New cytotoxic callipeltins from the Solomon Island marine sponge Asteropus sp.

Article abstract of DOI:10.1016/j.tet.2016.09.016

Four new callipeltin A derivatives (N–Q) have been isolated from the Solomon Island marine sponge Asteropus sp. Their structures were established by spectroscopic techniques followed by acid hydrolysis and derivatisation of the free amino acids, and subsequent LCMS analysis of the derivatives. The compounds were evaluated for their activity against cancer cell lines A2058 (melanoma), HT-29 (colorectal adenocarcinoma) and MCF-7 (breast adenocarcinoma) and non-malignant MRC-5 fibroblast cells. While the acyclic callipeltins P and Q were inactive the cyclic callipeltins N and O showed significant cytotoxicity against all exposed cell lines with IC₅₀values as low as 0.16?μM confirming the role of cyclic configuration in biological activity.

Full text of DOI:10.1016/j.tet.2016.09.016

Accepted Manuscript
New cytotoxic callipeltins from the Solomon Island marine sponge Asteropus sp.
Marc Stierhof, Kine Østnes Hansen, Mukesh Sharma, Klaus Feussner, Karolina
Subko, Fernando Fernandez Diazrullo, Johan Isaksson, Ignacio Pérez-Victoria, David
Clarke, Espen Hansen, Marcel Jaspars, Jioji N. Tabudravu
PII:
S0040-4020(16)30923-1
10.1016/j.tet.2016.09.016
TET 28087
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 13 June 2016
Revised Date: 26 August 2016
Accepted Date: 9 September 2016
Please cite this article as: Stierhof M, Hansen KE, Sharma M, Feussner K, Subko K, Diazrullo
FF, Isaksson J, Pérez-Victoria I, Clarke D, Hansen E, Jaspars M, Tabudravu JN, New cytotoxic
callipeltins from the Solomon Island marine sponge Asteropus sp., Tetrahedron (2016), doi: 10.1016/
j.tet.2016.09.016.
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ACCEPTED MANUSCRIPT
Graphical Abstract
New cyctoxic callipeltins from the Solomon
Leave this area blank for abstract info.
Island marine sponge Asteropus sp.
Marc Stierhofa, Kine Østnes Hansenb, Mukesh Sharmac, Klaus Feussnerc, Karolina Subkoa, Fernando Fernandez
Diazrullod, Johan Isakssone, Ignacio Pérez-Victoriaf, David Clarkeg, Espen Hansenb, Marcel Jasparsa, Jioji N.
Tabudravua
aMarine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, AB24 3UE, Scotland, UK
bMarbio, UiT the Arctic University of Norway, N-9037 Tromsø, Norway
cInstitute of Applied Sciences, Faculty of Science, Technology and Environment, The University of the South
Pacific, P.O Box 1168, Suva, Fiji Islands
dInstitute of Medical Sciences, University of Aberdeen, AB24 3UE, Scotland, UK
eDepartment of. Chemistry, UiT the Arctic University of Norway, Realfagsbygget, Breivika, 9037 Tromsø,
Norway
fFundación MEDINA, Parque Tecnológico de la Salud, Av. Conocimiento 34 - 18016 Granada, Spain
gSchool of Chemistry, Joseph Black Building, University of Edinburgh, David Brewster Road, Edinburgh, EH9
3FJ, UK

ACCEPTED MANUSCRIPT
2
Tetrahedron
Tetrahedron
journal homepage: www.elsevier.com
New cytotoxic callipeltins from the Solomon Island marine sponge Asteropus sp.
Marc Stierhofa, Kine Østnes Hansenb, Mukesh Sharmac, Klaus Feussnerc, Karolina Subkoa, Fernando
Fernandez Diazrullod, Johan Isakssone, Ignacio Pérez-Victoriaf, David Clarkeg, Espen Hansenb, Marcel
Jasparsa, Jioji N. Tabudravua
aMarine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, AB24 3UE, Scotland, UK
bMarbio, UiT the Arctic University of Norway, N-9037 Tromsø, Norway
cInstitute of Applied Sciences, Faculty of Science, Technology and Environment, The University of the South Pacific, P.O Box 1168, Suva, Fiji Islands
dInstitute of Medical Sciences, University of Aberdeen, AB24 3UE, Scotland, UK
eDepartment of. Chemistry, UiT the Arctic University of Norway, Realfagsbygget, Breivika, 9037 Tromsø, Norway
fFundación MEDINA, Parque Tecnológico de la Salud, Av. Conocimiento 34 - 18016 Granada, Spain
gSchool of Chemistry, Joseph Black Building, University of Edinburgh, David Brewster Road, Edinburgh, EH9 3FJ, UK
ARTICLE INFO
ABSTRACT
Four new callipeltin A derivatives (N-Q) have been isolated from the Solomon Island marine sponge
Asteropus sp. Their structures were established by spectroscopic techniques followed by acid
hydrolysis and derivatisation of the free amino acids, and subsequent LCMS analysis of the
derivatives. The compounds were evaluated for their activity against cancer cell lines A2058
(melanoma), HT-29 (colorectal adenocarcinoma) and MCF-7 (breast adenocarcinoma) and non-
malignant MRC-5 fibroblast cells. While the acyclic callipeltins P and Q were inactive the cyclic
callipeltins N and O showed significant cytotoxicity against all exposed cell lines with IC50 values as
low as 0.16 µM confirming the role of cyclic configuration in biological activity.
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
Callipeltin
Asteropus
NMR
LCMS
2016 Elsevier Ltd. All rights reserved
.
Derivatisation
Antiproliferative activity
———
Corresponding author. Tel.: +44-1224-273105; fax: +44-1224-272921; e-mail: j.tabudravu@abdn.ac.uk

ACCEPTED MANUSCRIPT
1.0 Introduction
and showed the doubly charged molecular ion at m/z 766.4563
[M+2H]2+ in the HRESIMS spectrum corresponding to the
molecular formula C70H120N18O20 (∆ +2.5 ppm). Extensive
analysis of 2D NMR data of COSY, Edited-HSQC,
HSQCTOCSY, HMBC and by comparison of chemical shifts
with those of callipeltin A1 revealed one residue each of alanine
(Ala), β-methoxytyrosine (β-MeOTyr), 3,4-dimethylglutamine
(3,4-diMeGln), N-methylglutamine (NMeGln), leucine (Leu),
arginine (Arg), threonine (Thr), 4-amino-7-guanidino-2,3-
dihydroxy heptanoic acid (AGDHE), and 3-hydroxy-2,4,6-
trimethylheptanoic acid (TMHEA). There was however one
amino acid that that was different from components of known
callipeltins. This was the amino acid homoproline (Hpr).
Identification of homoproline was carried out using
HSQCTOCSY correlations between the α-carbon at 51.8 ppm
and the methylene protons at 2.13/1.58, 1.66/1.50, 1.64/1.47 and
3.78/2.82 ppm respectively indicating that the carbons and
associated protons were part of the same spin system. The
deshielded methylene group at δH 3.78/2.82 ppm and δC 43.2
ppm suggest that this carbon is attached to a nitrogen atom to
form a ring thus accounting for the correct number of double
bond equivalents in the molecule. Even though homoproline has
been reported here for the callipeltins for the first time it has been
known to be part of the structurally related neamphamide,5
homophymine6 and papuamide.7
Marine invertebrate derived cyclic peptides continue to attract
considerable attention due to their strong anticancer and antiviral
activities.1,2 For example aplidine (dehydrodidemnim B) derived
from the ascidian Aplidium albicans, is currently in clinical trials
phase III for multiple myeloma and in phase II trials for T-cell
lymphomas.3 A group of related sponge derived cyclic peptides
such as the callipeltins4 from Latrunculia sp. the neamphamides5
from Neamphius huxleyi, homophymines6 from Homophymia sp.,
papuamides and theopapuamides7,8 from Theonella mirabilis and
T. swinhoei respectively, contain unusual non-proteinogenic units
such as (2S,3S,4R)-3,4-dimethylglutamine (3,4-diMeGln),
(2S,3R)-β-methoxytyrosine (β-OMeTyr), (2R,3R,4S)-4-amino-
7-guanidino-2,3-dihydroxyheptanoic acid (AGDHA) and
(2R,3R,4R)-3-hydroxy-2,4,6-trimethylheptanoic acid (HTMHA)
suggesting a common biosynthetic origin.
A sponge collected from a reef in the central province of the
Solomon Islands in 2006 was identified as Asteropus sp. A
methanolic extract of this sponge showed strong inhibition
against amphotericin B resistant (ATCC90873) and wild type
Candida albicans (ATCC32354). A bioassay-guided isolation of
this extract resulted in the isolation of cyclic callipeltins N and O
as the main components. Also isolated in smaller quantities were
their linear forms named callipeltin P and Q respectively.
The sequence of amino acids in callipeltin N (1) was determined
by HMBC correlations (Figure 1). It was consistent with those
found in callipeltin A1 with the exception of N-methylalanine
which has been replaced by homoproline in the structure of
callipeltin N (1).
The effects of callipeltin N – Q on in vitro survival of human
cancer cell lines A2058 (melanoma), HT-29 (colorectal
adenocarcinoma) and MCF-7 (breast adenocarcinoma), as well as
the non-malignant human fibroblast cell line MRC-5 were
evaluated.
The absolute stereochemistry of
D
-alanine,
D
-arginine,
D-
2.0 Results and Discussion
threonine, -homoproline and -leucine in callipeltin N (1) was
L
L
determined by complete acid hydrolysis of 1 and Marfey’s
derivatisation of the resulting amino acids using 1-fluoro-2,4-
dinitrophenyl-5-L-leucinamide (FDLA)12 and the comparison of
the LCMS data of the derivatives of 1 with the FDLA derivatives
of corresponding standards. The configurations of the remaining
The frozen sponge sample was thawed and extracted 3 times with
MeOH followed by dichloromethane. The combined crude
extract (4.2 g) was partitioned using a modified Kupchan
method9,10 followed by reversed phase HPLC to yield callipeltins
N-Q. Callipeltin N (1) was obtained as a white amorphous solid
of callipeltin N except for the presence of 3-methylglutamine
replacing 3,4-dimethylglutamine in callipeltin N (1) (Figure 2).
stereocentres have not been determined and are assumed to be the
same as in callipeltin A based on similarities of 1H and 13C NMR
chemical shifts.1
The stereochemistry of the amino acids was determined to be D-
alanine, D-arginine, L-homoproline, D-threonine, and L-leucine by
Callipeltin O (2) was obtained as a white amorphous solid and
showed the doubly charged molecular ion at m/z 759.4392
[M+2H]2+ (∆ +1.7 ppm) in the HRESIMS spectrum
corresponding to the molecular formula C69H118N18O20. The
primary structure of callipeltin O (2) was determined by
extensive analysis of 2D NMR data and was consistent with that
Marfey’s method.12 The configurations of the remaining
stereocentres of callipeltin O have not been determined and all
except 3-MeGln are assumed to be the same as in callipeltin A
based on similarities of 13C NMR chemical shifts.1

ACCEPTED MANUSCRIPT
4
Tetrahedron
Table 1. 1H and 13C NMR data (CD3OD) for compounds 1, 2, 3 and 4
aa/C-pos
1
aa/position
2
C-pos
3
C-pos
4
a
b
a
b
c
d
c
d
δH
δC
δH
δC
δH
δC
δH
δC
HPr
HPr
HPr
HPr
α
5.25 d (4.5)
2.13 bm, 1.58
ovl.
1.66 ovl., 1.50
ovl.
1.64 ovl., 1.47
d (8.1)
3.78 d (12.9),
2.82 m
51.8
25.1
19.9
24.9
α
5.24 d (5.7)
2.13 d (13.4), 1.58
ovl.
51.9
25.1
19.9
24.9
α
5.27 ovl.
2.34 d (13.8),
1.67 ovl.
1.77 ovl., 1.43 d
(4.0)
1.74 ovl., 1.53 d
(12.8)
52.2
26.2
20.5
24.8
α
5.29 ovl.
2.33 d (13.8), 1.67
ovl.
1.76 ovl., 1.44 d
(4.0)
1.72 ovl., 1.53 d
(12.8)
52.2
26.4
20.6
24.8
β
γ
δ
β
γ
δ
β
γ
δ
β
γ
δ
1.67 ovl., 1.46 ovl.
1.65 ovl., 1.46 ovl.
3.81 d (12.2), 2.87
m
4.12 ovl., 3.27
ovl.
ε
43.2
ε
43.3
ε
43.7
ε
4.11 ovl., 3.26 ovl.
43.8
CO
169.7
CO
169.7
COOH
β-MeOTyr
α
172.3
COOH
β-MeOTyr
α
172.2
β-MeOTyr
α
β-MeOTyr
α
4.95 d (9.3)
4.48 d (9.4)
51.8
82.7
4.99 d (9.5)
4.48 d (9.4)
51.8
82.7
5.27 ovl.
4.33 ovl.
52.4
83.7
5.29 ovl.
4.33 ovl.
52.5
83.7
β
β
β
β
C4
128.1
129.5
114.7
157.2
C4
128.1
129.5
114.5
157.2
C4
128
C4
127.9
129.1
114.8
158.0
C5/C9
C6/C8
C7
7.21 d (8.4)
6.76 d (8.3)
C5/C9
C6/C8
C7
7.22 d (8.3)
6.78 d (8.5)
C5/C9
C6/C8
C7
7.21 d (8.4)
6.80 d (8.2)
129.1
114.8
157.5
C5/C9
C6/C8
C7
7.20 d (8.4)
6.79 d (8.2)
OH
n.a.
OH
n.a.
OH
n.a.
OH
n.a.
OMe
CONH
3.13 s
55.57
169.7
OMe
CONH
3.13 s
55.6
OMe
CONH
3.17 s
55.7
OMe
3.17 s
55.7
8.37 d (10.0)
8.33 d (10.1)
170.0
7.63 d (9.8)
170.6
CONH
N-
7.63 d (9.8)
170.6
N-MeGln
N-MeGln
N-MeGln
MeGln
4.81 dd (9.4,
6.1)
1.58 m, 1.46 d
(7.3)
α
β
55.1
23.9
α
β
4.80 dd (8.9, 6.5)
1.59 ovl., 1.51 ovl.
55.3
24.0
α
β
4.94 m
1.94 ovl., 1.69
ovl.
55.9
23.3
α
β
4.93 m
55.9
23.3
1.92 ovl., 1.70 ovl.
1.81 ovl., 1.63
ovl.
γ
30.9
29.2
γ
1.80 ovl., 1.63 ovl.
31.0
29.3
γ
1.98 m
2.82 s
n.a.
31
29.7
γ
1.97 m
2.81 s
n.a.
31.1
29.9
NMe3
CONH
CONH2
Leu
2.97 s
n.a.
NMe3
CONH
CONH2
Leu
2.98 s
n.a.
NMe3
CONH
CONH2
Leu
α
NMe3
CONH
CONH2
Leu
169.8
175.9
169.9
175.9
169.2
176.1
169.2
176.2
n.a.
n.a.
n.a.
n.a.
α
4.59 m
1.72 ovl., 1.29
d (6.7)
49.5
α
4.60 m
1.75 ovl., 1.29 d
(6.6)
49.4
4.73 d (9.6)
1.70 ovl., 1.44 d
(7.4)
48.5
α
4.72 d (9.6)
1.69 ovl., 1.43 d
(7.4)
48.7
β
38.5
24.7
β
38.5
24.7
22.2
19.9
173.3
β
39.2
24.5
β
39.2
24.6
γ
1.72 ovl.
γ
1.73 ovl.
γ
1.69 ovl.
γ
1.69 ovl.
Me-δ
Me-ε
CONH
Arg
α
0.94 d (6.7)
0.90 d (6.4)
7.28 d (7.6)
22.2
Me-δ
Me-ε
CONH
Arg
α
0.95 d (6.5)
0.91 d (6.4)
7.28 d (7.8)
Me-δ
Me-ε
CONH
Arg
α
0.96 d (6.6)
0.94 d (6.4)
8.00 d (8.4)
22.4
Me-δ
Me-ε
CONH
Arg
α
0.96 d (6.7)
0.93 d (6.5)
8.00 d (7.8)
22.3
19.9
20.1
20.2
173.3
174.1
174.2
4.36 ovl.
1.91 m, 1.63
ovl.
51.9
4.35 ovl.
52.0
4.37 d (4.8)
1.93 ovl., 1.71
ovl.
52.7
4.37 d (4.8)
52.7
β
26.8
24.9
β
1.94 m, 1.65 ovl.
1.58 ovl.
26.8
24.8
β
28
25
β
1.93 ovl., 1.65 ovl.
1.69 ovl.
28.1
25.0
γ
1.63 ovl
γ
γ
1.72 ovl.
3.20 ovl.
7.43 ovl.
8.22 d (7.2)
γ
δ
3.14 ovl.
7.32 t (5.7)
7.76 ovl.
40.6
δ
3.15 ovl.
40.6
δ
40.4
157.2
172.4
δ
3.2 ovl.
40.4
Guan
CONH
Thr-1
α
157.2
172.6
Guan
CONH
Thr-1
α
7.31 t (5.8)
7.84 d (8.4)
157.1
172.5
Guan
CONH
Thr-1
α
Guan
CONH
Thr-1
α
7.43 ovl.
157.2
172.3
8.23 d (7.4)
3.90 ovl.
4.31 ovl.
1.27 d (6.2)
n.a.
62.3
65.5
18.8
3.94 ovl.
4.29 ovl.
1.28 d (6.7)
n.a.
62.2
65.6
18.9
4.24 d (6.4)
4.09 d (7.4)
1.31 d (5.9)
n.a.
59.8
67.1
19.1
4.23 d (6.5)
4.09 d (7.4)
1.31 d (5.1)
n.a.
59.8
67.1
19.2
β
β
β
β
γ
γ
γ
γ
OH
OH
OH
OH
CONH
8.32 bs
170.6
CONH
8.21 d (4.4)
170.7
CONH
8.22 d (7.2)
171.3
CONH
8.23 d (7.4)
171.5

ACCEPTED MANUSCRIPT
Table 1 (continued)
aa/C-pos
1
aa/position
2
C-pos
3
C-pos
4
a
b
a
b
c
d
c
d
δH
δC
δH
δC
δH
δC
δH
δC
Thr-2
Thr-2
Thr-2
Thr-2
α
5.41 m
55.2
70.9
α
5.36 d (8.9)
5.58 m
55.3
70.8
α
4.38 d (6.3)
4.13 ovl.
1.27 d (6.4)
n.a.
59
67
19
α
β
γ
4.38 d (6.3)
4.13 ovl.
1.27 d (6.4)
n.a.
59.1
67.1
19.0
β
5.57 m
β
β
γ
1.2 ovl.
8.84 d (9.9)
13.8
γ
1.22 d (4.3)
8.76 d (9.7)
13.3
γ
CONH
171.8
CONH
171.6
CONH
171.7
CONH
3-
3,4-diMeGln
3-MeGln
3,4-diMeGln
MeGln
α
4.18 d (9.7)
2.39 m
57.6
36.4
14.1
α
4.22 m
59.3
31.9
16.2
α
4.44 d (4.3)
2.23 m
55.6
38.2
12.4
α
4.44 d (4.3)
2.55 m
57.3
32.9
15.9
β
β
2.59 ovl.
β
β
β-Me
1.21 ovl.
β-Me
1.21 d (4.5)
2.52 dd (14.4,
5.4)
β-Me
1.03 d (7.2)
β-Me
1.06 d (6.9)
2.40 dd (14.4,
5.8)
γ
2.75 m
41.9
γ
38.8
γ
2.67 ovl
40.6
γ
38.1
2.31 dd (14.4,
7.4)
2.20 m
γ-Me
1.27 d (6.7)
n.a.
13.2
179.4
172.3
n.a.
γ-Me
1.20 d (7.3)
8.66 d (8.2)
14.4
178.6
172.1
n.a.
CONH2
CONH
AGDHE
α
CONH2
CONH
AGDHE
α
n.a.
176.2
172.1
CONH2
CONH
AGDHE
α
CONH2
CONH
AGDHE
α
8.36 s
n.a.
176.1
171.9
9.36 d (3.0)
8.81 d (5.7)
3.91 d ovl.
3.61 dd (9.0,
1.8)
71.2
3.93 ovl.
3.62 dd (8.6,
2.0)
71.3
4.00 d (7.7)
3.72 dd (7.5,
2.9)
71.5
4.00 d (7.4)
71.5
β
γ
73.9
49.5
β
γ
73.7
49.5
β
γ
74
β
γ
3.71 d (7.5)
4.15 ovl.
74.0
49.6
4.17 ovl.
1.71 ovl., 1.57
ovl.
4.18 m
1.70 ovl., 1.58
ovl.
4.16 ovl.
49.5
δ
28.5
25.2
40.8
δ
28.5
25.1
40.8
δ
1.66 ovl.
1.63 ovl.
3.20 ovl.
7.77 d (9.8)
7.43 ovl.
n.a.
28.3
25.1
40.6
δ
1.65 ovl.
1.63 ovl.
3.20 ovl.
7.74 d (9.6)
7.43 ovl.
n.a.
28.3
25.0
40.8
ε
1.58 ovl.
3.20 ovl.
7.74 ovl.
7.43 t (5.6)
n.a.
ε
1.59 ovl.
ε
ε
ζ
ζ
3.19 ovl
ζ
ζ
γ-NH
γ-NH
7.68 d (9.4)
7.38 t (5.7)
γ-NH
γ-NH
Guan
157.2
174.9
Guan
157.2
175.1
Guan
157.2
174.5
Guan
157.2
174.4
CO
CO
CO
CO
Ala
Ala
Ala
Ala
α
4.31 ovl.
49.8
16.6
α
4.30 ovl.
49.8
16.5
α
4.33 ovl.
49.6
16.3
α
4.32 ovl.
49.8
16.4
β
1.42 d (7.3)
8.25 d (6.3)
β
1.41 d (7.2)
8.23 d (6.8)
β
1.43 d (7.4)
8.27 d (7.0)
β
1.42 d (7.4)
8.27 d (7.0)
CONH
174.9
CONH
174.8
CONH
174.6
CONH
174.6
TMHEA
TMHEA
TMHEA
TMHEA
1
2
n.a.
177.4
43.5
1
2
n.a.
177.5
43.5
1
2
n.a.
177.6
43.4
1
2
n.a.
177.5
43.4
2.60 m
3.47 dd (8.8,
3.1)
2.60 ovl.
3.47 dd (8.7,
3.2)
2.64 m
3.51 dd (8.5,
2.2)
2.63 m
3
78.2
32.3
38.2
24.9
23.4
13.3
16.1
20.2
3
78.3
32.4
38.2
25.0
23.4
13.3
16.1
20.3
3
78.3
32.3
38.1
25
3
3.51 d (8.8)
1.76 ovl.
78.4
32.4
38.2
25.0
23.4
13.3
16.1
20.3
4
1.73 ovl.
4
1.73 ovl.
4
1.77 ovl.
4
5
1.16 m
5
1.17 m
5
1.21 m
5
1.20 m
6
1.61 ovl.
6
1.58 ovl.
6
1.63 ovl.
6
1.66 ovl.
7
0.92 d (6.6)
1.07 d (6.9)
0.96 d (7.0)
0.85 d (6.5)
7
0.93 d (6.5)
1.07 d (6.9)
0.97 d (6.9)
0.86 d (6.5)
7
0.96 d (6.6)
1.09 d (7.1)
1.01 d (7.0)
0.90 d (6.6)
23.4
13.1
16
7
0.96 d (6.7)
1.09 d (7.1)
1.00 d (7.0)
0.89 d (6.6)
8
8
8
8
9
9
9
9
10
10
10
20.2
10
a600 MHz; b150 MHz; c500 MHz; d125 MHz. 1H and 13C assignments aided by HSQC, COSY, HMBC and HSQCTOCSY experiments.
Coupling constants are in parantheses and given in Hz. Overlap (ovl.); Not available (n.a.).
Callipeltin P (3) was obtained as a white amorphous solid
showing the doubly charged molecular ion at m/z 775.4505
- 0.72 ppm) in the HRESIMS spectrum
corresponding to the molecular formula C70H122O21N18. The
molecular formula indicated an addition of 18 units to the mass
of callipeltin N (1). It also indicated an unsaturation number one
less than that of callipeltin N suggesting that callipeltin P (3) was
the linear form of callipeltin N (1). The structure was fully
supported by both 1D and 2D NMR data (Table 1, Figure S25)
and MS/MS fragmentation data (Figure S18). The
[M+2H]2+ (∆

ACCEPTED MANUSCRIPT
6
Tetrahedron
stereochemistry of each of the amino acid has not been
Table 2
determined and are assumed to be the same as in callipeltin N (1).
Callipeltin C the acyclic version of callipeltin A was found to
have the same amino acid configurations as callipeltin A.2
Cytotoxicity data of callipeltin N – Q (1 – 4) towards normal cell
line MRC-5 and cancer cell lines A2058, HT-29 and MCF-7.
Values are means ± SD based on 3 independent determinations.
IC50 (µM)
Compound
A2058
0.17±0.04
0.67±0.03
n.a.
HT-29
0.19±0.05
2.08±0.08
n.a.
MCF-7
MRC-5
1
2
3
4
0.21±0.21 0.16±0.14
0.48±0.08 1.37±0.23
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
an.a. = not active at 20 µM.
In summary, we have discovered four new depsipeptides
callipeltins N-Q that add to the class first exemplified by
callipeltin A. Callipeltins N and O showed cytotoxic activities
similar to those observed for other related macrocyclic
depsipeptides such as callipeltins A and B2, homophyamine6,
theopapuamide8a, neamphamide A5, pipecolidepsin A12
underlying the importance of macrocyclisation as well amino
acid composition in biological activity. Of the three cell lines that
were tested the results showed that callipeltin N containing the
3,4-dimethylGln was on average six times more cytotoxic than
Figure 1 Selected HMBC and some observed ROE correlations
in CD3OD for callipeltin N (1)
Callipeltin Q (4) was obtained as a white amorphous solid
showing the doubly charged molecular ion at m/z 768.4437
[M+2H]2+ (ꢀ 0.66 ppm) in the HRESIMS spectrum
corresponding to the molecular formula C69H120O21N18. The
molecular formula indicated an addition of 18 units to the mass
of callipeltin O (2), with an unsaturation number one less
suggesting that callipeltin Q (4) was the linear form of callipeltin
O (2). The structure was supported by both 1D and 2D NMR data
(Table1, Figure S36) and MS/MS fragmentation data (Figure
S29. The stereochemistry of each of the amino acids has not been
determined and is assumed to be the same as in callipeltin O (2).
callipeltin
O containing the 3-methylGln suggesting the
significance of that position to bioactivity. The strong antifungal
activities observed in the crude extracts may have been attributed
to the presence of macrocyclic callipeltins (N and O) and linear
callipeltins (P and Q) as both macrocyclic and liner callipeltins
are known for their antifungal activities.1,4 The compound map13
(Figure S38) shows the close distribution of the depsipeptides
that share a high degree of structural homology to the callipeltins
where differences are due to different amino acids introduced at
selected positions. The variability could be explained by the
presence of various isoforms of the bacterial NRPS enzymes or
alternatively enzymes that have broad substrate specificity14
allowing the introduction of new amino acids into the peptidic
frame work in response to selection pressure.
3.0.
Experimental Section
3.1 General experimental proceduresNMR data, both 1D and
2D were recorded on a Varian Unity Inova spectrometer at 600
1
and 150 MHz for H and 13C respectively using an ID probe.
High resolution mass spectrometry data were recorded using a
Waters LCT Premier coupled to a Waters Acquity UPLC system
and a ThermoScientific LTQXL-Discovery Orbitrap coupled to
an Accela HPLC system. Optical rotation measurements were
recorded using a Perkin Elmer, Model 343 Polarimeter at 589
nm. Semi-preparative HPLC purifications were performed on an
Agilent 1100 HPLC system consisting of a binary pump,
degasser, DAD and a preparative fraction collector. All solvents
used were of HPLC grade.
Figure 2. Selected HMBC and some observed ROE correlations
in CD3OD for callipeltin O (2).
The compounds were evaluated for their antiproliferative activity
against A2058 (melanoma), HT-29 (colorectal adenocarcinoma),
MCF-7 (breast adenocarcinoma) and human lung fibroblast
MRC-5 cell lines. The cyclic compounds, 1 and 2, showed
antiproliferative activity against all cell lines, while their linear
forms, 3 and 4, had no apparent effect on the cells up to a
concentration of 20 µM (Table 2). This clearly suggests that
peptide cyclisation is essential for the observed activity. With
IC50-values of 0.17, 0.19, 0.21 and 0.16 µM against A2058, HT-
29, MCF-7 and MRC-5 respectively, compound 1 showed a 2- to
11-fold higher cytotoxic potency than 2, which displayed IC50-
values of 0.67, 2.08, 0.48 and 1.37 µM against the same cell
lines. No distinction could be seen for the cytotoxic potency of
compound 1 and 2 between the non-malignant and the malignant
cell lines indicating high cellular non-specificity.
3.2 Collection
The sponge Asteropus sp. was collected by hand using SCUBA
at a depth of 7-15 m from Mary Island, Russell group, Solomon
Islands (9.02°S, 158.74°E) in June 2006 during a sample
collection expedition in the central province of the Solomon
Islands. The sponge (greenish exterior and light brown inside)
was spongy and soft to touch and had a porous interior. Oscules
were very small (0.5 mm). It was identified by Dr J. Hooper,
Queensland Museum, Australia. A voucher specimen (SI06-5s-

ACCEPTED MANUSCRIPT
has been preserved at the Marine Reference Collection, The
University of the South Pacific, Suva, Fiji Islands.
at a temperature of 110 °C for 24 h. The reaction mixture was
subsequently cooled and dried under nitrogen. To remove the
residual HCl, water (0.4 mL) was added and subsequently
removed three times and the hydrolysate was dried under N2 for
24 h. To 50 µL of each of 50 mM aqueous solution of standard
3.3 Extraction and isolation
amino acids and the hydrolysate, 1N NaHCO3 (20µL) and 1% L-
150 g frozen sample was thawed and extracted 3 times with
MeOH followed by dichloromethane. The combined crude
extract (4.2 g) was partitioned using a modified Kupchan
method.9,10 The antifungal activities were found to be in the
dichloromethane fraction. Further purification of this fraction
was performed using a semi-preparatory C18 HPLC column (Ace
5 C18-HL, 50 x 10 mm) using water-acetonitrile (with 0.05%
TFA) as solvent and a solvent gradient system from 0 to 100%
acetonitrile in 30 min to afford 1 (38 mg), 2 ( 7.7 mg), 3 (2.9 mg)
and 4 (2.8 mg).
FDLA (1-fluoro-2,4-dinitrophenyl-5-L-leucinamide) (100 µL) in
acetone were added. The reaction mixtures were incubated at 40
°C for 1 h, with frequent mixing. After cooling at room
temperature, the reactions were quenched by the addition of 10
µL of 2 N HCl. The samples were diluted by making the volume
up to 1 mL with MeOH. The derivatised amino acids were
analysed by LC-MS (Table 2).
3.4.2 General procedure for LCMS analysis of Marfey’s
FDLA derivatives
20
3.3.1 Callipeltin N (1). White amorphous solid, 38.5 mg; [α]D
-
2 µL of each of the derivatised mixture of hydrolysed 1 and 2
and standards were injected into a Waters Acquity Xylene
bridged hybrid (BEH) C18 column (2.1 × 150 mm, 1.7 ꢁm) using
a water-acetonitrile solvent system, starting from 10-100%
acetonitrile in 4.5 min and flushing the column using 100%
acetonitrile with a further 1.5 minutes before returning to the
starting gradient. The eluted compounds were detected by a
Waters LCT Premier MS system using the following conditions:
Capillary voltage 2600 V, cone voltage 50 V, desolvation
temperature 350 °C, desolvation gas 650 L/h, cone gas 5 L/h,
mass range 150-1500 amu. The instrument was tuned to a
resolution of 10,000 (FWHM) and leucine-enkephaline were
used as lock mass for internal calibration.
17.2° (c 0.0015 M, methanol) IR (MeOH, cm-1) 3338, 3213,
2959, 2936, 2871, 1657, 1532, 1520, 1452, 1384, 1201, 1186,
1137, 837, 803, 719. H and 13C NMR data in CD3OD are given
1
in Table 1. HRESIMS m/z 766.4563 [M+2H]2+ ꢀ +2.5 ppm from
calculated for C70H120O20N18.
20
3.3.2 Callipeltin O (2). White amorphous solid, 7.7 mg; [α]D
-
11.2° (c 0.00023 M, methanol); IR (MeOH, cm-1) 3338, 3213,
2959, 2936, 2871, 1657, 1532, 1520, 1452, 1384, 1201, 1186,
1137, 837, 803, 719. H and 13C NMR data in CD3OD are given
1
in Table 1. HRESIMS m/z 759.4385 [M+2H]2+ ꢀ 1.7 ppm from
calculated for C69H118O20N18.
20.0
3.3.3 Callipeltin P (3). White amorphous solid, 2.9 mg; [α]D
14.8° (c 0.000048 M, methanol); IR (MeOH, cm-1) 3338, 3213,
-
3.5 Biological activities
3.5.1 Antifungal
2959, 2936, 2871, 1657, 1532, 1520, 1452, 1384, 1201, 1186,
1
1137, 837, 803, 719. H NMR (MeOD-δ4) are given in Table 1.
The crude extract showed inhibition zones at 20 and 21 mm for
amphotericin resistant (ATCC90873) and wild type Candida
albicans (ATCC32354) respectively using susceptibility discs (6
mm in diameter) and at a concentration of 2.5 mg crude extract
per disc.
HRESIMS m/z 775.4505 [M+2H]2+ ꢀ -0.72 ppm from calculated
for C70H122O21N18.
20.4
3.3.4 Callipeltin Q (4). White amorphous solid, 2.8 mg; [α]D
-20.0 (c 0.000016 M, methanol) IR (MeOH, cm-1) 3338, 3213,
2959, 2936, 2871, 1657, 1532, 1520, 1452, 1384, 1201, 1186,
3.5.2 Cell growth inhibitory assay.
1
1137, 837, 803, 719. H NMR (CD3OD) are given in Table 1.
HRESIMS m/z 768.4437 [M+2H]2+ ꢀ 0.66 ppm from calculated
The cytotoxicity of compound 1 – 4 were evaluated at ranging
concentrations against the malignant cell lines A2058 (ATTC
CRL-11147TM), HT-29 (ATCC HTB-38) and MCF-7 (ATCC
HTB-22) and the non-malignant cell line MRC-5 (ATCC CCL-
171) using a previously reported MTS assay. RPMI containing
the maximum DMSO concentration used in each assay (0.4%)
was used as negative control and Triton® X-100 (Sigma-Aldrich)
as positive control.
for C69H120O21N18.
3.4 Determination of absolute stereochemistry
3.4.1. General procedure for peptide hydrolysis
To determine the absolute stereochemistry of callipeltins N
and O, 0.2 mg of the compounds were hydrolysed by 6N HCl aq
Table 2. HPLC retention time in minutes of derivatised callipeltins N (1), O (2) amino acids and standards
Amino acid
aStandard
D
-HPr
2.73
424.18 424.18 384.15 384.15 426.20 426.20 469.21 469.21 414.16 414.16
L
-HPr
D
-Ala
L
-Ala
D
-Leu
L
-Leu
D
-Arg
L
-Arg
D
-Thr
L
-Thr
btR
m/z
tR
2.85
2.63
2.37
3.26
2.77
1.79
1.88
2.39
2.10
Callipeltin N
Callipeltin O
-
-
-
-
2.85
424.18 384.15
2.85 2.63
424.18 384.15
2.63
-
-
-
-
-
-
-
-
2.75
426.20 469.21
2.76 1.82
426.20 469.21
1.82
-
-
-
-
2.28
414.16
2.28
-
-
-
-
m/z
tR
m/z
414.16
aAmino acid standards; bLCMS retention time

ACCEPTED MANUSCRIPT
8
Tetrahedron
Acknowledgements
J.T, E.H and M.J acknowledge the financial support of the EU 7th
Framework Programme Project ‘PharmaSea’ (Contract no.
312184) and acknowledge the support of the RSC for use of the
MarinLit Data Base. K.F and M.C acknowledge the support of
the NCDDG (Grant no. U19 CA67786) awarded to Professor C.
M. Ireland, University of Utah, USA. J.T and M.S thank Dr
Somayah Elsayed Sameer of the University of Aberdeen for
advice on amino acid hydrolysis and derivatisation.
Supplementary data
HRESIMS, LC-MS/MS, and NMR data of 1, 2, 3 and 4 including
1H, 13C, COSY, HSQC, HMBC, HSQCTOCSY and ROESY are
in the supplementary material.
References and notes
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