Synthesis and histone deacetylase inhibitory activity of largazole analogs: Alteration of the zinc-binding domain and macrocyclic scaffold

Article abstract of DOI:10.1021/ol900078k

Fourteen analogs of the marine natural product largazole have been prepared and assayed against histone deacetylases (HDACs) 1, 2, 3, and 6. Olefin cross-metathesis was used to efficiently access six variants of the side-chain zinc-binding domain, while adaptation of our previously reported modular synthesis allowed probing of the macrocyclic cap group.

Full text of DOI:10.1021/ol900078k

ORGANIC
LETTERS
2009
Vol. 11, No. 6
1301-1304
Synthesis and Histone Deacetylase
Inhibitory Activity of Largazole Analogs:
Alteration of the Zinc-Binding Domain
and Macrocyclic Scaffold
Albert A. Bowers,† Nathan West,§,| Tenaya L. Newkirk,† Annie
E. Troutman-Youngman,† Stuart L. Schreiber,|, Olaf Wiest,# 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, DiVision of
Hematologic Neoplasia, Dana-Farber Cancer Institute, Boston, Massachusetts 02115,
Chemical Biology Program, Broad Institute of HarVard & MIT, Cambridge,
Massachusetts 02142, Howard Hughes Medical Institute, Chemistry & Chemical
Biology, HarVard UniVersity, Cambridge, Massachusetts 02138, and Walther Cancer
Research Center and Department of Chemistry and Biochemistry, UniVersity of Notre
Dame, Notre Dame, Indiana 46556
rmw@lamar.colostate.edu; james_bradner@dfci.harVard.edu
Received January 14, 2009
ABSTRACT
Fourteen analogs of the marine natural product largazole have been prepared and assayed against histone deacetylases (HDACs) 1, 2, 3, and
6. Olefin cross-metathesis was used to efficiently access six variants of the side-chain zinc-binding domain, while adaptation of our previously
reported modular synthesis allowed probing of the macrocyclic cap group.
Histone deacetylases (HDACs) control gene transcription via
regulation of lysine acetylation, and their selective inhibition
is currently a major area of research in cancer chemotherapy.1
The 18 known HDACs are divided into four classes based
on sequence homology to yeast counterparts.2 The signifi-
cance of individual classes and isoforms to cancer growth
and inhibition is not well understood, yet the majority of
data suggests that class I HDACs are the most relevant,
tractible cancer targets.3
(1) (a) Somech, R.; Israeli, S.; Simon, A. Cancer Treat. ReV. 2004, 30,
461. (b) Synthesis of 7200 small molecules based on a substructural
analysis of the histone deacetylase inhibitors trichostatin and trapoxin.
Sternson, S. M.; Wong, J. C.; Grozinger, C. M.; Schreiber, S. L. Org. Lett.
2001, 3, 4239–4242. (c) Moradei, O.; Maroun, C. R.; Paquin, I.; Vaisburg,
A. Curr. Med. Chem., Anti-Cancer Agents 2005, 5, 529. (d) Bolden, J. E.;
Peart, M. J.; Johnstone, R. W. Nat. ReV. Drug DiscoVery 2006, 5, 769.
(2) (a) Taunton, J.; Hassig, C. A.; Schreiber, S. L. Science 1996, 272,
408–11. (b) Grozinger, C. M.; Hassig, C. A.; Schreiber, S. L. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 4868. (c) Johnstone, R. W. Nat. ReV. Drug
DiscoVery 2002, 1, 287.
† Colorado State University.
‡ University of Colorado Cancer Center.
§ Dana-Farber Cancer Institute.
| Broad Institute of Harvard & MIT, Cambridge.
Harvard University.
# University of Notre Dame.
10.1021/ol900078k CCC: $40.75
Published on Web 02/24/2009
2009 American Chemical Society

The pharmacophore model for HDAC inhibition consists
of three elements: (1) a surface recognition unit which
interacts with the rim of the binding pocket, (2) a metal-
binding domain which coordinates to the active site zinc ion,
and (3) a linker that connects the surface recognition site to
the zinc-binding domain.1b Numerous HDAC inhibitors
(HDACi), both natural and synthetic, are known, and
variations in all three features have variably contributed to
potency and selectivity in new HDACi’s.4,5
amide isosteres with insight into the key contacts and
associated spatial determinants that provide this remarkable
level of activity.9 Herein we describe our continuing efforts
to modify the structural scaffold of largazole with the goal
of further defining and expanding structure-activity relation-
ships within the family of macrocyclic HDACi’s. Our over-
arching aim in this context is to perturb the class- and isoform-
selectivity as initially observed in robust biochemical assays
such that differences in specific enzymatic activity might later
be correlated with phenotypic responses in cell-based assays.
Our reported route to largazole proved highly reproducible,
and we were able to rapidly adapt it to simple variants of
the macrocyclic core.7 Thus we were able to easily access
milligram quantities of the C-2 epimer (3) and the enantiomer
(2) of largazole. Additionally, we sought to perturb the
conformation of the macrocycle by imparting greater rigidity;
our initial foray in this capacity replaced the valine residue
with proline (4). Compound 4 could be obtained in only
slightly diminished overall yield via the same synthetic route
we deployed in the total synthesis of largazole (see Sup-
porting Information).
Figure 1. Selected largazole analogs and azumamide E.
To date, the most potent and selective HDACi known is
largazole (1a, Figure 1), a densely functionalized macrocyclic
depsipeptide isolated from the cyanobacterium Symploca sp.
by Luesch and co-workers.6 We have recently disclosed a
concise, modular, and scalable total synthesis of largazole
and demonstrated its picomolar activity against HDACs 1,
2, and 3, as well as low nanomolar cytotoxicity against a
number of chemoresistant cancer cell lines.7,8 Additionally,
we have disclosed a detailed conformation-activity relation-
ship model for largazole, FK228, and their corresponding
Scheme 1. Synthesis of the Largazole-Azumamide Hybrid
(3) Karagiannis, T. C.; El-Osta, A. Leukemia 2007, 21, 61–5, and
references therein.
(4) (a) Richon, V. M.; Emiliani, S.; Verdin, E.; Webb, Y.; Breslow, R.;
Rifkind, R. A.; Marks, P. A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3003–
7. (b) Yoshida, M.; Kijima, M.; Akita, M.; Beppu, T. J. Biol. Chem. 1990,
265, 17174–9. (c) Darkin-Rattray, S. J.; Gurnett, A. M.; Myers, R. W.;
Dulski, P. M.; Crumley, T. M.; Allocco, J. J.; Cannova, C.; Meinke, P. T.;
Colletti, S. L.; Bednarek, M. A.; Singh, S. B.; Goetz, M. A.; Dombrowski,
A. W.; Polishook, J. D.; Schmatz, D. M. Proc. Natl. Acad. Sci. U.S.A. 1996,
93, 13143–7. (d) Kijima, M.; Yoshida, M.; Sugita, K.; Horinouchi, S.;
Beppu, T. J. Biol. Chem. 1993, 268, 22429–35. (e) Nakao, Y.; Yoshida,
S.; Matsunaga, S.; Shindoh, N.; Terada, Y.; Nagai, K.; Yamashita, J. K.;
Ganesan, A.; van Soest, R. W.; Fusetani, N. Angew. Chem., Int. Ed. Engl.
2006, 45, 7553–7. (f) Izzo, I.; Maulucci, N.; Bifulco, G.; De Riccardis, F.
Angew. Chem., Int. Ed. Engl. 2006, 45, 7557–60. (g) Wen, S.; Carey, K. L.;
Nakao, Y.; Fusetani, N.; Packham, G.; Ganesan, A. Org. Lett. 2007, 9,
1105–8. (h) Ueda, H.; Nakajima, H.; Hori, Y.; Fujita, T.; Nishimura, M.;
Goto, T.; Okuhara, M. J. Antibiot. (Tokyo) 1994, 47, 301–10. (i) Shigematsu,
N.; Ueda, H.; Takase, S.; Tanaka, H.; Yamamoto, K.; Tada, T. J. Antibiot.
(Tokyo) 1994, 47, 311–4. (j) Ueda, H.; Manda, T.; Matsumoto, S.;
Mukumoto, S.; Nishigaki, F.; Kawamura, I.; Shimomura, K. J. Antibiot.
(Tokyo) 1994, 47, 315–23. (k) Yurek-George, A.; Habens, F.; Brimmell,
Two methods were employed to alter side chain functionality
and access a series of largazole chimeras. For the largazole-
azumamide hybrid (9), the cis-geometry of the alkene residue
necessitated its early introduction. Thus, aldol condensation of
aldehyde 510 with thiazolidine-2-thione 6 provided the necessary
ꢀ-hydroxy acid building block (7, Scheme 1 and Supporting
Information). For other variants investigated, late-stage intro-
duction of the zinc-binding side arms via cross metathesis
proved expedient. Cross metathesis had previously been used
by Luesch, Phillips, and Cramer to attach the natural side chain
in their syntheses of largazole itself and was investigated
independently in our laboratories.8a-d
Cramer et al. have demonstrated that, at least where
largazole is concerned, the four-atom linker length relative
to the thiol is optimal for maximum HDAC inhibition.8d
However, literature precedent has shown that a four- to five-
atom chain is optimal in small molecules bearing alternative
zinc-binding functionality. Therefore, in the series of analogs
M.; Packham, G.; Ganesan, A. J. Am. Chem. Soc. 2004, 126, 1030–1
.
(5) For seminal work on HDACi hybrids:(a) Furumai, R.; Komatsu, Y.;
Nishino, N.; Khochbin, S.; Yoshida, M; Horinouchi, S. Proc. Natl. Acad.
Sci. U.S.A. 2000, 98, 87–92. (b) Nishino, N.; Jose, B.; Okamura, S.;
Ebisusaki, S.; Kato, T.; Sumida, Y.; Yoshida, M. Org. Lett. 2003, 5, 5079–
82.
(6) Taori, K.; Paul, V. J.; Luesch, H. J. Am. Chem. Soc. 2008, 130,
13506.
(7) Bowers, A. A.; West, N.; Taunton, J.; Schreiber, S. L.; Bradner,
J. E.; Williams, R. M. J. Am. Chem. Soc. 2008, 130, 11219–22
.
(8) For additional syntheses of largazole, see: (a) Nasveschuk, C. G.;
Ungermannova, D.; Liu, X.; Phillips, A. J. Org. Lett. 2008, 10, 3595–98.
(b) Ghosh, A. K.; Kulkarni, S. Org. Lett. 2008, 10, 3907–9. (c) Ying, Y.;
Taori, K.; Kim, H.; Hong, J.; Luesch, H. J. Am. Chem. Soc. 2008, 130,
8455–59. (d) Seiser, T.; Kamena, F.; Cramer, N. Angew. Chem., Int. Ed.
2008, 47, 6483–85. (e) Ren, Q.; Dai, L.; Zhang, H.; Tan, W.; Xu, Z.; Ye,
(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., DOI: 10.1021/
ja807772w.
(10) Wen, S.; Carey, K. L.; Nakao, Y.; Fusetani, N.; Packham, G.;
Ganesan, A. Org. Lett. 2007, 9, 1105–8.
T. Synlett 2008, 15, 2379
.
1302
Org. Lett., Vol. 11, No. 6, 2009

Scheme 2
.
Metathesis Route to Largazole Hybrids
Scheme 3
.
Synthesis of Cysteine and Thiazole-Thiazole
Analogs
prepared via metathesis, both the four- and the five-atom
tethers were synthesized.
Compounds 11 and 12 bear the well-known R-aminoben-
zamide group present in MS-275.11 Meanwhile, compounds
13, 14 and 15, 16 contain R-thioamides and R-thioketones,
respectively. These two motifs were identified as potential
candidates in a computational study done by Vanommeslae-
ghe and have demonstrated promise in subsequent medicinal
efforts.12 Of note, our yields in the metathesis reaction
conform to the results reported by Cramer.8d Our initial yields,
employing Grubb’s second-generation ruthenium catalyst, were
low with poor conversion (Scheme 2). The Hoveyda-Grubbs
second-generation catalyst proved much more efficient. All Boc-
and Trityl-protecting groups were removed prior to biological
assay (see Supporting Information).
synthetic strategy (Scheme 4). Known chloro-nitrile 24 could
be Boc-protected and then condensed with R-methyl cysteine
to provide acid 27. Subsequent deprotection, coupling, and
cyclization provided analog 29.
Scheme 4. Synthesis of Thiazole to Pyridine Substitution
We also sought to probe the significance of the methyl
substituent on the thiazoline ring. Condensation of nitrile 17
with L-cysteine proved remarkably facile, proceeding in near
quantitative yield (Scheme 3). Initial efforts at coupling to
ester 19 provided poor yields of the desired acyclic precursor
20. The major product was thiazole-thiazole 21, resulting
from in situ oxidation. Optimization of conditions for this
coupling eventually allowed for up to 62% yield of the
desired product. Oxidation could be the cause of the
somewhat diminished yields in the cyclization of 20.
Compound 23 could not be detected in NMR spectra of the
crude reaction mixtures from cyclization of 20. Moreover,
23 could not be prepared directly from its acyclic precursor
21. Instead, oxidation of 22 under standard conditions provided
23. This macrocycle clearly contains some added constraint as
We also sought to perform additional single-atom replace-
ments within the largazole macrocyclic scaffold to interrogate
very small structural and attendant conformational changes
manifest. Due to the inherent acid instability of oxazolines,
additional protecting group manipulations were required for
synthesis of oxazoline-oxazole substrate 39 (Scheme 5).
Thus, oxazole 31 could be saponified and coupled to
R-methyl serine.13 Switching the nitrogen-protecting group
then allowed for cyclization and deprotection/acylation with
thiazolidine-2-thione 35 to obtain alcohol 36. Coupling to
Fmoc-L-valine then provided the acyclic precursor 38.
Finally, deprotection under basic conditions and cyclization
gave macrocycle 39a. In this case, removal of the trityl group
1
demonstrated by the presence of rotamers in the H NMR
spectrum in CDCl3. Both substrates 22a and 23a could be
deprotected in good yield using our standard conditions.
Replacement of the thiazole moiety with a pyridine residue
in the heterocyclic backbone was readily ammenable to our
(11) (a) Wang, D.-F.; Helquist, P.; Wiech, N. L.; Wiest, O. J. Med.
Chem. 2005, 48, 6936–47. (b) Ryan, Q. C.; Headlee, D.; Acharya, M.;
Sparreboom, A.; Trepel, J. B.; Ye, J.; Figg, W. D.; Hwang, K.; Chung,
E. J.; Murgo, A.; Melillo, G.; Elsayed, Y.; Monga, M.; Kalnitskiy, M.;
Zwiebel, J.; Sausville, E. A. J. Clin. Oncol. 2005, 23, 3912–22. (c) Bouchain,
G.; Delorme, D. Curr. Med. Chem. 2003, 10, 2359–72.
(12) (a) Vanommeslaeghe, K.; Loverix, S.; Geerlings, P.; Tourwe, D.
Bioorg. Med. Chem. 2005, 13, 6070–82. (b) Vanommeslaeghe, K.; De Proft,
F.; Loverix, S.; Tourwe, D.; Geerlings, P. Bioorg. Med. Chem. 2005, 13,
3987–92.
(13) R-Methylserine provided as a generous gift by Ajinomoto Co.,
Japan.
Org. Lett., Vol. 11, No. 6, 2009
1303

Scheme 5
.
Synthesis of the Oxazoline-Oxazole Analog
Table 1. Biochemical Inhibition of Human HDACs (IC50, µM)
compound
HDAC1 HDAC2 HDAC3 HDAC6
largazole thiol (1b)
enantiomer (2)
0.0012
1.2
0.0035
3.1
0.0034
1.9
0.049
2.2
C-2 epimer (3)
0.030
0.11
0.082
0.80
0.084
0.58
0.68
13
proline substitution (4)
largazole-azumamide
hybrid (9b)
>30
0.27
23
0.67
1
>30
4.1
>30
>30
>30
>30
0.7
0.24
0.13
>30
benzamide (11b)
benzamide (12b)
thioamide (13b)
thioamide (14b)
4.1
29
14
1.6
0.96
1.5
1.9
cysteine substitution 22b 0.0019
0.0048
0.12
0.0038
0.085
thiazole-thiazole (23b)
thiazole-pyridine
substitution (29b)
oxazoline-oxazole (39c)
SAHA
0.077
0.00032 0.00086 0.0011
0.029
0.045
0.013
>30
0.00069 0.0017
0.0015
0.017
0.17
0.010
0.045
0.026
0.13
MS-275
(as for 11b, 12b, 13b, and 14b). While none of the zinc-binding
replacements for the natural 3-hydroxy-7-mercaptohept-4-enoic
acid moiety present in largazole, FK228, and spiruchostatin
yielded more potent analogs, many additional zinc-binding
replacements are evident and will be evaluated in due course.
We were pleased to observe a significant increase in potency
with pyridine substitution of the thiazole; this compound (29b)
possesses subnanomolar activity against Class I HDACs and
will be studied further in translational models of cancer.
Compound 29b now constitutes the most biochemically potent
Class I HDAC inhibitor known being between three to four
times more potent than largazole itself against HDACs 1, 2,
and 3. Most notably, we have demonstrated that the methyl
substituent of the thiazoline ring is nonessential for the dramatic
potency of the natural product (cf. 22b). The commercial
availability of relatively inexpensive cysteine, compared with
that of the R-methylcysteine residue of natural largazole,
permitted for a reduction in the overall synthetic approach to
22b by four steps, establishing a high-yielding, scalable, five-
step synthesis of this agent. This highly efficient synthesis is
compatible with further derivitization and potential for practical
scale-up endeavors. The present study provides additional
insight into the structural, functional, stereochemical, and
conformational aspects of the largazole molecular scaffold that
constitutes the basis for the further design and synthesis of
extraordinarily potent HDAC inhibitors with potential thera-
peutic significance. Studies along these lines are under intensive
investigation in our laboratories.
was performed with iodine in methanol, yielding exclusively
the disulfide homodimer (39c) resulting from spontaneous
autoxidation of the incipient thiol (39b). The dimer is reduced
to the active thiol (39b) under the reducing conditions (TCEP)
of our biochemical assay. We next performed comparative
profiling of largazole thiol and the thiol synthetic derivatives
described herein, for inhibitory potency and selectivity against
HDAC1, HDAC2, HDAC3/NCoR, and HDAC6. A complete
description of the assay has been provided previously7 and is
described in detail in the Supporting Information.
In brief, small-molecule inhibitors are arrayed at twelve-point
dose-response (3-fold increments) in 384-well library plates
and transferred by a robotic pin device to replicate assay plates
containing assay buffer under reducing conditions (TCEP 200
µM). A liquid handling device then transfers a tripeptide
substrate terminating in acetyl-lysine and amide conjugated to
4-methyl-7-aminocoumarin (AMC), recombinant human histone
deacetylase (BPS Bioscience, San Diego, CA), and recombinant
human trypsin (Sigma-Aldrich, St. Louis, MO). Following
deacetylase hydrolysis of acetyl-lysine, trypsin cleavage liberates
the AMC fluorophore. Kinetic (fluorescence per unit time) and
end-point (total fluorescence) data are captured by a multilabel
plate reader. Replicate data are analyzed by curve-fit using
logistic regression (Spotfire Decision-Site). A summary of assay
data is in Table 1.
Several striking observations emerge from this data set. The
largazole enantiomer (2) exhibits a decrease in potency by
almost exactly 3 orders of magnitude for all isoforms tested,
underscoring the obligate, stereochemical, and conformation-
activity relationship between the natural product and its protein
targets. This is further substantiated by the intermediate potency
of the C-2 epimer (3), the valine-to-proline substitution (4), and
the oxdized and strained thiazole-thiazole derivative (23b). It
is significant to note that the single-atom substitutions of the
sulfur atoms for oxygen atoms in the oxazoline-oxazole
derivative (29b) provided a compound equipotent to largazole
itself. Our synthetic approach has allowed rapid diversification
of the zinc-binding arm, modulating both potency and specificity
Acknowledgment. We thank the CSU Cancer Supercluster
and the NIH for financial support and for a postdoctoral
fellowship for AAB (NCI Grant CA136283). Mass spectra
were obtained on instruments supported by the NIH Shared
Instrument Grant GM49631. We thank Ralph Mazitschek
for the provision of acetylated substrate. J.E.B. acknowledges
supportbygrantsfromtheNationalCancerInstitute(1K08CA128972)
and the Burroughs-Wellcome Foundation (CAMS).
Supporting Information Available: Spectroscopic data
and experimental details for the preparation of all new
compounds as well as procedures for the biochemical HDAC
assay used in these experiments are provided. This material
is available free of charge via the Internet at http://
pubs.acs.org.
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Org. Lett., Vol. 11, No. 6, 2009