Spirofungin A: Stereoselective synthesis and inhibition of isoleucyl-tRNA synthetase

Article abstract of DOI:10.1002/anie.200702440

(Chemical Equation Presented) Lock it in: A temporary silicon-based configurational lock (see scheme) has enabled an efficient and fully diastereo-selective assembly of the spiroketal subunit of spirofungin A. This complex natural product was synthesized in 20 steps, including a rapid polyketide assembly based on the ring-opening metathesis of a cyclopropenone acetal. It was established that spirofungin A elicited notable anti-proliferative activity against several human cancer cell lines and selectively inhibited isoleucyl-tRNA synthetase in vitro.

Full text of DOI:10.1002/anie.200702440

Communications
DOI: 10.1002/anie.200702440
Natural Product Synthesis
Spirofungin A: Stereoselective Synthesis and Inhibition of Isoleucyl-
tRNA Synthetase**
Jasmina Marjanovic and Sergey A. Kozmin*
Spirofungins A and B constitute a family of secondary
metabolites from Streptomyces violaceusniger Tü 4113.[1]
The two natural products arise from epimerization of the
spiroketal subunit[2] and were initially isolated as a mixture,
which was reported to inhibit growth of Candida albicans.[1]
While the antifungal activity of each congener has not been
assessed, the structure–activity relationship of the closely
related reveromycins[3,4] strongly suggested that spirofun-
gin A (1) should display antiproliferative activity not only in
yeast, but also in mammalian cells, possibly by specific
inhibition of isoleucyl-tRNA synthetase.[5,6] The first synthe-
ses of spirofungins A and B were recently reported by
Shimizu et al.[7] The assembly process, however, required
chromatographic separation of the two diastereomeric spi-
roketals en route to the final targets. Our synthetic strategy
was uniquely designed to solve a challenging spiroketalization
problem and to provide a fully stereoselective access to
spirofungin A (1). Herein we report the development of a
highly stereocontrolled and efficient synthesis of this natural
product. We further demonstrate that spirofungin A (1)
displays notable antiproliferative activity in a panel of
cancer cell lines, and selectively inhibits the activity of
isoleucyl-tRNA synthetase in mammalian cells.
The retrosynthetic analysis of spirofungin A (1) involves
the initial detachment of the two unsaturated side arms from
À
the spiroketal subunit at the C(20) C(21) alkene and the
diene fragments at C(7) and C(8) (Scheme 1). Control of the
spiroketalization event entailed the most challenging aspect
of the synthesis.[8] While the desired spiroketal 2 is favored
stereoelectronically, the axial disposition of the C(19) sub-
stituent leads to significant steric congestion. As a result, a
mixture of two spiroketals 2 and 3 is expected to form upon
spontaneous spiroketalization.[9] To enable the exclusive
formation of spiroketal 2, we exploited different spatial
orientation of the side arms (R1 and R2) in the two spiroketal
units. Indeed, if the two arms were held by a temporary
connection (that is, using a cyclic silane 4),[10] this would force
Scheme 1. Retrosynthetic analysis of spirofungin A (1).
the spiroketalization of the 15-membered silacyclic ketone 5
to produce spiroketal 4 exclusively.[11] Cyclic ketone 5, in turn,
would derive from dienone 6, which would be assembled from
four simple building blocks (7–10) by employing our cyclo-
propenone acetal metathesis-based approach for polyketide
assembly, which was initially developed during the synthesis
of bistramide A.[12]
[*] J. Marjanovic, Prof. Dr. S. A. Kozmin
Department of Chemistry
University of Chicago
5735 South Ellis Avenue, Chicago, IL 60637 (USA)
Fax: (+1)773-702-0805
The synthesis began by subjecting alkene 11[13] to cyclo-
propenone acetal 12 in the presence of the Grubbs catalyst
13,[14] which promoted the ring-opening metathesis to give
diene 14 upon subsequent desilylation (Scheme 2). Sequential
exposure of a mixture of alcohols 14 and 15[13] to dichlor-
odiisopropylsilane and imidazole introduced the requisite
dialkoxysilane connector. Chemoselective removal of the 1,3-
dioxane was efficiently achieved using oxalic acid to give
ketone 16. Exposure of 16 to the Grubbs catalyst 13 resulted
E-mail: skozmin@uchicago.edu
[**] S.A.K. thanks the Sloan Foundation, the Dreyfus Foundation,
Amgen, and GlaxoSmithKline for financial support of this work. We
thank Prof. Wool and Dr. Chan (University of Chicago, Department
of Biochemistry and Molecular Biology) for assistance with amino-
acyl-tRNA synthetase assays.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Angewandte
Chemie
Scheme 2. Stereocontrolled synthesis of spirofungin A (1). Bn = benzyl, Mes = 2,4,6-trimethyl phenyl, Cy = cyclohexyl, TBAF =
tetrabutylammonium fluoride, TBSCl = tert-butyldimethylsilyl chloride, dba = dibenzylideneacetone, LHMDS = lithium hexamethyldisilazanide,
DMPU = N,N’-dimethyl-N,N’-propylene urea.
in the cyclization to afford the 15-membered dienone 17 in
85% yield.[15] The ring-closing metathesis proceeded with
complete chemoselectivity only at the two terminal alkenes of
trienone 16. Hydrogenation of 17 with concomitant hydro-
genolysis of the two benzyl ethers resulted in a spontaneous
formation of tricyclic spiroketal 18 as a single detectable
diastereomer in 98% yield. The structure of 18 was estab-
lished by a combination of COSY and NOESY NMR
spectroscopy, as well as X-Ray crystallographic analysis of
the structurally analogous spiroketal 26 obtained during our
removal of the primary TBS ether. The final diene subunit
was introduced by Dess–Martin oxidation of alcohol 23 and
treatment of the resulting aldehyde with phosphonate 24.[7]
Completion of the synthesis of spirofungin A entailed the
saponification of the two methyl esters in 25, followed by
removal of the TBS group according to the protocol
developed by Shimizu et al.[7] The NMR and mass spectra,
as well as the optical rotations of (À)-spirofungin A (1) and
diester 25 were in agreement with those reported.[7]
While the inhibition of the yeast growth by spirofungin A
(1) has been previously reported,[1] we were interested in
probing the antiproliferative activity of this natural product in
mammalian cells. By using the standard ATP-monitoring
luciferase-based protocol,[13] we established that spirofun-
gin A inhibited the growth of several human cancer cell lines,
including HL-60 (leukemia), HCT116 (colon), PC3 (pros-
tate), and A549 (lung), with IC50 values of 1.0, 0.64, 1.9, and
6.4 mm, respectively (Figure 1). It is noteworthy that this
activity profile was consistent with the previously observed
cell-based behavior of reveromycin A.[3b]
exploratory study.[16] Fluoride-mediated cleavage of three O
À
Si bonds afforded the corresponding triol, which was treated
with NaIO4 to give hydroxy aldehyde 19. Installation of the
TBS protecting group, and conversion of the aldehyde into
dibromoalkene 20 allowed the introduction of the diene
subunit. The first stage of this process entailed the E-selective
Stille cross-coupling reaction[17] of dibromide 20 with stan-
nane 21.[13] The resulting bromodiene was subjected to
Negishi cross-coupling[18] using Me2Zn and [Pd(tBu3P)2] to
give triene 22, which was further elaborated via cross-
metathesis with methyl acrylate[19] and chemoselective
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Communications
act as a specific inhibitor of isoleucyl-tRNA synthetase. To
test this hypothesis we examined the ability of spirofungin A
to inhibit the synthesis of isoleucyl-tRNA and leucyl-tRNA
in vitro by monitoring the incorporation of the corresponding
[3H]-labeled amino acids into the aminoacyl-tRNAs in HL-60
cell lysates. We found that spirofungin A (1) inhibited
isoleucyl-tRNA synthetase in a dose-dependent manner
(Figure 2a). Importantly, no effect of spirofungin A (1) on
the activity of a homologous leucyl-tRNA synthetase was
observed (Figure 2b), which strongly indicated a highly
selective mode of inhibition of isoleucyl-tRNA synthetase
by spirofungin A (1).
In closing, we have developed a fully stereocontrolled
synthesis of (À)-spirofungin A (1) with a longest linear
sequence of 20 steps. The strategy exploits our approach
based on cyclopropenone acetal metathesis for rapid polyke-
tide assembly. We further demonstrated that the natural
product suppressed proliferation of several human cancer cell
lines and selectively inhibited the activity of isoleucyl-tRNA
synthetase in vitro. This study sets the stage for determining
the nature of the remarkably specific inhibition of isoleucyl-
tRNA synthesis by spirofungin A (1) at the molecular level
and the investigation of potential therapeutic applications of
this natural product.[6]
Figure 1. Dose-dependent suppression of cancer cell proliferation by
spirofungin A (1). The cell viability assays were performed with 1000
cells per well by using an ATP-monitoring luciferase-based protocol at
a variable concentration of spirofungin A (1).
Reveromycin A has been previously demonstrated to
specifically inhibit the activity of isoleucyl-tRNA synthetase,
which was identified by a combination of yeast genetics and
biochemical methods to be the cellular target of this natural
product.[5,6] The significant degree of structural homology
between reveromycin A and spirofungin A (1), as well as the
similar cell-based antiproliferative activities of the two
natural products, strongly suggested that spirofungin A may
Received: June 5, 2007
Revised: August 27, 2007
Published online: October 11, 2007
Keywords: metathesis · natural products · spiroketals ·
.
stereocontrolled synthesis · tRNA synthetase
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Figure 2. Dose-dependent effects of spirofungin A (1) on the activity of
isoleucyl-tRNA synthetase (a) and leucyl-tRNA synthetase (b) in HL-60
cell lysates. The data is expressed as mean values from two cultures.
[9] A nearly equimolar mixture of spiroketals 2 and 3 is typically
obtained upon spontaneous spiroketalization (see Refs. [7] and
[8]).
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lographic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
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[16] ORTEP representation of tricyclic silane 26 prepared during our
initial studies of spiroketalization; for details, see the Supporting
Information. CCDC-658800 contains the supplementary crystal-
[17] W. Shen, L. Wang, J. Org. Chem. 1999, 64, 8873 – 8879.
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