Improved total synthesis of the potent HDAC inhibitor FK228 (FR-901228)

Article abstract of DOI:10.1021/ol702957z

A scaleable synthesis of the potent histone deacetylase (HDAC) inhibitor FK228 is described. A reliable strategy for preparing the key β-hydroxy mercapto heptenoic acid partner was accomplished in nine steps and 13% overall yield. A Noyori asymmetric hydrogen-transfer reaction established the hydroxyl stereochemistry in >99:1 er via the reduction of a propargylic ketone.

Full text of DOI:10.1021/ol702957z

ORGANIC
LETTERS
2008
Vol. 10, No. 4
613-616
Improved Total Synthesis of the Potent
HDAC Inhibitor FK228 (FR-901228)
Thomas J. Greshock,† Deidre M. Johns,† Yasuo Noguchi,† and
Robert M. Williams*,†,‡
Department of Chemistry, Colorado State UniVersity, Fort Collins, Colorado 80523,
and The UniVersity of Colorado Cancer Center, Aurora, Colorado 80045
rmw@lamar.colostate.edu
Received December 6, 2007
ABSTRACT
A scaleable synthesis of the potent histone deacetylase (HDAC) inhibitor FK228 is described. A reliable strategy for preparing the key â-hydroxy
mercapto heptenoic acid partner was accomplished in nine steps and 13% overall yield. A Noyori asymmetric hydrogen-transfer reaction
established the hydroxyl stereochemistry in >99:1 er via the reduction of a propargylic ketone.
The depsipeptide FK228 (1) (previously named FR-901228)
was isolated from the fermentation broth of Chromobacte-
rium Violaceum (No. 968) by Fujisawa Pharmaceutical Co.,
Ltd. in 1991 along with a closely related depsipeptide, FR-
901375 (2) (Figure 1).1 The potent activity of FK228 (IC50
) 1.0 nM for FK228 with DTT for HDAC1) against a range
of murine and human solid tumor cells and its ability to target
epigenetic silencing mechanisms crucial to carcinogenesis
have culminated in its advancement into clinical trials as a
potential anticancer therapy.2 During a phase II study, serious
adverse cardiac events were observed, which precipitated
termination of the trial and indicate that a better understand-
ing of the biochemistry and cell biology of this unique natural
product is critical.3 These investigations have been hindered
Figure 1. Structures of FK228 (1) and FR-901375 (2).
† Colorado State University.
‡ The University of Colorado Cancer Center.
(1) Fujisawa Pharmaceutical Co., Ltd., Jpn. Kokai Tokkyo Koho JP,
03141296, 1991.
(2) 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, 4916-4921.
by a lack of available material within the scientific com-
munity. The only reported total synthesis of FK228, by
Simon et al., has proven to be capricious and difficult to
10.1021/ol702957z CCC: $40.75
© 2008 American Chemical Society
Published on Web 01/19/2008

reproduce, and we were thus compelled to develop a new
synthesis amenable to a larger scale preparation.4-6
of synthesizing FR-901375 (2), Wentworth and Janda noted
that they encountered difficulties reproducing the enantio-
selectivity in the key Carreira-type asymmetric aldol reac-
tion.5 Likewise, Ganesan noted the same difficulty in their
recent synthesis of spiruchostatin A.6 Both groups developed
alternative asymmetric aldol approaches to 5. Wentworth and
Janda mimicked an acetate aldol by using 2-chloroacetyl-
oxazolidinone in an asymmetric aldol reaction. This provided
5 in six steps and 26% yield from commercially available
materials. Although efficient, it requires chromatographic
separation of diastereomers (>90% de), which may be
tedious on a large scale. Ganesan’s synthesis of 5 using a
SmI2-promoted Reformatsky reaction of an N-(R-bromoacetyl)-
oxazolidin-2-one (Seebach’s chiral auxiliary) and also man-
dates chromatographic separation of diastereomers (86%
de).12 These workers obtained 5 in nine steps and ap-
proximately 9.5% yield from commercially available materi-
als.13 It is apparent from the literature that compound 5 is a
deceptively challenging substance to access on a prepara-
tive scale. We report herein a catalytic, enantioselective, and
scaleable synthesis of â-hydroxy acid 5, which is comparable
in the number of steps to that reported by others and, in our
hands, constitutes a robust and reproducible method.
FK228, acting as a prodrug, undergoes a disulfide reduc-
tion within the cell to release a zinc-binding thiol. The
butenyl thiol is known to interact with zinc in the binding
pocket of Zn-dependent histone deacetylase (HDAC) revers-
ibly.7 Inhibitors of HDAC are under intense investigation
as potential “transcriptional therapies” for the treatment of
cancer.8 They are able to restore normal expression of genes,
which may result in cell cycle arrest, differentiation, and
apoptosis. HDAC inhibitors are known to affect expression
of the following genes: VEGF, ErbB2, cyclin D1, cyclin E,
and gelsolin.9 The mechanism of action for FK228 has been
linked to the reversal of prodifferentiation of the ras
oncogene pathway via blockade of p21 protein-mediated
signal transduction. Its cytotoxicity is associated with the
upregulation of Rap1, a small GTP-binding protein of the
Ras family.10 Rap1 is an intrinsic regulator of the Ras-Raf-
MAP kinase signaling pathway in melanoma cells.
The structure of FK228 is intriguing when compared to
other HDAC inhibitors. It contains a 16-membered cyclic
depsipeptide bridged by a 15-membered macrocyclic disul-
fide. The D-Cys residue of the tetrapeptide portion enables
FK228 to act as a prodrug, which is reduced in vivo by
glutathione reductase to reveal the Zn-binding butenyl thiol.2
This thiol is connected to the depsipeptide by a four-carbon
tether, which is shorter than the typical tether length of five
to six atoms observed to be optimal.11 The potent biological
activity of FK228 with respect to its unique structure as an
HDAC inhibitor has yet to be thoroughly elucidated.
The key challenges in preparing FK228 are (1) the
formation of the delicate macrocyclic depsipeptide and (2)
preparation of the â-hydroxy mercapto heptenoic acid (5)
intermediate. Simon and co-workers reported in their total
synthesis of FK228 that a Mitsunobu-based macrolacton-
ization provides a good yield of the 16-membered cyclic
depsipeptide.4 The other key challenge, the synthesis of
â-hydroxy acid 5, has been addressed by two other research
groups.5,6,12 Acid 5 is a key intermediate for the synthesis of
FR-901375 and spiruchostatin A. Simon applied Carreira’s
catalytic asymmetric aldol reaction to establish the C3-hy-
droxyl stereochemistry (99% yield, >98% ee).4 In the course
Our synthesis of FK228 follows the pioneering route
established by Simon for completion of the synthesis, in
which the disulfide of FK228 is disconnected to the trityl-
protected bis-mercaptan 3, followed by opening of the macro-
cyclic depsipeptide at the lactone. Intermediate 3 is further
disconnected to reveal tetrapeptide 4 and â-hydroxy mercapto
acid 5 (Scheme 1). Our synthesis of 5 involves late-stage
Scheme 1. Retrosynthetic Analysis
(3) Shah, M. H.; Binkley, P.; Chan, K.; Xiao, J.; Arbogast, D.; Collamore,
M.; Farra, Y.; Young, D.; Grever, M. Clin. Cancer Res. 2006, 12, 3997-
4003.
(4) Li, K. W.; Wu, J.; Xing, W.; Simon, J. A. J. Am. Chem. Soc. 1996,
118, 7237-7238.
(5) Chen, Y.; Gambs, C.; Abe, Y.; Wentworth, P., Jr.; Janda, K. D. J.
Org. Chem. 2003, 68, 8902-8905.
(6) Yurek-George, A.; Habens, F.; Brimmell, M.; Packham, G.; Ganesan,
A. J. Am. Chem. Soc. 2004, 126, 1030-1031.
(7) (a) Ueda, H.; Nakajima, H.; Hori, Y.; Fujita, T.; Nishimura, M.; Goto,
T.; Okuhara, M. J. Antibiot. 1994, 47, 301-310. (b) Shigematsu, N.; Ueda,
H.; Takase, S.; Tanaka, H.; Yamamoto, K.; Tada, T. J. Antibiot. 1994, 47,
311-314. (c) Ueda, H.; Manda, T.; Matsumoto, S.; Mukumoto, S.;
Nishigaki, F.; Kawamura, I.; Shimomura, K. J. Antibiot. 1994, 47, 315-
323.
(8) Somech, R.; Izraeli, S.; Simon, A. J. Cancer Treat. ReV. 2004, 30,
mercaptan installation and conversion of a dimethyl acetal
to the desired acid. The asymmetric allylic alcohol is derived
from a propargylic ketone (7) by a Noyori catalytic asym-
metric ketone reduction and an (E)-selective alkyne reduc-
tion.
461-472.
(9) Marks, P. A.; Richon, V. M.; Miller, T.; Kelly, W. K. AdV. Cancer
Res. 2004, 5, 137-168.
(10) Kobayashi, Y.; Ohtsuki, M.; Murakami, T.; Kobayashi, T.; Suthee-
sophon, K.; Kitayama, H.; Kano, Y.; Kusano, E.; Nakagawa, H.; Furukawa,
Y. Oncogene 2006, 25, 512-524.
614
Org. Lett., Vol. 10, No. 4, 2008

Our synthesis of â-hydroxy mercapto acid 5 commences
with the preparation of amide 9 from commercially available
methyl 3,3-dimethoxypropionate (Scheme 2). Standard con-
Scheme 3. Preparation of â-Hydroxy Mercapto Acid 5
Scheme 2. Preparation of Propargylic Ketone 11
ditions14 provide Weinreb amide 9. Addition of the lithium
acetylide of 10 to amide 9 gives propargylic ketone 11 in a
straightforward manner.
in 16% yield from D-cysteine. Furthermore, in our hands,
the overall yield using the existing synthetic route starting
from 15, N-Fmoc-D-cysteine(STrt), Fmoc-L-Thr, and Fmoc-
D-Val resulted in a mere 23% yield of 4 rather than the
reported 78% overall yield. The most significant decrease
in yield was observed for the simultaneous threonine
dehydration-Fmoc deprotection step, in which we obtained
a 39% yield of 4 and could not realize the reported 96%
yield. To address these low-yielding and irreproducible steps,
we then examined an alternative protecting group strategy.
Fortunately, N-Alloc-D-cysteine(STrt) can be obtained in high
yield from D-cysteine as described by Kruse,18 which
prompted us to investigate its use in the synthesis of 4
(Scheme 4). Coupling of N-Alloc-D-cysteine(STrt) with
L-Thr-L-Val-OMe affords a 67% yield of 16. We observed
an improved yield (95% over two steps) by dehydrating the
threonine residue of tripeptide 16 earlier in the synthesis,
Noyori’s asymmetric hydrogen transfer conditions reduced
ketone 11 to the (R)-propargylic alcohol in 71% yield and
99:1 er as determined by HPLC.15 Treatment with sodium
bis(2-methoxyethoxy)aluminum hydride (Vitride) selectively
affords (E)-alkene 13.16 It was determined that conversion
of the silyl ether to tosylate 14 must be done prior to dimethyl
acetal hydrolysis. Similarly, oxidation of the mercaptan is
avoided by revealing the carboxylic acid before introduction
of the trityl mercaptan. Therefore, the dimethyl acetal of
tosylate 14 was hydrolyzed to the aldehyde using LiBF4,
which was immediately oxidized to the carboxylic acid using
Pinnick oxidation conditions.17 Finally, the trityl mercaptan
was introduced by tosylate displacement to provide 5 in nine
steps (13.3% overall yield) from commercially available
materials and has been prepared on a multigram scale
(Scheme 3).
We initially envisioned preparing tetrapeptide 4 using
Simon’s strategy. Dipeptide 15, prepared as previously
described, was to be coupled with N-Fmoc-D-cysteine(STrt).
However, N-Fmoc-D-cysteine(STrt) could only be obtained
Scheme 4. Synthesis of Tetrapeptide 4
(11) (a) TSA A analogues: Woo, S. H.; Frechette, S.; Khalil, E. A.;
Bouchain, G.; Vaisburg, A.; Bernstein, N.; Moradei, O.; Leit, S.; Allan,
M.; Fournel, M.; Trachy-Bourget, M.-C.; Li, Z.; Besterman, J. M.; Delorme,
D. J. Med. Chem. 2002, 45, 2877-2885. (b) SAHA analogues: Remisze-
wski, S. W.; Sambucetti, L. C.; Atadja, P.; Bair, K. W.; Cornell, W. D.;
Green, M. A.; Howell, K. L.; Jung, M.; Kwon, P.; Trogani, N.; Walker, H.
J. Med. Chem. 2002, 45, 753-757. FK228 analogues: (c) Yurek-George,
A.; Cecil, A. R. L.; Mo, A. H. K.; Wen, S.; Rogers, H.; Habens, F.; Maeda,
S.; Yoshida, M.; Packham, G.; Ganesan, A. J. Med. Chem. 2007, 50, 5720-
5726.
(12) Doi, T.; Iijima, Y.; Shin-ya, K.; Ganesan, A.; Takahashi, T.
Tetrahedron Lett. 2006, 47, 1177-1780.
(13) The yield for N-acylation of Seebach’s chiral auxiliary was not
reported. A yield of 80% was reported for a similar N-acylation in the
following article: Fukuzawa, S.-i.; Matsuzawa, H.; Yoshimitsu, S.-i. J. Org.
Chem. 2000, 65, 1702-1706.
(14) Denmark, S. E.; Fujimori, S. J. Am. Chem. Soc. 2005, 127, 8971-
8973.
(15) (a) Matsumura, K.; Hashiguchi, S.; Ikariya, T.; Noyori, R. J. Am
Chem. Soc. 1997, 119, 8738-8739. (b) Haack, K.-J.; Hashiguchi, S.; Fujii,
A.; Ikariya, T.; Noyori, R. Angew. Chem., Int. Ed. 1997, 36, 285-287.
(16) Jones, T. K.; Denmark, S. E. Organic Syntheses; Collect. Vol. 7;
John Wiley & Sons: New York, 1990; p 524; 1986, 64, 182-186.
(17) (a) Wong, L. S. M.; Sherburn, M. S. Org. Lett. 2003, 5, 3603-6.
(b) Raach, A.; Reiser, O. J. Prakt. Chem. 2000, 342, 605-608.
Org. Lett., Vol. 10, No. 4, 2008
615

in 24% yield after multiple purifications to remove triphen-
ylphosphine oxide. It was treated with I2/MeOH, which
promoted disulfide formation and afforded FK228 (1)
(Scheme 5).
Scheme 5. Completion of the Synthesis of FK228 (1)
In conclusion, an improved synthesis of FK228 (1) has
been completed from simple, commercially available materi-
als. An efficient, multigram scale synthesis of the tetrapeptide
portion (4), using N-Alloc-D-cysteine, was developed. The
key â-hydroxy mercapto acid (5) was prepared in nine steps
and >99:1 er via an asymmetric Noyori hydrogen transfer
reaction and late-stage mercaptan introduction. The weak step
in this synthesis, which follows the pioneering route first
developed by Simon,4 is the macrolactone-forming cycliza-
tion reaction. We are currently investigating alternative
conditions and synthetic strategies for closing the bicyclic
ring system with the objective of further improving the
throughput to the natural product.
Acknowledgment. This paper is dedicated to the memory
of Professor Albert I. Meyers. We thank the National
Institutes of Health (GM068011) for financial support. D.M.J.
thanks the American Cancer Society, New England Division
Broadway on Beachside for a postdoctoral fellowship. We
also acknowledge Dr. Julian Simon of the Fred Hutchinson
Cancer Research Center for many helpful discussions and
for graciously providing additional experimental details.
prior to coupling with N-Fmoc-D-valine. The N-Alloc depro-
tection and coupling with N-Fmoc-D-Val proceeded smoothly
to provide 10 g of tetrapeptide 18. Deprotection of the
terminal nitrogen quantitatively affords tetrapeptide 4 in
seven steps and 53% overall yield from 15 and readily
available amino acid derivatives.
With tetrapeptide 4 and â-hydroxy mercapto acid 5 in
hand, we were poised to complete the synthesis of FK228.
Following Simon’s precedent, the two portions were coupled
using BOP to provide 19 in quantitative yield. After methyl
ester hydrolysis, the linear FK228 precursor was subjected
to known Mitsunobu macrolactonization conditions. Despite
extensive efforts, we were unable to realize the 62% yield
reported4 for this cyclization. Depsipeptide 3 was isolated
Note Added after ASAP Publication. In the Abstract and
Table of Contents graphics, the hydroxy acid side chain was
missing a carbon in the version published ASAP January
19, 2008; the corrected version was published January 30,
2008.
Supporting Information Available: Experimental pro-
cedures and characterization of all key compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(18) Kruse, C. H.; Holden, K. G. J. Org. Chem. 1985, 65, 1192-1194.
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