Enantioselective synthesis of spliceostatin e and evaluation of biological activity

Article abstract of DOI:10.1021/ol503127r

An enantioselective total synthesis of spliceostatin E has been accomplished. The δ-lactone unit A was constructed from readily available (R)-glycidyl alcohol using a ring-closing olefin metathesis as the key reaction. A cross-metathesis of ring A containing δ-lactone and the functionalized tetrahydropyran B-ring provided spliceostatin E. Our biological evaluation of synthetic spliceostatin E revealed that it does not inhibit splicing in vitro and does not impact speckle morphology in cells. Spliceostatin E was reported to possess potent antitumor activity.

Full text of DOI:10.1021/ol503127r

Letter
pubs.acs.org/OrgLett
Enantioselective Synthesis of Spliceostatin E and Evaluation of
Biological Activity
Arun K. Ghosh,*,† Anne M. Veitschegger,† Venkata Reddy Sheri,† Kerstin A. Effenberger,‡
Beth E. Prichard,‡ and Melissa S. Jurica‡
†Department of Chemistry and Department of Medicinal Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana
47907, United States
‡Department of Molecular Cell and Developmental Biology and Center for Molecular Biology of RNA, University of California, Santa
Cruz, California 95064, United States
S
* Supporting Information
ABSTRACT: An enantioselective total synthesis of spliceostatin E
has been accomplished. The δ-lactone unit A was constructed from
readily available (R)-glycidyl alcohol using a ring-closing olefin
metathesis as the key reaction. A cross-metathesis of ring A containing
δ-lactone and the functionalized tetrahydropyran B-ring provided
spliceostatin E. Our biological evaluation of synthetic spliceostatin E
revealed that it does not inhibit splicing in vitro and does not impact
speckle morphology in cells. Spliceostatin E was reported to possess potent antitumor activity.
Splicing is an essential step in gene expression in which
noncoding sequences (introns) are removed from RNA
transcripts and coding sequences (exons) are ligated to form
messenger RNA (mRNAs).1 A complex ribonucleoprotein
machinery, known as spliceosome, facilitates this splicing
event.2 The splicing levels are generally increased in cancer
cells, and as a consequence, spliceosome inhibitors have been
implicated in anticancer drug development.3−5 Pladienolide B,
1 (Figure 1), and their derivatives have been shown to inhibit
spliceosome by binding to the SF3b subunit of spliceosome.6 A
semisynthetic pladienolide B derivative has entered in human
clinical trials.7 Structurally distinct, FR901464, 2, and its
methylated derivative, spliceostatin A, 3, have also been shown
to inhibit in vitro splicing by binding to the SF3b subunit of the
spliceosome.8 FR901464, 2, was isolated by Nakajima and co-
workers at the Fujisawa Pharmaceutical Co. from the
fermentation broth of strain no. 2663 in 1996.9,10 It exhibited
potent anticancer activity with IC50 values ranging from 0.6 to
3.4 nM against numerous human cancer cell lines. Yoshida and
co-workers then reported spliceostatin A with similar activity as
FR901464.11,12 Not surprisingly, both FR901464 and spliceos-
tatin A have attracted considerable synthetic interest.13−15 We
recently reported the synthesis and structure−activity studies of
FR901464 and spliceostatin A.16,17 FR901464 and spliceostatin
A are structurally quite complex and thus posed a challenge as a
starting point for structural modification.
Figure 1. Structures of pladienolide B, FR901464, and spliceostatins A
and E.
Very recently, He and co-workers reported the isolation of a
number of spliceostatin class natural products from the
fermentation broth of FERM BP3421, which was later renamed
as Burkholderia sp.18 Among these new spliceostatins,
structurally less complex spliceostatin E, 3, exhibited good
potency against multiple human cancer cell lines with IC50
values ranging from 1.5 to 4.1 nM. The structure of
spliceostatin E was elucidated by extensive spectroscopic
studies.18 Spliceostatin E structure contains a 5, 6-dihydro-α-
pyrone unit (A ring) and a common highly functionalized B
Received: October 25, 2014
© XXXX American Chemical Society
A
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Organic Letters
Letter
ring. The structural features of spliceostatin E appear to be very
attractive for structural variation and optimization of potency.
Herein, we report an enantioselective synthesis of spliceostatin
E. We have also evaluated its spliceosome inhibitory activity
and effect on cells.
Scheme 2. Synthesis of (S)-Dihydropyranone, 5
Our synthetic strategy for spliceostatin E is shown in Scheme
1. We planned to carry out a cross-metathesis of vinyl
Scheme 1. Retrosynthesis of Spliceostatin E
To provide rapid access to (S)-dihydropyranone ent-5, we
have explored an alternative route. As shown in Scheme 3, allyl
Scheme 3. Synthesis of Racemic Dihyropyranone
dihydropyranone 5 and tetrahydropyranyl diene 6 to construct
spliceostatin E. Optically active 5,6-dihydro-α-pyrone ring A
would be synthesized from (S)-glycidyl silyl ether 7, which can
be readily prepared from commercially available (R)-glycidyl
alcohol. The tetrahydropyranyl ring B fragment would be
obtained by formation of an amide between previously
prepared amine 816,17 and optically active acid 9.
The synthesis of (S)-dihydropyranone 5 is shown in Scheme
2. Opening of the epoxide 7 with isopropenylmagnesium
bromide in the presence of a catalytic amount of CuCN (10
mol %) at −20 to +23 °C for 30 min provided the
corresponding homoallylic alcohol.19 Reaction of the resulting
alcohol with acrolein diethyl acetal in the presence of a catalytic
amount of PPTS in benzene at 23 °C for 12 h afforded ethyl
bromide 14 was synthesized by bromination of 3,3-dimethyl
acrylate with N-bromosuccinimide as described by Fallis and
Lei.23 The E/Z mixture (1:1) was exposed to vinylogous
Reformatsky reaction with acrolein to provide allylic alcohol 15
and racemic dihydropyranone ( )-5.24 These products were
separated by silica gel chromatography (54% yield, 1:1
mixture). The racemic mixture was separated by chiral HPLC
using a Chiralpak IA column to provide access to both (R)- and
(S)-dihydropyranone derivatives.25
The synthesis of Z-allylic acetate 9 is outlined in Scheme 4.
Commercially available (S)-3-butyn-2-ol was protected as TES-
ether with TESOTf and Et3N in CH2Cl2. Treatment of this
TES-ether with nBuLi followed by reaction with ethyl
chloroformate afforded alkylated product 17 in 81% yield.26
Removal of the TES group followed by saponification of the
ethyl ester with aqueous LiOH provided the corresponding
acid. Acylation of this acid with acetyl chloride at 23 °C for 5 h
furnished acetate 18. Catalytic hydrogenation of acetate 18 over
Lindlar’s catalyst as described by us previously16,17 provided
optically active Z-alkene acetate.
1
acetal 10 as a mixture (1:1 by H NMR) of diastereomers in
74% yield. This mixture was subjected to ring-closing
metathesis using Grubbs’ second-generation catalyst20,21 in
the presence of NaHCO3 in CH2Cl2 at reflux. This condition
resulted in a 1:1 mixture of dihydropyran derivative in 86%
yield. These isomers could not be separated by silica gel
chromatography. However, treatment of this mixture with a
catalytic amount of camphorsulfonic acid (CSA) (10 mol %) in
ethanol for 2 h provided 11 as a major diastereomer (ratio
>20:1). Exposure of 11 to nBu4N+F− in THF for 3 h at 23 °C
provided the corresponding alcohol in near quantitative yield.
Swern oxidation of the alcohol afforded the aldehyde which was
subjected to Wittig olefination at 0 °C to furnish olefin 13 in
54% yield over three steps. Oxidation of ethyl acetal 13 with
22
PCC in CH2Cl2 at 23 °C for 5 h afforded optically active
dihydropyranone derivative 5 in 73% yield.
B
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Scheme 4. Synthesis of Z-Alkene Acid 9
Figure 2. Impact of analogues on in vitro splicing. Average splicing
efficiency vs inhibitor concentration normalized to DMSO control.
SSE, spliceostatin E; SSA, spliceostatin A.
The synthesis of spliceostatin E is shown in Scheme 5. Amide
coupling of acid 9 and amine 8, which was prepared as
Scheme 5. Synthesis of Spliceostatin E
Figure 3. Changes in nuclear speckle morphology. SFRS2 (SC35)
immunofluorescence in HeLa cells nuclei treated with (A) DMSO, (B)
1 μM pladienolide B, and (C) 0.1, 1, and 10 μM spliceostatin E, left to
right.
not reported for spliceostatin E). Enantioselective synthesis of
dihydropyranone ring A was achieved from commercially
available (R)-glycidyl alcohol using a ring-closing olefin
metathesis as the key reaction. A Reformatsky reaction of
methyl 3-bromomethyl crotonate with acrolein also provided
rapid access to racemic dihydropyranone which was separated
by chiral HPLC. A cross-metathesis of two fragments using
Grubbs catalyst efficiently provided the final product. The
synthesis is convergent and amenable to the synthesis of
structural variants. We have also evaluated spliceosome
inhibitory activity of spliceostatin E and compared its activity
with spliceostatin A. Surprisingly, spliceostatin E does not
inhibit splicing in vitro and does not impact speckle
morphology in cells. The design and synthesis of structural
variants of spliceostatins are in progress. These analogs will be
important to clarify the link between splicing inhibition and
changes in cellular function induced by these remarkable
compounds.
described by us previously, provided amide derivative 6 in 60%
yield. Cross-metathesis of diene 6 and alkene 5 with Grubbs’
second-generation catalyst in CH2Cl2 at reflux afforded
1
spliceostatin E, 4, in 71% yield. The H and 13C NMR of our
synthetic spliceostatin E [[α]23D −70 (c 0.2, CHCl3)] are in full
agreement with the reported spectra of natural spliceostatin
E.18
The biological properties of synthetic spliceostatin E, 4, were
evaluated (Figure 2) in an in vitro splicing system as previously
described.27 Strikingly, the compound showed no inhibition of
splicing in this system, even at 200 μM concentration. In
contrast, spliceostatin A in the same assay shows strong splicing
inhibition. This result was counter to our expectations because
of the recent demonstration of comparable cytotoxicity for both
compounds.18 Therefore, we examined the effect of spliceos-
tatin E on the structure of nuclear speckles in HeLa cells
(Figure 3). Potent splicing inhibitors including pladienolide B
and spliceostatin A12 cause speckles to increase in size. We see
no strong difference between cells treated with DMSO and
spliceostatin E, which correlates with the in vitro splicing
results.
ASSOCIATED CONTENT
■
S
* Supporting Information
General experimental procedures and characterization data for
all new products. This material is available free of charge via the
Internet at http://pubs.acs.org.
In summary, we have accomplished the first enantioselective
synthesis of reported spliceostatin E (the optical rotation was
C
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(24) Ghosh, A. K.; Cheng, X. Org. Lett. 2011, 13, 4108−4111.
AUTHOR INFORMATION
Corresponding Author
■
(25) For details, see the Supporting Information.
(26) Crimmins, M. T.; Choy, A. L. J. Am. Chem. Soc. 1997, 119,
10237−10238.
*E-mail: akghosh@purdue.edu.
Notes
(27) Effenberger, K. A.; Anderson, D. D.; Bray, W. M.; Prichard, B.
E.; Ma, N.; Adams, M. S.; Ghosh, A. K.; Jurica, M. S. J. Biol. Chem.
2014, 289, 1938−1947.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Financial support from the National Institutes of Health and
Purdue University is gratefully acknowledged.
■
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