Total synthesis and antimalarial activity of symplostatin 4

Article abstract of DOI:10.1021/ol1024663

The first total synthesis of symplostatin 4, a marine cyanobacterium- derived natural product, is described. Notable features of the route include the efficient preparation of three key fragments and final assembly to the natural product via sequential imide and amide couplings. Symplostatin 4 was also demonstrated to possess significant antimalarial activity (ED₅₀ of 74 nM against Plasmodium falciparum, strain 3D7).

Full text of DOI:10.1021/ol1024663

ORGANIC
LETTERS
2010
Vol. 12, No. 23
5576-5579
Total Synthesis and Antimalarial Activity
of Symplostatin 4
Trent Conroy,† Jin T. Guo,‡ Nicholas H. Hunt,‡ and Richard J. Payne†,*
School of Chemistry, The UniVersity of Sydney, NSW 2006, Australia, and School of
Medical Sciences, Sydney Medical School, The UniVersity of Sydney, NSW 2006, Australia
richard.payne@sydney.edu.au
Received October 12, 2010
ABSTRACT
The first total synthesis of symplostatin 4, a marine cyanobacterium-derived natural product, is described. Notable features of the route
include the efficient preparation of three key fragments and final assembly to the natural product via sequential imide and amide couplings.
Symplostatin 4 was also demonstrated to possess significant antimalarial activity (ED50 of 74 nM against Plasmodium falciparum, strain 3D7).
Symplostatin 4 (1, Figure 1) is a linear depsipeptide natural
product of cyanobacterial origin, first isolated in 2009 from
the genus Symploca sp. by Luesch and co-workers in Key
Largo, Florida Keys.1 Structurally, it comprises a peptidic
backbone bearing a dimethylated N-terminal amino acid and
C-terminal pyrrolinone moiety. Symplostatin 4 (1) has been
shown to possess the same planar structure as gallinamide
A (2) (Figure 1), isolated from the Schizothrix species of
cyanobacteria collected off the Carribean coast of Panama
in 2009,2 but it has a diastereomeric configuration at the
N-terminal amino acid (25S, 26S). In the case of gallinamide
A, assignment of the absolute configurations of C25 and C26
has, to date, remained elusive.1,2
phase II clinical trials as anticancer agents (Figure 1).8,9
Unique structural features of symplostatin 4 and gallinamide
A include the depsipeptidic linkage, which in this instance
is formed through a 2-hydroxyisocaproic acid residue and
an unusual 4(S)-amino-2(E)-pentenoic acid unit. Initial
biological screening of symplostatin 4 indicated only moder-
ate cytotoxic activity (IC50 values of 12 and 53 µM against
HeLa cervical carcinoma cells and HT-29 colon adenocar-
cinoma cells, respectively).1 However, significantly, this
natural product was also shown to synergize with another
Symploca-derived natural product, largazole,10 which may
therefore present opportunities for the use of symplostatin 4
in the development of combination anticancer therapies.
Gallinamide A has been reported to possess antimalarial
Both symplostatin 4 and gallinamide A also share struc-
tural features with dolastatin 15 (3),3,4 dolastatin 10 (4),5,6
and symplostatin 1 (5),7 analogues of which have entered
(5) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Tuinman, A. A.; Boettner,
F. E.; Kizu, H.; Schmidt, J. M.; Baczynskyj, L.; Tomer, K. B.; Bontems,
R. J. J. Am. Chem. Soc. 1987, 109, 6883–6885
.
(6) Pettit, G. R.; Singh, S. B.; Hogan, F.; Lloyd-Williams, P.; Herald,
† School of Chemistry.
D. L.; Burkett, D. D.; Clewlow, P. J. J. Am. Chem. Soc. 1989, 111, 5463–
‡ School of Medical Sciences.
5465.
(1) Taori, K.; Liu, Y.; Paul, V. J.; Luesch, H. ChemBioChem 2009, 10,
1634–1639.
(7) Harrigan, G. G.; Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Nagle,
D. G.; Paul, V. J.; Mooberry, S. L.; Corbett, T. H.; Valeriote, F. A. J. Nat.
(2) Linington, R. G.; Clark, B. R.; Trimble, E. E.; Almanza, A.; Urena˜,
L.-D.; Kyle, D. E.; Gerwick, W. H. J. Nat. Prod. 2009, 72, 14–17.
(3) Pettit, G. R.; Kamano, Y.; Dufresne, C.; Cerny, R. L.; Herald, C. L.;
Prod. 1998, 61, 1075–1077
(8) Simmons, T. L.; Andrianasolo, E.; McPhail, K.; Flatt, P.; Gerwick,
W. H. Mol. Cancer Ther. 2005, 4, 333–342
(9) Tan, L. T. Phytochemistry 2007, 68, 954–979
.
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Schmidt, J. M. J. Org. Chem. 1989, 54, 6005–6006
(4) Pettit, G. R.; Herald, D. L.; Singh, S. B.; Thornton, T. J.; Mullaney,
J. T. J. Am. Chem. Soc. 1991, 113, 6692–6693
.
.
(10) Ying, Y.; Taori, K.; Kim, H.; Hong, J.; Luesch, H. J. Am. Chem.
Soc. 2008, 130, 8455–8459.
.
10.1021/ol1024663  2010 American Chemical Society
Published on Web 11/04/2010

Scheme 1. Retrosynthesis of Symplostatin 4
Scheme 2. Synthesis of Depsipeptide 6
Figure 1. Symplostatin 4 (1) and structurally related marine natural
products.
activity (IC50 of 8.4 µM against Plasmodium falciparum, W2
strain), moderate activity against mammalian Vero cells and,
surprisingly, no detectable cytotoxicity to NCI-H460 human
lung tumor or neuro-2a mouse neuroblastoma cell lines.2 This
is in stark contrast to dolastatin 10 and 15, which possess
antimalarial activity but signficant cytotoxicity,11 and sug-
gests that symplostatin 4 and gallinamide A may serve as
promising lead structures for the elucidation of antimalarial
agents with low cytotoxicity.
The interesting structural features of symplostatin 4,
coupled with the intriguing pharmacological profile of this
class of natural products,12-14 prompted us to investigate
the development of a succinct synthetic route. It was also
anticipated that the approach described herein would prove
amenable to the preparation of structurally related analogues,
including gallinamide A (2). We envisaged that symplostatin
4 (1) could be assembled in a rapid and convergent manner
by coupling three key fragment building blocks, namely
depsipeptide 6, pyrrolinone 7 and Boc-protected 4(S)-amino-
2(E)-pentenoic acid 8 (Scheme 1).
The preparation of N-terminal ester 6 began from 2-hy-
droxy-L-isocaproic acid 9, which upon treatment with DIC,
CuCl, and tert-butyl alcohol, was converted to the corre-
sponding tert-butyl ester 10 in good yield (78%, Scheme
2).15 From here, reaction with the acid chloride of Fmoc-L-
Ile-OH (11) proceeded smoothly and without epimerization
(as determined by NMR spectroscopy) to give depsipeptide
12 in 67% yield. Following esterification, removal of the
N-terminal Fmoc carbamate (20% piperidine/MeCN) yielded
the corresponding free amine 13 in 81% yield. At this stage,
reductive amination of 13, using formaldehyde and
NaBH3CN, afforded the desired dialkylated ester 14. Facile
acidolysis of the C-terminal tert-butyl ester of 14 with TFA
gave the requisite amino acid 15 in essentially quantitative
(11) Fennell, B. J.; Carolan, S.; Pettit, G. R.; Bell, A. J. Antimicrob.
Chemother. 2003, 51, 833–841.
(12) Burja, A. M.; Banaigs, B.; Abou-Mansour, E.; Burgess, J. G.;
Wright, P. C. Tetrahedron 2001, 57, 9347–9377
(13) Kaur, K.; Jain, M.; Kaur, T.; Jain, R. Bioorg. Med. Chem. 2009,
17, 3229–3256
.
.
(14) Fattorusso, E.; Taglialatela-Scafati, O. Marine Drugs 2009, 7, 130–
152
.
Org. Lett., Vol. 12, No. 23, 2010
5577

yield. Coupling of 15 to 2-chlorotrityl chloride resin pre-
loaded with L-Leu 16 was readily achieved using PyBOP
and NMM and was followed by mild acidolysis with
hexafluoro-2-propanol (HFIP) to complete the desired key
depsipeptide fragment 6 (77%).
Scheme 4. Completion of Symplostatin 4 (1) Synthesis
With N-terminal depsipeptide fragment 6 now in hand,
attention turned to the preparation of the required pyrrolinone
fragment 7 (Scheme 3). Toward this end, Boc-L-alanine 17
Scheme 3. Synthesis of Pyrrolinone Fragment 3
was condensed with Meldrum’s acid using EDC and DMAP.
Subsequent reflux in ethyl acetate facilitated the anticipated
thermal cyclization16 to the desired pyrrolinone 18 in 87%
yield over two steps. Alkylation of the enol functionality
under Mitsunobu conditions selectively generated the desired
4-methoxypyrrolinone and was followed by acidolysis of the
Boc-carbamate with TFA to complete the preparation of 7
in good yield (63%).
4). We chose to perform this coupling at 0 °C in a
concentrated solution with PyBOP and NMM to reduce
epimerization of the C-terminal L-isoleucine residue of 6.
Pleasingly, under these conditions, the desired product was
formed with minimal epimerization (<2%). The small
quantity of epimer was successfully removed during puri-
fication by preparative reversed-phase HPLC to afford
symplostatin 4 (1) in 85% yield. Gratifyingly, the spectro-
scopic data obtained for synthetic symplostatin 4 (1H and
Preparation of the unusual 4(S)-amino-2(E)-pentenoic acid
subunit 8 commenced from Boc-L-Ala-OH (17) (Scheme 4).
Conversion to the corresponding Weinreb amide, followed
by reduction with LiAlH4, provided Boc-L-alaninal 19 in 87%
yield over the two steps.17 From here, completion of the
desired R,ꢀ unsaturated amino acid was neatly achieved using
a Horner-Wadsworth-Emmons reaction18 between 19 and
trimethylphosphonoacetic acid (TMPAA) to give 8 in 66%
yield and with complete selectivity for the desired E-isomer.
In order to facilitate acylation of the pyrrolinone moiety,
fragment 8 was first activated as the corresponding pen-
tafluorophenyl (Pfp) ester using pentafluorophenyl trifluo-
roacetate and pyridine (Scheme 4).19 Deprotonation of 7,
followed by addition of the Pfp ester, successfully generated
the desired coupled product 20 with no detectable epimer-
ization as determined by NMR spectroscopy.16 Removal of
the residual Boc protecting group was then readily achieved
by treatment with TFA to afford 21 in excellent yield (99%).
Our attention now turned to the key coupling between
C-terminal fragment 21 and N-terminal fragment 6 (Scheme
13C NMR, MS) and the measured specific rotation, [R]20
D
-47 (c ) 0.06, MeOH) cf. -53 correlated fully with that
provided for the isolated material,1 providing convincing
evidence that the relative and absolute configurations of 1
are identical to those reported by Luesch and co-workers
(see the Supporting Information). Furthermore, the spectro-
scopic data for synthetic 1 were significantly different from
those reported for gallinamide A (2).1,2 Specifically, reso-
nances corresponding to the N-terminal dimethylated iso-
leucine residue showed the greatest differences in chemical
shift, providing convincing evidence for the diastereomeric
relationship of this natural product.
With synthetic symplostatin 4 now in hand, and given
the promising antimalarial activity exhibited by gallina-
mide A against P. falciparum, we were next interested in
assessing the biological activity of our synthetic material.
Toward this end, 1 was screened against the drug sensitive
(15) Seyberth, T.; Voss, S.; Brock, R.; Wiesmuller, K.-H.; Jung, G.
J. Med. Chem. 2006, 49, 1754–1765.
3
3D7 strain of P. falciparum using a H hypoxanthine
incorporation assay (see the Supporting Information).20,21
Gratifyingly, symplostatin 4 exhibited potent inhibition of
P. falciparum growth, with an ED50 of 74 nM. Furthermore,
it was demonstrated that symplostatin 4 did not cause lysis
of red blood cells up to a concentration of 25 µM, suggesting
(16) Hosseini, M.; Kringelum, H.; Murray, A.; Tonder, J. E. Org. Lett.
2006, 8, 2103–2106.
(17) Wu, Y.; Xu, J.-C. Tetrahedron 2001, 57, 8107–8113.
(18) Grison, C.; Gene`ve, S.; Halbin, E.; Coutrot, P. Tetrahedron 2001,
57, 4903–4923.
(19) Formica, M.; Fusi, V.; Macedi, E.; Paoli, P.; Piersanti, G.; Rossi,
P.; Zappia, G.; Orlando, P. New J. Chem. 2008, 32, 1204–1214.
5578
Org. Lett., Vol. 12, No. 23, 2010

that the antimalarial activity was due to targeting of the
parasite (see the Supporting Information). Future work will
involve a detailed investigation of the mode of action of
symplostatin 4.
Award for PhD funding (T.C.). The work in N.H.H.’s
laboratory was supported by the National Health and Medical
Research Council of Australia. We also acknowledge Dr.
Ian Luck, School of Chemistry, The University of Sydney,
for assistance with NMR spectroscopy.
In summary, we have completed the first total synthesis
of the cyanobacterium-derived depsipeptide natural product
symplostatin 4 (seven steps in the longest linear sequence,
23% overall yield). It is anticipated that the synthetic route
described should provide access to related members of this
class of compounds, including gallinamide A and structural
analogues thereof. This may prove to be extremely valuable
for the development of selective antiparasitic agents against
drug resistant P. falciparum and other related organisms with
low cytotoxicity toward human cells. Studies toward this end
will be the focus of future work in these laboratories.
Supporting Information Available: Experimental pro-
cedures and characterization for all new compounds. Ex-
perimental details and raw data for biological assays and 1H
and 13C NMR spectra for all novel compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL1024663
(20) Golenser, J.; Casuto, D.; Pollack, Y. Exp. Parasitol. 1981, 52, 371–
377.
Acknowledgment. We thank the University of Sydney
for start-up funding (R.J.P.) and an Australian Postgraduate
(21) Becker, K.; Christopherson, R. I.; Cowden, W. B.; Hunt, N. H.;
Schirmer, R. H. Biochem. Pharmacol. 1990, 39, 59–65.
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