Synthesis of clionamine B, an autophagy stimulating aminosteroid isolated from the sponge Cliona celata

Article abstract of DOI:10.1021/ol4016783

Clionamine B (2), an aminosteroid isolated from the marine sponge Cliona celata, has been synthesized starting from the plant sapogenin tigogenin (5). A key step in the synsthesis is the stereoselective introduction of the C-20 α-hydroxyl substituent via oxidation of a γ-lactone enolate with molecular oxygen. Synthetic clionamine B (2) strongly stimulated autophagy in human breast cancer MCF-7 cells.

Full text of DOI:10.1021/ol4016783

ORGANIC
LETTERS
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Synthesis of Clionamine B, an Autophagy
Stimulating Aminosteroid Isolated from
the Sponge Cliona celata
Roberto Forestieri,† Elizabeth Donohue,‡ Aruna Balgi,‡ Michel Roberge,*,‡ and
Raymond J. Andersen*,†
Departments of Chemistry and Earth Ocean & Atmospheric Sciences, University of
British Columbia, 2036 Main Mall, Vancouver, B. C., Canada, V6T 1Z1, and
Department of Biochemistry and Molecular Biology, University of British Columbia,
2350 Health Sciences Mall, Vancouver, B. C., Canada V6T 1Z3
raymond.andersen@ubc.ca
Received June 13, 2013
ABSTRACT
Clionamine B (2), an aminosteroid isolated from the marine sponge Cliona celata, has been synthesized starting from the plant sapogenin
tigogenin (5). A key step in the synsthesis is the stereoselective introduction of the C-20 R-hydroxyl substituent via oxidation of a γ-lactone
enolate with molecular oxygen. Synthetic clionamine B (2) strongly stimulated autophagy in human breast cancer MCF-7 cells.
Autophagy is a cellular process that captures organelles
and proteins in a double membrane vesicle called an
autophagosome and delivers them to the lysosome for
degradation and recyling of the amino acids, monosac-
charides, and lipids.1 Formation of autophagosomes from
nascent double membrane fragments called phagophores,
fusion of autophagosomes with the lysosome, and the
subsequent degradation and recycling of their organelle
and protein cargo is a complex multistep process that is
under the control of more than 35 autophagy-related (Atg)
proteins.2 The basal level of autophagy in cells plays a
quality control role in cellular homeostasis by eliminating
old and damaged organelles and proteins that are essential
cellular components but are no longer functioning reliably.
During starvation, autophagy (Greek for “self-eating”) is
upregulated to provideenergyfor the nutrient deprivedcell
from degraded self-components. Autophagy is also an
important mechanism used by cells to clear pathogenic
viruses3 and bacteria4 and deal with microenvironment
stresses such as exposure to reactive oxygen species.5
Defective autophagy has been implicated in many diseases
suchasneuronaldegeneration and cancer, and itisthought
to play a role in aging. Pharmacological stimulation or
inhibition of autophagy have both been proposed as
approaches to treating a variety of diseases6 and promot-
ing healthy aging.7
(3) Yordy, B.; Tai, M. C.; Hayashi, K.; Arojo, O.; Iwasaki, A. Intl.
Immunol. 2012, 25, 1–10.
(4) (a) Mostowy, S. Cell. Microbiol. 2013, 15, 395–402. (b) Campoy,
E.; Colombo, M. I. Biochim. Biophys. Acta 2009, 1793, 1465–1477. (c)
Songane, M.; Kleinnijenhuis, J.; Netea, M. G.; van Creval, R. Tubercu-
losis 2012, 92, 388–396.
(5) Jones, S. A.; Mills, K. H. G.; Harris, J. Immunol. Cell. Biol. 2013,
91, 250–258.
(6) (a) Rubinsztein, D. C.; Cogogno, P.; Levine, B. Nat. Rev. Drug
Discovery 2012, 11, 709–730. (b) Floto, R. A.; Sarkar, S.; Perlstein, E. O.;
Kampmann, B.; Schreiber, S. L.; Rubinsztein, D. C. Autophagy 2007, 3,
620–622.
† Chemistry and EOAS.
‡ Biochemistry and Molecular Biology.
(1) (a) Oczypok, E. A.; Oury, T. D.; Chu, C. T. Am. J. Pathol. 2013,
182, 612–622. (b) Codogno, P.; Mehrpour, M.; Proikas-Cezanne, T.
Nat. Rev. Mol. Cell. Biol. 2012, 13, 7–12.
(2) (a) Yang, Z.; Klionsky, D. J. Curr. Opin. Cell. Biol. 2010, 22, 124–
131. and (b) Subramani, S.; Malhotra, V. EMBO rep. 2013, 14, 143–151.
r
10.1021/ol4016783
XXXX American Chemical Society

Although basic knowledge about the role of autophagy
in cell biology and disease progression is rapidly increas-
ing, many specific details about the roles of individual Atg
proteins remain unknown.2 It has been proposed that
selective small molecule modulators of Atg protein func-
tion that work in a cellular context would be extremely
useful chemical genetics/cell biology tools for the further
study of autophagy.8 Despite the recognized need for
chemical tools to study autophagy, the number of effective
compounds available for this purpose is extremely limited.
undertook a total synthesis of clionamine B (2), one of the
least abundant analogues in the sponge extract. Since the
ultimate goal was to be able to generate sufficient quan-
tities of a natural clionamine or a more potent analogue for
invivostudies inanimalmodels, the synthesiswasdesigned
to use cheap starting materials and reagents.
The retrosynthetic analysis for a practical general synthe-
sis of clionamines is shown in Scheme 1. Steroidal saponins
are common metabolites of plants and their aglycones
(sapogenins) have been extensively used as starting materi-
als for the production of steroidal hormone drugs.12 As a
consequence, sapogenins are cheap reagents that are read-
ily available in kg quantities. Tigogenin (5) and sarsasa-
pogenin (6) (Scheme 1) are two of the most easily accessed
sapogenins, but variants with substituents at C-12 such as
hecogenin are also available. Methodology for the degra-
dation of sapogenin side chains to give the E ring γ-lactone
found in the clionamines has been known for decades,12
but continues to be refined.13
Scheme 1. Retrosynthetic Analysis for a Clionamine Synthesis
Prompted by the need for novel small molecule mod-
ulators of autophagy as chemical tools and drug leads,9
a library of marine organism crude extracts were screened
in a cell-based high content assay designed to find both
stimulators and inhibitors10 of autophagy. A MeOH ex-
tract of the sponge Cliona celata collected on the Wild
Coast of South Africa showed promising autophagy stim-
ulation in the screen. Assay-guided fractionation of the
extract revealed that the amino steroids clionamines A (1)
to D (4) were responsible for the desired biological
activity.11 Clionamine A (1), the major component in the
extract, strongly stimulated autophagy in human breast
cancer MCF-7 cells in the screening assay.
The clionamines contain structural features not pre-
viously encountered in naturally occurring steroids. Fore-
mostamong theseisthecombination ofanE ringγ-lactone
and C-20 hydroxylation found in all of the analogues and
the spirobislactone side chain found in clionamine D (4).
The clionamines are also 3β-amino steroids, a structural
variation that has precedent in natural steroids, but is rare.
Only single digit milligram quantities of most of the
clionamines were available from the sponge extracts. This
severely limited the range of biological evaluation that
could be carried out on the compounds. In order to solve
the supply issue for the clionamines and to facilitate the
generation of photoaffinity probes for molecular target
identification and provide analogues for SAR studies, we
As proposed in Scheme 1, the last step in the clionamine B
synthesis would be a standard reductive amination of ketone
intermediate IV, which would come from C-20 hydroxyla-
tion of intermediate III. Intermediate III could be prepared
via conjugate addition of R1 to the R-methylene lactone II,
which could arise from dehydrogenation of the R-methyl-γ-
lactone in the sapogenin degradation product I. Degradation
(7) Pallauf, K.; Rimbach, G. Ageing Res. Rev. 2013, 12, 237–252.
(8) Rubinsztein, D. C.; Gestwicki, J. E.; Murphy, L. O.; Klionsky,
D. J. Nat. Rev. Drug Discovery 2007, 6, 304–312.
(9) Lam, K. K. Y.; Zheng, X.; Forestieri, R.; Balgi, A. D.; Nodwell,
M.; Vollett, S.; Anderson, H. J.; Andersen, R. J.; Av-Gay, Y.; Roberge,
M. PLoS Pathogens 2012, 8, e10022691.
(10) Carr, G.; Williams, D. E.; Dıaz-Marrero, A. R.; Patrick, B. O.;
´
Bottriell, H.; Balgi, A. D.; Donohue, E.; Roberge, M.; Andersen, R. J.
J. Nat. Prod. 2010, 73, 422–427.
(11) Keyzers, R. A.; Daoust, J.; Davies-Coleman, M. T.; Van Soest,
R.; Balgi, A.; Donohue, E.; Roberge, M.; Andersen, R. J. Org. Lett.
2008, 10, 2959–2962.
(12) (a) Marker, R. E.; Rohrmann, E. J. Am. Chem. Soc. 1940, 62,
518–520. (b) Ibid. 898À900. (c) Marker, R. E.; Krueger, J. J. Am. Chem.
Soc. 1940, 62, 3349–3350. (d) Djerassi, C. Steroids 1992, 57, 631–641.
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B
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Scheme 2. Degradation of the Side Chain of Tigogenin (5)
Scheme 3. Preparation of the E Ring R-Methylene Lactone
of the sapogenin starting material to give I would follow
literature procedures.12,13 The commercial availability of a
variety of sapogenin starting materials [e.g., tigogenein (5)
and sarsasapogenin (6)] would allow for the synthesis of a
library of analogues (varying in the colored functionalities)
for SAR optimization studies. Variations in R1, R2, and R3
would facilitate the incorporation of the photophore and
alkyne or azide Click chemistry components required to
make probes for cellular protein target identification.
hemiketal 10 was carried out by treatment with PCC in
DCM at rt in the presence of 4 A molecular sieves to give
the ketone 11, containing the E ring γ-lactone found in the
clionamines, in 90% yield.13
Installation of the C-20 hydroxyl group was expected to
be a key transformation in the synthesis of clionamine B
(2). Therefore, we decided to explore methodology for
carrying out C-20 hydroxylation on ketone 11 before
continuing the further elaboration of the side chain carbon
skeleton. Our plan for C-20 hydoxylation involved oxida-
tion of the γ-lactone enolate. To set the stage for this
transformation, the C-3 ketone in 11 was protected as the
cyclic ketal 12 in 92% yield as shown in Scheme 3 to
prevent unwanted base catalyzed condensation and ketone
R-oxidation side reactions. Formation of the enolate of
lactone 12 with t-BuOK in DME at À10 °C in the presence
of a stream of oxygen gas and triethyl phosphite14 followed
by a 10% HCl workup successfully installed the desired
hydroxyl group at C-20 with the R-orientation found in the
clionamines and removed the cyclic ketal at C-3 to give 13
in 51% yield.
The synthesis of clionamine B (2) started with acetylation
of tigogenin (5) to give the acetate 7 in nearly quantitative
yield as shown in Scheme 2.13 Treatment of 7 with Br2 in
glacial acetic acid led to bromination at C-23 on the
dihydropyran ring adjacent to the spiro ketal. A mixture
of the axial and equatorial brominated products 8 was
obtained in 80% combined yield. It is well documented that
when the methyl substituent at C-25 is axial, as in sarsasa-
pogenin (6), bromination gives almost exclusively the equa-
torial bromine at C-23 due to steric interactions with the
methyl, but when the C-25 methyl substituent is equatorial,
as in tigogenin (5), bromination gives a mixture of epimeric
bromides at C-23 as we observed for 8.13
Treatment of the epimeric mixture of bromides 8 with
aqueous ammonia in n-butanol at reflux for 7 days led to
solvolysis and rearrangement to give the desired product 9
in a modest 26% yield.13 The low yield was anticipated
because it was known that the C-23 axial bromides do not
effectively rearrange to give C-22 hemiketals such as 9
under these conditions. Even though the transformation of
8 to 9 proceeded in low yield, the ready availability of 5 and
the ability to carry out the steps from 5 to 9 on multigram
scalesusing cheap reagentsmeant that these first steps were
still a viable entry point to a practical clionamine synthesis.
Hydrolysis of the acetate 9 with K2CO3 in MeOH gave
the alcohol 10 in 91% yield. Oxidative degradation of the
With the C-20 hydroxyl group in hand, we turned our
attention to dehydration as a route to an R-methylene
lactone intermediate. Mesylation of 13 gave the sulfonate
ester 14 in 96% yield.15 In anticipation of using a strong
base to promote elimination of the mesylate to give the
(14) Coburn, C. E.; Anderson, D. K.; Swenton, J. S. J. Org. Chem.
1983, 48, 1455–1461.
(15) Crossland, R. K.; Servis, K. L. J. Org. Chem. 1970, 35, 3195–
3196.
Org. Lett., Vol. XX, No. XX, XXXX
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Scheme 4. Final Steps in the Synthesis of Clionamine B (2)
Figure 1. Autophagy stimulation by clionamines A (1) and B (2).
MCF-7 cells expressing the autophagy marker EGFP-LC3 were
incubated for 4 h with 1 or 2, and autophagosomes (green
puncta) were measured (Supporting Information). Cell nuclei
are in blue.9,11
.
bioactive marine invertebrate secondary metabolites. Clio-
namines are novel amino steroids isolated in small quan-
tities from a marine sponge.11 They stimulate autophagy at
low μM concentrations, making them potentially interest-
ing drug leads for a variety of human diseases.6
conjugated alkene,16 the C-3 ketone was again protected as
a cyclic ketal. Fortuitously, during the ketalization reac-
tion on 14, the mesylate also eliminated to give the desired
R-methylene lactone 15 in 92% yield (Scheme 3).17
Herein, we describe a total synthesis of clionamine B (2)
that starts with a readily available and low cost starting
material tigogenin (5) and makes use of simple reagents. A
key transformation in the synthesis is the stereoselective
installation of the R-hydroxyl substituent at C-20 that uses
molecular oxygen as the oxidant. The synthetic route is flexible
in design and scalable and should, therefore, be able to provide
natural clionamines and unnatural analogues in sufficient
quantities for further biological evaluation. The synthesis of
clionamine B (2) represents the first synthesis of a natural
clionamine. It has confirmed the proposed structure of 2
and allowed us to show for the first time that 2 strongly
stimulates autophagy, with similar potency to clionamine
A (1)11 (Figure 1). Synthesis of additional clionamines and
unnatural analogues is ongoing, and their autophagy-
stimulating and potential therapeutic properties will be re-
ported elsewhere.
The isopentyl Grignard reagent 16 was reacted with
excess CuI, and the resulting organocuprate intermediate18
was added to the R-methylene lactone 15 to give 17 as a 4:1
mixture of β and R epimers in 50% overall yield (Scheme 4).
Hydroxylation of the mixture of C-20 epimers 17 using the
same enolate oxidation conditions developed for lactone
11 gave the C-20 hydroxy derivative 18 as a single epimer
having the clionamine configuration in 65% yield. The
approach of molecular oxygen to the β-face of C-20 is
completely blocked by steric interaction with Me-18. Re-
ductive amination of 18 using ammonium acetate and
sodium cyanoborohydride gave clionamine B (2) in 90%
yield.19 The proton NMR spectrum recorded for synthetic
clionamine B (2) at 600 MHz in MeOD was identical to the
spectrum of natural clionamine B (2) recorded in the same
solvent (Supporting Information).
Acknowledgment. Financial support was provided by
NSERC(R.J.A.) andtheCanadianCancerSociety(M.R.).
We thank A. Lewis of Simon Fraser University for collection
of NMR data on synthetic clionamine B (2).
The lack of an adequate and sustainable supply of
compound is most often a limiting factor in the preclinical
biological evaluation and eventual clinical development of
(16) Chiao, W.-B.; Saunders, W. H. J. Org. Chem. 1980, 45, 1319–
1320.
(17) Bolster, M. G.; Jansen, B. J. M.; de Groot, A. Tetrahedron 2001,
57, 5663–5679.
(18) House, H. O.; Respess, W. L.; Whitesides, G. M. J. Org. Chem.
1966, 31, 3128–3141.
(19) Borch, R. F.; Bernstein, M. D.; Durst, H. D. J. Am. Chem. Soc.
Supporting Information Available. 1H and 13C NMR
spectra of synthetic intermediates and clionamine B (2).
This material is available free of charge via the Internet at
http://pubs.acs.org.
1971, 93, 2897–2904.
The authors declare no competing financial interest.
D
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