overlapped proton resonances, hampered the ap-
plication of J-based analysis to define the stereochemistry at C-6.
Starting from the encouraging results recently obtained from
our research group in the solving of stereochemical ambiguities on
Fig. 5. NMR solution state representative conformations for 3a and 3b obtained by
restrained MD calculations, using ROESY data collected at 700 MHz in DMSO-d .
6
the two diastereoisomers. Through evaluation of the MAE values,
13
1
the GIAO calculated C and H NMR chemical shifts of nuclei of
AHMOA residue indicated the best fit between stereoisomer
2R,3R,6S (3b) and the experimental data.
10,11
marine natural products from T. swinhoei,
quantum chemical (QM) calculation of the NMR chemical shifts
to establish the absolute configuration of AHMOA residue in 3.
we decided to apply
1
2,13
Even though small differences in the MAE values of calculated
13
Two diastereoisomers of perthamide E (3a and 3b, Fig. 4) were
built, varying the configuration at C-6 of AHMOA residue and fixing
the absolute configuration of all residues previous established by
Marfey’s analysis with perthamide C as standard.
C chemical shifts were observed (4.2 for 3a vs 3.9 for 3b, Table 2),
1
MAE values of calculated H chemical shifts were relatively enough
different (0.40 for 3a vs 0.24 for 3b, Table 3) to propose the 2R,3R,6S
absolute configuration for AHMOA residue. Definitive confirmation
R
S
H2N
O
O
H2N
O
O
O
O
O
O
R
R
R
O
R
O
N
H
N
H
N
H
N
H
NH
NH
OH
O
N
NH
NH
OH
O
N
HN
O
HN
O
O
O
H
N
H
N
OH
OH
O
N
NHCONH2
OSO3H
O
N
NHCONH2
OSO3H
O
O
MeO
MeO
3
a
3b
Fig. 4. AHMOA-C6 epimers (3a,b) of perthamide E (3).
The adopted protocol consists of four fundamental steps: (a)
conformational search and preliminary geometry optimization of
all the significantly populated conformers of each stereoisomer; (b)
geometry optimization of all the species at MPW1PW91 level; (c)
GIAO 13C and H NMR calculations of all the so obtained structures;
to the above tentatively assigned absolute configuration could de-
rive from synthetic studies already ongoing in our laboratories.
Table 2
1
13
Experimental and calculated C NMR chemical shifts (ppm), DD [(
d
calcd
dexptl)],
ꢀ
P
13
1
DDabs [j(
d
3
calcd
ꢀd
exptl)j] and MAE (Mean average error as [j
d
exptl
ꢀd
calcdj]/n) on se-
(
d) comparison of the Boltzmann averaged C and H NMR
lected sp atoms of AHMOA residue in 3
chemical shifts calculated for each stereoisomer with those mea-
sured for perthamide E.
2
R,3R,6R
2R,3R,6S
The preliminary conformational search was performed by em-
pirical force field molecular dynamics (OPLS force field, Macro-
model 8.5 software).1 A set of distance restraints were collected
from 2D-ROESY NMR experiments (tmix¼150 ms) and used in mo-
lecular dynamics calculations (500 K) of two diastereoisomers.
Subsequently, all the conformers found were optimised at DFT
level by using the MPW1PW91 functional and 6-31G(d) basis set.
By discarding all of the conformations higher in energy than
a threshold of 10 kcal/mol from the most stable species, five major
conformers were found for stereoisomer 3a and five for 3b. These
showed a well defined macrocycle core, but differed in the con-
formational structure of the side chain of AHMOA residue (Fig. 5).
Finally, GIAO 13C and H chemical shift calculations were per-
formed at the MPW1PW91 functional and 6-31G (d,p) basis set on
each set of conformers relative to the epimers 3a,b, and calculated
dexptl
dcalcd
DD
ꢀ5.9
DDabs
dcalcd
DD
ꢀ4.5
DDabs
C2
C3
C4
C5
C6
C7
C8
72.8
51.9
23.6
31.8
33.4
28.7
11.0
18.5
78.7
58.8
31.6
38.5
34.1
30.7
14.2
18.7
5.9
6.9
8.0
6.7
0.7
2.0
3.2
0.2
33.6
77.3
53.2
36.8
31.6
36.5
32.3
14.0
21.5
4.5
1.3
13.2
0.2
3.1
3.6
3.0
3.0
31.9
4
ꢀ6.9
ꢀ8.0
ꢀ6.7
ꢀ0.7
ꢀ2.0
ꢀ3.2
ꢀ0.2
ꢀ33.6
ꢀ4.2
ꢀ1.3
ꢀ13.2
0.2
ꢀ3.1
ꢀ3.6
ꢀ3.0
ꢀ3.0
ꢀ31.5
ꢀ3.9
C9
P
[
[
d
d
exptl
ꢀ
d
d
calcd
]
P
exptl
a
ꢀ
calcd]/n
MAE
4.2
3.9
Mean average error¼P
[j
d
exptl
ꢀdcalcdj]/n.
a
1
Perthamide F (4) showed a molecular weight 16 mass units (one
15
1
by simulating the presence of DMSO (IEF-PCM), using Gaussian 09
oxygen atom) smaller than that of perthamide E (3). The H NMR
spectrum, which is very similar to that of 3, clearly revealed the
absence of the signal ascribable to exchangeable OH proton located
on the o-Tyr residue in perthamides C and E and a perturbation in
the aromatic region.
software.16 For each stereoisomer, the C and H chemical shifts
were obtained as the weighted average chemical shift values in all
of the conformers sampled by the initial conformational search.
MAE (Mean Absolute Error) parameters were obtained for each of
13
1

7784
C. Festa et al. / Tetrahedron 67 (2011) 7780e7786
Table 3
Moreover we demonstrated that perthamides C and E are
endowed with anti-inflammatoryactivitymediated viaup-regulation
1
Experimental and calculated H NMR chemical shifts (ppm), DD [(
d
calcd
dexptl)],
ꢀ
P
DDabs [j(
d
3
calcd
ꢀd
exptl)j] and MAE (Mean average error as [j
d
exptl
ꢀd
calcdj]/n) on se-
of TNF-a, a key cytokine in inflammatory skin diseases, such as
lected sp atoms of AHMOA residue in 3
psoriasis.
2
R,3R,6R
2R,3R,6S
d
exptl
d
calc.
DD
DDabs
d
calc.
DD DDabs
3
3
. Experimental section
H2
H3
H4a
H4b
H5
4.14
4.01
1.09
1.44
1.06
1.17
1.03
1.22
0.76
0.74
4.18
3.88
1.96
2.26
1.29
1.94
0.92
1.86
0.99
0.92
ꢀ0.04
0.13
0.04
0.13
0.87
0.82
0.23
0.77
0.11
0.64
0.23
0.18
4.0
4.36
4.03
1.60
1.77
1.54
1.22
1.40
1.10
0.91
0.85
ꢀ0.22
ꢀ0.02
ꢀ0.51
ꢀ0.33
ꢀ0.48
ꢀ0.05
ꢀ0.37
0.12
0.22
0.02
0.51
0.33
0.48
0.05
0.37
0.12
0.15
0.11
2.4
.1. General experimental procedures
ꢀ0.87
ꢀ0.82
ꢀ0.23
ꢀ0.77
0.11
Specific rotations were measured on a Jasco P-2000 polarimeter.
H6
The melting points were measured on a B u€ chi Melting Point B-440
apparatus. IR spectra were recorded on a PerkineElmer FT-IR 100
spectrometer. High-resolution ESIMS spectra were performed with
a Micromass QTOF spectrometer. ESIMS experiments were per-
formed on an Applied Biosystem API 2000 triple-quadrupole mass
spectrometer. NMR spectra were obtained on Varian Inova 500 and
H7a
H7b
H8
ꢀ0.64
ꢀ0.23
ꢀ0.18
ꢀ3.5
ꢀ0.15
ꢀ0.11
ꢀ2.1
H9
P
[
[
d
d
exptl
ꢀ
d
d
calcd
]
P
exptl
a
ꢀ
calcd]/n
ꢀ0.35
ꢀ0.21
MAE
0.40
0.24
1
13
Varian Inova 700 NMR spectrometers ( H at 500 and 700 MHz,
at 125 and 175 MHz, respectively) equipped with a Sun hardware,
(ppm), J in Hz, spectra referred to DMSO-d as internal standard
2.50, 39.5). HPLC was performed using a Waters 510 pump
C
Mean average error¼P
[j
d
ꢀdcalcdj]/n.
a
exptl
d
6
(d
H
d
C
Interpretation of 2D NMR data, including COSY, HSQC and HMBC
equipped with Waters Rheodine injector and a differential re-
fractometer, model 401.
data (Table 1) indicated that perthamide F (4) possesses similar
primary structure to that of perthamide E (3), with the differences
traced to substitution of o-Tyr in 3 with Phe in 4. As for in per-
1
Through-space H connectivities were evidenced using a ROESY
experiment with mixing time of 150 ms.
thamide E, the presence of
L-Asn,
L
-Phe, cis-
L-gMePro, L-ThrOMe,
d
DCCC was performed using a DCC-A (Rikakikai Co. Di Tokyo)
equipped with 250 columns (internal diameter 3 mm).
The purities of compounds were determined to be greater than
erythro-D-N -c-b-OSO Asn was established by application of Mar-
3
fey’s method,17 whereas the complete agreement of chemical shifts
and coupling constant pattern of AHMOA and
suggested the same configuration.
D-Abu residues
95% by HPLC, MS and NMR.
Starting from the anti-inflammatory activity demonstrated for
perthamide C, we decided to investigate the eventual antipsoriatic
3.2. Sponge material and separation of individual peptides
effect of these natural compounds on TNF-
a and IL-8 release in
primary human keratinocytes (PHK) cells (Fig. 6).
T. swinhoei (order Lithistida, family Theonellidae) was collected
at a depth of 22 m, on an isolated reef off the western coast of
Fig. 6. Dose-dependent inhibition of TNF-a and IL-8 production by perthamide C (1) and E (3) in PHK cells. B: non stimulated cells. C: stimulated cells. Dx: Dexamethasone.
Dexamethasone, a well known antipsoriatic drug, was used as
a reference tool. Perthamides C and E were devoid of significant
cytotoxic effects on PHK cells at concentrations up to 10 mM, as
assessed by the mitochondrial-dependent reduction of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to
Malaita Island, Solomon Islands, in July 2004. The samples were
frozen immediately after collection and lyophilized to yield 400 g of
dry mass. Taxonomic identification was performed by Dr John
Hooper at Queensland Museum, Brisbane, Australia, where
a voucher specimen is deposited under the accessing number
G322662.
The lyophilized material (400 g) was extracted with methanol
(3ꢁ1.5 L) at room temperature and the crude methanolic extract was
subjected to a modified Kupchan’s partitioning procedure as follows.
formazan (data not shown). Their ability to inhibit TNF-
release on PHK cells stimulated with 12-O-tetradecanoylphorbol-
3-acetate (TPA) was also investigated (Fig. 6). Interestingly, per-
thamide C (1) showed a dose-dependent capability to inhibit TNF-
and IL-8 release, whereas perthamide E (3) significantly inhibited
a and IL-8
1
a
The methanol extract was dissolved in a mixture of MeOH/H
containing 10% H O and partitioned against n-hexane (15.2 g). The
water content (% v/v) of the MeOH extract was adjusted to 30% and
partitioned against CHCl (5.83 g). The aqueous phase was concen-
2
O
only IL-8 release. The ability of perthamide C to inhibit both key
biomarkers upregulated in the inflammatory response of psoriatic
skin, could be interesting to provide promising antipsoriatic agents.
In conclusion in this paper we report the isolation and the
stereostructural characterization of two new cyclic peptides from
the marine sponge T. swinhoei.
2
3
trated to remove MeOH and then extracted with n-BuOH (6.0 g).
The n-BuOH extract (6.0 g) was chromatographed in three runs
by DCCC using n-BuOH/Me CO/H O (3:1:5) in the descending mode
2 2

C. Festa et al. / Tetrahedron 67 (2011) 7780e7786
7785
(
4
the upper phase was the stationary phase), flow rate 8 mL/min;
mL fractions were collected and combined on the basis of their
a standard. The hydrolysate of perthamide F contained
(45.0 min).
L-Phe
similar TLC retention factors.
Fractions 6e12 (1.1 g) were purified in many runs by HPLC on
3.5. Computational details
a reverse phase C-12 Jupiter Proteo column (Phenomenex, 4
m,
2
50ꢁ4.6 mm, 1.0 mL/min), eluting in isocratic mode with 59%
Molecular mechanics (MM) calculations were performed using
MeOH/H
2
O (0.1% TFA) to afford 265.3 mg of perthamide C
¼6.9 min).
Fractions 13e14 (113 mg) were purified by HPLC on a reverse
phase C-12 Jupiter Proteo column (Phenomenex, 4
, 250ꢁ4.6 mm,
.0 mL/min), with 59% MeOH/H O (0.1% TFA). The peak at
¼6.9 min was analyzed by H NMR and revealed a mixture of
perthamides E and F. This mixture was purified by HPLC on Vydac
C-18 column (5 O as
, 250ꢁ4.6 mm,1.0 mL/min) with 24% MeCN/H
eluent to give 2.3 mg of perthamide E (t
¼6.3 min) and 1.7 mg of
perthamide F (t
¼8.4 min).
14
MacroModel 8.5 and the MMFFs force field OPLS. MonteCarlo
Multiple Minimum (MCMM) method (10,000 steps) was used in
order to extensively explore the conformational space. All the
structures, so obtained, were optimised using the Polak-Ribiere
Conjugated Gradient algorithm (PRCG, 1000 steps, maximum de-
rivative less than 0.05 kcal/mol). The initial geometries of the
minimum energy conformers were optimised at the hybrid DFT
MPW1PW91 level using the 6-31G(d) basis set (Gaussian 09 soft-
(t
R
¼3.5 min) and 10.5 mg of perthamide E (t
R
m
1
2
1
t
R
m
2
16
13
1
R
ware). GIAO C and H NMR chemical shifts were performed
using the MPW1PW91 functional, the 6-31G(d, p) basis set and
DMSO as solvent, using as input the geometry previously optimised
at MPW1PW91/6-31G(d) level.
R
3
.3. Characteristic data for each peptide
2
2
3
.3.1. Perthamide C (1). White amorphous solid; ½
a
ꢂ
ꢀ87.7 (c 0.87,
D
3.6. Anti-inflammatory assays
ꢀ
1
MeOH); IR
n
max (KBr disc)/cm 3395, 2932, 1665, 1618, 1522, 1457,
234, 1130, 1083, 1028; mp 210.8e214.6 C; HRESIMS m/z
ꢃ
1
1
3.6.1. Isolation, culture and stimulation of primary human keratino-
066.4106 [MꢀH]ꢀ (calcd for C44
64 11
H N O18S: 1066.4151).
cytes (PHK). Foreskins from healthy young males were the source
of primary human keratinocytes. All protocols and procedures were
approved by the Institutional Ethical Committee. Isolation of ker-
atinocytes was performed essentially as described by Boyce and
Ham; briefly, skin samples were washed, cut, scraped, and treated
with a disperse solution to prepare a largely epidermal layer. The
tissue was then trypsinized, filtered, centrifuged, and the resultant
2
2
3
0
1
.3.2. Perthamide E (3). White amorphous solid; ½
a
ꢂ
ꢀ199.6 (c
D
ꢀ
1
.22, MeOH); IR nmax (KBr disc)/cm 3395, 2950, 1670, 1618, 1533,
457, 1251, 1130, 1080, 1031; mp 204.1e211.6 C; H and C NMR
data in DMSO-d given in Table 1; HRESIMS m/z 1080.4361 [MꢀH]
ꢃ
1
13
18
ꢀ
6
(
calcd for C45H N O18S m/z 1080.4308).
66 11
keratinocytes cultured and seeded as required. The cells were in-
2
2
3
.3.3. Perthamide F (4). White amorphous solid; ½
a
ꢂ
ꢀ10.5 (c 0.07,
D
ꢃ
cubated at 37 C in a humidified atmosphere of 5% CO
free low-Ca (<0.1 mM) defined Keratinocyte-SFM (Invitrogen).
2
in a serum-
1
13
MeOH); H NMR and C NMR data in DMSO-d
6
given in Table 1;
H N O17S m/z
66 11
2
þ
ꢀ
HRESIMS m/z 1064.4313 [MꢀH] (calcd for C45
The medium was changed every other day. For all experiments,
1
3
3
064.4359).
cells were seeded at passage numbers 1e3 into 24-well plates
5
(
25ꢁ10 cells per well) and were treated upon reaching 70e90%
.4. Determination of amino acid absolute configuration
.4.1. Hydrolysis of perthamides. Perthamides (500 g) CeF were
dissolved, respectively, in 0.5 mL of 6 N HCl in three evacuated glass
tubes and heated at 160 C for 16 h. The solvent was removed in
confluence. Before the experiments, cells were grown in basal
medium without growth factors for 24 h to avoid the effects of
supplements in the growth medium and to obtain quiescent cells
m
with low levels of activated NF-
stimulus, the basal medium was renewed (300
k
B. Prior to the addition of the
L) and the cells
ꢃ
m
vacuo and the resulting material was subjected to further
derivatisation.
were subjected to 30 min pretreatment with perthamide C or
perthamide E. 12-O-Tetradecanoylphorbol-13-acetate (TPA) (Sig-
maeAldrich) 1 mg/mL was then added to the culture and the cells
3
.4.2. LCeMS analysis of Marfey’s (FDAA) derivatives. A portion of
the hydrolysate mixture (300 g) was dissolved in 80 L of a 2:3
solution of TEA/MeCN and this solution was then treated with 75
were incubated for 7 h after which the supernatant was removed
m
m
ꢃ
and stored at 20 C until further use. The MTT assay was performed
mL
to the remaining cells to test the effect of the compounds on cell
viability.
of 1% 1-fluoro-2,4-dinitrophenyl-5-
MeCN/Me
L
-alaninamide (
L
-FDAA) in 1:2
ꢃ
2
CO. The vials were heated at 70 C for 1 h, and the
contents were neutralised with 0.2 N HCl (50
room temperature. An aliquot of the -FDAA derivative was dried
under vacuum, diluted with MeCN-5% HCOOH in H O (1:1) and
separated on a Proteo C18 column (25ꢁ1.8 mm i.d.) by means of
a linear gradient from 10% to 50% aqueous MeCN containing 5%
formic acid and 0.05% trifluoroacetic acid, over 45 min at 0.15 mL/
min. The RP-HPLC system was connected to the electrospray ion
source by inserting a splitter valve and the flow going into the mass
mL) after cooling to
3
.7. Cytotoxicity evaluation (MTT assay)
L
2
Cytotoxicity of the tested compounds was determined by the 3-
(
(
4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
MTT) uptake method as described. Briefly, at the end of the in-
bromide
19
cubation period (7 h), culture medium was withdrawn. The cell
culture was incubated for 1 h at 37 C and in the dark with 200
MTT at 0.5 mg/mL in basal medium. Formazan violet crystals, in-
duced by MTT cleavage by mitochondrial enzymes, were dissolved
in DMSO and analyzed by spectrophotometry at
ꢃ
mL of
spectrometer source was set at a value of 100
were acquired in positive ion detection mode (m/z interval of
20e900) and the data were analyzed using the suite of programs
mL/min. Mass spectra
l
¼490 nm.
3
Xcalibur; all masses were reported as average values. The capillary
ꢃ
temperature was set at 280 C, capillary voltage at 37 V, tube lens
3.8. ELISA assays
offset at 50 V and ion spray voltage at 5 V.
All FDAA derivatives of amino acid residues in perthamide E
were co-eluted with the corresponding ones in perthamide C.
To determine the absolute configuration of Phe residue in per-
thamide F, an authentic sample of perthamide D was used as
Human TNF-a levels in cell supernatants were measured using
6
an ELISA kit from R&D Systems. IL-8 ELISA kit was from Bender
MedSystems. Both ELISA analyses were performed in accordance
with the manufacturer’s instructions.

7786
C. Festa et al. / Tetrahedron 67 (2011) 7780e7786
3
.9. Statistical analysis
6. Festa, C.; De Marino, S.; Sepe, V.; Monti, M. C.; Luciano, P.; D’Auria, M. V.;
Debitus, C.; Bucci, M.; Vellecco, V.; Zampella, A. Tetrahedron 2009, 65,
0424e10428.
. Sepe, V.; D’Auria, M. V.; Bifulco, G.; Ummarino, R.; Zampella, A. Tetrahedron
1
The results are presented as meanSEM and n represents the
7
number of experiments. The level of statistical significance was
determined by analysis of variance (ANOVA) followed by Dunnett’s
t-test for multiple comparisons.
2010, 66, 7520e7526.
. Wegerski, C. J.; Hammond, J.; Tenney, K.; Matainaho, T.; Crews, P. J. Nat. Prod.
8
2
007, 70, 89e94.
. Kupchan, S. M.; Britton, R. W.; Ziegler, M. F.; Sigel, C. W. J. Org. Chem. 1973, 38,
78e179.
0. Festa, C.; De Marino, S.; Sepe, V.; D’Auria, M. V.; Bifulco, G.; Debitus, C.; Bucci,
M. R.; Vellecco, V.; Zampella, A. Org. Lett. 2011, 13, 1532e1535.
9
1
1
Acknowledgements
1
1. De Marino, S.; Sepe, V.; D’Auria, M. V.; Bifulco, G.; Renga, B.; Petek, S.; Fiorucci,
S.; Zampella, A. Org. Biomol. Chem. 2011, 9, 4856e4862.
12. Bifulco, G.; Dambruoso, P.; Gomez-Paloma, L.; Riccio, R. Chem. Rev. 2007, 107,
NMR spectra were provided by the CSIAS, Centro Inter-
dipartimentale di Analisi Strumentale, Faculty of Pharmacy, Uni-
versity of Naples. We thank the Solomon Islands government for
collection permits, the Fisheries Department, R. Sulu (University of
the South Pacific in Honiara) for their help and assistance, and Dr.
John Hooper for the identification of the sponges. R.M.A. was the
recipient of a research fellowship from Generalitat Valenciana
3
744e3779.
3. Di Micco, S.; Chini, M. G.; Riccio, R.; Bifulco, G. Eur. J. Org. Chem. 2010,
411e1434.
14. MacroModel, Version 8.5; Schr o€ dinger LLC: New York, NY, 2003.
5. Cances, E.; Mennucci, B.; Tomasi, J. J. Chem. Phys. 1997, 107, 3032e3041.
6. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.;
1
1
1
1
Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Na-
katsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng,
G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.;
Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery,
J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K.
N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.;
Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene, M.;
Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.;
Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.;
Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dan-
(
BFPI/2009/145). This work was supported in part by the Spanish
Ministry of Science and Innovation (SAF2009-10347).
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