Isolation, structure determination, and synthesis of galaxamide, a rare cytotoxic cyclic pentapeptide from a marine algae Galaxaura filamentosa

Article abstract of DOI:10.1021/ol801799d

(Chemical Equation Presented) Galaxamide (1), a rare cyclic pentapeptide, was Isolated from the marine algae Galaxaura filamentosa. A preliminary bioassay of Galaxamide showed remarkable in vitro antiproliferative activities against GRC-1 and HepG₂ cell lines. The first total synthesis of the cyclic peptide was achieved for further biological evaluation.

Full text of DOI:10.1021/ol801799d

ORGANIC
LETTERS
2008
Vol. 10, No. 20
4569-4572
Isolation, Structure Determination, and
Synthesis of Galaxamide, A Rare
Cytotoxic Cyclic Pentapeptide from a
Marine Algae Galaxaura filamentosa
Wen-Jie Xu,† Xiao-Jian Liao,‡ Shi-Hai Xu,*,‡ Jian-Zhong Diao,‡ Bin Du,§
Xu-Long Zhou,§ and Shan-Shan Pan‡
Institute of Hydrobiology, Department of Chemistry, and Medical College, Jinan
UniVersity, Huangpu Road West 601, Guangzhou,
Guangdong 510632, People’s Republic of China
txush@jnu.edu.cn
Received August 4, 2008
ABSTRACT
Galaxamide (1), a rare cyclic pentapeptide, was isolated from the marine algae Galaxaura filamentosa. A preliminary bioassay of Galaxamide
showed remarkable in vitro antiproliferative activities against GRC-1 and HepG2 cell lines. The first total synthesis of the cyclic peptide was
achieved for further biological evaluation.
Cyclopeptides have many physiological functions and curatorial
worthiness; for example, aplidine (dehydrodidemnin B) from
the ascidian Aplidium albicans.1,2 As part of our continuing
search for structurally and pharmacologically interesting
secondary metabolites from marine algae, Galaxaura fila-
mentosa, we found a rare cyclopeptide (1) composed of a
kind of amino acid and its derivates. Herein, we report the
isolation, structure elucidation, biological activity, and
synthesis of the peptide that we have named Galaxamide
(1).
Specimens of Galaxaura filamentosa were collected from
the Xisha Island of South China Sea in May of 2001. The
EtOH extract from Galaxaura filamentosa was concentrated
under vacuum to yield an orange-brown gum. The crude
residue was partitioned between water and petroleum ether.
The organic layer was concentrated under vacuum to yield
a greenish gum (20.5 g), and this residue was then subjected
to silica gel column chromatography using petroleum ether
(60-90) containing an increasing amount of EtOAc and
CHCl3 containing an increasing amount of MeOH as an
eluent,respectively.Thefractionelutedwithpetroleum-EtOAc
(5:5) was subjected to Sephadax LH-20 (MeOH:CH2Cl2 1:1),
yielding colorless needles, compound 1 (15 mg).
† Institute of Hydrobiology
‡ Department of Chemistry
§ Medical college
(1) Rinehart, K. L. Med. Res. ReV. 2000, 20, 1–27
(2) Schmitz, F. J.; Yasumoto, T. J. Nat. Prod. 1991, 54, 1469–1490
Galaxamide (1), [R]20 -130° (C 0.1, MeOH), mp
D
.
.
208-210 °C, was obtained as colorless needles and showed
10.1021/ol801799d CCC: $40.75
Published on Web 09/25/2008
2008 American Chemical Society

1
Table 1. H and 13C NMR Data for 1 at 600 MHz in Pyridine-d5
position
13C
1H (δH, J in Hz)
position
Leu2
13C
1H (δH, J in Hz)
N-MeLeu1
N-CH3
RCH
40.2
65.8
37.5
25.4
22.8
23.4
172.0
3.43 (s)
NH
8.59 (d, 9.8)
5.28 (m)
1.65 (m); 1.52 (m)
1.84 (m)
0.97 (d, 6.6)
0.83 (d, 6.7)
3.90 (dd, 10.7, 4.7)
2.41 (m); 1.86 (m)
1.69 (m)
RCH
ꢀCH2
γCH
δCH3
48.3
40.7
25.2
21.6
20.7
172.1
ꢀCH2
γCH
δCH3
0.87 (m)
0.76 (d, 6.6)
CdO
NH
RCH
ꢀCH2
γCH
δCH3
CdO
NH
RCH
ꢀCH2
γCH
δCH3
Leu1
8.33 (d, 9.1)
5.30 (m)
2.09 (m); 1.80 (m)
1.76 (m)
1.02 (d, 6.5)
0.88 (m)
Leu3
8.65 (d, 7.9)
4.88 (m)
2.15 (m); 1.97 (m)
1.92 (m)
0.89 (m)
0.87 (m)
49.3
42.2
24.7
22.9
22.7
173.0
39.4
71.4
39.2
26.3
23.0
21.9
53.5
40.3
25.3
21.7
23.2
174.7
CdO
N-CH3
RCH
ꢀCH2
γCH
δCH3
CdO
N-MeLeu2
3.27 (s)
3.81 (t, 8.0)
2.64 (m); 2.25 (m)
1.72 (m)
0.93 (d, 6.6)
0.89 (m)
negative reaction to ninhydrin reagent but positive reaction
after hydrolysis with 6 mol/L of HCl. The moleclular
formula of 1 was deduced as C32H59N5O5 by means of
spectral analysis and TOF-MS-ES+ [(M + H)+ 594.4597,
calcd m/z 594.4594]. The IR absorption bands of 1 at 3351,
3305, and 1638 cm-1 were characteristic of amino and
amide carbonyl groups. The structure of 1 was assigned
using a combination of one- and two-dimensional NMR
spectroscopic methods. The 1H and 13C NMR spectral data
acquired for 1 illustrated typical resonances for a cyclic
peptide (Table 1). The presence of five carbonyl resonances
at 172.0, 172.1, 173.0, 173.2, and 174.7 together with the
presence of three NH protons and two N-Me functionality
suggested Galaxamide was a monocyclic pentapeptide. The
13C NMR spectrum (Table 1) displayed signals for 32 unique
carbons, and the DEPT experiment indicated that 1 contained
12 methyl, 5 methylene, and 10 methine carbons. The five
signals at δH 3.81 (t, J ) 8.0), 3.90 (dd, J ) 10.7, 4.7), 4.88
(m), 5.28 (m), and 5.30 (m) were indicative of R-proton
resonances for amino acids. Analysis of 1D-TOCSY, HSQC,
and HMBC data established the presence of three leucine
(Leu) and two N-methylleucine (N-Me-Leu) residues (Table
1). The sequence of the cyclic peptide was established by
analysis of HMBC data, which generally showed correlations
of the carbonyl carbons of each amino acid with the
corresponding NH or N-CH3 proton of the adjacent residue.
Further evidence was provided by TOF-MS-MS-ES+ which
showed the fragments of I to V as follows:
The absolute stereochemistry for the amino acids of 1
was determined by hydrochloric acid hydrolysis of 1
followed by treatment of the hydrolysate with Marfey’s
reagent.3 Analysis of the mixture of FDAA derivatives
by HPLC, using retention times and coinjections with
standards, revealed all leucines of 1 were assigned to
L-configuration.
In vitro cytotoxic assay accomplished by the MTT
method4 following exposure of cells to the tested com-
pounds for 72 h showed that 1 was remarkably active
against the human renal cell carcinoma GRC-1 and human
hepatocellular carcinoma HepG2 cell lines with corre-
sponding IC50 values of 4.26 and 4.63 µg/mL, and those
of the commercially available Mitomycin C which tested
as a positive reference are 6.01 and 0.41 µg/mL, respec-
tively. Following the experiment that indicated Galaxa-
mide possesses potent antitumor properties, we thus
carried out the total synthesis of 1 to provide access to
additional material for further biological evaluation.
Compoud 1 is a high symmetry cyclopeptide, which is
composed of three leucines and two N-methyl leucines.
In the previous paper, Sakurai5 described the synthesis
of the cyclic peptide c (-Leu5-) which is a analogue of 1.
Here we designed a solution phase route for the synthesis
of 1 and chose to elaborate 3 from the tripeptide 4, then
synthesized 2 from the tripeptide 3. The target product 1
was to be produced in a final step of cyclization from
pentapeptide 2 (Scheme 1).
Following the strategy, our synthesis started from
dipeptide 4. Reaction of the commercially available tert-
butyl-L-leucine with N-methyl-L-leucine benzyl ester in
the presence of a coupling reagent, 3-(diethoxyphospho-
ryloxy)-3H-benzo[d][1,2,3] triazin-4-one (DEPBT), and
I
m/z 594 [Leu-N-MeLeu-Leu-N-MeLeu-Leu+H]+
m/z 481 [Leu-N-MeLeu-Leu-N-MeLeu+H]+
II
III m/z 368 [N-MeLeu-Leu-N-MeLeu+H]+
IV m/z 354 [Leu-N-MeLeu-Leu+H]+
V
m/z 241 [Leu-N-MeLeu+H]+
(3) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591–596.
(4) Mosmann, T. J. Immunol. Meth. 1983, 65, 55–63.
(5) Sakurai, A.; Okumura, Y. Bull. Chem. Soc. Jpn. 1979, 52 (2), 540–
543.
Therefore, the structure of compound 1, a new pentapetide,
was elucidated as cyclo(-N-Me-Leu1-Leu1-N-Me-Leu2-
Leu2-Leu3-).
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Org. Lett., Vol. 10, No. 20, 2008

Scheme 1
.
Retrosynthesis Strategy of 1
Scheme 2. Synthesis of 1
diisopropylethylamine (DIPEA) gave the monocrystal
dipeptide 46 in 89% yield.7 Removal of the Boc group in
4 using TFA was coupled with Boc-L-leucine using
DEPBT as a coupling reagent, leading to the tripeptide 3
in 85% yield (from 4 to 3). We removed the Boc group
of 3 using TFA to 5 and took off the benzyl from 4 using
hydrogen reduction with Pd/C8 to 6. The synthesis of the
pentapeptide 2 was achieved by coupling of 5 and 6 in
the presence of DEPBT and DIPEA in 82% yield (from 3
to 2) (Scheme 2). We found, upon completion, each
reaction was concentrated, needed not carry out the
postproccessing, and directly subjected to silica gel column
chromatography using n-hexane/acetone (20:1) isocratic
elution when we prepared the three intermediates 2-4
using DEPBT as the coupling reagent.
solvent by distillation under reduced pressure, and the
mixture was disssolved in EtOAc and washed with am-
monium chloride. The organic layers were combined, dried,
filtered, and concentrated. The crude mixture was purified
by reverse phase HPLC to the target product 1 in 42.5%
yield (from 2 to 1) (Scheme 2).
The cyclization is a key step in the synthesis of cyclic
peptides. After the Boc group and benzyl in pentapeptide 2
were removed using TFA and hydrogen reduction in Pd/C,
respectively, the dried, crude, free amine/free acid linear
pentapeptide was dissolved in a 2:2:1 ratio of THF/CH3CN/
CH2Cl2 (0.004 M). Then, three coupling agents, DEPBT,
[2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU), and O-(benzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU) (0.7
equiv each), and DIPEA (6 equiv) were added to the
reaction.9,10 The reaction was usually completed within about
4 days. The final product was generated after removing the
Figure 1. TOCSY and key HMBC (HfC) correlation of 1.
In summary, we have isolated the rare cytotoxic cyclic
pentapeptide 1 from the marine algae Galaxaura filamen-
tosa. The structure as well as the absolute configuration
were determined by a combination of its spectral data and
Marfey’s method. The efficient synthesis described herein
should allow for the preparation of various analogues of
this bioactive cyclic peptide. We believe that the charac-
teristic cyclopeptide composed of a kind of amino acid
will be a potential lead compound. Further studies on
analogues and bioactivity of 1 are underway.
(6) Liao, X. J.; Xu, W. J.; Xu, S. H.; Dong, F. F. Acta Crystallogr. E
2007, 63, O3313–U4398.
(7) Li, H. T.; Jiang, X. H.; Ye, Y. H.; Fan, C. X.; Romoff, T.; Goodman,
M. Org. Lett. 1999, 1 (1), 91–93.
(8) Zhu, J. D.; Ma, D. W. Angew. Chem., Int. Ed. Engl. 2003, 42 (43),
5348–5351.
(9) Belofsky, G. N.; Jensen, P. R.; Fenical, W. Tetrahedron Lett. 1999,
40 (15), 2913–2916
.
(10) Rodriguez, R. A.; Pan, P. S.; Pan, C. M.; Ravula, S.; Lapera, S.;
Singh, E. K.; Styers, T. J.; Brown, J. D.; Cajica, J.; Parry, E.; Otrubova,
K.; McAlpine, S. R. J. Org. Chem. 2007, 72 (6), 1980–2002
.
Org. Lett., Vol. 10, No. 20, 2008
4571

Acknowledgment. This work was supported by grants
from the National High Technology Development Project
(863 Project) (Nos. 2006AA09Z408 GDSFC 06025194,
2005A30503001, and 2006Z3-E4041) and National natural
Science Fund (Nos. 20772048).
Supporting Information Available: Experimental details,
NMR assignments for Galaxamide, NMR spectra of 1 and
synthetic intermediates, and Marfey’s method. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL801799D
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Org. Lett., Vol. 10, No. 20, 2008