A solid-phase total synthesis of the cyclic depsipeptide HDAC inhibitor spiruchostatin A

Article abstract of DOI:10.1016/j.tetlet.2009.04.005

Spiruchostatin A, a potent histone deacetylase inhibitor, was efficiently synthesized from (3S,4R)-4-amino-3-hydroxy-5-methylhexanoic acid utilizing solid-phase peptide elongation with d-cysteine, d-alanine, and (E)-3-hydroxy-7-thio-4-heptenoic acid and s

Full text of DOI:10.1016/j.tetlet.2009.04.005

Tetrahedron Letters 50 (2009) 2970–2972
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
A solid-phase total synthesis of the cyclic depsipeptide HDAC inhibitor
spiruchostatin A
a,
Yusuke Iijima a, Asami Munakata a,b, Kazuo Shin-ya c, A. Ganesan d, Takayuki Doi a,e, , Takashi Takahashi
*
*
a Department of Applied Chemistry, Tokyo Institute of Technology, 2-12-1, Ookayama, Meguro, Tokyo 152-8552, Japan
b Japan Biological Informatics Consortium (JBIC), 2-45 Aomi, Koto-ku, Tokyo 135-8073, Japan
c National Institute of Advanced Industrial Science and Technology, 2-42, Aomi, Koto-ku, Tokyo 135-0064, Japan
d School of Chemistry, University of Southampton, Southampton SO17 1BJ, United Kingdom
e Graduate School of Pharmaceutical Sciences, Tohoku University, Aza-Aoba, Aramaki, Aoba, Sendai 980-8578, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Spiruchostatin A, a potent histone deacetylase inhibitor, was efficiently synthesized from (3S,4R)-4-
amino-3-hydroxy-5-methylhexanoic acid utilizing solid-phase peptide elongation with -cysteine, -ala-
nine, and (E)-3-hydroxy-7-thio-4-heptenoic acid and solution-phase macrolactonization, followed by
intramolecular disulfide formation.
Received 27 February 2009
Revised 31 March 2009
Accepted 2 April 2009
Available online 7 April 2009
D
D
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Cyclic depsipeptide
Histone deacetylase
Natural product synthesis
Solid-phase synthesis
Histone deacetylases (HDACs) play an important role in the reg-
ulatory post-translational modification of chromatin, and are con-
sidered to be promising targets for cancer therapy.1 A variety of
both natural products and synthetic compounds have provided ac-
cess to potent HDAC inhibitors.2 SAHA (Vorinostat) produced by
Merck3 is a synthetic example, and is the first approved HDAC
inhibitor in the market as a therapeutic anticancer agent. Among
natural products, the cyclic depsipeptide FK2284 is the only one
in clinical development, and is currently in Phase II trials.5 Spiruc-
hostatin A (1), isolated from Pseudomonas sp., is a more recent dis-
covery with a structure similar to that of FK228 and with more
potent activity.6 Both compounds are bicyclic depsipeptides having
an intramolecular disulfide bridge. The postulated mechanism of
inhibition is that the disulfide bond of 1 is reduced in cells and
the resulting thiol in the longer side chain binds to the catalytic
zinc atom in the pocket of HDACs.4b We are interested in the po-
tency of the parent skeleton of 1 with diversification of the side
chains in the peptide components to discover subtype selective
inhibitors.7–9 As part of our studies on solid-phase combinatorial
synthesis based on natural products, we report a solid-phase total
synthesis of spiruchostatin A (1) suitable for the preparation of
analogues.10
We previously reported a solution-phase total synthesis of
1.11,12 Based on the synthesis, solid-phase assembly of 4–7 would
afford cyclization precursor 2, which can undergo macrolactoniza-
O
O
HN
N
H
HN
N
H
HN
O
S
S
S
OH
S
O
O
NH
NH
O
O
O
O
O
O
spiruchostatin A (1)
FK228
O
FmocHN
HO
FmocHN
OH
O
OH
TrtS
HN
N
5
H
O
OH
O
O
STrt
O
STrt
OH
4
NH
W
STrt
OH
NHFmoc
OH
X
Linker
O
6
HO
O
3
2
W = solid-support
or OH
7
* Corresponding authors. Fax: +81 22 795 6864, +81 3 5734 2884.
E-mail addresses: doi_taka@mail.pharm.tohoku.ac.jp (T. Doi), ttak@apc.tite-
ch.ac.jp (T. Takahashi).
Scheme 1. Strategy for the solid-phase-based total synthesis of spiruchostatin A
(1).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.04.005

Y. Iijima et al. / Tetrahedron Letters 50 (2009) 2970–2972
2971
tion, followed by intramolecular disulfide bond formation either on
the solid-phase or in the solution phase, leading to 1 (Scheme 1).
Our initial attempts for solid-phase total synthesis utilizing
safety-catch linkers, such as Kenner’s sulfonamide linker13 and a
hydrazinobenzoyl linker14 were unsuccessful.15 Then, we chose
the 2-chlorotrityl linker for the preparation of the cyclization pre-
cursor by an Fmoc strategy. The Fmoc derivative 4, readily pre-
pared from its Boc derivative,11 was immobilized on the 2-
chlorotrityl chloride resin (8) (Scheme 2).16 The loading amount
was determined to be 1.0 mmol gÀ1 by cleavage from the resin
(1% CF3COOH/CH2Cl2). Surprisingly, the Fmoc group in 9 could
not be removed by conventional methods (20% piperidine/DMF).
The complete deprotection, however, was performed by treatment
of the well-swollen resins with 2% DBU and 2% piperidine in DMF
five times.17 The magic mixture (20% piperidine in 1% TritonX100
CH2Cl2/DMF/NMP = 1:1:1),18 which is useful for deprotection of
the N-terminus of aggregating peptide sequences, was also effec-
tive in our case. Fortunately, the 4-amino-3-hydroxy-5-methyl-
hexanoate 10 was stable in such strongly basic conditions
without forming lactam 11, due to the steric hindrance of the trityl
group.
dation was successfully performed without observation of severe
epimerization (<5%).20 The coupling with Fmoc–
-Ala–OH (6)
D
(DIC/HOBt) and (E)-3-hydroxy-7-tritylthio-4-heptenoic acid
(7)11,21 (PyBOP/DIEA) was sequentially performed. The linear pep-
tide 2 (W = OH) was cleaved from the polymer-support with 30%
hexafluoroisopropyl alcohol in CH2Cl2.22 After simple filtration
through silica gel column, 2 (W = OH) was isolated in 56% overall
yield.23 According to our previous synthesis with modification of
the reaction conditions, macrolactonization using Shiina’s method
(MNBA/DMAP/CH2Cl2/1 mM/rt),24 followed by disulfide bond for-
mation (I2/MeOH) was performed in solution phase to furnish spir-
uchostatin A (1) in 89% overall yield. The spectral data of synthetic
1 were in good accordance with those of the natural product.11
In summary, we accomplished a solid-phase total synthesis of
spiruchostatin A. The linear peptide was assembled by an Fmoc
strategy using the 2-chlorotrityl linker on a polymer-support. After
cleavage from the polymer-support, macrocyclization and disulfide
formation in solution phase provided spiruchostatin A in a high
overall yield. This solid-phase procedure was adapted to the com-
binatorial synthesis of a library of spiruchostatin analogues based
on in silico design. The experimental details and their biological
investigation will be reported in due course. Furthermore, it can
be employed in the synthesis of other natural product HDAC inhib-
itors such as FK228 and the recently isolated largazole.21,25,26
The next step, involving condensation of 10 with Fmoc-D-
Cys(Trt)-OH (5), was also initially problematic. When the resin
10 was treated with a mixture of 5, diisopropylcarbodiimide
(DIC), and 1-hydroxybenzotriazole (HOBt) in DMF–CH2Cl2 (4:1),
the dipeptide 12 formed was a 77:23 mixture of diastereomers.
Acknowledgments
The
a
position of the cysteine residue was partially racemized19
prior to the acylation probably because the condensation of steri-
cally hindered amine 10 would be too slow. We needed to over-
come the problem by pre-incubation forming an activated ester
in an appropriate mixed solvent. When a mixture of 5, DIC, HOBt
in DMF–CH2Cl2 (1:1) was incubated for 5 min in advance, the ami-
This work was supported by New Energy and Industrial Tech-
nology Development Organization.
References and notes
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4, DIEA,
4. (a) Ueda, H.; Nakajima, H.; Hori, Y.; Fujita, T.; Nishimura, M.; Goto, T.; Okuhara,
M. J. Antibiot. 1994, 47, 301–310; (b) Furumai, R.; Matsuyama, A.; Kobashi, N.;
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Yoshida, M.; Horinouchi, S. Cancer Res. 2002, 62, 4916–4921.
Cl
CH2Cl2
O
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L
Cl
OH
O
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5. Piekarz, R. L.; Frye, A. R.; Wright, J. J.; Steinberg, S. M.; Liewehr, D. J.; Rosing, D.
R.; Sachdev, V.; Fojo, T.; Bates, S. E. Clin. Cancer Res. 2006, 12, 3762–3773.
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Hayakawa, Y.; Seto, H. Tetrahedron Lett. 2001, 42, 41–44; (b) Davidson, S. M.;
Townsend, P. A.; Carroll, C.; Yurek-George, A.; Balasubramanyam, K.; Kundu, T.
K.; Stephanou, A.; Packham, G.; Ganesan, A.; Latchman, D. S. ChemBioChem.
2005, 6, 162–170; (c) Crabb, S. J.; Howell, M.; Rogers, H.; Ishfag, M.; Yurek-
George, A.; Carey, K.; Pickering, B. M.; East, P.; Mitter, R.; Maeda, S.; Johnson, P.
W. M.; Townsend, P.; Shin-ya, K.; Yoshida, M.; Ganesan, A.; Packham, G.
Biochem. Pharmcol. 2008, 76, 463–475.
9
L = 2-chlorotrityl
(1.48 mmol g–1
)
(1.0 mmol g–1
)
8
2%DBU,
2%piperidine,
DMF
OH
O
HN
H2N
L
OH
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10 min x 5
O
7. Bhuiyan, M. P. I.; Kato, T.; Okauchi, T.; Nishino, N.; Maeda, S.; Nishino, T. G.;
Yoshida, M. Bioorg. Med. Chem. 2006, 14, 3438–3446.
11
10 L = 2-chlorotrityl
8. 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.
9. 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, 2900–2905.
10. For a solid-phase synthesis of FK228 analogues: Maro, S. D.; Pong, R.-C.; Hsieh,
J.-T.; Ahn, J.-M. J. Med. Chem. 2008, 51, 6639–6641.
O
5, DIC, HOBt,
O
FmocHN
TrtS
DMF–CH2Cl2(1:1)
N
H
L
OH
O
11. (a) Yurek-George, A.; Habens, F.; Brimmell, M.; Packham, G.; Ganesan, A. J. Am.
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Takahashi, T. Tetrahedron Lett. 2006, 47, 1177–1180.
L = 2-chlorotrityl
12
12. (a) Takizawa, T.; Watanabe, K.; Narita, K.; Kudo, K.; Oguchi, T.; Abe, H.; Katoh,
T. Heterocycles 2008, 76, 275–290; (b) Takizawa, T.; Watanabe, K.; Narita, K.;
Oguchi, T.; Abe, H.; Katoh, T. Chem. Commun. 2008, 1677–1679.
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(e) Rosenbaum, C.; Waldmann, H. Tetrahedron Lett. 2001, 42, 5677–5680.
15. In the case of a sulfonamide linker, immobilization of N-Boc (3S,4R)-3-amino-
4-hydroxy-5-methylhexanoic acid using various condensation reagents
1) MNBA, DMAP
1 mM CH2Cl2
rt
1) 2% DBU, 2% piperidine, DMF
2) 6, DIC, HOBt, DMF–CH2Cl2(1:1)
3) 2% DBU, 2% piperidine, DMF
1
2
2) I2, MeOH
CH2Cl2
(W = OH)
4) 7, PyBOP, DIEA, CH2Cl2
5) 30% (CF3)2CHOH, CH2Cl2
(89%)
(56% from 9)
Scheme 2. The solid-phase total synthesis of spiruchostatin A (1). DIEA = N,N-
diisopropylethylamine, DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene, DIC = diisopro-
PyBOP = (benzotriazol-1-
yloxy)tripyrrolidinophosphonium hexafluorophosphate, MNBA = 2-methyl-6-nitro-
benzoic anhydride, DMAP = 4-(dimethylamino)pyridine.
pylcarbodiimide,
HOBt = 1-hydroxybenzotriazole,

2972
Y. Iijima et al. / Tetrahedron Letters 50 (2009) 2970–2972
resulted in poor yields (<30%). On the other hand, with the hydrazine linker,
and the mixture was shaken for 2 h. The resin was filtered and washed with
DMF three times and with CH2Cl2 three times.
21. Numajiri, Y.; Takahashi, T.; Takagi, M.; Shin-ya, K.; Doi, T. Synlett 2008, 2483–
2486.
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immobilization and peptide elongation were successfully performed. However,
activation of the linker for cyclization-cleavage from the polymer-support
provided a complex mixture in the presence of trityl sulfide.
16. To a suspension of 2-chlorotrityl chloride resin (8) (250 mg, 1.48 mmol g–1
,
0.37 mmol) in CH2Cl2 (3 mL) in
a 6 mL syringe-shaped vessel (Varian
Reservoir) was added acetyl chloride (0.3 mL) at room temperature. After
being shaken for 3 h, the resin was filtered and washed with dry CH2Cl2 five
23. To the polymer-supported tripeptide was added
a solution of 7 (84 mg,
0.20 mmol), DIEA (35 L, 0.20 mmol), and PyBOP (104 mg, 0.20 mmol) in
l
times. To the resin were added acid 4 (567 mg, 1.48 mmol) and DIEA (517
l
L,
CH2Cl2 (1 mL) and the mixture was shaken for 24 h. The resin was filtered and
washed with CH2Cl2 three times, MeOH three times, and CH2Cl2 three times.
The resin was added 30% (CF3)2CHOH in CH2Cl2 (1 mL) and the mixture was
shaken for 30 min. The resin was filtered and washed with CH2Cl2 five times.
The filtrate was concentrated in vacuo. The residue was purified by
2.96 mmol) in CH2Cl2 (3 mL) at room temperature and the mixture was shaken
for 24 h. The resin was filtered and washed with CH2Cl2 three times, MeOH
three times, and CH2Cl2 three times.
17. General procedure for removal of the Fmoc group. To the resin (50 mg,
0.050 mmol) in a 3 mL syringe-shaped vessel (Varian Reservoir) was added
CH2Cl2 (1 mL) and the mixture was shaken for 1 h and filtered. To this resin
were added 2% DBU and 2% piperidine in DMF (1 mL). After being shaken for
10 min, the resin was washed with CH2Cl2 five times. After this procedure was
repeated five times, the resin was washed with CH2Cl2 five times and with DMF
five times.
18. (a) Zhang, L.; Goldammer, C.; Henkel, B.; Zühl, F.; Panhaus, G.; Jung, G.; Bayer,
E. In Innovation and Perspectives in Solid Phase Synthesis; Epton, R., Ed.;
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H.; Pillai, S. K.; Jensen, I.; Albericio, F.; Kates, S. A. J. Pept. Sci. 2000, 6, 512–518.
19. Han, Y.; Albericio, F.; Barany, G. J. Org. Chem. 1997, 62, 4307–4312.
20. General procedure for peptide formation using DIC–HOBt. To a solution of
Fmoc-protected amino acid (0.20 mmol) and HOBt (27 mg, 0.20 mmol) in
chromatography on silica gel (5% MeOH in CHCl3) to afford desired
2
(27.2 mg, 0.0278 mmol, 56% from 9).
24. Shiina, I.; Kubota, M.; Ibuka, R. Tetrahedron Lett. 2002, 43, 7535–
7539.
25. Taori, K.; Paul, V. J.; Luesch, H. J. Am. Chem. Soc. 2008, 130, 1806–1807.
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8455–8459; (b) Seiser, T.; Kamena, F.; Cramer, N. Angew. Chem., Int. Ed. 2008,
47, 6483–6485; (c) Bowers, A.; West, N.; Taunton, J.; Schreiber, S. L.; Bradner, J.
E.; Williams, R. M. J. Am. Chem. Soc. 2008, 130, 11219–11222; (d) Ren, Q.; Dai,
L.; Zhang, H.; Tan, W.; Xu, Z.; Ye, T. Synlett 2008, 2379–2383; (e) Ying, Y.; Liu,
Y.; Byeon, S. R.; Kim, H.; Luesch, H.; Hong, J. Org. Lett. 2008, 10, 4021–4024; (f)
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3595–3598; (g) Ghosh, A. K.; Kulkarni, S. Org. Lett. 2008, 10, 3907–3909; (h)
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CH2Cl2 (0.5 mL) and DMF (0.5 mL) was added DIC (31
lL, 0.20 mmol) and the
mixture was stirred for 5 min. This solution was added to the amine-free resin