The cytotoxic styryl lactone goniothalamin is an inhibitor of nucleocytoplasmic transport

Article abstract of DOI:10.1016/j.bmcl.2010.03.049

An in vivo nuclear export assay (immunostaining of Rio2 in HeLa cells) demonstrated that (R)-goniothalamin is an inhibitor of nucleocytoplasmic transport above 500 nM, which was rationalized also by molecular modeling. The cytotoxic styryl lactone natural product was prepared via an enantioselective Cr(III) catalyzed hetero Diels-Alder reaction and a Sonogashira coupling. A series of analogs was synthesized and only the oxidized goniothalamin derivative featuring an alkyne spacer was found active. Unsaturated lactones of natural origin other than leptomycin (LMB) are thus suggested to operate via a similar mechanism targeting the CRM1 nuclear receptor.

Full text of DOI:10.1016/j.bmcl.2010.03.049

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2843–2846
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The cytotoxic styryl lactone goniothalamin is an inhibitor of
nucleocytoplasmic transport
Jean-Yves Wach a,b, Stephan Güttinger c, Ulrike Kutay c, Karl Gademann a,b,
*
a Swiss Federal Institute of Technology (EPFL), Chemical Synthesis Laboratory (SB-ISIC-LSYNC), 1015 Lausanne, Switzerland
b University of Basel, Department of Chemistry, St. Johanns-Ring 19, CH-4056 Basel, Switzerland
c Swiss Federal Institute of Technology (ETH), Institut für Biochemie, 8093 Zürich, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
An in vivo nuclear export assay (immunostaining of Rio2 in HeLa cells) demonstrated that (R)-goniothal-
amin is an inhibitor of nucleocytoplasmic transport above 500 nM, which was rationalized also by molec-
ular modeling. The cytotoxic styryl lactone natural product was prepared via an enantioselective Cr(III)
catalyzed hetero Diels–Alder reaction and a Sonogashira coupling. A series of analogs was synthesized
and only the oxidized goniothalamin derivative featuring an alkyne spacer was found active. Unsaturated
lactones of natural origin other than leptomycin (LMB) are thus suggested to operate via a similar mech-
anism targeting the CRM1 nuclear receptor.
Received 19 February 2010
Revised 7 March 2010
Accepted 9 March 2010
Available online 24 March 2010
Keywords:
Natural products
Chemical biology
Target identification
Organic synthesis
Ó 2010 Elsevier Ltd. All rights reserved.
Goniothalamin (1 Fig. 1) is the prototypical example of many
styryl lactones that have been discovered in phytochemical stud-
ies of plants of the genus Goniothalamus (Annonaceae).1 For this
compound, a broad manifold of biological activities along the
general theme of cytotoxicity has been described in the litera-
ture, ranging from antifungal,2,3 antimicrobial,3 insecticidal4 to
anticancer properties.5 In the context of antiproliferative activity,
Xiao and co-workers reported selectivity of goniothalamin on
both drug-resistant and transformed hepatocytes when com-
pared to a primary cell line.6 Pilli and co-workers prepared ana-
logs of goniothalamin and evaluated them in a panel of eight
cancer cell lines leading to potent derivatives against kidney
cancer cells and adriamycin resistant breast cancer cells.7 Gonio-
thalamin has been reported to selectively trigger caspase in-
duced apoptosis in cancer cells, with little activity on normal
cell lines.8 Interestingly, up-regulation of p53 has been observed
in goniothalamin treated rats.9 In all these studies, the exact
mechanism of action and the target of goniothalamin (1) re-
mained unclear.
Both leptomycin and the closely related derivative callystatin
have been evaluated in clinical trials against cancer, but have failed
in phase II due to unfavorable toxicity issues.12 Recent studies sug-
gested these toxic effects to be the result of off-target effects, and
second generation analogs display promising results in cancer
xenograft models.13 In the context of our synthetic and biological
studies on the anguinomycins,14 it has been discovered that the
truncated analog 3 retained almost the full biological potency of
the parent compound. The structural similarity of this leptomycin
analog 3 to goniothalamin (1) thus led to the hypothesis that
goniothalamin (1) and similar naturally occurring lactones would
CH3
H
H
O
O
O
O
O
O
H3C CH3
3
goniothalamin (1)
The polyketide lactone leptomycin (2, LMB) has been demon-
strated to be a potent inhibitor of the CRM1-mediated nuclear ex-
port of proteins,10 and hundreds of studies document its usefulness
as tool compound in cell biology.11
CH3
H
CH3 CH3 CH3 CH3 CH3 O
OH
CH3 H3C
O
OH
leptomycin B (2)
* Corresponding author.
E-mail address: karl.gademann@unibas.ch (K. Gademann).
Figure 1. Goniothalamin (1), lemptomycin B (2, LMB) and the truncated analog 3.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.049

2844
J.-Y. Wach et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2843–2846
also constitute inhibitors of nucleocytoplasmic transport leading to
potent antiproliferative effects. In this study, we present a new
synthetic route to goniothalamin (1) and analogs, and analyze their
role as inhibitors of nucleocytoplasmic transport in an in vivo
transport assay in order to evaluate the above-mentioned
hypothesis.
for R = Ph (8): a-c
R
H
H
for R = CHCHPh (9): d-f
O
O
O
O
6
for R = Ph (10): g-i
for R = Bn (11): j-l
The Cr(III) catalyzed asymmetric hetero Diels–Alder reaction
for R = C6H4Ph (12): m-o
developed by Jacobsen and co-workers15 provides a rapid and
R
O
+
4
CH3
high-yielding synthetic entry into
a,b-unsaturated lactones. In
H
R
addition, the resulting intermediates can be rapidly functionalized
by cross-coupling to provide a range of derivatives. We chose thus
to utilize this synthetic approach for the preparation of goniothal-
amin (1) and analogs.
The key intermediate 6 was obtained by the reaction of meth-
oxybutadiene (4) with TES protected propargylic aldehyde (5), fol-
lowed by transacetalization and deprotection in 70% overall yield
over three steps (Scheme 1).14,15 The dihydropyran was then con-
verted to the goniothalamin lactol ether 7 by hydrozirconation,
transmetallation and Negishi cross-coupling.16 Deprotection and
oxidation furnished synthetic goniothalmin (1), the spectral prop-
erties of which were identical to those reported for the natural
product.17
Analogs 8 and 9 were accessed via Sonogashira cross-coupling
of intermediate 6 with bromobenzene or bromostyrene followed
by deprotection/oxidation in 53% and 47% overall yield, respec-
tively, over three steps. Finally, analogs 10–12 were directly
obtained via the Cr-catalyzed hetero Diels–Alder reaction involv-
ing 1-methoxy-1,3-butadiene 4 and different aromatic aldehydes
(phenylacetaldehyde, benzaldehyde or 4-biphenylcarboxalde-
hyde) followed by the deprotection/oxidation sequence
(Scheme 2).
N
O
O
Cr
13
Cl
Scheme 2. Synthesis of goniothalamin analogs 8–12. Reagents and conditions: (a)
iodobenzene, Pd(PPh3)4 (5 mol %), CuI, EtN(iPr)2, THF, room temperature, 1 h; (b)
PPTS, acetone/water (3:1), room temperature, 22 h; (c) PCC, 4 Å MS, AcOH, CH2Cl2,
room temperature, 3 h, 53% over three steps; (d) bromostyrene, Pd(PPh3)4
(5 mol %), CuI, EtN(iPr)2, THF, room temperature, 1 h; (e) PPTS, acetone/water
(3:1), room temperature, 22 h; (f) PCC, 4 Å MS, AcOH, CH2Cl2, room temperature,
3 h, 47% over three steps; (g) 4, benzaldehyde, 13 (5 mol %), 4 Å MS, room
temperature, 14 h; (h) PPTS, acetone/water (3:1), room temperature, 22 h; (i) PCC,
4 Å MS, AcOH, CH2Cl2, room temperature, 3 h, 70% over three steps; (j) 4,
phenylacetaldehyde, 13 (5 mol %), 4 Å MS, room temperature, 14 h; (k) PPTS,
acetone/water (3:1), room temperature, 22 h; (l) PCC, 4 Å MS, AcOH, CH2Cl2, room
temperature, 3 h, 61% over three steps; (m) 4, biphenyl-4-carboxaldehyde, 13
(5 mol %), 4 Å MS, room temperature, 14 h; (n) PPTS, acetone/water (3:1), room
temperature, 22 h; (o) PCC, 4 Å MS, AcOH, CH2Cl2, room temperature, 3 h, 59% over
three steps.
Goniothalamin (1) exhibited a strong inhibition of CRM1-
Overall, goniothalamin (1) and series of different lactones have
been prepared, which allow for the evaluation of the distance of
the aromatic ring to the lactone. Whereas in the truncated gonio-
thalamin analogs, the double bond was omitted (10) or replaced
by a CH2 group (11), extended analogs containing an additional
aromatic spacer (12) or an alkyne (9) were obtained. Lastly, in
the alkyne 8, the geometry of the side chain has changed by con-
verting the bent alkene to the linear alkyne. The natural product
1 and all these analogs 8–12 have been tested for their ability to
block CRM1-mediated nuclear export of the protein kinase Rio2
in HeLa cells (Fig. 2).
dependent nuclear export at higher concentrations (Fig. 2). The
compound fully blocked export of human Rio2 protein at 1 lM,
with partial inhibition starting between 200 and 500 nM. Of all
the other derivatives 8–12 tested, only 8 showed activity similar
to goniothalamin ( Fig. 2). All other compounds, namely 9–12,
did not show any significant inhibition of nuclear export up to a
concentration of 2 lM (data not shown).
These biological data can be rationalized by inspecting the
published model14b of LMB-mediated inhibition of CRM1, based
on the recently published X-ray crystal structures of this expor-
tin.18 Superposition of goniothalamin (1) to LMB reveals a com-
plementary fit of this natural product into the hydrophobic
binding grove of the export receptor (Fig. 3). Goniothalamin is ex-
pected to be covalently bound to CRM1 and the styryl group
mimics the hydrophobic diene part of LMB. Consequently, several
both smaller and larger analogs of goniothalamin lead to negative
steric interactions with this binding grove and thus to much low-
er (or no) activity towards CRM1. The exception is the alkyne 8,
which can still be accommodated by the substrate binding grove
of the exportin.
OiPr
OCH3
O
ref. 15
O
+
H
3 steps
TES
H
70% overall yield
H
4
5
6
Goniothalamin (1) has been reported to display cytotoxic activ-
a - c
ity in breast cancer cells with an IC50 value of about 1.46 lM, and
to induce cell cycle arrest and apoptosis.5 Based on our results, we
hypothesize that these effects, and therefore the cytotoxicity,
might be due to a block of CRM1-mediated nuclear export by
OiPr
O
goniothalamin at concentrations above 1 lM. This correlation is
d,e
supported by certain described phenotypes of goniothalamin ex-
posed cells, as for example up-regulation of p53 has been
reported.9
In conclusion, we report a new route to goniothalamin (1)
based on stereoselective hetero Diels–Alder and cross-coupling
1
H
7
Scheme 1. Synthesis of goniothalamin (1). Reagents and conditions: (a) Cp2ZrHCl,
0 °C to room temperature, THF, 1 h; (b) ZnCl2, THF, room temperature, 30 min; (c)
iodobenzene, Pd(PPh3)4 (5 mol %), DIBAH (1.0 M in hexane), room temperature to
40 °C, 12 h, 85% over three steps; (d) PPTS, acetone/water (3:1), room temper-
ature, 22 h; (e) PCC, 4 Å MS, AcOH, CH2Cl2, room temperature, 3 h, 61% over two
steps.
reactions. The versatility of the dihydropyran platform
6
allowed for the generation of several analogs probing the dis-
tance of the aromatic group to the lactone ring. Biological eval-
uation revealed that goniothalmin (1) is an inhibitor of

J.-Y. Wach et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2843–2846
2845
Figure 2. Goniothalamin (1) and the alkyne analog 8 inhibit CRM1-dependent nuclear export of Rio2 in HeLa cells.
nucleocytoplasmic transport, and structure–activity relationships
established the molecular requirements for binding, which could
also be rationalized by an atomistic description of the goniothal-
amin/CRM1 interaction.
The results documented in this study thus support the hypoth-
esis that a target of goniothalamin (1) is the exportin CRM1 and
that the antiproliferative activity of 1 might be due to a block
of CRM1-mediated nuclear export. With a mechanism of action
of this natural product revealed, the search for structurally sim-
pler LMB analogs useful for anticancer purposes might be
stimulated.
In addition, the arsenal of natural products capable of interfering
with nuclear transport receptors is thus expanded by goniothalamin
(1). It is reasonable to assume that other natural products, or even
endogenous compounds featuring similar structural elements such
as
a,b-unsaturated lactones with hydrophobic appendages, might
constitute inhibitors of CRM1-mediated nuclear export. Current ef-
forts in our laboratories are directed towards their identification.

2846
J.-Y. Wach et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2843–2846
2. (a) Martins, C. V. B.; de Resende, M. A.; Magalhães, T. F. F.; Lima, B. H. S.;
Watanabe, G. A.; Ruiz, A. L. T. G.; de Carvalho, J. E.; Pilli, R. A.; de Fátima, Ñ. Lett.
Drug Des. Disc. 2008, 5, 74; (b) Martins, C. V. B.; de Resende, M. A.; da Silva, D. L.;
Magalhães, T. F. F.; Modolo, L. V.; Pilli, R. A.; de Fátima, Ñ. J. Appl. Microbiol.
2009, 107, 1279.
3. Mosaddik, M. A.; Haque, M. E. Phytother. Res. 2003, 17, 1155.
4. Kabir, K. E.; Khan, A. R.; Mosaddik, M. A. J. Appl. Entomol. 2003, 127, 112;
Senthil-Nathan, S.; Choi, M.-Y.; Paik, C.-H.; Kaaivani, K. Chemosphere 2008, 72,
1393.
5. (a) Chen, W. Y.; Wu, C. C.; Lan, Y. H.; Chang, F. R.; Teng, C. M.; Wu, Y. C. Eur. J.
Pharmacol. 2005, 522, 20; (b) Pihie, A. H. L.; Stanslas, J.; Din, L. B. Anticancer Res.
1998, 18, 1739; (c) de Fátima, Ñ; Modolo, L. V.; Conegero, L. S.; Pilli, R. A.;
Ferreira, C. V.; Kohn, L. K.; de Carvalho, J. E. Curr. Med. Chem. 2006, 13, 3371; (d)
Marquissolo, C.; de Fátima, Ñ.; Kohn, L. K.; Ruiz, A. L. T. G.; de Carvalho, J. E.;
Pilli, R. A. Bioorg. Chem. 2009, 37, 52.
6. Tian, Z.; Chen, S.; Zhang, Y.; Huang, M.; Shi, L.; Huang, F.; Fong, C.; Yang, M.;
Xiao, P. Phytomedicine 2006, 13, 181.
7. de Fátima, Ñ.; Kohn, L. K.; de Carvalho, J. E.; Pilli, R. A. Bioorg. Med. Chem. 2006,
14, 622.
8. (a) Inayat-Hussain, S. H.; Annuar, B. O.; Din, L. B.; Ali, A. M.; Ross, D. Toxicol. In
Vitro 2003, 17, 433; (b) Inayat-Hussain, S. H.; Osman, A. B.; Din, L. B.; Ali, A. M.;
Snowden, R. T.; MacFarlane, M.; Cain, K. FEBS Lett. 1999, 456, 379.
9. Meenakshii, N.; Lee, A.; Azimahtol Hawariah, L. P.; Hasidah, S. Malays. Appl. Biol.
2000, 29, 121.
10. (a) Fornerod, M.; Ohno, M.; Yoshida, M.; Mattaj, I. W. Cell 1997, 90, 1051; (b)
Fukuda, M.; Asano, S.; Nakamura, T.; Adachi, M.; Yoshida, M.; Yanagida, M.;
Nishida, E. Nature 1997, 390, 308; (c) Wolff, B.; Sanglier, J. J.; Wang, Y. Chem.
Biol. 1997, 4, 139; (d) Nishi, K.; Yoshida, M.; Fujiwara, D.; Nishikawa, M.;
Horinouchi, S.; Beppu, T. J. Biol. Chem. 1994, 269, 6320; (e) Kalesse, M.;
Christmann, M. Synthesis 2002, 981.
11. Review: Kutay, U.; Güttinger, S. Trends Cell Biol. 2005, 15, 121–124.
12. Newlands, E. S.; Rustin, G. J. S.; Brampton, M. H. Br. J. Cancer 1996, 74,
648.
Figure 3. Superposition of goniothalamin (1, blue) and the model of leptomycin B
(2, LMB, yellow) binding to CRM114b based on the X-ray structures.18
13. (a) Santi, D. V.; Zhou, Y. WO Patent 2005086944, 2005.; (b) Santi, D. V.; Myles,
D. C.; Dong, S.; Brian, H. U. S. Patent 2005272727, 2005.; (c) Dong, S.; Santi, D.
V. WO Patent 2007033214, 2007.; (d) Dong, S.; Myles, D. C.; Santi, D. V.; Brian,
H. CN Patent 1964718, 2008.; (e) Mutka, S. C.; Yang, W. Q.; Dong, S. D.; Ward, S.
L.; Craig, D. A.; Timmermans, P. B. M. W. M.; Murli, S. Cancer Res. 2009, 69, 510;
(f) Van Neck, T.; Pannecouque, C.; Vanstreels, E.; Stevens, M.; Dehaen, W.;
Daelemans, D. Bioorg. Med. Chem. 2008, 16, 9487.
Acknowledgments
K.G. is a European Young Investigator (EURYI). We thank the
SNF for support of this work (3100AO-118053, 200021-115918/1
and PE002-117136/1).
14. (a) Bonazzi, S.; Güttinger, S.; Zemp, I.; Kutay, U.; Gademann, K. Angew. Chem.,
Int. Ed. 2007, 46, 8707; (b) Bonazzi, S.; Eidam, O.; Güttinger, S.; Wach, J.-Y.;
Zemp, I.; Kutay, U.; Gademann, K. J. Am. Chem. Soc. 2010, 132, 1432.
15. (a) Dossetter, A. G.; Jamison, T. F.; Jacobsen, E. N. Angew. Chem., Int. Ed. 1999, 38,
2398–2400; (b) Chavez, D. E.; Jacobsen, E. N. Angew. Chem., Int. Ed. 2001, 40,
3667.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.03.049.
16. Huang, Z.; Negishi, E. Org. Lett. 2006, 8, 3675.
17. de Fátima, Ñ.; Pilli, R. A. Tetrahedron Lett. 2003, 44, 8721.
18. (a) Dong, X.; Biswas, A.; Suel, K. E.; Jackson, L. K.; Martinez, R.; Gu, H.; Chook, Y.
M. Nature 2009, 458, 1136; (b) Monecke, T.; Güttler, T.; Neumann, P.;
Dickmanns, A.; Görlich, D.; Ficner, R. Science 2009, 324, 1087.
References and notes
1. Jewers, K.; Davis, J. B.; Dougan, J.; Manchanda, A. H.; Blunden, G.; Kyi, A.;
Wetchapinan, S. Phytochemistry 1972, 11, 2025.