Total synthesis of spiruchostatin A via chemoselective macrocyclization using an accessible enantiomerically pure latent thioester

Article abstract of DOI:10.1021/ol900436f

HDAC inhibitor Spiruchostatin A was synthesized via a route that differs significantly from previously reported routes. The key step involves a latent thioester that initiates a chemoselective transformation similar to native chemical ligation to form the

Full text of DOI:10.1021/ol900436f

ORGANIC
LETTERS
2009
Vol. 11, No. 9
1971-1974
Total Synthesis of Spiruchostatin A via
Chemoselective Macrocyclization using
an Accessible Enantiomerically Pure
Latent Thioester
Nicole A. Calandra, Yim Ling Cheng, Kimberly A. Kocak, and Justin S. Miller*
Department of Chemistry, Hobart and William Smith Colleges, GeneVa, New York 14456
jsmiller@hws.edu
Received March 2, 2009
ABSTRACT
HDAC inhibitor Spiruchostatin A was synthesized via a route that differs significantly from previously reported routes. The key step involves
a latent thioester that initiates a chemoselective transformation similar to native chemical ligation to form the macrocyclic alanine-cysteine
amide bond. The easily prepared latent thioestersthe first such moiety reported in enantiomerically pure formsis designed with a pendant
carboxylic acid to serve as a solid-phase linker for the synthesis of cyclic, cysteine-containing, peptidic materials.
Spiruchostatin A (1a)1 is a member of a class of cyclic,
cysteine-containing, depsipeptidic natural products shown in
Figure 1 that also includes FK228 (2)2 and FR901,375 (3),3
and can also include Largazole (4).4 Due to their histone
deacetylase (HDAC) inhibitory activity, tremendous effort
has been thrust into the synthesis of these compounds and
their analogs as potential chemotherapeutic targets.5-7 Struc-
tural features shared by these molecules include an (S,E)-
3-hydroxy-7-mercapto-4-heptenoate (Hmh) residue shown in
blue, in most cases a D-Cys residue illustrated in green, and
an Ala or Val residue drawn in red appended to the Hmh
(7) (a) Crabb, S. J.; Howell, M.; Rogers, H.; Ishfaq, M.; Yurek-George,
A.; Carey, K.; Pickering, B. M.; East, P.; Mitter, R.; Maeda, S.; Johnson,
P. W.; Townsend, P.; Shin-ya, K.; Yoshida, M.; Ganesan, A.; Packham, G.
Biochem. Pharmacol. 2008, 76, 463. (b) Shigematsu, N.; Ueda, H.; Takase,
S.; Tanaka, H.; Yamamoto, K.; Tada, T. J. Antibiot. 1994, 47, 311. (c)
Ueda, H.; Manda, T.; Matsumoto, S.; Mukumoto, S.; Nishigaki, F.;
Kawamura, I.; Shimomura, K. J. Antibiot. 1994, 47, 315. (d) Bowers, A. A.;
West, N.; Taunton, J.; Schreiber, S. L.; Bradner, J. E.; Williams, R. M.
J. Am. Chem. Soc. 2008, 130, 11219. (e) Nasveschuk, C. G.; Ungermannova,
D.; Liu, X.; Phillips, A. J. Org. Lett. 2008, 10, 3595. (f) Numajiri, Y.;
Takahashi, T.; Takagi, M.; Shin-ya, K.; Doi, T. Synlett 2008, 16, 2483. (g)
Seiser, T.; Kamena, F.; Cramer, N. Angew. Chem., Int. Ed. 2008, 47, 6483.
(h) Ying, Y.; Liu, Y.; Byeon, S. R.; Kim, H.; Luesch, H.; Hong, J. Org.
Lett. 2008, 10, 4021. (i) Bowers, A. A.; Greshock, T. J.; West, N.; Estiu,
G.; Schreiber, S. L.; Wiest, O.; Williams, R. M.; Bradner, J. E. J. Am.
Chem. Soc. 2009, 131, 2900. (j) Montero, A.; Beierle, J. M.; Olsen, C. A.;
Ghadiri, M. R. J. Am. Chem. Soc. 2009, 131, 3033. (k) Greshock, T. J.;
Johns, D. M.; Noguchi, Y.; Williams, R. M. Org. Lett. 2008, 10, 613.
(1) Masuoka, Y.; Nagai, A.; Shin-ya, K.; Furihata, K.; Nagai, K.; Suzuki,
K.; Hayakawa, Y.; Seto, H. Tetrahedron Lett. 2001, 42, 41.
(2) Ueda, H.; Nakajima, H.; Hori, Y.; Fujita, T.; Nishimura, M.; Goto,
T.; Okuhara, M. J. Antibiot. 1994, 47, 301.
(3) Chen, Y.; Gambs, C.; Abe, Y.; Wentworth, P., Jr.; Janda, K. D. J.
Org. Chem. 2003, 68, 8902.
(4) Taori, K.; Paul, V. J.; Luesch, H. J. Am. Chem. Soc. 2008, 130,
1806.
(5) Yurek-George, A.; Habens, F.; Brimmell, M.; Packham, G.; Ganesan,
A. J. Am. Chem. Soc. 2004, 126, 1030.
(6) Yurek-George, A.; Cecil, A. R.; Mo, A. H.; Wen, S.; Rogers, H.;
Habens, F.; Maeda, S.; Yoshida, M.; Packham, G.; Ganesan, A. J. Med.
Chem. 2007, 50, 5720.
10.1021/ol900436f CCC: $40.75
Published on Web 03/30/2009
 2009 American Chemical Society

moiety via an amide bond (except in Largazole). The Hmh
residue has been shown to be involved in HDAC inhibitory
activity.8
Ganesan, et al. have noted the shortcomings of the macro-
lactonization routes10b and have recently developed a dif-
ferent route toward FK228 (2) that involves macrolactam-
ization via standard peptide coupling methods and the
concomitant required protecting group manipulations.
We envisioned instead a route, illustrated in Scheme 1,
that employs the D-Cys residue in a key macrolactamization
involving the N-terminal D-Cys residue and the C-terminal
D-Ala latent thioester of 5. Macrocyclizations such as this
Scheme 1.
Retrosynthesis of Spiruchostatin A (1a)a
Figure 1. Various macrocyclic depsipeptides, including Spirucho-
statin A (1a), containing Hmh (blue), cysteine (green), and Ala or
Val residues (red).
Several syntheses of 1a have been published, with each
reported synthesis employing a key macrolactonization to
form the despsipeptidic linkage between the D-Ala and Hmh
residues.5,9 Such synthetic routes have been shown to be
effective in generating both the parent compound and even
analogs of 1a.6 However, similar macro-lactonizations en
route to FK228 (2) have been reported to be difficult due to
the sensitivity of the reacting allylic alcohol moieties or steric
concerns.7k,9a,10 Issues regarding the reaction conditions for
these macrolactonizations apparently render such routes ill-
suited to the more demanding environment of solid-phase
synthesis, given the lack of reported solid-phase syntheses
for any of these compounds. Peptidic isosteres of FK228
were synthesized on the solid phase, but only when the
lactone functionality was replaced with a lactam.11 A solid-
phase synthetic route toward 1a, and by extension 2-4,
would facilitate the generation of their analogs for SAR.
a Abbreviations: NCL ) native chemical ligation; Sta ) statine.
one that are similar to native chemical ligation (NCL)12 have
been demonstrated using C-terminal thioesters13 and even a
latent aryl thioester.14 Moreover, since latent thioesters have
been shown previously to function as solid-phase linkers,15
if the latent thioester here were designed to serve as a solid-
phase linker, then the synthetic route would have the clear
advantage over previous routes of making the transition from
solution phase to the solid phase manageable. The remainder
(12) (a) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H.
Science 1994, 266, 776. (b) Tam, J. P.; Lu, Y. A.; Liu, C. F.; Shao, J.
Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 12485.
(8) Furumai, R.; Matsuyama, A.; Kobashi, N.; Lee, K.-H.; Nishiyama,
M.; Nakajima, H.; Tanaka, A.; Komatsu, Y.; Nishino, N.; Yoshida, M.;
Horinouchi, S. Cancer Res. 2002, 62, 4916
.
(13) (a) Tam, J. P.; Lu, Y. A. Tetrahedron Lett. 1997, 38, 5599. (b)
Zhang, L. S.; Tam, J. P. J. Am. Chem. Soc. 1997, 119, 2363. (c) Camarero,
J. A.; Muir, T. W. Chem. Commun. 1997, 1369. (d) Camarero, J. A.; Cotton,
G. J.; Adeva, A.; Muir, T. W. J. Pept. Res. 1998, 51, 303.
(14) Chen, J.; Warren, J. D.; Wu, B.; Chen, G.; Wan, Q.; Danishefsky,
S. J. Tetrahedron Lett. 2006, 47, 1969.
(9) (a) Doi, T.; Iijima, Y.; Shin-Ya, K.; Ganesan, A.; Takahashi, T.
Tetrahedron Lett. 2006, 47, 1177. (b) Takizawa, T.; Watanabe, K.; Narita,
K.; Kudo, K.; Oguchi, T.; Abe, H.; Katoh, T. Heterocycles 2008, 76, 275
.
(10) (a) Li, K. W.; Wu, J.; Xing, W. N.; Simon, J. A. J. Am. Chem.
Soc. 1996, 118, 7237. (b) Wen, S.; Packham, G.; Ganesan, A. J. Org. Chem.
2008, 73, 9353
.
(15) (a) Botti, P.; Villain, M.; Manganiello, S.; Gaertner, H. Org. Lett.
2004, 6, 4861. (b) George, E. A.; Novick, R. P.; Muir, T. W. J. Am. Chem.
Soc. 2008, 130, 4914.
(11) Di Maro, S.; Pong, R.-C.; Hsieh, J.-T.; Ahn, J.-M. J. Med. Chem.
2008, 51, 6639.
1972
Org. Lett., Vol. 11, No. 9, 2009

of the strategy involves peptide synthesis using commercially
available amino acids (7) or materials that can be accessed
through previously described routes (8 and 9).3,5,16
alcohols to give TBS ether 12, and etherification of the
remaining alcohol with bromoacetic acid to give 13 in
70% combined yield for the three steps. To occupy the
position designed for a solid-phase resin, acid 13 was
coupled with 2-methoxyethylamine (to simulate a PEG-
resin), which upon TBAF removal of the silyl ether
afforded alcohol 6. In a step equivalent to resin loading,
Fmoc-D-Ala-OH was coupled under standard esterification
conditions to give 14.
Further elaboration of the depsipeptide required that two
intermediates, 8 and 9, be synthesized. Trityl-protected
hydroxymercaptoheptenoate 8 was synthesized as described,
and exibited spectroscopic properties identical to those
reported.3 Compound 9 was prepared from known 15
(Scheme 3), which also displayed spectroscopic properties
To serve ultimately as a solid-phase linker for a synthesis
that will precisely mimic the solution-phase version described
here, the latent thioester requires an attachment point to the
solid phase, and therefore a stereocenter, somewhere along
its ꢀ-mercaptoalcohol functionality.17 The enantiomeric
purity of this functional group is critical for the spectroscopic
analysis required during the solution-phase synthesis of 1a
and would also facilitate similar analysis of cleaved inter-
mediates that still contain the linker during the envisioned
solid-phase synthesis. Disulfide protection of the thiol is
valuable because this protecting group is generally stable,
but is readily removed under the reducing conditions of NCL.
To fulfill the structural requirements of an enantiomerically
pure, disulfide protected ꢀ-mercaptoalcohol, we developed
the short, efficient route displayed in Scheme 2 toward
Scheme 3.
Completing the Synthesis of Spiruchostatin A (1a)a
Scheme 2. Synthesis of an Enantiopure Latent Thioester
internally disulfide protected ꢀ-mercaptoalcohol 13, which
is a potential solid-phase linker due to its pendant carboxylic
acid. The starting material for this linker is (L)-(-)-1,4-
dithiothreitol (10), which is a commercially available enan-
tiomerically pure compound with much of the essential
functionality. The linker synthesis requires only three high-
yielding steps: oxidation to form disulfide 11,18 selective
protection of one of the two C2-symmetrical secondary
a Abbreviations: MES-Na ) sodium 2-mercaptoethanesulfonate; TCEP
) tris(carboxyethyl)phosphine; Tce ) tricholorethyl.
identical to those reported.5 Conversion of 15 into 9 involved
protection of the secondary alcohol as a silyl ether followed
by removal of the Tce protecting group, as shown in
Scheme 3.
Protected Ala fragment 14 was extended by removal of
Fmoc using piperidine followed by standard carbodiimide
peptide coupling with carboxylic acid 8 to give alcohol 16.
Yamaguchi-type esterification19 of carboxylic acid 9 with
alcohol 16 afforded protected depsipeptide 17, which contains
(16) Liang, B.; Richard, D. J.; Portonovo, P. S.; Joullie, M. M. J. Am.
Chem. Soc. 2001, 123, 4469
.
(17) Although alkyl ꢀ-mercaptoalcohol linkers with all of the appropriate
functionality can be designed without a stereocenter, these would generally
require a tertiary thiol or alcohol, which would confer unwanted reactivity
or steric hindrance. Disulfide-protected aryl ꢀ-mercaptoalcohols (o-mer-
captophenols) are not suitable as Fmoc solid-phase linkers because their
esters are cleaved by the secondary amines used for Fmoc deprotection.
(18) Evans, C. A.; Bernier, L.; Dugas, J.; Mansour, T. S. Tetrahedron
Lett. 1997, 38, 7657.
(19) Du, Y.; Chen, Q.; Linhardt, R. J. J. Org. Chem. 2006, 71, 8446.
Org. Lett., Vol. 11, No. 9, 2009
1973

all of the functionality of 1a but is absent in both macro-
cycles. In elaborating 16, the statine and D-Cys residues were
first joined into a single unit to prevent the expected16
unproductive cyclization of the statine γ-amine onto the
newly formed allylic ester. Although a Boc-protected statine
derivative could in principle circumvent this issue through
in situ neutralization,20 removal of the Boc group in the
presence of the trityl thioether proved difficult in this case,
and such a route was not pursued further.
Because it involves only coupling and deprotection reac-
tions, the linear route from 13 to 17 will likely benefit from
adaptation of this synthesis to the solid phase, where excess
reagents and their byproducts are easily removed. For
example, optimizing the Yamguchi reaction affording 17 was
challenging because 17 and excess 9 were difficult to separate
chromatographically. Because no products from elimination
of the allylic alcohol (or ester, after the fact) were observed,
and 17 was stable for weeks, the other issues with this
coupling may largely stem from side reactions of 9 (e.g.,
premature cyclization of the activated acid), and such issues
are readily addressed during a solid-phase synthesis using
excess 9.
Conclusion of the synthesis from 17 required global
deprotection followed by the key chemoselective macrocy-
clization and further oxidative macrocyclization.5 TBAF-
mediated removal of both the silyl ether and the Fmoc
protecting group,10b followed immediately by trityl depro-
tection with TFA, yielded a compound consistent21 with 5
(Scheme 1). Purification of this relatively unstable (oxidation)
and likely water- and organic-soluble material was not
attempted; the intermediate was instead subjected im-
mediately to the aqueous conditions required for NCL.15b
The products of this reaction were not isolated, thus
preventing intermolecular disulfide formation of the still
readily oxidized unprotected thiols. The crude product
mixture was treated immediately under dilute conditions with
iodine,5 affording the desired 1a and also disulfide 6, as
expected. All of the spectroscopic and analytical properties
of our synthetic 1a were identical to those reported previously
for Spiruchostatin A,1 thus confirming a successful synthesis.
We have clearly demonstrated an effective new strategy
for generating Spiruchostatin A. Furthermore, the isolation
of both 1a and 6, along with the stability of the alaninyl
ester of 6 throughout the synthesis, indicates that the ester
of 6 functioned as expected for a latent thioester. This proof
of principle not only provides a new method for generating
the Spiruchostatins and their analogs; it also opens the door
to new syntheses of FK228 and FR901,375 and their analogs,
as well. Moreover, carboxylic acid 13 is designed to be
loaded onto suitable peptide synthesis resins using standard
coupling conditions and, following removal of the silyl ether
protection, used as would any other peptide ester resin. This
work will hopefully thus facilitate the solid-phase synthesis
of analog libraries for SAR and drug development of these
cysteine-containing, cyclic depsipeptides. Finally, the easily
synthesized 13 is a single enantiomer, which enabled the
analysis of the resulting diastereomerically homogeneous
latent thioester intermediates described here, and will by
extension do the same for intermediates containing 13 that
are cleaved for analysis during solid-phase synthesis using
13 as a linker. Results from our solid-phase synthetic efforts
will be reported in due course.
Acknowledgment. We thank Dr. Robert Boeckman
(University of Rochester) and his research group, including
Greg Frattini, for the use of their polarimeter, and Dr. Ivan
Keresztes (Cornell University) for acquiring mass spectral
data. We gratefully acknowledge support from the Research
Corporation for Science Advancement (CC6250/5873), a
Camille and Henry Dreyfus Faculty Start-up Award (SU-
04-007), and the NSF-MRI program (CHE-0722178) for the
purchase of an NMR spectrometer.
Supporting Information Available: Experimental pro-
cedures along with spectroscopic and analytical data for all
new compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
(20) Schno¨lzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S. B. H.
Int. J. Pept. Prot. Res. 1992, 40, 180.
(21) During TLC, the color change and rate of oxidation (little heat
required) with ceric ammonium molybdate stain were consistent with the
presence of free thiol, and ninhydrin stain indicated a free amine.
OL900436F
1974
Org. Lett., Vol. 11, No. 9, 2009