Enantioselective Synthesis of Spliceostatin G and Evaluation of Bioactivity of Spliceostatin G and Its Methyl Ester

Article abstract of DOI:10.1021/acs.orglett.7b03456

An enantioselective total synthesis of spliceostatin G has been accomplished. The synthesis involved a Suzuki cross-coupling reaction as a key step. The functionalized tetrahydropyran ring was constructed from commercially available optically active tri-O-acetyl-d-glucal. Other key reactions include a highly stereoselective Claisen rearrangement, a Cu(I)-mediated 1,4 addition of MeLi to install the C8 methyl group, and a reductive amination to incorporate the C10 amine functionality of spliceostatin G. Biological evaluation of synthetic spliceostatin G and its methyl ester revealed that it does not inhibit splicing in vitro.

Full text of DOI:10.1021/acs.orglett.7b03456

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Enantioselective Synthesis of Spliceostatin G and Evaluation of
Bioactivity of Spliceostatin G and Its Methyl Ester
Arun K. Ghosh,*,† Guddeti Chandrashekar Reddy,† Andrew J. MacRae,‡ 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 G has been
accomplished. The synthesis involved a Suzuki cross-coupling reaction as a key step.
The functionalized tetrahydropyran ring was constructed from commercially
available optically active tri-O-acetyl-D-glucal. Other key reactions include a highly
stereoselective Claisen rearrangement, a Cu(I)-mediated 1,4 addition of MeLi to
install the C8 methyl group, and a reductive amination to incorporate the C10
amine functionality of spliceostatin G. Biological evaluation of synthetic spliceostatin G and its methyl ester revealed that it does
not inhibit splicing in vitro.
n mammalian cells, the splicing of pre-mRNAs is a
1,2
Ifundamental process for gene expression.
Splicing is
carried out by a complex ribonucleoprotein machinery, called
the spliceosome, which upon recognition of splicing signals
catalyzes the removal of intervening sequences (introns) and
assembles protein coding sequences (exons) to form messenger
RNA (mRNA) prior to export and translation.3,4 Spliceosome
assembly and catalysis are generally very complicated. Recent
studies have revealed that splicing is pathologically altered in
many different ways in cancer cells.5,6 Therefore, manipulation
or inhibition of splicing events by targeting the spliceosome
may be an effective strategy for anticancer drug development.
Pladienolides (pladienolide B, 1, Figure 1) isolated from
Streptomyces have been shown to be potently cytotoxic.7,8 They
inhibit the spliceosome by binding to the SF3B subunit of the
spliceosome.9,10 While pladienolides are unsuitable for clinical
use due to inadequate physicochemical properties, a semi-
synthetic derivative E707, 2, with improved properties
underwent clinical trials.11,12 Subsequently, other natural
products, such as FR901464, 3, and its methylated derivative
spliceostatin A, 4, were shown to potently inhibit spliceosome
through binding to the SF3B subunit of the spliceosome.13,14
Total synthesis and further design of structural isomers were
pursued for these natural products to improve stability and
reduce structural complexities of these agents.15−17 Recently,
Figure 1. Structures of pladienolide B, E707, FR901464, and
spliceostatins A and G.
He and co-workers reported a series of spliceostatin classes of
natural products isolated from the fermentation broth FERM
BP-3421 of Burkholderia sp.18 Among these natural products, a
more active natural products like spliceostatins A and E.18 As
part of our continuing interest in the chemistry and biology of
spliceostatins, we have devised an enantioselective synthesis of
less complex structure, spliceostatin G, was isolated, and the full
1
structure of spliceostatin G was confirmed by detailed H and
13C NMR studies.18 Spliceostatin G did not exhibit potent
cytotoxicity inherent to other spliceostatins. Spliceostatin G
does not contain an epoxy alcohol on a tetrahydropyran
framework nor a 5,6-dihydro-α-pyrone subunit present in the
Received: November 7, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b03456
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
spliceostatin G using readily available tri-O-acetyl-D-glucal as
the key starting material. The current synthesis will provide
ready access to highly functionalized tetrahydropyrans and the
diene frameworks of the spliceostatins.
Scheme 2. Synthesis of Dihydropyranone 18
Our strategy for an enantioselective synthesis of spliceostatin
G is shown in Scheme 1. To construct the diene component of
Scheme 1. Retrosynthesis of Spliceostatin G
spliceostatin G, we planned a Suzuki cross-coupling reaction
between the boronate segment 7 and iodoacrylate 6 at a late
stage of the synthesis. The boronate derivative 7 would be
obtained by a cross-metathesis of the amide derivative of olefin
8 and commercially available pinacol boronate. Coupling of the
amine functionality of 8 with acid 9 would provide the requisite
amide for cross-metathesis. The functionalized tetrahydropyran
ring 8 could be constructed from dihydropyran derivative 10.
Asymmetric synthesis of this dihydropyran derivative would be
carried out from commercially available tri-O-acetyl-D-glucal 11.
The preparation of dihydropyranone 18 is shown in Scheme
2. The synthesis of dihydropyran derivative 12 was carried out
on a multigram scale from commercially available tri-O-acetyl-
D-glucal 11 as reported in the literature.19 This was subjected to
heating in a sealed tube in toluene at 190 °C for 18 h to provide
the Claisen rearrangement product, the corresponding
aldehyde. Wittig olefination of the aldehyde with methylene-
triphenylphosphorane at 0 °C afforded dihydropyran derivative
13 in 90% yield. The removal of the silyl group was carried out
by exposure to tetrabutylammonium fluoride (TBAF) in THF
at 0−23 °C for 12 h. The resulting diol was initially treated with
p-toluenesulfonyl chloride in pyridine at 0 °C to 23 °C for 12 h
to furnish a mixture (6:1) of tosylate derivatives 14 and 15. For
regioselective formation of the primary sulfonate derivative, we
chose the sterically bulkier 2,4,6-triisopropylbenzenesulfonyl
chloride (TPSCl). Reaction of the diol with TPSCl in pyridine
at 0−23 °C for 24 h afforded primary sulfonate derivative 16 in
92% yield. Reduction of sulfonate 16 by LAH in THF at 0 °C
to 65 °C for 1.5 h provided the reduced product, the methyl
derivative. Oxidation of the resulting allylic alcohol with Dess−
Martin periodinane at 0−23 °C for 1 h furnished enone
derivative 17 in 80% yield. For stereoselective installation of the
C8-methyl group, we carried out a Cu(I)-mediated 1,4-addition
as developed by us previously.17 Thus, treatment of 17 with
MeLi in the presence of CuBr·SMe2 complex at −78 °C for 2 h
provided dihydro-2H-pyranone 18 in excellent yield and
excellent diastereoselectivity (25:1 by 1H NMR and 13C
NMR analysis).
Elaboration of dihydropyranone 18 to the boronate
derivative 7 is shown in Scheme 3. A substrate-controlled
stereoselective reduction of ketone 18 with ammonium acetate
and NaBH(OAc)3 in the presence of trifluoroacetic acid (TFA)
at 23 °C for 12 h afforded primary amine 8 with high
17,20
1
diastereoselectivity (96:4 by H NMR analysis).
Coupling
of optically active acid 9 and amine 8 using HATU in the
presence of diisopropylethylamine (DIPEA) resulted in amide
derivative 19 in 85% yield. Cross-metathesis of the allyl
derivative 19 with commercially available pinacol boronate 20
B
DOI: 10.1021/acs.orglett.7b03456
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Organic Letters
Letter
Scheme 3. Synthesis of Boronate Derivative 7
Scheme 4. Synthesis of Spliceostatin G
in the presence of Grubb’s second-generation catalyst (10 mol
%) in 1,2-dichloroethane at 80 °C for 1 h afforded boronate
derivative 7 in 41% yield.21−23
The synthesis of spliceostatin G is shown in Scheme 4.
Initially, we conducted a Suzuki coupling of boronate 7 and
iodocarboxylic acid 6a using Pd(dppf)Cl2·DCM catalyst (20
mol %) in the presence of aqueous K3PO4 in a mixture of
dioxane and acetonitrile at 23 °C for 30 min. However, this
condition only provided trace amount of coupling product
spliceostatin G. We have also carried out this coupling using
Pd(Ph3P)4 catalyst (10 mol %) in the presence of Cs2CO3 at 55
°C. This condition only provided a trace amount of desired
product.24 We then carried out this Suzuki reaction25 with
methyl iodoacrylate 6b using Pd(dppf)2Cl2·DCM (20 mol %)
catalyst in the presence of aqueous K3PO4 at 23 °C for 30 min
to furnish coupling product 21 in 75% yield after silica gel
chromatography. The methyl ester 21 was converted to
spliceostatin G by saponification with aqueous LiOH in THF
at 23 °C for 6 h, followed by reaction of the resulting hydroxyl
acid with acetyl chloride in CH2Cl2 at 23 °C for 3 h.
Spliceostatin G was obtained in 68% yield over two steps. The
1H NMR and 13C NMR of our synthetic spliceostatin G
[[α]D23 −71.7 (c 0.53, CHCl3)26] are in full agreement with the
reported spectra of natural spliceostatin G.18
Figure 2. Impact of spliceostatin G on in vitro splicing. Average
splicing efficiency relative to inhibitor concentration normalized to
DMSO control. Key: SSG, spliceostatin G; compound 21; SSA,
spliceostatin A.
In summary, we have achieved an enantioselective synthesis
of spliceostatin G and confirmed the assignment of relative and
absolute stereochemistry of spliceostatin G. The synthesis
involved a Suzuki cross-coupling reaction as the key step.
Enantioselective synthesis of the functionalized tetrahydropyran
ring was achieved from commercially available optically active
tri-O-acetyl-D-glucal using a highly stereoselective Claisen
rearrangement.
The biological properties of synthetic spliceostatin G (5) and
the precursor 21 were evaluated in an in vitro splicing system as
previously described27 (Figure 2). Neither compound showed
inhibition of splicing in this system, even at 100 μM
concentration. In contrast, spliceostatin A in the same assay
shows strong splicing inhibition. This result is consistent with
previous reports showing that spliceostatin G (5) does not
affect the growth of several cancer cell lines.18
A cross-metathesis of commercially available pinacol
boronate using Grubbs’ catalyst provided the vinyl boronate
C
DOI: 10.1021/acs.orglett.7b03456
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Organic Letters
Letter
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derivative for the cross-coupling reaction with iodoacrylic acid.
The other stereoselective transformations include a highly
stereoselective 1,4 addition to construct the C8 methyl group
and reductive amination to incorporate the C10 amine
functionality of spliceostatin G. The synthesis is convergent
and amenable to the synthesis of structural variants. We have
also evaluated spliceosome inhibitory activity of spliceostatin G
and compared its activity with spliceostatin A. Spliceostatin G
does not inhibit in vitro splicing assembly or chemistry. The
design and synthesis of structural variants of spliceostatins are
in progress. These analogues will be important to clarify the
link between splicing inhibition and changes in cellular function
induced by these remarkable compounds.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.7b03456.
(18) He, H.; Ratnayake, A. S.; Janso, J. E.; He, M.; Yang, H. Y.;
Loganzo, F.; Shor, B.; O’Donnell, C. J.; Koehn, F. E. J. Nat. Prod. 2014,
77, 1864−1870.
Experimental procedures in addition to 1H and 13C
NMR spectra (PDF)
́ ́ ́
(19) (a) Pazos, G.; Perez, M.; Gandara; Gomez, G.; Fall, Y.
Tetrahedron Lett. 2009, 50, 5285−5287. (b) Hoberg, J. O. Carbohydr.
Res. 1997, 300, 365−367. (c) Mori, Y.; Hayashi, H. J. Org. Chem. 2001,
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: akghosh@purdue.edu.
ORCID
(20) Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C.
A.; Shah, R. D. J. Org. Chem. 1996, 61, 3849−3862.
(21) Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R. H. Org. Lett. 1999,
1, 953−956.
Arun K. Ghosh: 0000-0003-2472-1841
Notes
(22) Trnka, T. M.; Morgan, J. P.; Sanford, M. S.; Wilhelm, T. E.;
Scholl, M.; Choi, T.-L.; Ding, S.; Day, M. W.; Grubbs, R. H. J. Am.
Chem. Soc. 2003, 125, 2546−2558.
The authors declare no competing financial interest.
(23) Nicolaou, K. C.; Rhoades, D.; Lamani, M.; Pattanayak, M. R.;
Kumar, S. M. J. Am. Chem. Soc. 2016, 138, 7532−7535.
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Monteiro, A. L. J. Braz. Chem. Soc. 2014, 25, 2186−2214.
(26) The optical rotation of natural spliceostatin G has not been
reported (see ref 18).
(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.
ACKNOWLEDGMENTS
■
Financial support of this work was provided by the National
Institutes of Health (GM122279) and Purdue University. We
thank Dr. Margherita Brindisi (Purdue University) for helpful
discussions.
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DOI: 10.1021/acs.orglett.7b03456
Org. Lett. XXXX, XXX, XXX−XXX