forward motion. Amoeboid migration involves a path-finding
process where cells change their shape to move through
existing spaces in the ECM rather than enzymatically creating
a new path. It has been shown in vitro that if the activity of
ECM degrading proteases is inhibited by drugs, tumor cells
can switch from mesechymal invasion to amoeboid invasion.
Removal of the protease inhibitors in wash-out experiments
results in reversion to mesenchymal invasion. This mesen-
chymal to ameoboid transition (MAT) has been offered as a
rationale for the relative lack of efficacy of MMP inhibitors
in mouse models of metastasis and in clinical trials. Tumor
cells faced with the blockage of one form of migration might
simply switch to the other.
The discovery of MAT has shown that simultaneous
inhibition of both forms of tumor cell motility will likely be
required to effectively control metastasis in vivo.3 Small
molecule MMP inhibitors4 that effectively block mesenchy-
mal migration in vitro3 are known. In contrast, there are no
documented small molecule inhibitors of amoeboid migra-
tion.
Recently, we described the development of a cell-based
assay5 that has proven effective for discovering novel
inhibitors of mesenchymal tumor cell invasion and migra-
tion.6 A modification of this assay employing LS174T human
colon cancer cells, that are known to migrate exclusively
via the amoeboid form, provided a screening method to detect
noncytotoxic inhibitors of amoeboid invasion of the ECM.
This modified assay has been used to screen a library of
marine invertebrate extracts for natural product inhibitors of
amoeboid invasion. Crude MeOH extracts of the marine
sponge Neopetrosia sp. (order Haplosclerida, family Petro-
siidae)7 collected in Papua New Guinea showed promising
activity in the assay. Bioassay guided fractionation of the
extracts identified the tricyclic peptides neopetrosiamides A
(1) and B (2) as the compounds responsible for inhibition
of amoeboid invasion. Details of the isolation and structure
elucidation of the neopetrosiamides are presented below.
Specimens of Neopetrosia sp. (450 g wet wt) were
harvested by hand using scuba near Milne Bay, Papua New
Guinea. Freshly collected sponge samples were frozen on
site and transported to Vancouver over dry ice. Thawed
sponge tissue was repeatedly extracted with MeOH (4 × 500
mL) and the combined MeOH extracts were concentrated
in vacuo to give a brown gum. The gum was suspended in
water (400 mL), and the aqueous suspension was sequentially
extracted with EtOAc (3 × 100 mL) and n-BuOH (4 × 100
mL). Antiinvasive activity was observed in the n-BuOH
soluble fraction. Bioassay-guided separation of the n-BuOH
soluble materials via sequential application of Sephadex LH-
20 chromatography (eluent: MeOH), flash reversed-phase
column chromatography (eluent: step gradient from 1:1
MeOH/H2O to MeOH), and repetitive reversed-phase HPLC
[first eluent: 3:7 MeCN/(0.05% TFA/H2O); second eluent:
3:2 MeOH/(0.05% TFA/H2O)] gave pure samples of neo-
petrosiamides A (1) and B (2).
Neopetrosiamide A (1) was obtained as an optically active
clear glass ([R]D ) -65.2 (c 4.2, MeOH)). The MALDITOF-
MS of 1 gave an [M + H]+ ion at m/z 3071, indicating a
molecular weight of 3070. Peptide 1 gave a sharp well-
resolved peak when analyzed by reversed-phase HPLC using
a variety of solvent systems and it gave a single clean
molecular ion in the MALDITOF-MS. However, in numer-
ous NMR solvents (e.g., MeOH-d4, DMSO-d6, MeCN-d3,
1
etc.) many or all of the resonances in the H and 13C NMR
spectra of 1 were broadened, resulting in poorly resolved
spectra that were not suitable for structural studies. Attempts
to simplify the NMR spectra by heating (to 50 °C) or
adjusting the pH by addition of TFA or Et3N did not alleviate
the problem. Eventually, it was found that acceptable spectra,
with only a single set of well-resolved resonances, could be
obtained using 4:1 MeCN-d3/H2O as the NMR solvent.
The 1H/13C/DEPT/HSQC NMR data recorded for 1 in 4:1
MeCN-d3/H2O identified 129 carbon atoms. Both the 1H and
13C NMR spectra (Supporting Information) of 1 had features
that suggested a peptide structure. Detailed analysis of the
COSY, HSQC, and HMBC NMR data (Tables 1 and 2,
Supporting Information) showed that neopetrosiamide A (1)
contained one alanine, two arginine, one asparagine, three
aspartic acid, three glycine, one leucine, one methionine
sulfoxide, four phenylalanine, three proline, one serine, one
threonine, and one valine residues, plus six 3-substituted -2-
aminopropionic acid residues, for a total of 28 amino acids.
The 13C NMR resonances assigned to the substituted meth-
ylenes of the 2-aminopropionic acid residues had chemical
shifts (δ 37.1-41.2 ppm) appropriate for S substitution.
Standard amino acid analysis, with and without performic
acid oxidation prior to hydrolysis (to convert cysteine and
cystine to cysteic acid), confirmed the presence of the amino
acids identified by NMR. In both of these analyses, Asn was
converted to Asp, and in the analysis without prior oxidation,
methionine sulfoxide was converted to methionine. A control
experiment showed that under the hydrolytic conditions used
in the amino acid analysis methionine sulfoxide is converted
to methionine and cystine is partially converted to cysteic
acid if excess cystine is present. Marfey’s analysis of the 6
N HCl hydrolysate of neopetrosiamide A (1) showed that
all of the amino acids had the L configuration. In the Marfey’s
analysis, Asn was detected as Asp, methionine sulfoxide was
detected as methionine, and cysteic acid was detected along
with cystine.
(1) Sahai, E.; Marshall, C. J. Nat. Cell Biol. 2003, 5, 711-719.
(2) Friedl, P. Curr. Opin. Cell Biol. 2004, 16, 14-23.
(3) Wolf, K.; Mazo, I.; Leung, H.; Engelke, K.; von Andrian, U. H.;
Deriyugina, E. I.; Strongin, A. Y.; Brocker, E.-B.; Friedl, P. J. Cell Biol.
2003, 160, 267-277.
(4) Heath, E. I.; Grochow, L. B. Drugs 2000, 59, 1043-1055.
(5) Roskelley, C. D.; Williams, D. E.; McHardy, L. M.; Leong, K. G.;
Troussard, A.; Karsan, A.; Andersen, R. J.; Dedhar, S.; Roberge, M. Cancer
Res. 2001, 61, 6788-6794.
(6) (a) Williams, D. E.; Craig, K. S.; Patrick, B.; McHardy, L. M.; van
Soest, R.; Roberge, M.; Andersen, R. J. J. Org. Chem. 2002, 67, 245-258.
(b) Warabi, K.; McHardy, L. M.; Matainaho, L.; van Soest, R.; Roskelley,
C. D.; Roberge, M.; Andersen, R. J. J. Nat. Prod. 2004, 67, 1387-1389.
(c) McHardy, L. M.; Warabi, K.; Andersen, R. J.; Roskelley, C. D.; Roberge,
M. Mol. Cancer Ther. 2005, 4, 772-778.
The 28 amino acids present in neopetrosiamide A (1)
contain 129 carbon atoms, which is equivalent to the number
identified from the NMR data. A linear peptide constructed
(7) A voucher sample has been deposited at the Zoological Museum,
University of Amsterdam (ZMA POR 18339).
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Org. Lett., Vol. 7, No. 19, 2005