Total synthesis of largazole and its biological evaluation

Article abstract of DOI:10.1055/s-2008-1078263

We achieved a total synthesis of largazole. The optically active β-hydroxycarbonyl unit was prepared from a modified Nagao's N-acetylthiazolidinethione. A 4-methylthiazoline-thiazole amino ester was prepared by both a step-by-step method and tandem cyclization from Cys-2-MeCys-containing tripeptide. Amidation of the activated β-hydroxycarbonyl unit and 4-methylthiazoline-thiazole-containing amino ester, followed by esterification with N-Fmoc valine afforded cyclization precursor after selective removal of the methyl ester at the C-terminus and the Fmoc group at the N-terminus. Macrolactamization, deprotection of thiol, and S-acylation provided largazole. Biological evaluation of its S-modified derivatives as well as the synthetic largazole exhibited strong inhibitory activity against histone deacetylases (HDAC). Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2008-1078263

LETTER
2483
Total Synthesis of Largazole and Its Biological Evaluation
T
otal
S
ynthesis of
o
Largazole shitaka Numajiri,a Takashi Takahashi,a Motoki Takagi,b Kazuo Shin-ya,c Takayuki Doi*d
a
Department of Applied Chemistry, Tokyo Institute of Technology, Ookayama, Meguro, Tokyo 152-8552, Japan
Biomedicinal Information Research Center (BIRC), Japan Biological Informatics Consortium (JBIC),
2-42 Aomi, Koto-ku, Tokyo 135-0064, Japan
National Institute of Advanced Industrial Science and Technology, 2-42 Aomi, Koto-ku, Tokyo 135-0064, Japan
Graduate School of Pharmaceutical Sciences, Tohoku University, Aza-Aoba, Aramaki, Aoba, Sendai 980-8578, Japan
Fax +81(22)7956864; E-mail: doi_taka@mail.pharm.tohoku.ac.jp
b
c
d
Received 10 June 2008
S
Abstract: We achieved a total synthesis of largazole. The optically
active b-hydroxycarbonyl unit was prepared from a modified Na-
gao’s N-acetylthiazolidinethione. A 4-methylthiazoline-thiazole
amino ester was prepared by both a step-by-step method and tan-
dem cyclization from Cys-2-MeCys-containing tripeptide. Amida-
tion of the activated b-hydroxycarbonyl unit and 4-methyl-
thiazoline-thiazole-containing amino ester, followed by esterifica-
tion with N-Fmoc valine afforded cyclization precursor after selec-
tive removal of the methyl ester at the C-terminus and the Fmoc
group at the N-terminus. Macrolactamization, deprotection of thiol,
and S-acylation provided largazole. Biological evaluation of its S-
modified derivatives as well as the synthetic largazole exhibited
strong inhibitory activity against histone deacetylases (HDAC).
S
N
N
RS
O
HN
NH
O
O
O
largazole 1 R = Me(CH2)6CO
2 R = H
3 R = Ac
4 R =
S
N
S
S
Key words: HDAC inhibitor, macrolactamization, natural prod-
ucts, peptides, total synthesis
S
S
N
N
N
N
CO2H
TrtS
TrtS
O
or
HN
H2N
NH
NH
CO2H
The cyclic depsipeptide largazole (1, Scheme 1), isolated
from a cyanobacterium of the genus Symploca, exhibits
strongly potent growth-inhibitory activity for cancer cell
lines.1 Largazole is a cyclic depsipeptide that possesses a
unique structure consisting of a substituted 4-methylthia-
zoline fused to a thiazole,2 (E)-3-hydroxy-7-octanoylthio-
4-heptenoic acid and valine. The (E)-3-hydroxy-7-thio-4-
heptenoic acid moiety is the same component as that
found in FK-228 and spiruchostatin A and B (Figure 1),
known as potent histone deacetylase (HDAC) inhibi-
tors.3,4 We are interested in the HDAC inhibitory activity
of 1 and its S-substituted derivatives 2–4 (Scheme 1) be-
cause an alkyl thiol can be released within a cell and acts
as a zinc ion binder in the pocket of the HDAC.5 We
planned syntheses of 1 and its derivatives 2–4 and evalu-
ated their HDAC inhibitory activity.6
OH
O
O
O
O
5
6
S
XHN
Y
S
N
CO2Me
N
O
NH2
N
7
9 X = H, Y = OMe
10 X = Fmoc, Y = OH
O
OH
S
STrt
S
8
Scheme 1 Synthetic strategy for largazole (1) and its derivatives
2–4
An outline of the synthetic strategy is illustrated in
Scheme 1. The macrocyclic ring of 1 can be constructed by
macrolactonization of 5 as we previously reported in the
synthesis of spiruchostatin A.7 We also planned macrolac-
tamization of 6 because it is reported that the macrolacton-
ization under Keck conditions did not proceed in the
synthesis of FK-228.8 Both cyclization precursors 5 and 6
would be prepared by coupling of 4-methylthiazoline-thia-
zole-containing amino ester 7 and activated b-hydroxycar-
bonyl unit 8, followed by amidation with H-Val-OMe (9) in
the C-terminus or O-acylation with N-Fmoc-valine (10).
R
O
O
HN
NH
N
HN
NH
N
H
H
S
S
O
OH
S
O
S
O
HN
O
O
O
O
O
O
spiruchostatin A (R = Me)
spiruchostatin B (R = Et)
FK-228
Figure 1
SYNLETT 2008, No. 16, pp 2483–2486
x
x
.x
x
.2
0
0
8
Advanced online publication: 22.08.2008
DOI: 10.1055/s-2008-1078263; Art ID: U06008ST
© Georg Thieme Verlag Stuttgart · New York

2484
Y. Numajiri et al.
LETTER
PyAOP21 and DIPEA and hydrolysis of the resulting
methyl ester furnished hydroxy acid 5. Macrolactoniza-
tion of 5, however, did not proceed by Shiina method us-
ing 2-methyl-6-nitrobenzoic anhydride (MNBA)22 that is
a significantly effective reagent for the macrolactoniza-
tion in the synthesis of spiruchostatin A in our hand.7
OH
TrtS
S
a
O
HCl•NH2
+
CO2Me
N
88%
NHFmoc
12
11
STrt
O
S
O
S
N
b
O
S
S
S
CO2Me
N
CO2Me
a
N
H
90%
+
N
8
N
STrt
94%
(94:6)
NHFmoc
NHFmoc
17
13
14
c
16
TrtS
O
40% overall yield
S
S
TrtS
N
CO2Me
CO2Me
b
c, 8
N
N
TrtS
7
H
14
O
NH
NH
15
NHFmoc
OH
O
18 (90%)
Scheme 2 Reagents and conditions: a) EDCI, HOAt, DIPEA,
CH2Cl2, 88%; b) Ph3PO (3 equiv), Tf2O (1.5 equiv), CH2Cl2, 0 °C, 1
h, 90%; c) i) TFA, Et3SiH, CH2Cl2, ii) cat. (NH4)2MnO4, toluene,
120 °C, 12 h, 40% in 2 steps. HOAt = 1-hydroxy-7-azabenzotriazole.
d, 9
e, 10
S
N
S
S
S
CO2H
N
The synthetic unit 14 was efficiently prepared as follows
(Scheme 2): Coupling of thiazole-containing amino acid
119,10 and (R)-H-2-MeCys(Trt)-OMe·HCl (12)11,12 using
EDCI and HOAt afforded peptide 13 in 88% yield. Thia-
zoline formation was performed by Kelly’s method
(Ph3PO, Tf2O)13 to provide the desired 4-methylthiazo-
line-thiazole 14 in 90% yield. One-pot bisthiazoline for-
mation was also carried out using Ishihara’s method.14
After removal of the two trityl groups in protected Gly-
Cys-2-MeCys tripeptide 15, tandem dehydrative cycliza-
tion was performed in the presence of a catalytic amount
of (NH4)2MnO4 in refluxing toluene with azeotropic re-
moval of water to form a bisthiazoline intermediate,
which was rapidly oxidized to thiazoline-thiazole 14 in
40% overall yield.15,16
N
TrtS
N
TrtS
O
XHN
HN
NH
NH
O
CO2H
OH
O
O
O
19 X = Fmoc (73%)
6 X = H
5
f
g
S
i
j
1 (90%)
S
N
N
TrtS
h
O
2
3 (95%)
4 (61%)
HN
k
NH
O
O
O
20 (65%)
Asymmetric aldol reaction of aldehyde 17 with various
acetate derivatives having a chiral auxiliary has been per-
formed by several groups including us.7,8,17,18 Here, we
utilized a modified Nagao reagent,19 N-acetylthiazoli-
dinethione 16 recently reported by Olivo.20 Aldol reaction
of enal 1717 with the titanium enolate generated from 16
Scheme 3 Reagents and conditions: a) TiCl4, DIPEA, CH2Cl2,
–78 °C, 94% (dr = 94:6); b) Et2NH, MeCN; c) DIPEA, CH2Cl2, 90%
in 2 steps; d) i) LiOH, THF, H2O, MeOH; ii) 9, PyAOP, DIPEA,
CH2Cl2, 98%; iii) LiOH, THF, H2O, MeOH, 90%; e) i) 10, 2,4,6-tri-
chlorobenzoyl chloride, DMAP, toluene, quantitative yield; ii)
Me3SnOH, DCE, 70 °C, 73%, recovery 15%; f) Et2NH, MeCN; g)
HATU, DIPEA, CH2Cl2, 1 mM, 65% in 2 steps; h) 1% TFA, CH2Cl2,
using TiCl4 and DIPEA was performed in dichlo- Et3SiH; i) octanoyl chloride, DIPEA, CH2Cl2, 90% in 2 steps; j)
Ac2O, DIPEA, DMAP, CH2Cl2, 95% in 2 steps; k) (2-Py)-S-S-(2-Py),
CH2Cl2, MeOH, 61% in 2 steps.
romethane at –78 °C (Scheme 3). The desired aldol prod-
uct
8
was provided in 94% yield with high
diastereoselectivity (94%). The major diastereomer was
readily separated by silica gel column chromatography.
Toward macrolactamization, the cyclization precursor 19
Moreover, the thiazolidinethione derivative can be direct- was prepared by esterification of the hydroxy group in 18
ly used for the next amidation as Nagao originally with Fmoc-Val-OH (10) using Yamaguchi method23 in
reported19 and Ganesan applied in the total synthesis of
spiruchostatin A.18 In fact, amidation of 8 with amine 7
readily prepared from 14 by removal of the Fmoc group
afforded 18 in 90% yield. Hydrolysis of methyl ester 18,
followed by coupling with H-Val-OMe (9) using
quantitative yield. Selective removal of the methyl ester in
the presence of Fmoc-Val-ester proceeded by treatment
with Me3SnOH at 70 °C.24 The desired acid 19 was ob-
tained in 73% yield accompanied with recovery of the
substrate (15%) and a small amount of alcohol (5%) pro-
Synlett 2008, No. 16, 2483–2486 © Thieme Stuttgart · New York

LETTER
Total Synthesis of Largazole
2485
(7) Doi, T.; Iijima, Y.; Shin-ya, K.; Ganesan, A.; Takahashi, T.
Tetrahedron Lett. 2006, 47, 1177.
(8) Li, K. W.; Wu, J.; Xing, W.; Simon, J. A. J. Am. Chem. Soc.
1996, 118, 7237.
(9) Thiazole 11 was prepared from commercially available
Fmoc-Gly-NH2 as follows: (i)Lawesson’s reagent, toluene,
80 °C;(ii) bromopyruvic acid, 1,4-dioxane, 56% in 2 steps.
(10) Videnov, G.; Kaiser, D.; Kempter, C.; Jung, G. Angew.
Chem., Int. Ed. Engl. 1996, 35, 1503.
(11) Compound 12 was prepared from previously reported (R)-2-
methylcysteine (ref. 12) as follows: (i) TrtCl, DMF, 24 h,
75%; (ii) AcCl, MeOH, 80 °C, 15 h.
duced by the cleavage of the Val-ester. After removal of
the Fmoc group in 19, macrolactamization of 6 was per-
formed using HATU25 and DIPEA under high dilution
conditions (1 mM). The desired cyclic depsipeptide 20
was obtained in 65% overall yield after purification by sil-
ica gel column chromatography.26 Removal of the trityl
group in 20 provided thiol 2, which was acylated with oc-
tanoyl chloride to furnish largazole (1) in 90% yield. Thiol
2 was further converted to acetate 3 and 2-pyridyl disul-
fide 427 by treatment with acetic anhydride and 2,2¢-dipy-
ridyl disulfide, respectively. The spectral data of synthetic
1 were in good accordance with those of the natural prod-
uct largazole (1).1,6,28
(12) Pattenden, G.; Thom, S. M.; Jones, M. F. Tetrahedron 1993,
49, 2131.
(13) You, S.-L.; Razavi, H.; Kelly, J. W. Angew. Chem. Int. Ed.
2003, 42, 83.
The HDAC inhibitory activity of synthetic natural product
1 and its derivatives 2–4 was evaluated.29 Reporter gene
assay in HEK293 cell promoted by cytomegalovirus
(CMV) exhibited that ED50 values of 1–4 were deter-
mined to be 60 nM, 38 nM, 50 nM, and 70 nM, respective-
ly, when the ED50 value of trichostatin A (TSA) was found
to be 300 nM. Actually, largazole (1) acts as a potent
HDAC inhibitor. The S-octanoyl, S-acetyl, and disulfide
derivatives 1, 3, and 4 possess similar inhibitory activity
whereas thiol derivative 2 is slightly more potent than the
S-substituted compounds. Therefore, the thiol derivative
2 could be released within cell and that is probably a real
active form as reported in the system of FK-228.5,30
(14) Sakakura, A.; Kondo, R.; Ishihara, K. Org. Lett. 2005, 7,
1971.
(15) Raman, P.; Razavi, H.; Kelly, J. W. Org. Lett. 2000, 2, 3289.
(16) The tandem dehydrative cyclization of S-trityl derivative 15
did not proceed by the use of either TiCl4 (ref. 15) or Ph3PO–
Tf2O (ref. 13).
(17) Chen, Y.; Gambs, C.; Abe, Y.; Wentworth, P., Jr.; Janda,
K. D. J. Org. Chem. 2003, 68, 8902.
(18) (a) Yurek-George, A.; Habens, F.; Brimmell, M.; Packham,
G.; Ganesan, A. J. Am. Chem. Soc. 2004, 126, 1030.
(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.
(19) Nagao, Y.; Hagiwara, Y.; Kumagai, T.; Ochiai, M.; Inoue,
T.; Hashimoto, K.; Fujita, E. J. Org. Chem. 1986, 51, 2391.
(20) Osorio-Lozada, A.; Olivo, H. F. Org. Lett. 2008, 10, 617.
(21) PyAOP = (7-azabenzotriazol-1-yloxy) tripyrrolidino-
phosphonium hexafluorophosphate: Albericio, F.; Cases,
M.; Alsina, J.; Triolo, S. A.; Carpino, L. A.; Kates, S. A.
Tetrahedron Lett. 1997, 38, 4853.
In summary, we have demonstrated an efficient total syn-
thesis of largazole (1) and its S-modified derivatives 2–4.
Biological evaluation of those derivatives exhibited that
they are potent HDAC inhibitors as naturally occurring
FK-228 and spiruchostatin A and B that possess the same
(E)-3-acyloxy-7-thio-4-heptenamide. Further study for
selective inhibitory activity against HDAC is under way
in our laboratory.
(22) (a) Shiina, I.; Ibuka, R.; Kubota, M. Chem. Lett. 2002, 31,
286. (b) Shiina, I.; Kubota, M.; Oshima, H.; Hashizume, M.
J. Org. Chem. 2004, 69, 1822.
(23) Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi,
M. Bull. Chem. Soc. Jpn. 1979, 52, 1989.
(24) Nicolaou, K. C.; Estrada, A. A.; Zak, M.; Lee, S. H.; Safina,
B. S. Angew. Chem. Int. Ed. 2005, 44, 1378.
(25) HATU = O-(7-azabenzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate: Carpino, L. A.
J. Am. Chem. Soc. 1993, 115, 4397.
Acknowledgment
This work was supported by New Energy and Industrial Technolo-
gy Development Organization.
(26) Spectral Data for 20
References and Notes
Mp 97–99 °C. 1H NMR (400 MHz, CDCl3): d = 7.76 (s, 1
H), 7.37 (m, 6 H), 7.28 (m, 6 H), 7.17–7.26 (m, 4 H), 6.55
(dd, J = 9.3, 3.0 Hz, 1 H), 5.73 (dt, J = 15.6, 6.8 Hz, 1 H),
5.62 (m, 1 H), 5.41 (dd, J = 15.6, 6.3 Hz, 1 H), 5.21 (dd,
J = 17.6, 9.3 Hz, 1 H), 4.56 (dd, J = 9.3, 3.9 Hz, 1 H), 4.13
(dd, J = 17.6, 3.0 Hz, 1 H), 4.05 (d, J = 11.7 Hz, 1 H), 3.29
(d, J = 11.7 Hz, 1 H), 2.82 (dd, J = 16.1, 9.3 Hz, 1 H), 2.65
(dd, J = 16.1, 2.9 Hz, 1 H), 2.16–2.27 (m, 2 H), 1.99–2.12
(m, 3 H), 1.85 (s, 3 H), 0.69 (d, J = 6.8 Hz, 3 H), 0.53 (d,
J = 6.8 Hz, 3 H). 13C NMR (100 MHz, CDCl3): d = 173.3,
169.5, 168.7, 168.1, 165.3, 147.2, 144.9, 133.2, 129.7,
128.1, 128.0, 126.7, 124.6, 84.1, 72.0, 66.7, 58.1, 43.4, 41.1,
40.8, 34.1, 31.5, 31.3, 24.1, 18.9, 16.9. IR (neat): 3373,
2930, 1734, 1674, 1511, 1245, 751, 701 cm–1. [a]D25 +4.0
(c 0.25, CHCl3). HRMS (ESI–TOF): m/z calcd for
[C40H42N4O4S3 + H]+: 739.2446; found: 739.2447.
(27) Nishino, N.; Jose, B.; Okamura, S.; Ebisusaki, S.; Kato, T.;
Sumida, Y.; Yoshida, M. Org. Lett. 2003, 5, 5079.
(1) Taori, K.; Paul, V. J.; Luesch, H. J. Am. Chem. Soc. 2008,
130, 1806.
(2) For other synthetic approaches of 4-methylthiazoline-
thiazole-containing molecules, see: (a) Han, F. S.;
Tokuyama, H.; Fukuyama, T. Chem. Commun. 2007, 3444.
(b) Boyce, R. J.; Pattenden, G. Tetrahedron 1995, 51, 7313.
(3) Ueda, H.; Nakajima, H.; Hori, Y.; Fujita, T.; Nishimura, M.;
Goto, T.; Okuhara, M. J. Antibiot. 1994, 47, 301.
(4) Masuoka, Y.; Nagai, A.; Shin-ya, K.; Furihata, K.; Nagai,
K.; Suzuki, K.; Hayakawa, Y.; Seto, H. Tetrahedron Lett.
2001, 42, 41.
(5) 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.
(6) During the preparation of this manuscript, another total
synthesis of largazole was reported. See: Ying, Y.; Taori, K.;
Kim, H.; Hong, J.; Luesch, H. J. Am. Chem. Soc. 2008, 130,
8455.
Synlett 2008, No. 16, 2483–2486 © Thieme Stuttgart · New York

2486
Y. Numajiri et al.
LETTER
(28) Spectral Data for 1
40.6, 34.2, 32.4, 31.7, 29.0, 29.0, 28.0, 25.7, 24.2, 22.7, 18.9,
16.8, 14.1. IR (neat): 3374, 2927, 2855, 1735, 1681, 1507,
1258, 1031 cm–1. [a]D24 +39 (c 0.38, MeOH) {lit.1 [a]D20 +22
(c 0.1, MeOH), lit.6 [a]D23.5 +37.8 (c 0.027, MeOH)}. HRMS
(ESI–TOF): m/z calcd for [C29H42N4O5S3 + H]+: 623.2396;
found: 623.2396.
1H NMR (400 MHz, CDCl3): d = 7.77 (s, 1 H), 7.16 (d,
J = 9.3 Hz, 1 H), 6.47 (dd, J = 9.8, 2.9 Hz, 1 H), 5.83 (dt,
J = 15.6, 6.8 Hz, 1 H), 5.66 (m, 1 H), 5.52 (dd, J = 15.6, 6.8
Hz, 1 H), 5.29 (dd, J = 17.6, 9.8 Hz, 1 H), 4.60 (dd, J = 9.3,
3.4 Hz, 1 H), 4.27 (dd, J = 17.6, 2.9 Hz, 1 H), 4.06 (d,
J = 11.2 Hz, 1 H), 3.28 (d, J = 11.2 Hz, 1 H), 2.90 (t, J = 7.3
Hz, 2 H), 2.87 (dd, J = 16.6, 10.2 Hz, 1 H), 2.68 (dd,
J = 16.6, 2.9 Hz, 1 H), 2.53 (t, J = 7.8 Hz, 2 H), 2.32 (dt,
J = 7.3, 6.8 Hz, 2 H), 2.10 (m, 1 H), 1.87 (s, 3 H), 1.64 (m, 2
H), 1.25–1.28 (m, 8 H), 0.87 (t, J = 6.8 Hz, 3 H), 0.68 (d,
J = 6.8 Hz, 3 H), 0.51 (d, J = 6.8 Hz, 3 H). 13C NMR (100
MHz, CDCl3): d = 199.4, 173.5, 169.6, 168.9, 168.1, 165.1,
147.4, 132.8, 128.5, 124.5, 84.3, 72.2, 57.9, 44.2, 43.4, 41.2,
(29) Dressel, U.; Renkawitz, R.; Baniahmad, A. Anticancer Res.
2000, 20, 1017.
(30) Hong and Luesch also concluded that the thiol group is the
pharmacophore of largazole by SAR studies based on the
biological evaluation of its thiol-free and S-acetyl
derivatives and the hydroxy analogue instead of the S-
octanoyl group. See ref. 6.
Synlett 2008, No. 16, 2483–2486 © Thieme Stuttgart · New York