Total synthesis and biological mode of action of largazole: A potent class I histone deacetylase inhibitor

Article abstract of DOI:10.1021/ja8033763

The efficient total synthesis of the recently described natural substance largazole (1) and its active metabolite largazole thiol (2) is described. The synthesis required eight linear steps and proceeded in 37% overall yield. It is demonstrated that largazole is a pro-drug that is activated by removal of the octanoyl residue from the 3-hydroxy-7-mercaptohept-4-enoic acid moiety to generate the active metabolite 2, which is an extraordinarily potent Class I histone deacetylase inhibitor. Synthetic largazole and 2 have been evaluated side-by-side with FK228 and SAHA for inhibition of HDACs 1, 2, 3, and 6. Largazole and largazole thiol were further assayed for cytotoxic activity against a panel of chemoresistant melanoma cell lines, and it was found that largazole is substantially more cytotoxic than largazole thiol; this difference is attributed to differences in the cell permeability of the two substances.

Full text of DOI:10.1021/ja8033763

Published on Web 07/19/2008
Total Synthesis and Biological Mode of Action of Largazole: A
Potent Class I Histone Deacetylase Inhibitor
Albert Bowers,† Nathan West,§ Jack Taunton,| Stuart L. Schreiber,
James E. Bradner,§ and Robert M. Williams*,†,‡
Department of Chemistry, Colorado State UniVersity, Fort Collins, Colorado 80523, UniVersity
of Colorado Cancer Center, Aurora, Colorado 80045, Dana-Farber Cancer Institute, 44 Binney
Street, Boston, Massachusetts 02115, Department of Cellular and Molecular Pharmacology,
UniVersity of CaliforniasSan Francisco, San Francisco, California 94158, Howard Hughes
Medical Institute, Chemistry & Chemical Biology, HarVard UniVersity, Broad Institute of
HarVard and MIT, Cambridge, Massachusetts 02142
Received May 6, 2008; E-mail: rmw@lamar.colostate.edu
Abstract: The efficient total synthesis of the recently described natural substance largazole (1) and its
active metabolite largazole thiol (2) is described. The synthesis required eight linear steps and proceeded
in 37% overall yield. It is demonstrated that largazole is a pro-drug that is activated by removal of the
octanoyl residue from the 3-hydroxy-7-mercaptohept-4-enoic acid moiety to generate the active metabolite
2, which is an extraordinarily potent Class I histone deacetylase inhibitor. Synthetic largazole and 2 have
been evaluated side-by-side with FK228 and SAHA for inhibition of HDACs 1, 2, 3, and 6. Largazole and
largazole thiol were further assayed for cytotoxic activity against a panel of chemoresistant melanoma cell
lines, and it was found that largazole is substantially more cytotoxic than largazole thiol; this difference is
attributed to differences in the cell permeability of the two substances.
Introduction
have been identified, which are generally divided into four
classes on the basis of their sequence homology to yeast
Largazole (1) is a densely functionalized macrocyclic dep-
sipeptide, recently isolated from the cyanobacterium Symploca
sp. by Luesch and co-workers.1 This natural product exhibits
exceptionally potent and selective biological activity, with 2-
to 10-fold differential growth inhibition in a number of
transformed and non-transformed human- and murine-derived
cell lines. The remarkable selectivity of this agent against cancer
cells prompts particular interest in its mode of action and its
value as a potential cancer chemotherapeutic.
counterparts.9 With respect to cancer therapy, there is an
emerging consensus that Class I HDACs are clinically relevant
and that the undesirable toxicity associated with the first
generation of HDAC inhibitors may be related to class indis-
criminancy. As a result, our laboratories have recently initiated
programs aimed at the synthesis and modification of peptide-
and depsipeptide-based HDACi with the objective of optimizing
structures for class- and even isoform-specific inhibition.
In their isolation paper, Luesch, et al. stated that “the
3-hydroxy-7-mercaptohept-4-enoic acid unit in 1 is unprec-
edented in natural products.” 1 In contrast to this assertion, the
(S)-3-hydroxy-7-mercaptohept-4-enoic acid is in fact an essential
motif in several cytotoxic natural products, including FK228
(FR901228),2 FR901375,2 and spiruchostatin3 (Figure 1), all
of which are known histone deacetylase inhibitors (HDACi).4,5
The histone deacetylase enzymes are zinc metalloenzymes
that catalyze the hydrolysis of acetylated lysine residues in
chromatin, and thereby regulate transcription in eukaryotic
cells.6,7 Their selective inhibition has recently become a major
area of research in cancer chemotherapy.8 To date, 18 HDACs
(2) (a) Fujisawa Pharmaceutical Co., Ltd. Jpn. Kokai Tokkyo Koho
JP,03141296, 1991. (b) Ueda, H.; Nakajima, H.; Hori, Y.; Fujita, T.;
Nishimura, M.; Goto, T.; Okuhara, M. J. Antibiot. 1994, 47, 301. (c)
Shigematsu, N.; Ueda, H.; Takase, S.; Tanaka, H. J. Antibiot. 1994,
47, 311. (d) Ueda, H.; Manda, T.; Matsumoto, S.; Mukumoto, S.;
Nishigaki, F.; Kawamura, I.; Shimomura, K. J. Antibiot. 1994, 47,
315.
(3) Masuoka, Y.; Nagai, A.; Shin-ya, K.; Furihata, K.; Nagai, K.; Suzuki,
K.; Hayakawa, Y.; Seto, H. Tetrahedron Lett. 2001, 42, 41.
(4) Townsend, P. A.; Crabb, S. J.; Davidson, S. M.; Johnson, P. W. M.;
Packham, G.; Ganesan, A. The bicyclic depsipeptide family of histone
deacetylase inhibitors. In Chemical Biology; Schreiber, S. L., Kapoor,
T. M., Wess, G., Eds.; Wiley-VCH Verlag GmbH & Co.: Weinheim,
Germany, 2007; Vol. 69, pp 3-720..
(5) Reference 1b substantially corrected the error in ref 1awith respect to
the novelty of the (S)-3-hydroxy-7-mercaptohept-4-enoic acid moiety
and also reported IC50 HDAC inhibitory data for largazole.
(6) Somech, R.; Israeli, S.; Simon, A. Cancer Treat. ReV. 2004, 30, 461.
(7) (a) Miller, T. A.; Witter, D. J.; Belvedere, S. J. Med. Chem. 2003, 46,
5097. (b) Moradei, O.; Maroun, C. R.; Paquin, I.; Vaisburg, A. Curr.
Med. Chem.; Anti-Cancer Agents 2005, 5, 529. (c) Bolden, J. E.; Peart,
M. J.; Johnstone, R. W. Nat. ReV. Drug DiscoVery 2006, 5, 769.
(8) Minucci, S.; Pelicci, P. G. Nature ReV. Cancer 2006, 6, 38.
(9) (a) Taunton, J.; Hassig, C. A.; Schreiber, S. L. Science 1996, 272,
408. (b) Grozinger, C. M.; Hassig, C. A.; Schreiber, S. L. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 4868. (c) Johnstone, R. W. Nature ReV.
Drug DiscoVery 2002, 1, 287.
† Colorado State University.
§ Dana-Farber Cancer Institute.
| University of CaliforniasSan Francisco.
Howard Hughes Medical Institute.
‡ University of Colorado Cancer Center.
(1) (a) Taori, K.; Paul, V. J.; Luesch, H. J. Am. Chem. Soc. 2008, 130,
1806–1807. (b) Subsequent to the submission of the present manuscript
on May 6, 2008, Luesch et al. independently published a distinct
synthesis of largazole: Ying, Y.; Taori, K.; Kim, H.; Homg, J.; Luesch,
H. J. Am. Chem. Soc. 2008, 130, 8455–8459.
9
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2008 American Chemical Society
J. AM. CHEM. SOC. 2008, 130, 11219–11222 11219

A R T I C L E S
Bowers et al.
Figure 2. Synthetic strategy to assemble largazole.
Scheme 1. Total Synthesis of Largazole (1) and Largazole Thiol
(2)
Figure 1. Structure of largazole (1), largazole thiol (2), and several known
macrocyclic HDACi natural products containing the (S)-3-hydroxy-7-
mercaptohept-4-enoic acid moiety.
The three natural substances FK228, FR901375, and spiru-
chostatin are all activated in Vitro and in ViVo by reductive
cleavage of a disulfide bond to expose the free sulfhydryl residue
of the pendant (S)-3-hydroxy-7-mercaptohept-4-enoic acid
moiety that coordinates to the active-site Zn2+ residue of the
HDACs, resulting in a potent inhibitory effect.10 Given that
largazole contains this well-known Zn2+-binding arm, it seemed
evident to us that largazole is simply a pro-drug that is activated
by hydrolytic removal of the octanoyl residue by cellular lipases
and/or esterases to produce the putative cytotoxic species 2 (the
“largazole thiol”). Two laboratories have previously demon-
strated that thioester analogues of FK228 retain their antipro-
liferative activity in cell-based assays.11 As part of our program
on the synthesis and chemical biology of FK228 and ana-
logues,12 we report herein an efficient total synthesis of largazole
and the largazole thiol and demonstrate that 2 is an extraordinar-
ily potent HDACi.
challenge turned out to be the macrocyclization strategy. Due
to the anticipated susceptibility of the ꢀ-carboxylate linkage to
undergo elimination, our initial efforts focused on installing this
linkage last. However, all methods, both direct (macrolacton-
ization Via Yamaguchi, Mukaiyama, Keck, and Shiina proce-
dures) and indirect (inversion Via Mitsunobu reaction), failed
to provide the desired macrocycle. An additional attempt at
closure of the depsipeptide ring Via a late-stage thiazoline-
forming reaction also failed to provide the desired macrocyclic
product. Thus, we turned to a strategy involving early installation
of the ester and subsequent closure about the least-hindered
amide bond.
We were able to readily access the necessary R-methylcys-
teine subunit with the requisite (R)-stereochemistry Via the
Pattenden modification of the Seebach protocol on L-cysteine
methyl ester (Scheme 1).14 Alternatively, we obtained R-me-
thylserine as a generous gift from Ajinomoto Co., Japan, and
converted this substance into R-methylcysteine by a published
procedure.15 Gram quantities of this amino acid were obtained
in high enantiomeric purity and condensed with the known
nitrile 716 to provide the thiazoline-thiazole subunit 8 in high
yield.
Results and Discussion
We envisioned disconnection of the macrocycle to the four
key subunits illustrated in Figure 2; that is, R-methylcysteine
(3), thiazole (4), (S)-valine (5), and (S)-3-hydroxy-7-mercap-
tohept-4-enoic acid (6). Given the ready availability of these
building blocks from prior efforts,13,14 the underlying synthetic
(10) (a) Yoshida, M.; Kijama, M.; Akita, M.; Beppu, T. J. Biol. Chem.
1990, 265, 17174. (b) Yoshida, M.; Hoshikawa, Y.; Koseki, K.; Mori,
K.; Beppu, T. J. Antibiot. 1990, 43, 1101.
(11) (a) Or, Y. S.; Verdine, G. L.; Keegan, M. Preparation of Metabolite
Derivatives of the HDAC Inhibitor FK228. PCT Int. Appl. WO
2007061939, 2007. (b) 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.
(12) (a) Johns, D. M.; Greshock, T. J.; Noguchi, Y.; Williams, R. M. Org.
Lett. 2008, 10, 613. (b) Greshock, T. J.; West, N.; Schreiber, S. L.;
Bradner, J. E.; Williams, R. M.,unpublished results.
(13) (a) Li, K. W.; Wu, J.; Xing, W.; Simon, J. A. J. Am. Chem. Soc.
1996, 118, 7237. (b) Chen, Y.; Gambs, C.; Abe, Y.; Wentworth, P.;
Janda, K. D. J. Org. Chem. 2003, 68, 8902. (c) Yurek-George, A.;
Habens, F.; Brimmell, M.; Packham, G.; Ganesan, A. J. Am. Chem.
Soc. 2004, 126, 1030.
(14) (a) Jeanguenat, A.; Seebach, D. J. Chem. Soc., Perkin Trans. 1 1991,
2291. (b) Mulqueen, G. C.; Pattenden, D.; Whiting, D. A. Tetrahedron
1993, 49, 5359.
(15) Smith, N. D.; Goodman, M. Org. Lett. 2003, 5, 1035.
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Synthesis and Biological Mode of Action of Largazole
A R T I C L E S
Figure 3. Inhibition of HDAC1 by largazole (b), largazole thiol (O), SAHA
(9), and FK228 (0).
We recently disclosed a new synthetic route to the ꢀ-hydroxy
acid 10 based on a Noyori asymmetric transfer hydrogenation.12a
In the present work, we found a recently published synthesis
of this subunit to also be expedient and high yielding.17
Thiazolidinethione (9) was treated with 2-(trimethylsilyl)ethanol
to provide the TSE-protected acid 10, which was subsequently
coupled to N-Fmoc-L-valine to afford 11. Due to the sluggish
reactivity of allylic alcohol 10, we found it necessary to use an
excess (5 equiv) of the commercially available amino acid.
Removal of the Fmoc group and PyBOP-mediated coupling to
the thiazoline-thiazole carboxylic acid (8) furnished the acyclic
precursor 12.
Cyclization was effected under high dilution in the presence
of 2 equiv each of HOBt and HATU, furnishing the desired
macrocycle 13 in 77% isolated yield from 12. Removal of the
S-trityl protecting group was accomplished with iPr3SiH and
TFA to provide an authentic sample of 2 in excellent yield.
Acylation of 2 with octanoyl chloride under standard condi-
tions afforded synthetic largazole in 89% yield from 13. The
spectroscopic data (1H NMR, 13C NMR, and HRMS) for the
synthetic substance were in excellent agreement with those
published for the natural product (see Supporting Information).1
We next interrogated the biochemical activity of synthetic
largazole (1) and 2 against HDACs 1, 2, 3, and 6, for which
we have developed robust, kinetic biochemical assays. To
measure the inhibitory effect on deacetylase function in Vitro,
we optimized a continuous kinetic biochemical assay miniatur-
ized to a 384-well plate format. In this assay, purified, full-
length HDAC protein (HDAC1 1.67 ng/µL, HDAC2 0.067 ng/
µL, HDAC3/NCor2 0.033 ng/µL, HDAC6 0.67 ng/µL; BPS
Biosciences) was incubated with a commercially available
fluorophore-conjugated substrate at a concentration equivalent
to the substrate Km (Upstate 17-372; 6 µM for HDAC1, 3 µM
for HDAC2, 6 µM for HDAC3/NCoR2, and 20 µM for
HDAC6). As presented in Figures 3, 4 and Table 1, 2 is an
extraordinarily potent inhibitor of HDAC1 and HDAC2 (Ki )
70 pM).
Figure 4. Comparative profiling of FK228 and the largazole thiol (2). Dose-
ranging studies of FK228, largazole, and 2 were performed against Class I
HDAC proteins using the described kinetic, homogeneous assay (see
Supporting Information). As a comparative measure of potency, compounds
were studied in triplicate at a standard concentration (0.6 nM). Averaged
data are presented for inhibition of HDAC1 (black), HDAC2 (gray), and
HDAC3/NCoR2 (white). Error bars reflect one standard deviation from the
mean.
Table 1. HDAC Inhibitory Activity (Ki, nM) of Largazole (1) and
Largazole Thiol (2) As Compared to Pharmaceutical Hdac
Inhibitors
compound
HDAC1
HDAC2
HDAC3
HDAC6
1
2
20
21
48
>1000
0.07
0.12
10
0.07
0.14
10
0.17
0.28
15
25
35
9
FK228a
SAHA
a The FK228 sample used in this study was synthesized in these
laboratories12a and purified by PTLC to homogeneity.
aqueous assay conditions or by trypsin (present in this enzyme-
coupled reaction) could account for the substantial decrease in
enzyme potency observed.
Detailed studies of FK228 isoform selectivity by our labo-
ratories previously identified a strong bias favoring the Class I
enzymes, HDAC1, HDAC2 and HDAC3, over the Class IIb
enzyme, HDAC6.12b Similarly, the active depsipeptide largazole
thiol (2) exhibits substantial potency against HDAC1, HDAC2
and HDAC3 in the picomolar range (Table 1). Indeed, this
degree of inhibitory potency against HDAC1, HDAC2 and
HDAC3 is, to the best of our knowledge, unprecedented. Only
FK228 itself has HDACi potency approaching that of 2.
Comparative profiling of FK228 and 2 demonstrates superior
inhibitory potency of the thiol derivative against HDAC1,
HDAC2, and HDAC3 (Figure 4). The comparatively diminished
potency of largazole itself in these homogeneous assays supports
the hypothesis of pro-drug activation of largazole, the specific
catalysis of which is the focus of our immediate follow-up
studies.
The parent natural product largazole itself, on the other hand,
is a comparatively weak HDAC inhibitor with potency ap-
proximating that of the nonselective pharmaceutical product
SAHA (Vorinostat, Merck Research Laboratories). In fact, the
measurement of potency obtained in these studies of largazole
defines the maximal possible HDAC inhibitory effect. That is,
even a trace contamination of 2 or free thiol liberated under
We have initiated studies aimed at determining the potential
utility of largazole as an HDAC inhibitor-based therapeutic
agent. This work comprises studies of physiochemical properties,
metabolic stability in serum and liver microsomes, and phar-
macokinetics in rodents. Immediately, we have initiated studies
to determine the antineoplastic effects of 1 and 2 on cultured
human cancer cells. Predicting a potent antiproliferative effect
of largazole on the basis of the biochemical potency for Class
I HDACs as described above, we selected a panel of malignant
melanoma cell lines for study due to the typically extreme
chemoresistance of this tumor. As demonstrated in Figure 5,
largazole exhibits submicromolar inhibitory effect on melanoma
(16) (a) Videnov, G.; Kaiser, D.; Kempter, C.; Jung, G. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 1503. (b) Lange, U. E. W.; Scha¨fer, B.;
Baucke, D.; Buschmann, E.; Mack, H. Tetrahedron Lett. 1999, 40,
7067.
(17) Yurek-George, A.; Habens, F.; Brimmell, M.; Packham, G.; Ganesan,
A. J. Am. Chem. Soc. 2004, 126, 1030.
9
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A R T I C L E S
Bowers et al.
Figure 5. Antiproliferative effects of largazole (red) and largazole thiol (blue). Effects on cell viability were evaluated using a panel of human malignant
melanoma cell lines, using the standard, surrogate measurement of ATP content (Cell TiterGlo; Promega) in 384-well plate format. Replicate measurements
were normalized to vehicle-only controls and IC50 calculations were performed by logistic regression (Spotfire DecisionSite). Largazole demonstrates a
potent antiproliferative effect (IC50 ) 45-315 nM) compared to largazole thiol (IC50 ) 360-2600 nM).
cell proliferation. Importantly, 1 has a consistent, superior
potency (IC50 ) 45-315 nM) compared to 2 (IC50 ) 360-2600
nM).18
inhibition of HDACs. Since FK228, FR901375, and spirucho-
statin mask the common and key 3-hydroxy-7-mercaptohept-
4-enoic acid unit as a reductively labile disulfide,19 other protect-
and-release strategies for exploiting this potent zinc-binding arm
in the context of new molecular scaffolds are readily apparent.
In addition, the molecular scaffold of largazole provides yet
another macrocyclic template20 from which a myriad of
potentially active and isoform-selective HDAC inhibitors might
be designed and synthesized. Studies along these lines are under
investigation in these laboratories.
Conclusion
In conclusion, we have completed an efficient total synthesis
of largazole (1) in eight linear steps and 37% overall yield, and
its active metabolite, the largazole thiol (2), in seven linear steps.
This serves to corroborate the structure assigned by Luesch and
co-workers.1 The synthesis recorded here provided 12 mg of 1
and 19 mg of 2 on the first pass and should be readily scaleable
to gram quantities. This amount of synthetic material has
allowed for further investigation of the biological activity of
this potential cancer chemotherapeutic.
We have demonstrated that largazole is, in fact, a pro-drug,
which must be converted to its active form, free thiol 2. The
combination of cap group and zinc-binding motif present in this
thiol provides the most potent and selective HDACi reported
to date. The octanoyl residue in largazole apparently serves a
dual role, imparting better cell-permeability and allowing facile
presentation of the free thiol within the cell. We attribute the
observed inverse difference in cytotoxicity to the superior cell-
permeability of the thioester 1 as compared to the thiol 2. These
studies reveal that numerous opportunities exist for capitalizing
on the masked thiol pro-drug manifold inherent in 1. The data
presented here should prove useful in the design and develop-
ment of potent and therapeutically active agents that target
Acknowledgment. We thank the Colorado State University
Cancer Supercluster for financial support (to R.M.W. and A.B.).
J.E.B. acknowledges support by grants from the Burroughs-
Wellcome Foundation and National Cancer Institute (1K08
CA128972-01A1). This project has been funded in part with Federal
funds from the National Cancer Institute’s Initiative for Chemical
Genetics, National Institutes of Health, under Contract No. N01-
CO-12400 (N.W., S.L.S., and J.E.B.). Mass spectra were obtained
on instruments supported by the NIH Shared Instrument Grant
GM49631. We thank Ajinomoto Co. for the generous gift of
R-methylserine which was also deployed to construct R-methyl-
cysteine utilized in this study.
Supporting Information Available: Complete experimental
details for the synthesis of largazole (1) and largazole thiol (2)
and for the HDAC inhibition assays. This material is available
free of charge via the Internet at http://pubs.acs.org.
JA8033763
(18) We note that there are significant discrepancies between what is
reported in ref 1b and our results reported here with respect to the
biochemical HDAC inhibitory data obtained and supporting mechanism
of action. The biochemical data provided herein reflect activity in
highly robust, miniaturized homogeneous assays, with Z′ calculations
compatible with high-throughput screening. In this assay, we observe
high concordance with published kinetic measurements of enzyme
inhibition (Ki). Thus, the accuracy of our reported HDAC inhibitory
data would be expected to be markedly improved. This is important
due to our observation of the unusual, perhaps unprecedented potency
of 2 for HDAC1 and the direct comparison provided to FK228. The
data offered in ref 1b are IC50 data, which have substantial variation
between assays and are thus of limited utility and extensibility. We
also note that the present synthesis is significantly higher yielding than
that reported in ref 1b.
(19) 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.
(20) For several naturally occurring macrocyclic HDAC inhibitors, see :(a)
Singh, S. B.; Zink, D. L.; Polishook, J. D.; Dombrowski, A. W.;
Darkin-Rattray, S. J.; Schmatz, D. M.; Goetz, M. A. Tetrahedron Lett.
1996, 37, 8077. (b) Nakao, Y.; Yoshida, S.; Matsunaga, S.; Shindoh,
N.; Terada, Y.; Nagai, K.; Yamashita, J. K.; Ganesan, A.; van Soest,
R. W. M.; Fusetani, N. Angew. Chem., Int. Ed. 2006, 45, 7553. (c)
Kijima, M.; Yoshida, M.; Sugita, K.; Horinouchi, S.; Beppu, T. J. Biol.
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